Top 5 Applications of Recombinant DNA Technology in Medicine

This article throws light upon the top five applications of recombinant DNA technology in medicine.

The top five applications are: (1) Diagnosis of Genetic Diseases (2) DNA Typing (DNA Fingerprinting) (3) Gene Therapy (4) Recombinant DNA Technology in the Synthesis of Human Insulin and (5) Hepatitis B Vaccine.

Application # 1. Diagnosis of Genetic Diseases:

Most of us do not suffer any harmful effects from our defective genes because we carry two copies of nearly all genes, one derived from our mother and the other from our father. The only exceptions to this rule are the genes found on the male sex chromosomes. Males have one X and one Y chromosome, the former from the mother and the latter from the father, so each cell has only one copy of the genes on these chromosomes.

In the majority of cases, one normal gene is sufficient to avoid all the symptoms of disease. If the potentially harmful gene is recessive, then its normal counterpart will carry out all the tasks assigned to both. Only if we inherit both copies of the same recessive gene from our parents, we will develop a disease. On the other hand, if the gene is dominant, it alone can produce the disease, even if its counterpart is normal. Clearly only the children of a parent with the disease can be affected, and then on average only half the children will be affected.

Huntington’s chorea, a severe disease of the nervous system, which becomes apparent only in adulthood, is an example of a dominant genetic disease. Finally, there are the X chromosome-linked genetic diseases. As males have only one copy of the genes from this chromosome, there are no others available to fulfill the defective gene’s function. Examples of such diseases are Duchenne muscular dystrophy and, perhaps most well known of all, hemophilia.

Queen Victoria was a carrier of the defective gene responsible for hemophilia, and through her it was transmitted to the royal families of Russia, Spain, and Prussia. Minor cuts and bruises, which would do little harm to most people, can prove fatal to hemophiliacs, who lack the proteins (Factors VIII and IX) involved in the clotting of blood, which are coded for by the defective genes. Sadly, before these proteins were made available through genetic engineering, hemophiliacs were treated with proteins isolated from human blood.

Some of this blood was contaminated with the AIDS virus, and has resulted in tragic consequences for many hemophiliacs. Use of genetically engineered proteins in therapeutic applications, rather than blood products, will avoid these problems in the future. Not all defective genes necessarily produce detrimental effects, since the environment in which the gene operates is also of importance. A classic example of a genetic disease having a beneficial effect on survival is illustrated by the relationship between sickle-cell anemia and malaria. Only individuals having two copies of the sickle-cell gene, which produces a defective blood protein, suffer from the disease.

Those with one sickle-cell gene and one normal gene are unaffected and, more importantly, are able to resist infection by malarial parasites. The clear advantage, in this case, of having one defective gene explains why this gene is common in populations in those areas of the world where malaria is endemic. The diagnosis of inherited disease at the genetic level makes possible for the individual to determine whether they are at risk. DNA analysis can be used for the identification of carriers of hereditary disorders, for prenatal diagnosis of serious genetic conditions, and for early diagnosis before the onset of symptoms.

Now-a-days these tests are at the DNA level and are definitive for determining the existence of specific genetic mutations, unlike previous genetic testing which were based on biochemical assay that used to determine either the presence or absence of a gene product.

Sickle Cell Anemia:

Sickle cell anemia is a genetic disease that is the result of a single nucleotide change in the codon for the sixth amino acids of the β-chain of the hemoglobin molecule (from valin to glutamic acid). In this disease the shape of the RBC becomes irregular (sickle shaped). The biological effects of this genetic alteration are severe anemia and progressive damage to the heart, lungs, brain, joints and major organ systems. The anemia is caused by the inability of the mutated hemoglobin to carry sufficient oxygen.

The life expectancy of S/S homozygotes is quite short. Heterozygous individuals A/S (genetic carriers) have normal shaped RBC and no symptoms unless subjected to extreme conditions, where there is low oxygen supply as on high altitude, or extremes of temperature. If both parents are heterozygous (A/S) then 25% of children are (S/S) homozygous.

One of the test systems to identify the individuals suffering from sickle cell anemia is as follows:

It is known that the single nucleotide change in the 0-globin gene causes sickle cell anemia. In the disease Cvn I restriction endo-nuclease site present in the gene gets diminished (Fig. 19.1). This restriction enzyme recognizes the sequence CCTGAGG and cleaves the DNA between the C and T. In the normal gene the DNA sequence is CCTGAGG, Genotype whereas, in sickle cell anemic gene the sequence is size (bp) AA AS SS CCTGTGG. This difference forms the basis for DNA diagnostic assay.

