PARLIAMENTARY INFORMATION AND RESEARCH SERVICE SERVICE D INFORMATION ET DE RECHERCHE PARLEMENTAIRES

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1 PRB 05-09E MOLECULAR FARMING Sonya Norris Science and Technology Division 4 July 2005 PARLIAMENTARY INFORMATION AND RESEARCH SERVICE SERVICE D INFORMATION ET DE RECHERCHE PARLEMENTAIRES

2 The Parliamentary Information and Research Service of the Library of Parliament works exclusively for Parliament, conducting research and providing information for Committees and Members of the Senate and the House of Commons. This service is extended without partisan bias in such forms as Reports, Background Papers and Issue Reviews. Analysts in the Service are also available for personal consultation in their respective fields of expertise. CE DOCUMENT EST AUSSI PUBLIÉ EN FRANÇAIS

3 TABLE OF CONTENTS Page INTRODUCTION... 1 TERMINOLOGY... 1 OVERVIEW OF MOLECULAR FARMING... 2 ADVANTAGES OF PLANTS AS EXPRESSION SYSTEMS... 3 CONCERNS REGARDING MOLECULAR FARMING... 5 A. Use of Food Crops... 5 B. Biodiversity... 6 C. Allergies... 7 CURRENT AREAS OF RESEARCH... 8 CANADIAN REGULATION OF MOLECULAR FARMING... 9 CONCLUSION SELECTED REFERENCES... 11

4 MOLECULAR FARMING INTRODUCTION The issue of genetically modified crops has been around for a number of years and continues to be a controversial subject. (1) Molecular farming is an application of this technology; it involves the use of plants, and potentially also animals, as the means to produce compounds of therapeutic value. This paper will analyze the technique of molecular farming, potential applications, and the regulatory framework in Canada, as well as the benefits and risks involved. TERMINOLOGY A number of different terms occur in reference to the use of genetically altered organisms for the production of therapeutics. Pharming is frequently used; in some instances it refers to the use of plants for the production of pharmaceuticals, but often it describes the use of animals, not plants, for the production of drugs. The Canadian Food Inspection Agency uses the term Plant-made Pharmaceutical (PMP) throughout its documentation. In some instances organizations will specify Plant Molecular Farming, to avoid confusion with the animal-sourced technique. Biomanufacturing (use of biological organisms to manufacture products of interest) and biopharmaceuticals (pharmaceuticals from biological organisms) are also terms that appear frequently in the literature. The terms plant-derived product of interest (PPI) and plants with novel traits (PNT) are also found in some cases. (1) For more information, see two publications by Frédéric Forge: Genetically Modified Foods, PRB 99-12E, Library of Parliament, Ottawa, 1 November 1999; and Genetically Modified Organisms, TIPS-2E, Library of Parliament, Ottawa, 11 June 2004.

5 OVERVIEW OF MOLECULAR FARMING 2 Historically, plants have been a primary source for medicinal products for many centuries. Many of the therapeutically active compounds in plants have been identified in the past century. Advances in molecular biology have resulted in the ability to produce some drugs by recombining the gene for the desired product within the genetic material of bacteria. A vast quantity of the transgenic bacteria is then grown and the desired protein purified from it. This recombinant technology, such as is used in the production of insulin, is molecular farming in its simplest form. Due to increased demand, however, and also to the impracticality, and sometimes impossibility, of purifying compounds from their natural source or transgenic bacteria or animal cell culture, it has become necessary to find alternative ways of producing these compounds. In addition, the recent completion of the Human Genome Project (2) has led to the identification of several genes, with many more to follow. It is very likely that, among the genes identified, there will be those that code for proteins of therapeutic value. The organism or material into which the new genetic information is inserted is often referred to as the expression system, (3) since it serves as the system for expressing the new product. Several expression systems have been explored, and each has advantages and disadvantages. The ideal expression system must: produce the desired, functional product; be cost-effective; allow for convenient storage and distribution of the desired product; come with little or no risk (perceived or real); and not be time-consuming. Expression systems that have been studied in addition to bacteria include plant viruses, yeast, animal cell cultures, transgenic plants and transgenic animals. Plants offer advantages over live animals and animal cell cultures in terms of safety, cost, time involved, and storage and distribution issues; plant expression systems are also believed to be better than microbes in terms of cost, protein complexity, and storage and distribution issues. Advantages and concerns regarding molecular farming are discussed later. (2) For more information on this subject, see Sonya Norris, The Human Genome Project and Beyond: Canada s Role, PRB 00-11E, Library of Parliament, Ottawa, rev. 4 July 2005; and Tim Williams, The Human Genome Project and Its Ethical, Legal and Social Implications, PRB 00-08E, Library of Parliament, Ottawa, 26 July (3) In molecular biology, expression is the process by which a gene s coded information is converted into the functioning protein.

