Biotechnology and its Applications

Plants, bacteria, fungi and animals whose genes have been changed by deliberate manipulation are called genetically modified organisms (GMOs). In agriculture, genetic modification has been used to make crops tolerant of abiotic stresses such as cold, drought, salt and heat; to cut down the need for chemical pesticides by building pest resistance into the plant itself; to reduce losses after harvest; to make plants use soil minerals more efficiently, so that soil fertility is not exhausted so quickly; and to raise the nutritional value of food, as in vitamin A enriched rice, usually called golden rice. Tailor-made plants have also been made to supply industry with starches, fuels and pharmaceuticals.

The best known example in India is Bt cotton. Bt stands for Bacillus thuringiensis, a soil bacterium. At a particular phase of its growth this bacterium forms protein crystals inside itself, and those crystals contain a protein that is toxic to insects. Different strains make different toxins, and each toxin is fairly specific to one insect group: some kill lepidopterans such as the tobacco budworm and armyworm, some kill coleopterans (beetles), and some kill dipterans (flies and mosquitoes).

The obvious question is why the bacterium is not killed by the poison it is carrying. The answer is that the crystal protein is not a toxin yet. It is stored as an inactive protoxin. When an insect eats the crystal, the gut of the insect is strongly alkaline, and this alkaline pH dissolves the crystal and converts the protoxin into the active toxin. The activated toxin then binds to the surface of the epithelial cells lining the midgut and creates pores in their membranes. Water rushes in, the cells swell and burst, the gut lining is destroyed, and the insect stops feeding and dies. Inside the bacterium, and inside the cotton plant, the pH never reaches the value needed for that conversion, so the protein simply sits there doing nothing.

To make Bt cotton, the gene coding for this protein is isolated from Bacillus thuringiensis and put into the cotton plant, so that the plant's own cells manufacture the protoxin. The genes are called cry genes, because the protein forms crystals. Which cry gene you choose depends on which pest you are fighting. The proteins encoded by cryIAc and cryIIAb control the cotton bollworms, while the protein of cryIAb controls corn borer. A caterpillar that starts chewing on such a cotton plant eats the protoxin along with the leaf or boll, activates it in its own gut, and is killed by it, so the crop protects itself and far fewer pesticide sprays are needed.

Two limits are worth noting. First, because each toxin acts on a narrow group of insects, a Bt crop is not a defence against every pest that attacks it, and sucking pests are untouched. Second, when a single toxin is used over a very large area for many seasons, insects can evolve resistance to it, and pink bollworm populations resistant to Bt cotton have in fact been reported from Indian cotton growing regions. Resistance management, such as planting some non-Bt plants alongside, is therefore part of using the technology sensibly rather than an afterthought.

The second strategy for building pest resistance into a plant does not use a poison at all. It uses the plant to switch off a gene inside the parasite. The parasite in the standard example is Meloidogyne incognita, a nematode (a roundworm) that infects the roots of tobacco plants and causes a large fall in yield.

RNA interference (RNAi) is not something invented for this purpose. It is a method of cellular defence that already takes place in all eukaryotic organisms. In it, a particular messenger RNA is silenced because a complementary double-stranded RNA (dsRNA) is present in the cell; the dsRNA leads to the target mRNA being bound and prevented from being translated, so the protein is never made. Naturally, the source of such complementary RNA can be infection by viruses that have RNA genomes, or mobile genetic elements called transposons that replicate through an RNA intermediate. In more detail, the long double-stranded RNA is chopped up inside the cell into very short RNA pieces, and one strand of each short piece is loaded into a protein complex that then searches out any mRNA whose sequence matches, and destroys or blocks it. Because the matching is by base pairing, the silencing is extremely specific: only the one gene whose sequence you copied is affected.