After two primer sequences that flank the Cvnl site are added, a small amount of sample DNA can be amplified by PCR. The amplified DNA is digested with CvnI, and the cleavage products are separated by gel electrophoresis and visualized by ethidium bromide staining of the DNA in the gel. If the intact CvnI site is present, then a specific set of DNA fragments is observed and if the Cvnl site is absent, different profile of DNA fragment occurs. By this procedure, the genetic makeup of a tested person can be determined (Fig. 19.2).

The PCR/OLA Procedure:

All genetic diseases do not produce defect in the restriction site, so they could not be determined easily as in the case of sickle cell anemia. For such diseases, other strategies are required to detect single nucleotide change. One of the procedures used is PCR combined with oligonucleotide ligation assay (OLA). To understand PCR/OLA procedure, assume that in a normal gene at a specific site (say 127th nucleotide) the nucleotide pair is A-T and in the mutant form the nucleotide pair at this site is G- C. Now, two DNA oligonucleotides are synthesized which are 20 bp long and their nucleotide sequence is complementary to the DNA.

First probe (oligonucleotide) X is 20 bp long and is synthesized in such a fashion that its nucleotides sequences are complementary to the adjacent side of 127th nucleotide, including 127th nucleotide (i.e., its last base is at the 3′ end, at position 127). The other probe Y starts at 5′ end is again 20 bp long and its nucleotides are complementary to the other immediately adjacent side of 127th nucleotide, excluding it. When these two probes are hybridized with target DNA (amplified by PCR) having normal sequence, the nucleotide at the 3′ end of probe X base pairs with 127th nucleotide of the target DNA and probe Y is aligned in such a way that its 5′ end lies next to the 3′ end of probe X.

The addition of DNA ligase to the reaction covalently joins probe X and Y. When these two probes are hybridized with mutant gene in which the nucleotide at 127th position is altered as G-C. The nucleotide at the 3′ end of probe X which is ‘A’ is mismatched and is not able to pair with 127th nucleotide in the target DNA sequence. Probe Y however, is perfectly aligned. In this case, DNA ligase cannot join probe X and probe Y because of the single nucleotide misalignment.

After this procedure, the ligation product are determined by the use of two indicator probes, probe X is labeled at its 5′ end with biotin and probe Y is labeled at its 3′ end with digoxigenin (antibody binding indicator). These molecules help in identifying the ligated product. The sample in which the ligated product are seen, it means it carries the normal genes sequence and the sample in which the ligation product are not seen, they have the defective gene sequence.

Overall, PCR/OLA system is rapid, sensitive and highly specific. This procedure has now also been automated. The ligase chain reaction is a less sensitive variant of PCR/OLA system. Sample DNA is mixed with an excess amount of a pair of OLA indicator probes in the presence of heat resistance DNA ligase. After an initial ligation reaction at 65 °C, the temperature is raised to 94 °C to denature the probe-target DNA hybrid and then lowered to 65 °C to allow hybridization of the free non-ligated OLA indicator probes to the target DNA.

The cycle is repeated 20 times. If the OLA indicator probes match the target DNA perfectly, then ligation will occur at 65 °C during each cycle and after 20 cycles enough ligation products (i.e., probe X joined to Y) will accumulate to be observed by either gel electrophoresis or an ELISA detection system. If no ligation occurs because of a mismatch, then no joined probe product will be produced or detected.

Genotyping with Fluorescence-Labelled PCR Primers:

PCR primers labelled with different fluorescent dyes can be used in the development of non-­radioactive color-based detection systems. To distinguish between mutant and wild type DNA, PCR is performed with two different primers. One is exactly complementary to the wild type DNA and is labeled at its 5′ end with rhodamine (red). The other is complementary to the mutant DNA and its is labeled at its 5′ end with fluorescein (green).

In both cases, amplification is programmed a third unlabeled primer that is complementary to the opposite strand. Since PCR amplification can take place only when the primer is exactly complementary to the target DNA, the presence of these three primers in the same reaction mixture will result in the amplification of either the wild type or the mutant DNA or both, depending on which target DNAs are initially present to act as PCR template.

