INFORMATION SYSTEMS FOR BIOTECHNOLOGY


September 2003
COVERING AGRICULTURAL AND ENVIRONMENTAL BIOTECHNOLOGY DEVELOPMENTS


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IN THIS ISSUE:



USDA TO REQUIRE PERMITS FOR ALL INDUSTRIAL BIOTECH PLANTS

On August 5, 2003, the U.S. Department of Agriculture's Animal and Plant Health Inspection Service announced it is amending its biotechnology regulations as they pertain to plants designed to produce industrial compounds. Entities wishing to move, field test, or import these types of engineered plants must apply for a permit.

Previously, APHIS allowed companies and institutions to field test, move or import plants genetically engineered to produce industrial compounds under its notification process, which is an expedited permitting procedure. The notification process was originally added to the biotechnology regulations in 1993 in order to expedite introductions for genetically engineered plants considered low risk and developed using genetic modifications with which APHIS was already familiar. Previous notifications issued for genetically engineered industrial plants involved plants in which nutritional components, such as oil, were being engineered.

Recently, requests involving genetically engineered industrial plants have utilized new, less familiar processes and non-food, non-feed traits that no longer qualify for the notification process. This interim rule strengthens APHIS regulations for field testing of genetically engineered industrial plants in anticipation of an increase in requests to move, import or field test these types of plants.

Notice of the interim rule appeared in the Aug. 6 Federal Register and was effective upon publication. APHIS documents published in the Federal Register and related information, including the names of organizations and individuals who have commented on APHIS dockets, are available on the Internet at http://www.aphis.usda.gov/ppd/rad/webrepor.html. Consideration will be given to comments received on or before Oct. 6. Send an original and three copies of postal or commercial delivery comments to Docket No. 03-038-1, Regulatory Analysis and Development, PPD, APHIS, Station 3C71, 4700 River Road, Unit 118, Riverdale, Md. 20737-1238. If you use e-mail, address your comments to regulations@aphis.usda.gov. Your comments must be contained in the body of the message; do not send attached files. Please include your name and address in the message and use "Docket No. 03 038 1" on the subject line.

Comments may be reviewed at USDA, Room 1141, South Building, 14th Street and Independence Avenue, S.W., Washington, D.C., between 8 a.m. and 4:30 p.m., Monday through Friday, except holidays. Persons wishing to review comments are requested to call ahead on (202) 690-2817 to facilitate entry into the comment reading room.

Source:

Press Release: http://www.aphis.usda.gov/lpa/news/2003/08/bioplants_brs.html

Meghan Thomas (301) 734-3266
Jerry Redding (202) 720-6959



ASSESSING THE RISKS OF TRANSGENE ESCAPE: A CASE STUDY IN SUNFLOWERS
John M. Burke

In recent years, the potential impact of transgenic crops on the environment has been a topic of intense international debate. Arguments in favor of genetic engineering point to the possible environmental benefits of genetically modified (GM) crops. These include a reduction in the amount of chemicals applied to agricultural systems, a transition to less toxic chemical treatments, and the facilitation of zero-till agriculture. Environmental objections to GM crops, on the other hand, are largely based on factors such as the possible negative effects of transgenes on non-target organisms and the potential for transgene escape via crop-wild hybridization to facilitate the evolution of increasingly weedy or invasive plants.

Although it may take years for the true environmental effects of transgene escape to be known, predictions regarding the particular crops or traits that are likely to pose the greatest environmental risks can be made. For example, crops that hybridize readily with wild relatives represent greater risks than those that do not. Likewise, transgenes that are advantageous in wild or weedy forms of a plant are most likely to pose a significant risk, whereas those that are neutral or disadvantageous will do little to disrupt the evolutionary dynamics of the recipient population(s). Current concern stems from the fact that many of the traits that are the target of genetic manipulation—such as pest or pathogen resistance and tolerance of various abiotic stresses—may be highly advantageous in the wild.

In many cases, the conditions necessary for hybridization between crop plants and their wild relatives are met, and hybridization appears to be frequent. For example, there is evidence that twelve of the world's thirteen most important food crops hybridize with at least one wild relative in at least part of their range of cultivation1. Thus, the question of whether or not genes will ultimately escape from cultivation has been largely answered; in most cases, they will. Research on the risks associated with transgene escape should, therefore, focus on the fitness consequences of the gene(s) in question, rather than on rates of gene flow. Until recently, however, virtually nothing was known about the fitness effects of pest or pathogen resistance transgenes in wild plant populations.