The strategy for the tobacco plant works like this. Nematode-specific genes, that is, stretches of DNA taken from Meloidogyne incognita itself, are introduced into the tobacco plant using Agrobacterium as the vector. The DNA is introduced in such a way that the plant cell transcribes both a sense RNA and an antisense RNA from it. Since these two RNA molecules are complementary to each other, they base pair inside the plant cell and form double-stranded RNA. When the nematode invades the root and starts feeding on the plant cell contents, it takes up this dsRNA. Inside the parasite, that dsRNA initiates RNA interference against the parasite's own matching mRNA and silences it. The nematode has lost the product of a gene it needs, and it cannot survive in that transgenic host. The plant is therefore protected, and it is protected without spraying anything.

Notice the direction of the whole thing carefully, because it is very easy to state it backwards. The gene that gets silenced belongs to the nematode, not to the plant. The dsRNA is manufactured by the plant, not by the nematode. And the transfer happens because the parasite feeds on the plant, so the plant never has to attack anything.

Recombinant DNA technology gave medicine a way to mass produce safe human proteins. Before this, insulin for diabetic patients was extracted from the pancreas of slaughtered cattle and pigs. Animal insulin works, because the molecule is very similar across mammals, but it is not identical to the human protein, and some patients developed allergy or other reactions to the foreign protein. A supply that depended on slaughterhouses was also hard to scale as the number of patients rose.

To understand the technical problem, you have to look at how insulin is built. Mature insulin consists of two short polypeptide chains, chain A and chain B, held together by disulphide bridges. But in mammals, including humans, insulin is not synthesised in that finished form. It is synthesised as a pro-hormone called proinsulin: a single chain that contains, in addition to A and B, an extra stretch called the C peptide lying between them. Just as a pro-enzyme has to be processed before it can act, the pro-hormone has to be processed before it becomes fully mature and functional. During maturation in the body, the C peptide is cut out and discarded, and the A and B chains, already held by disulphide bridges, remain as the working hormone. The C peptide is not present in mature insulin.

So the main challenge in producing insulin by recombinant DNA methods was not getting a bacterium to make an insulin sequence. It was getting the insulin assembled into a mature form. A bacterium given the human proinsulin gene will make proinsulin, but a bacterium does not have the specialised processing machinery of a human pancreatic beta cell, so it will not reliably snip out the C peptide for you.

The route taken in 1983 by the American company Eli Lilly sidestepped the problem completely instead of solving it. Two DNA sequences were prepared corresponding to the A and B chains of human insulin. These were introduced separately into plasmids of Escherichia coli, and the two chains were therefore produced in separate bacterial cultures. The chains were then extracted, purified, and combined by creating disulphide bonds between them, giving human insulin. Because the sequence is the human sequence, the product does not carry the foreign-protein problem that animal insulin had, and because bacteria grow fast in bioreactors the supply is limited only by fermentation capacity rather than by slaughterhouses.

This is the general shape of every recombinant therapeutic. Around thirty recombinant therapeutics have been approved for human use worldwide, and a number of them are marketed in India. Their common advantages are that the protein is the true human sequence, that there is no risk of contamination from an animal or human tissue donor, and that production can be scaled up in a controlled way.

Gene therapy is a collection of methods that allow correction of a gene defect that has been diagnosed in a child or an embryo. Instead of giving a medicine that makes up for the missing function again and again, genes are inserted into the person's cells and tissues so that the cells themselves supply what is missing, and the defect is corrected.

The first clinical gene therapy was given in 1990 to a four-year-old girl with adenosine deaminase (ADA) deficiency. Adenosine deaminase is an enzyme of purine metabolism: it converts adenosine and deoxyadenosine into inosine and deoxyinosine so that they can be broken down further. When the enzyme is missing, these substrates and their phosphorylated products pile up, and they are particularly toxic to developing lymphocytes. Since lymphocytes are the cells of the immune response, the child is left with an immune system that cannot function, and ordinary infections become life threatening. The disorder arises from the loss of the gene for adenosine deaminase, so no working enzyme is made at all.

Before gene therapy there were two ways of managing it, and both fall short. In some children, bone marrow transplantation can cure the condition, since the transplanted marrow supplies stem cells that make normal lymphocytes. In others, the treatment is enzyme replacement therapy, in which functional ADA is given to the patient by injection. The problem is that neither approach is completely curative: a suitable marrow donor is not always available, and injected enzyme is used up and has to be given repeatedly.