If an individual is homozygous for the wild type DNA, after PCR and removal of unincorporated primer, the reaction mixture will fluoresce red; if a person is homozygous for the mutant DNA, the reaction mixture will fluoresce green; and if he has both mutant and wild type DNA (heterozygous). The reaction mixture will fluoresce yellow. This assay can be automated and can be adapted for any single nucleotide target site of any gene that has been sequenced.

Mutation at Different Sites within One Gene:

Not all genetic diseases are due to a single specific nucleotide change within a gene. In most cases, a variety of different intragenic sites can mutate, and each mutation can cause the same form of the genetic disease. For example, β-thalassemia is a genetic disease that is due to a loss in the activity of β-globin. Heterozygotes (carriers) tend to have only a mild form of anemia.

In contrast, people who are homozygous for one of eight or more possible mutations must receive regular blood transfusions and other treatment to survive. Because a mutation at any one of eight or more different sites within the β-globin gene can cause β-thalassemia, testing for a change at only one particular nucleotide site is insufficient; at least eight separate tests are necessary. Although this is feasible, it is costly.

Therefore, a PCR/hybridization strategy that uses one reaction assay system has been devised to screen, for mutant nucleotide sites at different locations within a single gene. A set of sequence specific oligonucleotide probes (each 20 nucleotides in length) is synthesized. Each oligonucleotide can form a perfect match with a segment of the target gene that corresponds to the site of a known mutation.

A thymidine homopolymer [poly (dT)] tail approximately 400 mucleotides long is added to the 3′ end of each of the probes. The tail enables the probe DNA to be physically bound to the rest of the ignited discrete spot on a nylon filter while ensuring that the rest of the probe is accessible for hybridization. Segments of the sample (test) DNA that spans each of the possible mutant sites within the gene are amplified simultaneously by PCR.

In each case, one of each of the pairs of primers is labeled at the 5′ end with biotin. The amplified target DNA is then hybridized to the filter-bound probes under conditions that allow only perfect matches to hybridize.

Streptavidin with attached alkaline phosphatase is added during the hybridization reaction. After hybridization, the filter is washed and a colorless substrate is added. A colored spot on the filter appears wherever there is a perfect nucleotide matched between an amplified target DNA segment and one of the specific oligonucleotide probes. One filter can be spotted with a number of sequence specific oligonucleotide probes. Thus, with a single filter assay, one of a number of different mutant sites can be identified.

Application # 2. DNA Typing (DNA Fingerprinting):

1. DNA typing is a technique in which biological samples help in solving forensic problems. This technique is used to establish that whether the suspected person has committed a crime or not.

2. For DNA typing, biological samples like blood, skin, semen or hair are collected from the site of crime. A portion of a sample is analyzed to confirm that there is sufficient amount of intact DNA (un-degraded) for the further analysis.

3. If there is sufficient amount of intact DNA, it is digested with a restriction enzyme, and the fragments are separated on an agarose gel and transferred by blotting onto a nylon membrane.

4. This nylon membrane is then hybridized sequentially with four or five separate radio-labeled probes that each recognizes a distinct DNA sequence. After each hybridization reaction, the bands where the probe has bound to the digested. DNA sample are visualized by autoradiography and the banding pattern for each sample is noted.

5. Before the next probe is used, the first probe is completely removed from the membrane. Since each hybridization and autoradiography step can take up to 10 to 14 days, the entire process may take many weeks and even several months.

6. A commonly used set of probes for this type of analysis consists of human mini-satellite DNAs. These sequences occur throughout the human genome and consist of tandemly repeated sequences. The length of the repeats range from 9 to 40bp, and the number of repeats in the mini-satellites ranges from about 10 to 30. Unrelated individuals generally have different length of mini-satellites.

However, a child will inherit one mini-satellite DNA sequence from mother and one from father. Even the same mini-satellites DNA sequences can have a different length in different individuals. This variability is due to either a gain or a loss of tandem repeats, probably during DNA replication. These changes do not have any biological effect because the mini-satellite DNA do not code for any protein.

7. The human mini-satellites DNA sequences are highly variable, and the chance of finding two individuals in the population with the same DNA fingerprint is about one in 10⁵ to 10⁸. An individual’s DNA banding pattern based on mini-satellites DNA sequences is almost as unique as his or her fingerprints.

8. In addition to forensic applications of DNA typing, this technique may be used to determine the paternity of a child. The DNA fingerprint banding pattern of the child should be a composite of the patterns of its mother and father.

9. In case when the amount of forensic sample collected is quite less but the DNA is un-degraded then in that case, it is possible to amplify small portions of DNA of the mini-satellite DNA by PCR (Fig. 19.4).