In a recent study2, we examined the fitness effects of a transgene that confers resistance to white mold (Sclerotinia sclerotiorum) following its `escape' from cultivated into common sunflower (Helianthus annuus). Of the more than three dozen pathogens that afflict sunflower, white mold is one of the most common and widespread, having been reported from all sunflower growing regions throughout the world. White mold infection, which typically begins at the base of the stem, results in the rapid wilting and death of cultivated sunflower plants, greatly reducing seed output. Infection rates as high as 100% have been reported in North American sunflower fields, and white mold has been known to reduce yield by as much as 70%. Attempts to develop resistant cultivars via traditional plant breeding techniques have met with little success in sunflower, and chemical control methods are often costly and ineffective. Attention has turned, therefore, to genetic modification. Because oxalic acid plays a key role in the pathogenicity of white mold, it has been hypothesized that the insertion of an oxalate oxidase (OxOx) transgene would provide otherwise susceptible plants with a mechanism of resistance3. This approach has now been used by Pioneer Hi-Bred, Intl. to successfully enhance white mold resistance in cultivated sunflower.

Unfortunately, the potential for transgene escape is especially high in sunflower. Nearly all of the cultivated sunflower acreage in the United States is contained within the geographic range of common sunflower, and range-wide surveys of the potential for reproductive contact have revealed that approximately two-thirds of all cultivated sunflower fields in the United States occur in close proximity to, and flower coincidentally with, common sunflower populations4. Moreover, the results of previous research indicate that, where they come into contact, cultivated and wild sunflower often hybridize5. Thus, crop-wild gene flow is a virtual certainty throughout the range of sunflower cultivation in the United States.

The efficacy of the OxOx transgene in cultivated sunflower, combined with the high likelihood of escape, raises the specter of transgene escape, leading to the evolution of a more weedy and invasive common sunflower. We simulated the early stages of transgene escape by crossing the OxOx transgene into common sunflower and growing the resulting plants at field sites located in California, Indiana, and North Dakota. The final result revealed a set of populations consisting of wild-like plants that were segregating for the OxOx transgene. By inoculating a subset of these plants at each location with white mold and keeping the remainder as controls, we were able to examine the fitness benefits afforded by the OxOx transgene in the face of a pathogen challenge, as well as any possible fitness costs associated with it in the absence of white mold.

Overall, our results indicated that there was no "cost of resistance" associated with the OxOx transgene in the absence of a pathogen challenge. This gene did appear, however, to protect its carriers from white mold infection. Although the effect varied across locations, the frequency of infection was generally lower in plants carrying the OxOx transgene than in those that lacked it. In terms of seed output, however, the story was somewhat different. Following inoculation, there was no detectable difference in the productivity of transgenic and non-transgenic individuals. Although the underlying mechanisms remain unknown, this seemingly paradoxical result has a relatively straightforward explanation: The rate and severity of infection were effectively decoupled in this experiment. In California, where the OxOx transgene provided the greatest degree of protection against infection, onset of the disease had virtually no effect on fitness. In contrast, while white mold infection had a major (and negative) impact on fitness in Indiana, infection rates at this location were unaffected by the OxOx transgene.

Taken together, our results suggest that the OxOx transgene will do little more than diffuse neutrally following its escape, and therefore, will have little effect on the evolutionary dynamics of wild sunflower populations. In other words, it appears that, by giving the OxOx transgene to wild sunflower, we effectively gave it something that it already had—i.e., some degree of white mold resistance. This conclusion must be tempered, of course, with the realization that our work was performed within a single growing season and on a single genetic background. It is therefore possible that our results are not generalizable over time or across common sunflower populations. Longer-term studies replicated across various wild genetic backgrounds will be necessary to shed light on these issues. Even long-term studies, however, have their limits; strong but episodic selection can have a major influence on the evolutionary trajectory of populations, yet may be rare enough to avoid detection.

In the broader context, our results illustrate the importance of quantifying transgene fitness more directly than through the use of a presumptive correlate such as disease incidence. Indeed, if we had relied solely upon infection rates, rather than looking directly at reproductive output (albeit only through female function) our conclusions would have been quite different. This work also represents an important counterpoint to a recently published report6 in which a Bt transgene was shown to decrease herbivore damage and increase fecundity in common sunflower grown under field conditions.

Our work, combined with the Bt findings, indicates a clear need to assess the relative risks and benefits of genetic modification on a case-by-case basis. Although increases in reproductive output do not necessarily translate into an increase in weediness or invasiveness, the fitness of an allele remains the best predictor of the likelihood and rate of its spread. Thus, the best means currently available for assessing the environmental risks associated with transgene escape are fitness-related measures. The time has come for us to move beyond hand wringing about the likelihood of transgene escape and to ask the more important question: What will happen if and when these genes gets out?

References

1. Ellstrand et al. (1999) Gene flow and introgression from domesticated plants into their wild relatives. Annual Review of Ecology and Systematics 30: 539-63.