The gene therapy approach works step by step. Lymphocytes are taken from the blood of the patient and grown in a culture outside the body. A functional ADA cDNA is then introduced into these cultured lymphocytes, using a retroviral vector, which is well suited to this job because a retrovirus naturally inserts its genetic material into the DNA of the cell it enters. The corrected lymphocytes are then returned to the patient, where they now make their own adenosine deaminase.

This is a treatment rather than a cure, and the reason is simple biology. The lymphocytes that were corrected are not immortal. They are mature cells with a limited life; they die and are replaced by new lymphocytes made from the patient's own uncorrected bone marrow stem cells, which still carry the original defect. So the patient needs periodic infusion of such genetically engineered lymphocytes. A permanent cure would require the corrected gene to be present in the cells that keep dividing and giving rise to lymphocytes. If a functional ADA gene taken from marrow cells that produce the enzyme were introduced into cells at an early embryonic stage, it could be a permanent cure.

Early detection is the whole point of molecular diagnosis. Conventional methods such as serum and urine analysis are useful, but they are not sufficient for early detection. The presence of a pathogen, whether a bacterium or a virus, is normally suspected only after it has produced a disease symptom, and by that time its concentration in the body is already very high. Recombinant DNA technology, PCR and ELISA are the techniques that let us look earlier than that.

PCR (polymerase chain reaction) works by amplification. Even a very low concentration of a bacterium or a virus, at a stage when no symptom of the disease is yet visible, can be detected because its nucleic acid can be copied over and over until there is enough of it to see. Each cycle roughly doubles the number of copies of the chosen segment, so a handful of starting molecules becomes millions within a couple of hours. PCR is now routinely used to detect HIV in suspected AIDS patients, and it is used to detect mutations in genes in suspected cancer patients. It is also a powerful technique for identifying many other genetic disorders. The logic is worth stating plainly: PCR is sensitive because it multiplies the evidence, and it is specific because the primers will only start copying where their sequence matches.

The second tool uses a probe. A single-stranded piece of DNA or RNA, tagged with a radioactive molecule, is allowed to hybridise with its complementary DNA in a clone of cells. Wherever the sequences match, the probe base pairs and stays bound; wherever they do not, it washes away. Detection is then done by autoradiography, in which the radioactivity exposes a photographic film. The result then reads the opposite way round from what you might expect: the clone that has the mutated gene will not appear on the film. That is not because the mutation destroys anything, but because the probe no longer has complementarity with the mutated sequence, so it fails to hybridise and there is no radioactivity left at that spot.

ELISA stands for enzyme linked immunosorbent assay, and it works on a completely different principle: antigen and antibody interaction. Infection by a pathogen can be detected either by finding the antigens of the pathogen, that is its proteins and glycoproteins, or by finding the antibodies the patient's own immune system has synthesised against that pathogen. In the assay, the antibody used for detection carries an attached enzyme; when a colourless substrate is added, that enzyme converts it into a coloured product, and the appearance of colour is the visible signal that the antigen or antibody being looked for is present.

Put the three side by side and the division of labour is clear. PCR and probes read nucleic acid, so they can find the pathogen's genome or a mutation in a gene. ELISA reads protein, so it finds either the pathogen's antigens or the patient's antibody response.

Animals that have had their DNA manipulated to possess and express an extra (foreign) gene are called transgenic animals. Transgenic rats, rabbits, pigs, sheep, cows and fish have all been produced, but the workhorse of the field is the mouse: over 95 per cent of all existing transgenic animals are mice. There are five broad reasons for making them.

Normal physiology and development. Transgenic animals are made so that we can study how genes are regulated, and how they affect the normal functions of the body and its development. Genes involved in these processes can be studied by altering them or by adding genes from another species and watching what changes. Work on complex factors that take part in growth, such as insulin-like growth factor, has been done this way.

Study of disease. Many transgenic animals are designed to increase our understanding of how genes contribute to the development of a disease. They are made to serve as models for human diseases, so that new treatments can be tested on them. Models exist for cancer, cystic fibrosis, rheumatoid arthritis and Alzheimer's disease.