Application # 3. Gene Therapy:

Much attention has been focused on the so-called genetic metabolic diseases in which a defective gene causes an enzyme to be either absent or ineffective in catalyzing a particular metabolic reaction effectively. A potential approach to the treatment of genetic disorders in man is gene therapy. This is a technique whereby a working gene replaces the absent or faulty gene, so that the body can make the correct enzyme or protein and consequently eliminate the root cause of the disease.

The most likely candidates for future gene therapy trials will be rare diseases such as Lesch- Nyhan syndrome, a distressing disease in which the patients are unable to manufacture a particular enzyme. This leads to a bizarre impulse for self-mutilation, including very severe biting of the lips and fingers. The normal version of the defective gene in this disease has now been cloned.

If gene therapy does become practicable, the biggest impact would be on the treatment of diseases where the normal gene needs to be introduced into only one organ. One such disease is phenylketonuria (PKU). PKU affects about one in 12,000 white children, and if not treated early can result in severe mental retardation. The disease is caused by a defect in a gene producing a liver enzyme. If detected early enough, the child can be placed on a special diet for their first few years, but this is very unpleasant and can lead to many problems within the family.

Before treatment for a genetic disease can begin, an accurate diagnosis of the genetic defect needs to be made. It is here that biotechnology is also likely to have a great impact in the near future. Genetic engineering research has produced a powerful tool for pinpointing specific diseases rapidly and accurately. Short pieces of DNA called DNA probes can be designed to stick very specifically to certain other pieces of DNA. The technique relies upon the fact that complementary pieces of DNA stick together.

DNA probes are more specific and have the potential to be more sensitive than conventional diagnostic methods, and it should be possible in the near future to distinguish between defective genes and their normal counterparts, an important development.

The transfer of DNA is usually achieved either by mixing the DNA with a substance that enhances its uptake or by packaging it inside a disabled virus (a virus that can carry DNA into a cell but cannot cause a disease). First removing cells from the patient and then re-introducing those that have been appropriately modified may carry out Gene therapy. Alternatively, the direct introduction of DNA into the patient’s body may be the only effective strategy.

There are several types of gene therapy:

1. Gene Augmentation Therapy:

This is appropriate for the treatment of inherited disorders caused by the loss of a functional gene product. The aim is to add a functional copy of the lost gene back into the genome and express it at sufficient levels to replace the missing protein. It is only suitable if the pathogenic effects of the disease are reversible.

2. Gene Inhibition Therapy:

This is suitable for the treatment of infectious diseases, cancer and inherited disorders caused by inappropriate gene activity. The aim is to introduce a gene whose product inhibits the expression of the pathogenic gene or interferes with the activity of its product.

3. Killing of Specific Cells:

This is suitable for diseases such as cancer that can be cured by eliminating certain populations of cells. The aim is to express within such cells a suicide gene, whose product is toxic. One approach is the expression of an enzyme that converts a harmless pro-drug into a highly toxic molecule. Another is the expression of a protein that makes the cells vulnerable to attack by the immune system. It is very important to ensure that suicide genes are appropriately targeted; otherwise the therapy would result in widespread cell death.

4. Somatic and Germ Line Gene Therapy:

There is an important distinction between somatic gene therapy (DNA transfer to our normal body tissue) and germ line gene therapy (DNA transfer to cells that produce eggs or sperm). The distinction is that the results of any somatic gene therapy are restricted to the actual patient and are not passed on to his or her children.

In most gene therapy studies, a “normal” gene is inserted into the genome to replace an “abnormal,” disease-causing gene. A carrier molecule called a vector must be used to deliver the therapeutic gene to the patient’s target cells. Currently, the most common vector is a virus that has been genetically altered to carry normal human DNA.

Viruses have evolved a way of encapsulating and delivering their genes to human cells in a pathogenic manner. Scientists have tried to take advantage of this capability and manipulate the virus genome to remove disease-causing genes and insert therapeutic genes.

Target cells such as the patient’s liver or lung cells are infected with the viral vector. The vector then unloads its genetic material containing the therapeutic human gene into the target cell. The generation of a functional protein product from the therapeutic gene restores the target cell to a normal state.

Adenovirus Vector System:

Adenoviruses infect a wide range of non dividing human cells and have been used extensively as live vaccines against respiratory infections and gastroenteritis without side effects. These features make adenovirus a likely prospect for delivering genes to target cells. For use as a vector for gene therapy, a cell line that produces the adenoviral E1 gene products, which are essential for adenoviral replication, is co-transfected with two portions of the adenovirus genome.