2. Burke & Rieseberg. (2003) Fitness effects of transgenic disease resistance in sunflowers. Science 300: 1250.

3. Thompson et al. (1995) Degradation of oxalic acid by transgenic oilseed rape plants expressing oxalate oxidase. Euphytica 85: 169-72.

4. Burke et al. (2002) The potential for gene flow between cultivated and wild sunflower (Helianthus annuus) in the United States. American Journal of Botany 89: 1550-2.

5. Arias & Rieseberg. (1994) Gene flow between cultivated and wild sunflowers. Theoretical and Applied Genetics 89: 655-60.

6. Snow et al. (2003) A Bt transgene reduces herbivory and enhances fecundity in wild sunflowers. Ecological Applications 13: 279-86.

John M. Burke
Department of Biological Sciences
Vanderbilt University, Nashville, TN
john.m.burke@vanderbilt.edu



A SHORT ROUTE TO GENE DISCOVERY IN TREES: CONTROL OF STATURE THROUGH GIBBERELLIN CATABOLISM
Victor B. Busov, Richard Meilan and Steven H. Strauss

Manipulation of plant stature has long been a major goal in agronomy, horticulture, and silviculture. Dwarf and semi-dwarf "Green Revolution" varieties in wheat and rice contributed to dramatic increases in cereal crop yields. The new varieties were shorter, more resistant to damage by wind and rain (lodging), and responded better to nitrogen fertilizers by increasing grain yield rather than straw biomass. Dwarfing in fruit trees has allowed dense field cultivation, facilitated mechanized maintenance, increased efficiency of fruit collection, and allowed more precise pesticide application, reducing spray drift1. Most of the dwarfing rootstocks induced precocious and profuse flowering. Dwarf ornamental cultivars have also been developed in numerous tree and shrub species, and can provide added safety and low-cost maintenance.

Control of plant stature and form has previously required the use of plant growth regulators or classical plant breeding. Plant growth regulators are exogenously applied to promote or retard elongation, often through chemical alteration of gibberelic acid (GA) biosynthesis. However, stature control through `anti-GA' plant growth retardants requires repeated application of synthetic chemicals that are costly, variable in effectiveness, and can have undesired environmental consequences, or at least perceptions thereof.

Dwarfing alleles that do not produce deleterious consequences for plant vigor, particularly dominant forms most useful in horticulture, are very rare in natural gene pools because they are readily eliminated by natural selection. Healthy dwarf plants, especially in the variety of backgrounds needed for large-scale commercial deployment, are therefore difficult to obtain through classical breeding. Transgenic manipulation of GA levels or signaling through the insertion of dominant dwarfing genes could therefore provide an important alternative approach to control of plant stature2.

Research in the last several years has clearly demonstrated that dwarfism is commonly associated with deficiencies in GA levels or signaling. Gibberellic acids are tetracyclic diterpenoid growth regulators that play a critical role in plant development. The level of bioactive GAs is precisely controlled by several mechanisms, including transcriptional regulation of the genes encoding enzymes from both biosynthetic and catabolic pathways. By modifying transcriptional regulation of genes controlling GA flux, it is possible to modify developmental processes regulated by GA and thus plant stature and form.

The roles various enzymes play in the regulation of bioactive GA levels vary widely. A small number of enzymes near the final biosynthetic and catabolic steps appear to determine the ultimate level of bioactive GAs2 (Figure 1). These enzymes are encoded by small gene families. Thus, these genes are logical targets for biotechnological manipulation of GA levels.

Figure 1. GA metabolic pathway Enzymes in the cytoplasmic stage exert strong control over the final levels of bioactive gibberellins. Overexpression of enzymes from the ER or chloroplast stages does not affect the final level of bioactive GAs. Note that GA2ox affects both the levels of bioactive GAs and the immediate precursors, thus providing strong control over GA influx.

Using activation tagging, we identified a dwarf transgenic hybrid poplar (Populus tremula x P. alba)3. The mutant, which we have dubbed stumpy because of its short, stout form, was approximately four-fold shorter than the corresponding wild type (WT) (Figure 2). The cause of the phenotype was a hyperactivated gene encoding GA 2-oxidase (GA2ox), the major gibberellin (GA) catabolic enzyme in plants. The vegetative characteristics of the stumpy mutant are similar to the phenotype of GA-deficient mutants that contain defective GA biosynthetic or hyper-activated catabolic genes. They show severely reduced stem elongation, decreased leaf size, and dark green foliage. Consistent with the expectation that the mutant phenotype was caused by perturbation of the GA metabolic pathway, we recovered a nearby genomic sequence and then isolated a corresponding cDNA copy of the gene that showed high homology to GA 2-oxidase genes from a number of plant species.


Figure 2. Wild type (WT, right) and stumpy mutant (left) plants after more than a year of growth in the greenhouse. The mutant plant is approximately four-fold shorter that WT. Diameter growth of stumpy is not significantly different than the WT plants. The mutants have a larger diameter at the apex, suggesting less taper. Stumpy also displayed profuse leaf and stem pubescent, most likely resulting from trichome outgrowth.