6 3 Since 1982, 95 therapeutic proteins expressed in microbial or animal cell cultures have been licensed for production in microbial or animal cell cultures. The bioreactors containing cell cultures that are required for large-scale production are extremely expensive, and increasing demand for therapeutics prompted researchers to seek new ways of producing large quantities of affordable and safe drugs. That search led them to explore the potential of plants. Human proteins were first expressed in plant systems as long as 20 years ago, but these were relatively simple proteins. Proteins are molecules composed of long chains of subunits call amino acids. Proteins can be relatively small and contain dozens of amino acids, or they can be very large and be composed of hundreds of the subunits. These chains of amino acids have characteristic folding patterns and may require extra additions and bond formations ( post-translational modification ). Insulin, although an indispensable compound, is quite a simple molecule, composed of two linked chains totalling 51 amino acids. Similarly simple proteins were successfully expressed in plants between 1986 and 1990, namely, a modified human growth hormone; interferon (used in treating a range of diseases); and serum albumin (used as a substitute for plasma in the treatment of shock). However, proteins are often complex, three-dimensional structures requiring the proper assembly of two or more subunits. Researchers demonstrated in 1989 and 1990 that plants were capable of expressing such proteins and assembling them in their active form when functional antibodies (4) were successfully expressed in transgenic plants. Bacteria do not have this capacity. In addition to antibodies, other compounds that have been successfully expressed include enzymes, hormones, interleukins, blood proteins and vaccines. Although none are yet commercially available, a number have progressed through development stages and on to pre-clinical and clinical trials. ADVANTAGES OF PLANTS AS EXPRESSION SYSTEMS Global demand for pharmaceuticals is at unprecedented levels, and current production capacity will soon be overwhelmed. Expanding the existing microbial systems, although feasible for some therapeutic products, is not a satisfactory option on several grounds. First, it would be very expensive for the pharmaceutical companies. Second, other proteins of (4) Antibodies are proteins that bind specifically to other molecules (antigens), typically foreign to the organism, to aid in eliminating them from the body or preventing them from binding to certain tissues.

7 4 interest are too complex to be made by microbial systems. These proteins are currently being produced in animal cell cultures, but the resulting product is often prohibitively expensive for many patients. Finally, although it is theoretically possible to synthesize protein molecules by machine, this works only for very small molecules, less than 30 amino acid residue in length. Virtually all proteins of therapeutic value are larger than this and require live cells to produce them. For these reasons, science has been exploring other options for producing proteins of therapeutic value. The use of plants offers a number of advantages over other expression systems. Table 1 compares various properties of the different approaches. Plants can be used in two ways. One way is to insert the desired gene into a virus that is normally found in plants, such as the tobacco mosaic virus in the tobacco plant. The other way is to insert the desired gene directly into the plant DNA to produce a transgenic plant. Expression System Cost of maintaining Type of storage Gene size (protein) restriction Production cost Yeast Bacteria Table 1 Plant Viruses Transgenic Plants Animal Cell Cultures Transgenic Animals inexpensive inexpensive inexpensive inexpensive expensive expensive -2.0 C -2.0 C -2.0 C RT* N 2 ** N/A Unknown Unknown Limited Not limited Limited Limited Medium Medium Low Low High High Protein yield High Medium Very high High Therapeutic risk Medium to high High Unknown yes Unknown Unknown yes yes * RT room temperature. ** N 2 culture must be maintained under nitrogen gas. Plant expression systems offer advantages over other systems in a number of areas. Like animals, plants are complex, multicellular organisms and therefore their process of protein synthesis is more similar to that of animals than those of bacteria or yeast, which are not capable of producing complex proteins. Another advantage of using plants is that proteins that are produced in a plant accumulate to high levels in its tissues. Additionally, the use of plants

