TOPIC: - PHARMING: RECOMBINANTS

PROTEINS FROM LIVE ANIMALS AND

PLANTS

SUB. NAME : CONCEPTS IN

BIOTECHNOLOGY

SUB. CODE: BTC 012

SUBMITTED TO: - SUBMITTED BY:-

MR. HARSH KUMAR KARAN GULARIA
(LECT.BIOTECHNOLOGY) R109XB-52

REG. NO-1040070167
ACKNOWLEDGEMENT

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.

THANK YOU
Contents:

♦ Introduction

♦ Pharming: Recombinant proteins from

Animals

♦ Pharming and Recombinant proteins in

Animals

♦ Conclusion

♦ References
Introduction :
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.[1] 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.[1] It differs from
genetic recombination, in that it does not occur through processes within the cell or
ribosome, but is exclusively engineered.[1] Recombinant protein is protein that is
derived from recombinant DNA.[2]

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",[3] 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-1-
antitrypsin. 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 T-
lymphomas. 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 sub-

sector 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.
Conclusion :

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:-

1. Alan Dove (2000). "Milking the Genome for

Profit". Nature Biotechnology
http://www.nature.com/nbt/journal/v18/n10/full/nb
t1000_1045.html.

2. Phillip B. C. Jones. "European Regulators Curdle

Plans for Goat Milk Human Antithrombin".

3. "Go-ahead for 'pharmed' goat drug".

4. http://en.wikipedia.org/wiki/Pharming_(genetics)