Analysis of the GA content in the mutant indicates a several-fold decrease in the bioactive GAs (GA1 and GA4) and a several-fold increase in their main C-hydroxylated inactive catabolites (GA8 and GA34). We also detected a nearly two (1.8)-fold increase in GA29, the catabolite of GA20 (the main GA1 precursor). The changes in GA profile were consistent with the expected biochemical function of the GA2ox enzyme. Exogenous application of GA3, which is resistant to catabolism by GA2ox, rapidly restored normal development to the mutant (Figure 3), strongly supporting the hypothesis that the mutant phenotype is a result of the deficiency of the bioactive GAs, GA1 and GA4.

The rapid reversion to normal growth by exogenous application of GA that is resistant to the action of the enzyme provides a potential method for control of transgenic plants overexpressing GA 2-oxidase genes for horticultural purposes. For example, the rate of growth during commercial propagation could be greatly increased by GA application, allowing rapid nursery production. Once GA application ceases after transplanting, the slow growth and dwarf form would resume. Landscape managers might also choose to speed early growth via GA application, thereby allowing growth rate to attenuate only after plants reach a desired size. It can also be used by owners of the plants to achieve a customized form and size.

Trees of short stature can provide substantial benefits for urban forestry and wood products industries. Due to their large size, trees require intensive pruning to avoid damage to homes and power lines. Tree maintenance costs comprise a significant proportion of electrical utility budgets. Utility companies in U.S. spend $1.5 billion per year trimming trees and controlling brush, including herbicide and growth retardant treatments4. Despite these expenditures, trees are the largest single cause of power outages5. Pruning and tree removal are two of the highest street-tree maintenance costs; approximately 58% of urban tree-care budgets are allocated to tree trimming, removal, and disposal6.

Figure 3. Phenotypic rescue of stumpy with GA3 application to the shoot apex. A droplet of 10mM GA3 was applied to the shoot apex of the plant to the left at four-day intervals for two weeks.

When trees are intensively cultured as wood fiber crops, they are likely to benefit from substantial alterations in form and structure that might be achieved by modification of GA metabolism, but are difficult to achieve via conventional breeding7. Domesticated trees may be substantially shorter and stouter, which could reduce the amount of low-quality "reaction wood" that forms in response to bending and leaning. They might also have a higher harvest index, show improved harvesting/handling efficiencies, and have greater unit-area fiber yields. Resistance to water movement, and thus onset of water stress, might be reduced by a shorter distance from soil to photosynthetic surfaces. Crowns could be engineered to be narrow, allowing for a greater number of stems per unit area. Wood properties may also be modified via GA alteration to increase product value, such as by increasing fiber length to yield stronger paper products8.

This work appears to be the first case of successful forward genetics—where a gene has been isolated based on a mutant phenotype—in a tree. Activation tagging, which produces dominant phenotypes, is particularly suitable for identifying genes in trees because of their long juvenile periods and high genetic loads, which make inbreeding to expose loss-of-function (recessive) alleles difficult. Because of the many developmental differences between annual and perennial plants—including vegetative dormancy, delayed onset of flowering, extended periods of secondary (woody) growth, and gradual vegetative maturation—activation tagging and other forward genetic approaches may uncover many more types of regulatory genes useful for tree biotechnology. Gene tagging approaches have become even more powerful in poplars of late; its newly published genome sequence (http://genome.jgi-psf.org/poplar0/poplar0.home.html) greatly speeds the identification of genes. 

References

1. Webster T. (2002) Dwarfing rootstocks: past, present and future. Compact Fruit Tree 35: 67-72.

2. Hedden P. and Phillips AL. (2000) Manipulation of hormone biosynthetic genes in transgenic plants. Current Opinion in Biotechnology 11: 130-137.

3. Busov VB et al. 2003. Activation tagging of a dominant gibberellin catabolism gene (GA 2-oxidase) from poplar that regulates tree stature. Plant Physiology 132: 1283-1291. http://www.plantphysiol.org/cgi/content/abstract/132/3/1283?ct

4. EPRI. (1995) The right tree for the right-of-way. Technical Report-TB-105029.

5. Simpson P and Bossuyt R. (1996) Tree caused electric outages. Journal of Arboriculture 22: 117-121.

6. Nowak DJ. (1990) Street tree pruning and removal needs. Journal of Arboriculture 16: 309-315.

7. Bradshaw HD and Strauss SH. (2001) Breeding strategies for the 21st century: Domestication of poplar. In D. I. Dickman, J. G. Isebrands, J. E. Eckenwalder, and J. Richardson, editors, Poplar Culture in North America, Part B, 383-394. NRC Research Press, Ottawa.