8 5 avoids the risk of contamination with animal pathogens, such as viruses, that could be harmful to humans. No plant viruses have been found to be pathogenic to humans. Purification of the desired product from plants is often easier than from bacteria, which can be labour- and cost-intensive. Whereas transgenic plants, or virus-infected plants, can be grown on an agricultural scale requiring only water, minerals and sunlight, mammalian cell cultivation is an extremely delicate process that is also very expensive, requiring bioreactors that cost several hundred million dollars when production is scaled up to commercial levels. CONCERNS REGARDING MOLECULAR FARMING While molecular farming is one application of genetic engineering, there are concerns that are unique to it. In the case of genetically modified (GM) foods, concerns focus on the safety of the food for human consumption. In response, it has been argued that the genes that enhance a crop in some way, such as drought resistance or pesticide resistance, are not believed to affect the food itself. Other GM foods in development, such as fruits designed to ripen faster or grow larger, are believed not to affect humans any differently from non-gm varieties. In contrast, molecular farming is not intended for crops destined for the food chain. It produces plants that contain physiologically active compounds that accumulate in the plant s tissues. Considerable attention is focussed, therefore, on the restraint and caution necessary to protect both consumer health and environmental biodiversity. A. Use of Food Crops In 2004, the National Academy of Sciences in the United States published a report entitled Biological Confinement of Genetically Engineered Organisms. The report noted that absolute containment of crops is virtually impossible, and made two significant observations: the need for bioconfinement, or crop containment, must be explored early in the development of the plant; and non-food crops should be considered for those products that need to be kept out of the food supply. Ensuring that transgenic crops do not contaminate crops destined for the food supply requires that there be no cross-pollination between the crops, that no transgenic seeds be

9 6 left behind in fields where food crops are to be planted, and that there be no contamination of a harvested food crop with a harvested transgenic crop. There have already been examples of these containment breaches. Cross-pollination of GM corn with traditional crops occurred in Iowa in 2002, despite the observance of the required distance between crops. Similarly in Nebraska in 2002, seeds from GM corn were left behind in acreage used subsequently for soybean crops. In that instance, half a million bushels of soybean had to be destroyed when the contaminated crop was harvested and combined with other uncontaminated yields. ProdiGene, a private, Texas-based biotechnology company, was responsible for both of these incidents. Recent interest in edible vaccines has also yielded to caution as to the prudence of this idea. While the concept is intriguing, in practice it could prove quite unsafe. Even the creator of the edible vaccine, Charles Arntzen, now concedes that the idea should be abandoned. Although the ease of administering a vaccine as a food is very appealing, the risk of the vaccine-producing food becoming integrated into the food supply is too great. Research in this area now focuses on non-food crops where the vaccine is subsequently purified and packaged as a pill or capsule. The development of an orally administered vaccine, packaged as a pill or capsule, is in itself novel. It permits wider distribution and easier storage of the product. (5) As a result, developing countries would be capable of distributing much-needed vaccines that are impractical in a liquid form, which requires refrigeration or must be administered via injection. B. Biodiversity Whether molecular farming ultimately uses food or non-food crops, certain environmental concerns need to be addressed. All field crops are subject to ingestion by wildlife. It would even be difficult to guarantee that all wildlife could be kept away from greenhouse crops. The products that accumulate in these plants could be toxic to an animal or could lead to more subtle physiological or behavioural effects. Another aspect that has not yet been adequately researched is whether the altered plants produce a change in their surrounding soil due to alterations in the exudates from the root systems. Such changes could require longer-term studies than those that have been conducted so (5) An oral polio vaccine has been in use for many years, but this is in liquid form that is administered by dropper and swallowed. Distribution to and storage within developing countries are still obstacles, as samples must be shipped and stored in a frozen state. After thawing, samples must be kept at 10 C or below. Unused portions must be discarded after 30 days.

