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SUB. NAME : CONCEPTS IN BIOTECHNOLOGY SUB. CODE: BTC 012
SUBMITTED TO: MR. HARSH KUMAR (LECT.BIOTECHNOLOGY)
SUBMITTED BY:KARAN GULARIA R109XB-52 REG. NO-1040070167
I gratefully acknowledge my indebtedness to my respected teacher HARSH SIR who is always a source of inspiration for me, for providing this opportunity.Under his able guidance, I have learned how to overcome the odds and trying circumstances. I am also very thankful to my colleagues with whom I could discuss and give the final shape to this term paper. At the same time, I am also thankful to my class incharge Dr.SANCHITA TIWARI who gave us an immense help in the completion of this project. Any constructive comments,sujjestions,criticism from students and colleagues will be highly appreciated and gratefully acknowledged.
Contents: ♦ Introduction ♦ Pharming: Recombinant proteins from Animals ♦ Pharming and Recombinant proteins in Animals ♦ Conclusion ♦ References
Pharming is a merger of "farming" and "pharmaceutical" and refers to the use of genetic engineering to insert genes that code for useful pharmaceuticals into host animals or plants that would not otherwise express those genes. As a consequence, the host animals or plants then make the pharmaceutical product in large quantity, which can then be purified and used as a drug product. Some drug products and nutrients may be able to be delivered directly by eating the plant or drinking the milk. For example Golden rice has been developed which contains Beta-carotene. Another potential product would be milk that already has vitamin D in it. Such technology has the potential to produce large quantities of cheap vaccines, or other important pharmaceutical products such as insulin.
Recombinant DNA is a form of synthetic DNA that is engineered through the combination or insertion of one or more DNA strands, thereby combining DNA sequences that would not normally occur together. In terms of genetic modification, recombinant DNA is produced through the addition of relevant DNA into an existing organismal genome, such as the plasmid of bacteria, to code for or alter different traits for a specific purpose, such as immunity. It differs from genetic recombination, in that it does not occur through processes within the cell or ribosome, but is exclusively engineered. Recombinant protein is protein that is derived from recombinant DNA. The Recombinant DNA technique was engineered by Stanley Norman Cohen and Herbert Boyer in 1973. They published their findings in a 1974 paper entitled "Construction of Biologically Functional Bacterial Plasmids in vitro", which described a technique to isolate and amplify genes or DNA segments and insert them into another cell with precision, creating a transgenic bacterium. Recombinant DNA technology was made possible by the discovery of restriction endonucleases by Werner Arber, Daniel Nathans, and Hamilton Smith, for which they received the 1978 Nobel Prize in Medicine
The products of pharming are recombinant proteins or their metabolic products. Drugs made from recombinant proteins potentially have greater efficacy and fewer side effects than small organic molecules (which are often screened as potential drugs) because their action can be more precisely targeted toward the cause of a disease rather than treatment of symptoms. Recombinant proteins are most commonly produced using bacteria or yeast in a bioreactor, but pharming offers the advantage to the producer that it does not require expensive infrastructure, and production capacity can be quickly scaled to meet demand.
Pharming of Recombinant Proteins from animals :
Animal pharming, the process of using transgenic animals to produce human drugs, is staking its claim in a lucrative world market. Transgenic animals are animals which have been genetically transformed by splicing and inserting foreign animal or human genes into their chromosomes. The inserted gene, when successful, enables an animal to make a certain pharmaceutical protein in its milk, urine, blood, sperm, or eggs, or to grow rejection-resistant organs for transplant.
Global demand continues to grow for human proteins and vaccines. These proteins serve numerous therapeutic purposes such as treatments for cystic fibrosis, hemophilia, osteoporosis, arthritis, malaria, and HIV. Transgenic animals can also produce monoclonal antibodies (antibodies specifically targeted towards disease proteins) which are used in vaccine development.
In 1998, less than one percent of the world supply of human therapeutic proteins came from production of recombinant proteins (proteins which are formed by laboratory manipulation of genes in plants, bacteria, or animals). That tiny percentage of overall production, however, was valued at almost $12 billion, or 50 percent, of a $24 billion global market for human proteins1. A recent Financial Times article reported that a herd of 600 transgenic cows could supply the worldwide demand of some pharmaceuticals, for example, human serum albumin used in the treatment of burns and traumatic injuries.
With greater integration of computers into laboratory functions, molecular biologists have drastically reduced the time needed to identify and isolate genes. As gene sequencing has become increasingly automated, each known sequence is recorded and stored in a data base. Animal producers are using the body of genetic information to produce transgenic animals. Automated gene sequencing and the biological advantages of animals, when compared to more traditional methods of recombinant protein production, have combined to make pharming a preferred alternative. Traditional methods of recombinant protein production use laboratory cell cultures of transgenic bacteria, yeast or animal cells to produce proteins. Inherent disadvantages in traditional methods, when compared to using animals as bioreactors, include (1) cell and bacterial cultures require constant monitoring and sampling, (2) expansion is more costly, because substantial plant machinery must be purchased and maintained, and
(3) isolating and purifying proteins is more difficult than purifying proteins from an animal’s milk or bodily fluid.