8. Eriksson ME et al. (2000) Increased gibberellin biosynthesis in transgenic trees promotes growth, biomass production and xylem fiber length. Nature Biotechnology 18: 784-788.

Victor B. Busov
Department of Forest Science
Oregon State University

Richard Meilan
Department of Forest Science
Oregon State University

Steven H. Strauss
Department of Forest Science
Oregon State University
steve.strauss@orst.edu


PRODUCTION OF HUMAN SERUM ALBUMIN IN TRANSGENIC CROPS WITHOUT INTERFERING WITH FOOD OR FEED PRODUCTION
Henry Daniell

Reuters News reported on June 25th, 2003, the following: U.S. food products tainted with traces of pharmaceutical crops would immediately be seized from grocery store shelves under a proposal being considered by the Bush administration. Eric Flamm, FDA senior science policy adviser, said the administration was considering a proposal that deems all food products containing medicinal crops as adulterated. ProdiGene Inc., a privately owned Texas biotech firm, agreed to pay about $3 million last year after the USDA accused it of mishandling its biopharmaceutical corn crop and contaminating other crops. The USDA in March implemented stricter rules on new plantings of pharmaceutical crops to prevent another ProdiGene incident. These incidents underscore the need to re-evaluate production of pharmaceutical proteins in food or feed crops.

On the other hand, the daily average income of nearly one billion people is less than one U.S. dollar. Globally, about 170 million people are infected with hepatitis C virus, with 3-4 million new infections each year (WHO fact sheet 164, October 2000). The WHO Department of Communicable Disease Surveillance and Response reports that more than one third of the world population is infected with hepatitis B. In Asia, the prevalence of chronic hepatitis B and C is very high (about 110 million infected by HCV and 150 million infected by HBV). A large majority of hepatitis C infected patients have severe liver cirrhosis and currently there is no vaccine available for this disease. The annual requirement of insulin-like growth factor I, per cirrhotic patient, is 600 mg (1.5–2 mg per day) and the cost of IGF-1 per mg is $30,000. Currently, the annual cost of interferon therapy for viral hepatitis is $26,000 per year (Cowley & Geoffrey, Newsweek, April 22, 2002). Therefore, agricultural scale production of therapeutic proteins and vaccines (especially for agents of bioterrorism) is necessary to meet such a large demand at a reasonable cost. However, this production should be achieved without contaminating the world's food supply and harming the environment. Therefore, one should explore alternate non-food and non-feed crops that could be used to produce therapeutic proteins.

Tobacco: An ideal crop for therapeutic proteins
Tobacco is a non-food and non-feed crop and an ideal choice for the production of therapeutic proteins because of its relative tractability to genetic manipulation and an impending need to explore alternate uses for this crop. Tobacco is an excellent biomass producer (in excess of 40 tons of leaf fresh weight/acre, based on multiple cuttings per season) and a prolific seed producer (up to one million seeds produced per plant), thus hastening the time in which a product can be scaled up and brought to market.

Tobacco is widely used as a model system to test the suitability of plant-based expression systems for production of therapeutic proteins and other transgenes. More transgenes have been introduced into the tobacco chloroplast or nuclear genome than all other crop species combined. Both nuclear and chloroplast genomes of tobacco have been transformed with relative ease. Tobacco is a self-pollinating crop and there are no known wild or cultivated relatives in North America. Several studies have established that transgenes are maternally inherited when introduced via the chloroplast genome in tobacco. In addition, both male sterility and seed sterility techniques have been developed already for tobacco. Thus, advanced technology is readily available for containment of gene flow through pollen and seeds.

Plant derived biopharmaceuticals and human proteins

Availability of recombinant human proteins has revolutionized the use of therapeutically valuable proteins in clinical medicine. Plants offer a suitable alternative to microbial or animal expression of biopharmaceutical proteins because of their inexpensive production costs and absence of human pathogens. However, there are some limitations. In particular, expression of human proteins in nuclear transgenic plants has been disappointingly low: e.g., human serum albumin, 0.02% of total soluble protein (tsp); human interferon-alpha, 0.000017% of fresh weight; human epidermal growth factor, 0.001% of tsp; and erythropoietin, 0.0026% of tsp. Therefore, it is important to increase levels of expression in order to exploit plant production of pharmacologically important proteins.

