Tuesday, August 3, 2010

Genetically modified animal/plant for hormone and antibiotics production

Group Member

Ooi Yiyin                 1000819537

Stephanie Aksali   1000921924

Lee Jian Wei         1000923569

Lawrence How Kai Hern 1000819922

 

         Genetically modified organism (GMO) is a result from the insertion of combining genes from different organisms into a new organism in which these genes are known to carry the desirable traits to enhance the traits in the new organism. Any undesirable genes will undergo deletion. This process is called recombinant DNA where plasmids are usually served as vectors to express and multiply the genes of interest.

Introduction

The inexpensive production for large quantities (many kilograms) of protein has led to a new industry to produce recombinant proteins in transgenic plants and animals. The potential of ‘molecular pharming’, using transgenic plants or animals as ‘bioreactors’ to produce therapeutic proteins, has been apparent for over a decade and several proteins produced in these systems are now in clinical trials. Depending on the production system used, there are several issues of concern. Animal systems suffer from long development timelines and possible contamination of purified proteins with animal viruses and prions. Plant produced proteins have altered post-translational modifications that introduce novel carbohydrates.

Is there a need for plant-derived vaccines?

Yes. The production of recombinant vaccines in plants may overcome some of the major difficulties encountered when using traditional or subunit vaccines in developing and developed countries. In developing countries difficulties include vaccine affordability, the need for “cold chains” from the producer to the site of use of the vaccine and the dependence on injection. Plant derived vaccines do not face these issue. Below the table shown the technical and social benefits envisaged in plant-derived edible vaccines:

Benefits

Characteristics

Oral delivery

The plant cell wall, consisting essentially of cellulose and sugars, provides protection in the stomach and gradual release of the antigen in the gut.

Use as raw food or dry powder

The vaccinogenic plant tissue may be used as raw food, dried or, alternatively, proteins may

be partially or fully purified and administered in capsules as dry powder

No need for “cold chain”

The vaccinogenic plant parts or plant extracts can be stored and shipped at room temperature

Mucosal and serum immune response

Plant-derived vaccines are primarily designed to trigger the mucosal immune system (IgA),

thus preventing pathogen entry at mucosal surfaces; they also elicit serum and, possibly,

cytotoxic responses

Cost efficiency

Production cost will be reduced 100–1000 times as compared with that of traditional vaccines

6 Optimised expression system

Plants may be engineered to accumulate the antigen in convenient intracellular compartments

(endoplasmic reticulum, chloroplast)

Ease of genetic manipulation

Procedures essentially rely on established molecular and genetic manipulation protocols; these

are already available in developing countries

Ease of production and scale-up

GM-plants can be stored as seeds. Unlimited vaccine quantity can be produced from these in

limited time; production and management is suitable for developing countries

Safer than conventional vaccines

Lack of contamination with mammalian pathogen

Ideal to face bio-weapons

Safety and cost efficiency propose plants plant-derived vaccines as an ideal tool to face

bio-terrorism

Ideal for veterinary use

Cost affordable

Ready for use as food additive

What are the targets for plant-derived vaccines?

1. Vaccines against infectious diseases

There is a large and fast growing list of protective antigens from microbial and viral pathogens that have been expressed by plants. The initial focus was upon human pathogens. However, today attention has also spread to animal pathogens (Newcastle and good and mouth disease). There is no limit to the number and range of antigens that can be produced in plants if the DNA sequences coding for the appropriate genes are available.

2. Vaccines against autoimmune diseases

Transgenic plants expressing autoantigens are being produced in attempt to cure diseases in which the immune system recognizes the body’s own proteins as foreign. The disease include arthritis, multiple sclerosis, myasthenia gravis, and type I diabetes. The rational is that an appropriate oral dose of a plant-derived autoantigens will inhibit the development of the autoimmune disease.

3. Vaccines against human tumors

Particular proteins have been shown to over-express on the cell surface of many tumours, including melanoma and breast cancer. Naturally acquired, actively induced or passively administered antibodies against these antigens have been able, in some cases, to eliminate circulating tumour cells and micrometastasis. However, cancer vaccine development is complicated due to the tumour antigens also being auto-antigens.

The biotechnological approach: construction of appropriate gene expression cassettes, plant transformation, and efficiency of antigen expression.