10 7 far. In addition, if a change in the soil composition is established, further investigations would be required as to whether and how such a change would affect biodiversity. Molecular farming could potentially occupy a significant portion of currently farmed land. Despite its many advantages, molecular farming requires much more space than any of the other expression systems. Its critics warn that the practice could lead to: more intensive or aggressive agricultural practices, such as reducing crop rotations or increased herbicide use; encroachment of cropland onto field margins; and conversion of natural habitats to cropland. These criticisms are also used to condemn research into biofuels, such as ethanol from corn, and it is unlikely that pharma-crops would be grown on nearly the same scale as biofuel crops. The practice of molecular farming could also lead to a change in the proportion of crops grown in certain regions. This might affect biodiversity, because different crop types are attractive to different wildlife. Also, soil depletion and pesticide use varies depending on crop type. For example, tobacco is seen as a very attractive candidate for molecular farming. However, tobacco crops are not attractive to many species of birds and are responsible for a high rate of soil nutrient depletion. In addition, tobacco is prone to many diseases and therefore requires several applications of pesticides that can be harsh on wildlife. C. Allergies Concern over allergies has been raised for PMPs, as it has for GM crops, but in a reverse sense. Many critics of GM crops argue that allergies could be triggered by inserting a gene from an allergen such as peanuts into another plant. Despite scientists assertion that the inserted gene does not code for the allergen, critics fear that expression of the transgenic peanut gene in the new host plant could provoke an allergic response. With respect to PMP, the concern is not with the transgene, but with the host plant itself. Critics point out that allergies exist to a wide variety of plants, and that purification of the PMP from the plant could include contaminants that might induce an allergic response. For example, many vaccines in use now are produced in eggs, and individuals with an allergy to eggs are advised not to get such vaccines. Therefore, regulations may include a requirement to disclose the source of the PMP.

11 CURRENT AREAS OF RESEARCH 8 Plant molecular farming is currently being pursued to address either the increased demand for proteins that cannot be produced in sufficient quantities in either microbial or animal cell cultures, or as a means to produce proteins that cannot be expressed in microbial or animal cell cultures. With respect to increased demands for products currently produced by transgenic microbes or animal cell culture, the arthritis medication Enbrel of Amgen is a prime example. This drug, a genetic copy of anti-inflammatory proteins, is currently being manufactured in bioreactors containing transgenic hamster cells. It was introduced in 1998, and by 2001 demand for the drug exceeded the company s capacity to manufacture it. Increasing manufacturing capacity would require about $450 million and five years to build additional 10,000-litre sterile bioreactor facilities. There are several human diseases for which the underlying cause has been determined to be an ineffective, deficient or absent enzyme. (6) Fabry disease, for example, is a rare disorder in which the affected enzyme is alpha galactosidase A and which affects metabolism and storage of fats and carbohydrates. Although recombinant forms of this enzyme are currently available to sufferers of Fabry disease, such forms are prohibitively expensive and production capacity is limited. Large Scale Biology Corporation has developed ENZAGAL, which the company claims can be biomanufactured more efficiently and in greater abundance than competing products currently produced in animal cell cultures. With regard to products that are too complex to lend themselves to microbial transgenics, there are several examples of efforts being focused on new and innovative therapeutic products. The world s first plantibody, a plant-produced antibody, has been developed by Planet Biotechnology Inc. to help prevent tooth decay. The product, called CaroRx, is an antibody that specifically binds to S. mutans, the bacteria that cause tooth decay, which prevents the bacteria from adhering to teeth. CaroRx is in clinical trials in the United States. Vaccines are another area of research in molecular farming. Early-stage clinical trials have been completed on customized, patient-specific vaccines for Non-Hodgkins (6) An enzyme is a catalytic protein that is produced by living cells and that mediates and promotes the chemical processes of life without itself being altered or destroyed.