Overall, animals as bioreactors are more cost effective because the product is efficiently passed through the milk, with an average yield of 53 percent and with 99 percent purity. The purifying process may become more simple if harvesting proteins from poultry eggs and urine becomes viable.
Using animals as bioreactors is also cost-effective and advantageous because animals naturally carry the cellular mechanisms needed to produce complex proteins. Genes require certain cellular mechanisms to help them produce proteins. These mechanisms are present in a living animal, but they may be difficult or impossible to replicate in a cell culture.
Unit cost per protein should be significantly less when animals are used as bioreactors to produce human proteins. Only a fraction of the raw material, capital equipment, and maintenance costs needed for traditional cell culturing are required with transgenic animals. It highlights one private firm’s estimates regarding the superiority of transgenic human protein production through eggs or goat milk.
The technology used to develop transgenic animals is somewhat mature, however, the industrialization of bio-pharming is new. The first transgenic animal, a mouse, was produced in 1981. In an effort to determine which genes were involved with cancer, a gene was inserted into the mouse that made it susceptible to cancer. In 1985, the first transgenic farm mammal was produced, a sheep called "Tracy". Tracy had a human gene that expressed high levels of the human protein alpha-1antitrypsin. The protein, when missing in humans, can lead to a rare form of emphysema.
Transgenic animals with alterations to the germ line are commonly produced through microinjection. Changes to the germ line are heritable from generation to generation within the herd, and this heritability has potential to facilitate long-term productivity gains. For example, fish can be bred for increased expression of a growth hormone, although the industries are currently wary of consumer preferences for gene-modified product. Another example is the recently developed "enviro pig", a pig which had a phytase gene placed in its salivary glands to allow
better utilization of phosphorus in feedstuffs. The genetically modified "enviro pig" may prove to be part of the solution to animal waste issues.
Due to the expense and difficulty in producing a transgenic animal, cloning is the method of choice to produce multiple transgenic animals without altering the animal’s genes as traditional breeding would do. Cloning is the process of replicating an identical gene, cell, or organism from a single ancestor. Since Dolly the sheep was cloned by nuclear transfer in 1997, advances in cloning technology have made it easier to build a genetically tailored livestock herd. Several farms of cloned transgenic animals have emerged throughout the world, including a Wisconsin operation with 37 cloned cows in 1999, 17 of them transgenic. Cloning an animal is still costly, ranging from $100,000 to $200,000 in 1999, but cloning costs are projected to decrease to $5000 in the next few years as the practice becomes more common2.
Concerns regarding transgenic animals have developed along with the new technologies. These concerns include safety of the food supply, safety of the pharmaceutical product, and keeping the transgenes out of non-transgenic animals.
As of December 1999, no specific legislation had been enacted to direct federal oversight of transgenic animals. Therefore, the U.S. Food and Drug Administration (FDA) has considered transgenic animals according to animal drug provisions of the Federal Food, Drug and Cosmetic Act. In 1994, the FDA issued a report titled "Points to consider in the manufacture and testing of therapeutic products for human use derived from transgenic animals." This report set guidelines for the development, maintenance and disposal of transgenic animals.
Because some transgenic animals are produced for food rather than for pharmaceuticals, the U.S. Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS) has issued guidance on food safety issues related to transgenics. In 1994, the FSIS published "Points to consider in the food safety evaluation of transgenic animals from transgenic animal research." This FSIS guidance, revised in 1997, highlighted animal regulations that must be met before animals may be submitted for slaughter. Regulations require the producer to supply information about the animal including any drugs, biologics or chemicals that were administered, or any genetic alterations. The FSIS also offers continuing education courses for USDA employees on food safety issues in transgenic animals.
Pharming and Recombinant Proteins in Plants :
A large number of technically and pharmaceutically important proteins have already been produced in transgenic plants. Now molecular farming is being considered for the commercial production of proteins. Tobacco plants have been shown to be particularly suitable production systems.
The majority of the over fifty genetically engineered drugs currently marketed in Germany is produced with genetically modified bacteria. The production of such drugs in plants has turned out to be an attractive alternative: it is safe and comparably cheap; instead of expensive culture media, only sunlight, water and a few minerals are required in order to produce any amount of substances that are able to improve human health.
The first synthesis of a pharmaceutically-relevant protein, human growth hormone, was described in transgenic tobacco plants in 1986. Now, molecular farming has become commercially interesting as a method for the production of recombinant pharmaceutical proteins, in particular antibodies. The application spectrum of molecular farming has broadened rapidly in recent years. Molecular farming is now used to produce industrial enzymes (for example laccase in transgenic maize), technical proteins for research purposes (for example avidin, which is also produced in maize), milk proteins such as human beta casein, which is produced in transgenic tomatoes, or new protein polymers (collagens, which are used for medical as well as industrial purposes). Many more new products are expected to become available in the new future.
Tobacco field in Otterstadt, Germany. Will these plants soon be used as pharmaceutical drugs rather than for smoking?