Gene constructs, expression signals and peptide design for optimal vaccine-production in GM-plants

Purpose

Approach and notes

Optimize codon usage

Adapt codon usage to that preferred by plant genes

Optimise epitope sequence

Adapt A + T composition to that found in plant genes

Eliminate sequences that destabilise or splice mRNA

Minimise secondary structure hairpins

Select promoter

This may be: plant constitutive, tissue specific, inducible by

environmental factors

Use leader and 3’-polyadenilation signals

Alternative signals affect protein accumulation

Use TEV (the 5_untranslated region of the tobacco etch virus)

Target protein to the chloroplast

Integrate the DNA sequence in the nuclear DNA and use an

N-terminal chloroplast transit peptide: the protein is accumulated

in the chloroplast

Target protein to the endoplasmic reticulum

Use an endoplasmic reticulum retention signal, such as SEKDEL

Integrate the epitope DNA in the chloroplast DNA

Integrate the DNA sequence in the chloroplast DNA under

appropriate expression signals: the protein will be synthesised

and accumulated in the chloroplast

Integrate the epitope DNA into a plant virus vector

Use a viral promoter when the epitope is integrated into a plant

virus

Use a defective virus for improving yield and for environmental

Safety

Express polycystronic mRNA

Integrate into the plant DNA a poly-epitope under a single

expression signals

Choose selectable marker genes

Use an appropriately selected gene

Remove the gene after selection

Representative therapeutic proteins produced in transgenic plants.

Recombinant proteins

Plants

Antibodies

 

SIgA anti-S. mutans

Tobacco

IgG anti-herpes simplex virus

Soybean

Various antibodies

Maize/rice

Anti-carcinoembryonic antigen

Rice and wheat

Lymphoma idiotypes

Tobacco

Vaccines

 

Hepatitis B surface antigen

Tobacco

Rabies vaccine

Tobacco

Norwalk capsid protein

Tobacco, Potato

Porcine transmissible gastroenteritis virus

Tobacco

E. coli toxin (LT-B)

Potato

Cholera toxin (CT-B)

Potato

Mouse GAD67

Potato

VP2 capsid protein of mink enteritis virus inserted into cowpea mosaic virus

Black-eyed bean, Vigna unguiculata

Other proteins

 

Collagen

Tobacco

Hirudin

Canola, (Brassica napus)

Lactoferrin

Potato

Lipase

Tobacco, maize

Growth hormone

Tobacco

Erythropoietin

Tobacco

Issue Regarding Protein Production in Plants

  1. Glycosylation

Differences in the glycosylation patterns of proteins produced in plants and humans give perhaps most cause for concern regarding therapeutic protein production.

  1. Post-transcriptional gene silencing

Post-transcriptional gene silencing (PTGS) is a sequence-specific RNA degradation mechanism that is widespread in eukaryotic organisms.

It is often associated with methylation of the transcribed region of the silenced gene and with the accumulation of small RNA molecules (21 to 25 nucleotides) homologous to the silenced gene.

In plants, PTGS can be triggered locally and then spread throughout the organism via a mobile signal that can cross a graft junction.

Therapeutic Advances Using Plant-Produced Proteins

1. Therapeutic proteins produced in plants have been monoclonal antibodies for passive immunotherapy and antigens for use in oral vaccines.

2. Products that have entered clinical trials include two antibodies, an oral vaccine and pancreatic lipase.

  1. At the present time, plants offer the only large-scale, commercially viable system for production of this unique form of antibody such as SIgA which is the most abundant antibody class produced by the body (>60% of total immunoglobulin) and is secreted onto mucosal surfaces to provide local protection from toxins and pathogens.
  2. SIgA is comprised of four different protein chains:

Heavy and light immunoglobulin chains that form the antigen binding hypervariable region

The J chain that dimerizes two IgA molecules (SIgA has four antigen-binding sites)

The secretory component that is derived from the polyimmunoglobulin receptor of mucosal epithelial cells.