12 9 Lymphoma. These plant-produced vaccines can be generated in 6 to 10 weeks, a much shorter time frame than conventional methods. As mentioned previously, edible vaccines, although enthusiastically discussed in recent years, have virtually been abandoned. Despite promising results from early clinical trials of an edible vaccine in potato against Hepatitis B, fears that the engineered crops could become mixed in with food crops have prompted researchers to turn to non-food crops instead, primarily tobacco. The issue of the use of food crops in plant molecular farming was discussed earlier in this paper. CANADIAN REGULATION OF MOLECULAR FARMING No plant-made pharmaceuticals are currently approved for sale in Canada, nor are any plants genetically engineered to produce such products approved for commercial field production at this time. However, several plants are currently in development or early field trials. Because this is a relatively new issue, regulations surrounding the development, testing and ultimately market approval are as yet incomplete. The Canadian Food Inspection Agency (CFIA) has authority over the plant crop, while Health Canada is responsible for any subsequent drug approval of PMPs. The CFIA enforces the regulations for plants with novel traits for those plants engineered to produce pharmaceuticals. PNTs are plant varieties that are not considered substantially equivalent, in terms of their specific use and safety both for environment and for human health, to plants of the same species, having regard to weediness potential, gene flow, plant pest potential, impact on non-target organisms and impact on biodiversity. PNTs may be produced by conventional breeding, mutagenesis, or more commonly by recombinant DNA techniques as is the case for plants that produce pharmaceuticals. Before any PNT, including plants engineered to manufacture therapeutic products, can be considered for commercial production in Canada, it must undergo confined research field trials to assess potential environmental impacts. The CFIA s Plant Biosafety Office assesses applications for these confined research field trials and sets out the rules for how they are to be done. In 2002 the CFIA amended the regulations for these trials so that the minimum isolation distances of PNTs intended for the production of pharmaceuticals are greater than the requirement for other PNTs. All confined research field trials of PNTs in Canada are evaluated by government scientists to determine their environmental impact. Furthermore, in the

13 10 case of PNTs for pharmaceuticals, disposal and destruction of all harvested plant materials must be witnessed by a CFIA inspector. Under CFIA regulations, plants engineered to produce pharmaceuticals are not eligible for unconfined environmental release, unlike other PNTs, regardless of the findings of the environmental impact assessment of the field trial. This restriction is because of the additional environmental and human/animal health concerns associated with these plants. Should any such plants be granted approval for commercial production in the coming years, it is reasonable to assume that they would be subject to additional regulations and conditions to be prescribed under new regulations currently under development. In March 2005 the CFIA held a Technical Workshop on Developing a Regulatory Framework for the Environmental Release of PNTs Intended for Plant Molecular Farming. Health Canada has not yet received any applications to approve a PMP. Although the drug approval process may be revised in the future to specifically address PMPs, the department considers the current process is sufficient to address these products. If an application were to be submitted at this time, the Therapeutic Products Directorate would consider it as a New Drug Submission. Currently all the plants in development involve an established product, such as interferon, hemoglobin, interleukin, apoprotin, etc. However, an application for approval of an established product manufactured in a novel manner would be treated as a new drug, not as a generic version of an existing product. In this way, a full safety review of the therapeutic product and the entire range of clinical trials would be required. CONCLUSION Increased pharmaceutical demands, as well as advances in gene identification following the completion of the Human Genome Project, have led to an interest in plants as expression systems for therapeutic products. Despite numerous benefits to this approach, however, the concerns that have been raised must be adequately addressed. Although molecular farming offers an exciting alternative for pharmaceutical production, industry and government must proceed cautiously in this area in order to gain public acceptance.

14 11 SELECTED REFERENCES Biotechnology Industry Organization. Information on the Web site: Canadian Food Inspection Agency. Plant Molecular Farming page on the Web site: Elbehri, Aziz. Biopharming and the Food System: Examining the Potential Benefits and Risks. AgBioForum, Vol. 8, 2005, pp Fischer, Rainer, and Neil Emmans. Molecular farming of pharmaceutical proteins. Transgenic Research, Vol. 9, 2000, pp Food Safety Network. Information on the Web site: Health Canada. Personal communication with the Therapeutic Products Directorate. National Academies of Science (U.S.). Biological Confinement of Genetically Engineered Organisms. The National Academies Press, Washington, D.C., 2004.