Biological products. Some medicines needed to treat human diseases contain biological products, and these are often very expensive to make. A transgenic animal can be given the portion of DNA that codes for a particular product, and then makes that product for us. The human protein alpha-1-antitrypsin, used to treat emphysema, is made this way, and similar attempts have been made for phenylketonuria (PKU) and cystic fibrosis. The famous case is Rosie, the first transgenic cow, who in 1997 produced human protein enriched milk containing 2.4 grams per litre of human alpha-lactalbumin. This milk was a nutritionally more balanced product for human babies than ordinary cow milk.

Vaccine safety testing. Transgenic mice are being developed for use in testing the safety of vaccines before those vaccines are given to humans. Transgenic mice were used to test the safety of the polio vaccine. If such mice prove reliable, they can replace the use of monkeys for this testing.

Chemical safety testing. This is also known as toxicity testing. Transgenic animals are made carrying genes that make them more sensitive to toxic substances than ordinary animals of the same species. They are then exposed to the substance and the effects are studied. Because they react to lower doses, results are obtained in a much shorter time.

Be careful about the word transgenic itself. An animal is transgenic only if a foreign gene has been introduced into it and is expressed by it. An animal that has simply been selectively bred for a desirable trait, however different it looks from its ancestors, is not transgenic, because no gene has been transferred from outside.

Genetic modification of organisms can have unpredictable results when those organisms are introduced into an ecosystem, so no country allows such work to go on unsupervised. Beyond safety there is a second set of questions, about who owns living things and the knowledge attached to them, since manipulating living organisms for human use has been going on far longer than modern genetics and cuts across many social and religious ideas of what is acceptable.

In India, the Government has set up bodies such as GEAC, the Genetic Engineering Appraisal Committee, which was set up under the name Genetic Engineering Approval Committee and is still often written that way. Its job is to judge the validity of genetically modified research and the safety of introducing GM organisms for public services. In practice this means that no GM crop or GM organism may be released in the country simply because a company says it is safe; the proposal, the trial data and the environmental risk have to be examined and cleared first.

Biopiracy is the term used to refer to the use of bio-resources by multinational companies and other organisations without proper authorisation from the countries and people concerned, and without compensatory payment. The imbalance behind it is easy to state. Most of the world's biodiversity, and almost all of the traditional knowledge about how to use it, is held in developing countries and by their farmers and communities. Most of the money, laboratories and patent lawyers are elsewhere. A company can therefore take a plant or a piece of long-standing knowledge, describe it in patent language, and claim exclusive rights over something the community that developed it has used freely for centuries.

The Basmati case is the standard example. There are 27 documented varieties of Basmati grown in India, and Basmati is referred to in ancient texts, folklore and poetry, since it has been grown here for centuries. In 1997, an American company, Rice Tec Inc., was granted a patent by the US Patent and Trademark Office to sell a supposedly new variety of Basmati in the United States and abroad. That variety had in fact been derived from Indian farmers' varieties: Indian Basmati had been crossed with semi-dwarf varieties, and the result was claimed as an invention or a novelty. India challenged the claim, and most of the patent's claims were eventually withdrawn or struck down.

The turmeric case ended even more clearly. In 1995, a United States patent was granted on the use of turmeric for wound healing, a use familiar in Indian households for generations. India's Council of Scientific and Industrial Research contested it, producing documentary evidence including an ancient Sanskrit text and a paper published in an Indian medical journal in 1953. Because the use was already recorded in published prior art, it could not be a novel invention, and the patent was revoked in 1997. A similar challenge later succeeded against a European patent on a fungicide derived from neem.

These disputes changed policy. Documenting traditional knowledge in searchable form so that patent offices can find it, and passing laws that recognise the rights of farmers and communities over the varieties and knowledge they have built, are now treated as defences against biopiracy. The Indian Parliament has also cleared the second amendment of the Indian Patents Bill, which takes such issues into consideration, including patent terms, emergency provisions, and research and development initiatives.