Transgenic Animals

Technological Advances Using Transgenic Animals

One of the more promising approaches to the large-scale production of recombinant proteins has been the secretion of proteins into the milk of transgenic mammals. This has been successfully demonstrated for over two dozen different proteins in either cows, goats, sheep, pigs, rabbits or mice and appears particularly promising for the production of large amounts of monoclonal antibodies

To date, most recombinant proteins produced in transgenic animals have involved the microinjection of a genetic construct the expression of which is driven by a mammarygland- specific promoter (e.g. for the production of caseins, whey acidic protein, lactalbumins and lactoglobulin). Although successful, this approach is inefficient and timeconsuming. The challenge is to improve the efficiency and speed of producing high-yield protein-expressing founder animals. One way to quickly determine the activity and stability of a recombinant protein in milk is to transfect mammary tissue in vivo using an expression plasmid encoding the protein, as shown with human growth hormone which was infused through the nipple canal. A similar strategy could also be used to potentially transfect bladder epithelial cells for expression of proteins in urine. However, neither of these approaches would result in the production of large amounts of protein..

Therapeutic Advances Using Proteins Produced in Animal Bioreactors

The expression of proteins in milk offers several uses: to produce large amounts of therapeutics for human or veterinary use; to improve the health of dairy livestock; and to produce large amounts of proteins used in commercial processes or in drug formulation, such as human serum albumin. Although an increasing number of proteins are being produced in milk, most are in preclinical development and few are yet in clinical testing. Pharming Group and Genzyme are developing recombinant human α-glucosidase to treat infants with Pompe’s disease, which results from a genetic deficiency in this enzyme. Enzyme produced in rabbits milk was well-tolerated and showed clinical benefit in treated patients. Pharming Group and Baxter Healthcare Corporation have also recently initiated clinical testing with a rabbit-milk-derived recombinant human C1 inhibitor to treat patients with hereditary angioedema who exhibit C1 inhibitor deficiency. Pharming Group has also just completed a phase I clinical study with recombinant human lactoferrin — the protein was found to be safe and well tolerated. Lactoferrin will be tested initially for heparin neutralization in coronary artery angioplasty or bypass surgery. Recently, human lactoferrin was also found to have potent antibacterial activity in animal models using antibiotic-resistant Staphylococci, which could broaden its clinical use. Genzyme Transgenics and Genzyme General have produced antithrombin III in goats milk; the protein is currently in phase III clinical trials to prevent blood clotting during cardiac surgery in heparin-resistant patients. Goat herds are also being developed by Genzyme Transgenics for the large-scale production of the tumor necrosis inhibitory monoclonal antibody, Remicade. Remicade is marketed by Centocor for the treatment of inflammatory conditions, including Crohn’s disease and rheumatoid arthritis. Another use for the expression of recombinant proteins in milk is to prevent mastitis caused by Staphylococcus in dairy cattle. For example, Kerr and colleagues showed that expression of recombinant lysostaphin in the milk of mice prevented Staphylococcus aureus infection.

Issue Regarding the Production of Proteins in Animals

The technologies above go some way towards addressing the two big issues facing animal production of recombinant proteins: efficiency and the speed with which a commercial product can be produced.

1. The current methods for producing transgenic animals lead to live births at a low rate and the success rate is generally low.

2. The second issues that need to be considered with animal production of recombinant proteins is that of infectious diseases. The potential for products to contain prions has been a concern for some time, and mose companies have designed a process and testing to assure that their products are prion-free.

3. The therapeutic proteins produced in transgenic animals undergo post-translational processing characteristic of that animal, although the protein itself is of human sequence. Because carbohydrates in recombinant glycoproteins are somewhat different from those of human origin, there is still a question about whether or not this will have adverse effects, such as eliciting an immune response or conferring altered biodistribution or retention.

Conclusion

In summary, the future production of recombinant proteins in plants or in the milk of animals looks promising. The number of applications will increase as more therapeutic proteins are required in quantities that cannot be attained economically in cell culture. With the advent of nuclear transfer technology, optimized production of recombinant proteins in milk should become even more efficient and inexpensive. In plants, efforts to modify glycosylation and control post-transcriptional gene silencing will enhance the value of this important technology. An increasing number of transgenic plant- and animal-derived proteins are entering clinical testing. The initial success of these development programs suggests an important role for costeffective and large-scale production technologies

 

Sample Video Explaining Genetically Modified Animal for Hormone and Antibiotics Production

 

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