Apart from maize, tobacco plants have proved to be very suitable for the production of recombinant proteins. It might not be long before tobacco loses its importance as the basis of cancer-causing cigarettes and other smoking products, but will be turned into a producer of substances that are beneficial for humans. In order to turn plants into bioreactors for foreign proteins, the genes expressing the desired proteins are introduced into the plant genome and the substance
produced during the growth of the plant. Alternatively, the plants can be infected with a gene shuttle, which also leads to the production of the desired active substance.
Both systems are used by the research group of Dr. Krczal at the AIPlanta Institute for Plant Research (see article: “A New Institute Focusing on Green GeneticEngineering ”). The scientists have transformed tobacco plants in a way that they now express single chain antibodyfragments (known as scFV) that were directed against different plant viruses and have conferred resistance to the tobacco plants.
Transgenic scFv expressing tobacco plants (in the background) and control plants (in the foreground) 14 days after infection with different plant viruses. Source: AIPlanta Institute. This method can also be used for the production of pharmaceutically-active substances. For example, the scientists succeeded in expressing an scFV antibody in tobacco plants, which was directed against the CD40 surface receptor and coupled with bryodin. Bryodin, a toxin found in the roots of bryonia (white bryony), is a protein that deactivates ribosomes and is used to treat HIV infections and Tlymphomas. Viral proteins, which are required for the production of vaccines, have already been produced in tobacco plants. The first such candidate was the surface antigen of the hepatitis B virus (HBV) and it is currently being tested in clinical trials. A recombinant vaccine made from the surface glycoprotein S of porcine TGEV (transmissible gastroenteritis virus) is produced in tobacco and maize plants. This is the first example of a successful protection provided by vaccination that is administered to the animals with their fodder. Arabidopsis is often used as a model organism to study expression in plants, while actual production may be carried out in maize, rice, potatoes, tobacco, flax or safflower. The advantage of rice and flax is that they are self-pollinating, and thus gene flow issues (see below) are avoided. However, human error could still result in pharm crops entering the food supply. Using a minor crop such as safflower or Tobacco, avoids the greater political pressures and risk to the food supply involved with using staple crops such as beans or rice.
Plant-Made Pharmaceuticals (PMPs), also referred to as Biopharming, is a subsector of the biotechnology industry that involves the process of genetically
engineering plants so that they can produce certain types of therapeutically important proteins and associate molecules such as peptides and secondary metabolites. The proteins and molecules can then be harvested and used to produce pharmaceuticals. There is much debate over the practicality of using plants to produce proteins. Some groups fear that contamination of conventional crops might occur; in several instances, companies have been fined for violating protocols, resulting in potential contamination. This leads to the question of "Why would biotechnology companies use plants to produce proteins?" Conventional production methods for pharmaceutical proteins involve substantial investments of both time and finances. Not only are there manufacturing challenges involved with conventional production methods, but there are also considerable regulatory challenges that must be met. There are currently about 30 protein-based medicines on the market, and close to 100 in late-stage human trials. Consequently, companies are motivated to provide a wider range of options for production of proteins used in these treatments. Biopharm proponents claim that using plants can offer an easily controllable, safe, and cost-effective method for manufacturing proteins, provided that proper regulatory safeguards are put into place to ensure that no outcrossing can occur. It is also important to note, that the global demand for particular pharmaceutical protein can easily be met from just a few acres of pharma-crop, which can be grown under high containment conditions (e.g. in the greenhouse). Some scientists even think that the term "gardening" is more appropriate than farming. Opponents are concerned that there are too many ways in which contamination of the food supply and the environment can occur to make this form of production socially desirable, or even economically feasible. Compared to conventional production methods, plant-made pharmaceuticals could save substantial time, money, and provide a system for producing proteins that could solve current production challenges. Companies in this industry hope that proteins made from plants can be used to develop treatments for some of the most serious diseases and conditions such as cancer, diabetes, HIV, heart disease, Alzheimer's disease, cystic fibrosis, multiple sclerosis, Hepatitis C, and arthritis, but no such products have as yet been approved.
As transgenic animals, pharming, and cloning become more mainstream, a small yet growing portion of the animal production industry will shift its operations from farming livestock for meat production, to pharming transgenic animals for pharmaceutical production. The world market is growing for human pharmaceutical products. Producing transgenic animals is still relatively expensive, however, costs are trending down and transgenic animals have certain advantages over traditional laboratory methods for producing human proteins. More commercial use of transgenic animals in food production is also likely.
Regulators will need to review existing policies and guidelines regarding transgenic animals. New policies regarding transgenic and cloned animals may be necessary to ensure the safety and health of humans and animals. Ongoing public debate regarding transgenic technologies will ensure that further research and analyses will be demanded by animal producers, regulators, environmentalists, and the general public.
References:Alan Dove (2000). "Milking the Genome for Profit". Nature Biotechnology http://www.nature.com/nbt/journal/v18/n10/full/nb t1000_1045.html.
Phillip B. C. Jones. "European Regulators Curdle Plans for Goat Milk Human Antithrombin".
"Go-ahead for 'pharmed' goat drug".
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