Chloroplast transgenic system
Chloroplast genetic engineering was conceived as a novel approach to increase expression level and overcome problems of nuclear genetic engineering (Daniell et al., U.S. Patents 5,932,479; 5,693,507, and others). Foreign genes have been integrated into the chloroplast genome of several crop plants, including tobacco, tomato, and potato (up to 10,000 copies of transgenes per cell), resulting in accumulation of recombinant proteins several hundred fold higher than nuclear transgenic plants (up to 47% of the total soluble protein). Targeted integration of transgenes at specific sites, into the chloroplast genomes, eliminates the "position effect" frequently observed in nuclear transgenic plants resulting from random integration of transgenes. In addition, gene silencing has not been observed in transgenic chloroplasts, in spite of extraordinarily high levels of transgene expression, whereas it is a common phenomenon in nuclear transformation. Because of these reasons, expression and accumulation of foreign proteins is uniform in independent chloroplast transgenic lines1.

It has been shown that multiple genes can be engineered in a single transformation event via the chloroplast genome, regulated by a single promoter; this facilitates coordinated expression of multi-subunit proteins, multi-component vaccines, or engineering new pathways. This was demonstrated by successful expression and assembly of monoclonal antibodies, or multigene bacterial operons in transgenic chloroplasts2. Yet another advantage is the lack of toxicity of foreign proteins to plant cells when they are compartmentalized within chloroplasts. Chloroplast genetic engineering is an environmentally friendly approach, minimizing several environmental concerns, including transgene containment3. Most importantly, chloroplasts are able to process eukaryotic proteins, including correct folding and formation of disulfide bridges. Accumulation of large quantities of a fully assembled form of human somatotropin with the correct disulfide bonds provides strong evidence for hyper-expression and assembly of pharmaceutical proteins using this approach. In addition, functional assays showed that chloroplast-synthesized cholera toxin B subunit (CTB) binds to the intestinal membrane GM1-ganglioside receptor, confirming correct folding and disulfide bond formation of the plant-derived CTB pentamers. Such folding and assembly of foreign proteins should eliminate the need for highly expensive in vitro processing of pharmaceutical proteins produced in recombinant organisms. For example, 60% of the total operating cost for the commercial production of human insulin in E. coli is associated with in vitro processing (formation of disufide bridges and cleavage of methionine). Human therapeutic proteins as small as 20 amino acids (magainin) or as large as 83 kDa, requiring formation of a heptamer for functionality (anthrax protective antigen), including human interferons, insulin-like growth factors, etc., have been expressed at very high levels (up to 27% total soluble protein or 7.5 mg/g fresh weight) in transgenic chloroplasts4. Here we discuss a recent report of human serum albumin production in transgenic chloroplasts.

Human Serum Albumin (HSA)
HSA is the most widely used intravenous protein and is prescribed in multi-gram quantities to replace blood volume in trauma and in various other clinical situations. HSA accounts for 60% of the total protein in blood serum. However, HSA is currently extracted only from blood because of the lack of commercially feasible recombinant expression systems. HSA is a monomeric globular pre-protein whose mature form consists of a single polypeptide chain of 585 amino acids (66.5 kDa with 17 disulfide bonds). It is highly susceptible to proteolytic degradation in recombinant systems and is expensive to purify. The annual world need exceeds 500 tons, representing a market value of more than $1.5 billion. Lack of glycosylation facilitates production of functional HSA in prokaryotic systems. Although the HSA gene and cDNA have been expressed in a wide variety of microbial systems, no system is yet commercially feasible. Sijmons et al. made the first reported attempt to express HSA in transgenic plants in 1990, but very low expression levels were attained (0.02% tsp). HSA could not be detected if expressed in the cytoplasm, suggesting that the protein is not stable in this compartment due to high susceptibility to proteolytic degradation. Estimates by industry suggest that the cost-effective yield for pharmaceutical production is 0.1 mg of HSA per g of fresh weight.

In addition, good recombinant systems are still not available for many human proteins that are expensive to purify or highly susceptible to proteolytic degradation. It is known that traditional purification of biopharmaceuticals using columns accounts for 30% of the production cost and 70% of the setup cost. The increasing production of proteins in heterologous hosts through the use of recombinant DNA technology has brought the problem of proteolytic degradation into focus; heterologous proteins appear to be more prone to proteolysis. Recombinant proteins are often regarded by a cell as foreign, and therefore, degraded much faster than most endogenous proteins. Proteolytic stability of recombinant proteins is a significant factor influencing the final yield.

Hyper-expression of HSA in transgenic chloroplasts
We have recently reported a more efficient method of recombinant HSA production, which may be used as a model system to enrich or purify biopharmaceutical proteins from transgenic plants that are highly susceptible to proteolytic degradation5. Expression of HSA in transgenic chloroplasts using Shine-Dalgarno sequence (SD), that usually facilitates hyper-expression of transgenes, resulted only in 0.02% HSA in total protein (tp). Modification of HSA regulatory sequences using chloroplast untranslated regions (UTRs) resulted in hyper-expression of HSA (up to 11.1% tp), compensating for excessive proteolytic degradation. This is 500-fold higher than previous reports on HSA expression in transgenic leaves. HSA was translated so rapidly using UTRs that aggregates were formed within transgenic chloroplasts and all such aggregates were in the pellet rather than in the supernatant when crude plant extracts were centrifuged. Electron micrographs of immunogold-labelled transgenic chloroplasts revealed such HSA aggregates, which provided a simple method for purification from other cellular proteins (see Figure 1). HSA aggregates could be readily solubilized to obtain a monomeric form using appropriate reagents. Regulatory elements used in this study should serve as a model system for enhancing expression of foreign proteins that are highly susceptible to proteolytic degradation and provide advantages in purification when inclusion bodies are formed.

Figure 1. Human serum albumin aggregates seen in transgenic chloroplasts, detected by immunogold labelling and transmission electron microscopy.

References

1. Daniell H, Khan MS, and Allison L. (2002) Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology. Trends Plant Sci. 7, 84-91.

2. Daniell H and Dhingra A. (2002) Multigene engineering: Dawn of an exciting new era in biotechnology. Curr. Opin. Biotechnol. 13, 136-141.

3. Daniell H. (2002) Molecular strategies for gene containment in transgenic crops. Nat. Biotechnol. 20, 581-586.

4. Daniell H. (2003) Medical molecular farming: expression of antibodies, biopharmaceuticals and edible vaccines via chloroplast genome (Vasil, I.K. ed.), pp 371-376. Kluwer Academic Publishers, Netherlands.

5. Fernandez-San millan A, Mingo-Castel A, Miller M, and Daniell H (2003) A chloroplast transgenic approach to hyper-express and purify human serum albumin, a protein highly susceptible to proteolytic degradation. Plant Biotechnology Journal 1: 71-79.

Henry Daniell
Dept. Molecular Biology & Microbiology
University of Central Florida
daniell@mail.ucf.edu



SIX BILLS TO TREAT GM ILLS
Phillip B.C. Jones

Just before the August recess, Presidential candidate Congressman Dennis J. Kucinich (D-OH) introduced six bills intended to provide a regulatory framework for genetically modified (GM) plants, animals, and bacteria. The legislation contains an assortment of changes, including mandatory labeling for GM food, increased liability and a new tax for agbiotech companies, extra hurdles for GM food approval, and a moratorium for GM crops that produce pharmaceuticals or industrial chemicals.

H.R. 2916 ("Genetically Engineered Food Right to Know Act") would amend the Federal Food, Drug and Cosmetic Act, the Federal Meat Inspection Act, and the Poultry Products Inspection Act to require the labeling of food that contains GM material or that is produced with GM material. Food would not have to be labeled, however, if it is served in restaurants or contains adventitious GM material amounting to one percent or less. This legislation is based on the theory that consumers have a "right to know" whether their food contains or was produced with GM material, a notion rejected by at least one federal appellate court. H.R. 2916's labeling requirement also runs against the grain of the Bush administration's dispute at the World Trade Organization against the European Union's similar mandatory labeling regulation.

In an attempt to raise the bar for approval of GM food, H.R. 2917 ("Genetically Engineered Food Safety Act") would amend the Federal Food, Drug and Cosmetic Act to require the Food and Drug Administration to regulate GM material in food as a "genetic food additive." According to the bill's drafters, this classification would give the FDA discretion in applying additional factors to evaluate the safety of GM food.

H.R. 2918 ("Genetically Engineered Crop and Animal Farmer Protection Act of 2003") is intended to grant extra protection for farmers and ranchers who have been harmed economically by GM seeds, plants, or animals, and to ensure fairness in dealings between these individuals and agbiotech companies. The bill lists four actions of biotech companies that have allegedly eliminated farmers' basic rights: patenting seeds, depriving farmers the right to save seed (presumably, derived from patented material), unreasonable seed contracts, and intrusion into everyday farm operations. A loss of markets and a potential increased liability caused by GM crops are also a concern here.

To address these matters, H.R. 2918 would prohibit certain provisions in a contract for the sale of a GM animal, GM plant, or GM seed to a purchaser for use in agricultural production, including: a provision that prohibits the purchaser from retaining a portion of the harvested crop for future planting by the purchaser or that charges a fee to retain a portion of the harvested crop for future planting; a provision that shifts any liability from the biotech company to the purchaser; and a provision that requires the purchaser to grant the seller's agents access to the purchaser's property. The bill also requires the Secretary of Agriculture to issue rules that compel effective mitigation strategies for any GM crop that is a predominately outcrossed pollinator. H.R. 2918 would further amend the Federal Insecticide, Fungicide, and Rodenticide Act to establish a plan to prevent the development of pests resistant to Bacillus thuringiensis toxin. After establishing the "resistance plan," the agency must revoke all registrations for Bt toxin-producing plants that are not in compliance.

H.R. 2919 ("Genetically Engineered Organism Liability Act of 2003") establishes that a biotech company is liable to any party injured by the release of a GM organism into the environment if that injury is the consequence of the genetic engineering of the organism. "Injury" includes any liability of a person who uses the company's GM organism in accordance with applicable Federal and State law. For the purpose of this bill, a biotech company is not only a company (or individual) engaged in the business of genetically engineering an organism, but also a company (or individual) that is obtaining the patent rights to such an organism for the purposes of commercial exploitation.

Modestly titled "Real Solutions to World Hunger Act of 2003," H.R. 2920 aims to ensure that efforts for ending world hunger through the use of GM animals and GM crops actually help developing countries while protecting human health and the environment. The bill would make it unlawful for a person to ship to a foreign country any GM animal, GM plant, or GM seed that the person knows, or has reason to believe, will be used by the ultimate purchaser to produce an agricultural commodity if: (1) the GM material was denied Federal approval for commercial marketing in the United States, or the GM material was the subject of a withdrawn application for Federal approval; or (2) the government of the foreign country has not certified that ecological impacts related to the importation of the GM material have been mitigated to the satisfaction of the foreign government. H.R. 2920 would also create a tax for agbiotech companies, which would be used to promote the development of sustainable agriculture techniques that do not use any GM material.

H.R. 2921 ("Genetically Engineered Pharmaceutical and Industrial Crop Safety Act of 2003") would prohibit the open-air cultivation of GM pharmaceutical and industrial crops, prohibit the use of common human food or animal feed as the host plant for a GM pharmaceutical or industrial chemical, and establish a tracking system to regulate the growing, handling, transportation, and disposal of pharmaceutical and industrial crops and their byproducts. The bill would forbid the cultivation of a pharmaceutical crop or industrial crop until the final regulations and tracking system are in effect. The Secretary of Agriculture would have to identify the "common foods," which cannot be genetically engineered to produce pharmaceuticals or industrial chemicals, and to establish the tracking system. H.R. 2921 would derail the U.S. Department of Agriculture's current efforts to regulate GM plants that produce pharmaceutical and industrial compounds.

All bills have been referred to House committees for review. The Thomas website (http://thomas.loc.gov) is a good place to keep track of the bills' progress.

Defragmenting Public Sector Patent Rights
Four years ago, Professor Ingo Potrykus (Swiss Federal Institute of Technology, Zurich) and Dr. Peter Beyer (University of Freiburg, Germany) announced their creation of Beta-carotene-enhanced golden rice. They had developed the GM rice to help prevent vitamin A deficiency in the poor and disadvantaged of developing countries, a goal that could be realized if subsistence farmers obtained the rice free of charge and restrictions. But then Potrykus and Beyer found themselves entangled in a patent thicket. The International Service for the Acquisition of Agri-Biotech Applications ran an audit of intellectual property rights and found that the researchers had used 70 patented inventions belonging to 32 companies and universities, and that some of the materials had been used under agreements that restricted further dissemination. Eventually, Potrykus and Beyer overcame their limited freedom to operate by acquiring free licenses for humanitarian use to cover all of the intellectual and technical property. The lesson was not forgotten, however.

A recent study by the University of California, Berkeley, revealed that public sector researchers have created about 25 percent of patented agbiotech inventions. This intellectual property is highly fragmented across institutions and much has been licensed under terms that might restrict access to the technologies. Representatives from 14 universities, foundations, and non-profit research institutions decided that a collective management regime was needed to assess freedom to operate issues, and to overcome the fragmentation of public sector intellectual property rights. To develop a strategy and to implement it, they established the Public-sector Intellectual Property Resource for Agriculture.

PIPRA has three near-term objectives. One objective is to review public sector intellectual property licensing practices and to seek a series of best practices that will encourage the greatest commercial development of publicly funded research innovations while retaining rights for public research institutions.

The second objective is to develop a public intellectual property asset database that will provide an overview of intellectual property rights currently held by the public sector, including current information about licensing status. This should enable public sector researchers to evaluate freedom to operate obstacles at the initiation of their projects.

The third objective is to explore the possibility of making technology "packages" available to member institutions and to the private sector for commercial licensing or for designated humanitarian use. These patent packages could include complementary sets of key technologies, which reduce the transaction costs associated with negotiating a large number of licenses. Various U.S. industries have used this patent pooling strategy during the past 150 years, not always with the federal government's approval.

Additional information about the initiative can be found on the PIPRA website (http://www.pipra.org).

Selected References

Atkinson RC, Beachy RN, Conway G et al. (2003) Public sector collaboration for agricultural IP management. Science 301:174-175, July 11.

Potrykus I. 2001. Golden rice and beyond. Plant Physiol. 125(3):1157-1161.

Phillip B. C. Jones, PhD., J.D.
Spokane, Washington
PhillJones@nasw.org





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