One of these segments, which are maintained in Escherichia coli as a plasmid, carries a therapeutic gene in place of the E1 region and is flanked by adenovirus DNA sequences. The E1 region is located near the 5′ end of the adenovirus genome. The second transfected DNA segment is an adenovirus DNA molecule that lacks part of its 5′ end, including the E1 region, but shares a region of adenoviral DNA with the plasmid that carries the therapeutic gene.

A recombination event in the region of overlap between the two transfected pieces of DNA reconstitutes a full length adenovirus genome that contains the therapeutic gene and lacks the E1 region. The E1 gene products that are supplied by the host cell initiate virus production; virus particles are assembled and then released after cell lysis. The DNA cloning capacity of this adenovirus vector system is about 7.5 kb.

If recombination does not occur, both transfected DNA molecules are too small to be packaged properly. Moreover, recombination between the El DNA region in the genome of the host cell and the recombinant adenovirus DNA to form a replication -competent virus is extremely unlikely.

After infection of a target cell with a recombinant adenovirus, the DNA is passed into the cell nucleus, where the therapeutic gene is expressed. The recombinant DNA construct does not integrate into a chromosome, and consequently, it does not persist for long periods. Therefore, adenovirus based gene therapy requires periodic administration with additional recombinant viruses. Adenovirus gene delivery systems have been used in gene therapy trials for treating cystic fibrosis.

Adeno-Associated Virus Vector System:

Adeno- associated virus is a small, non pathogenic, single-stranded human DNA virus (4.7kb) that can integrate into a specific site on chromosome 19. Productive infection by adeno-associated virus depends on proteins from another virus (helper virus) such as adenovirus, hence the name adeno-associated virus. After the adeno-associated virus enters the nucleus, the polymerase of a host cell converts the adeno-associated virus genome to a double-stranded DNA that is then transcribed.

Its absence of pathogenicity makes adeno-associated virus a good candidate as a vector for the delivery of therapeutic genes. Recombinant adeno associated virus is generated by cotransfection of two plasmids into a host cell that has been infected with adenovirus (helper virus). One of the plasmids carries a therapeutic gene flanked by the inverse terminal repeat sequences (~125bp each) from the adeno-associated virus. The second plasmid contains the two genes (rep and cap) of the adeno-associated virus genome that are responsible for replication of the adeno-associated virus and its capsid, respectively.

After lysis of infected host cells, recombinant adeno-associated virus particles are purified from adenovirus by centrifugation and dialysis. Any residual adenovirus (helper virus) in the sample is killed by heat treatment. In the absence of the rep gene, the recombinant adeno-associated virus DNA is unlikely to integrate into chromosome 19. On the other hand, without any adeno-associated virus genes, the recombinant vector will not evoke an immune response.

Besides virus-mediated gene-delivery systems, there are several non-viral options for gene delivery. The simplest method is the direct introduction of therapeutic DNA into target cells. This approach is limited in its application because it can be used only with certain tissues and requires large amounts of DNA. Another non-viral approach involves the creation of an artificial lipid sphere with an aqueous core. This liposome, which carries the therapeutic DNA, is capable of passing the DNA through the target cell’s membrane.

Therapeutic DNA also can get inside target cells by chemically linking the DNA to a molecule that will bind to special cell receptors. Once bound to these receptors, the therapeutic DNA constructs are engulfed by the cell membrane and passed into the interior of the target cell. This delivery system tends to be less effective than other options.

Researchers also are experimenting with introducing a 47th (artificial human) chromosome into target cells. This chromosome would exist autonomously alongside the standard 46 not affecting their workings or causing any mutations.

It would be a large vector capable of carrying substantial amounts of genetic code, and scientists anticipate that, because of its construction and autonomy, the body’s immune systems would not attack it. A problem with this potential method is the difficulty in delivering such a large molecule to the nucleus of a target cell.

Current Status of Gene Therapy Research:

Gene therapy is “the use of genes as medicine”. It involves the transfer of a therapeutic or correct gene into specific cells of an individual in order to repair a faulty gene. Thus it may be used to replace a faulty gene, or to introduce a new gene whose function is to cure or to favourably modify the clinical course of a disease.

The scope of this new approach to the treatment of disease is broad, with potential in the treatment of many genetic conditions, some forms of cancer and certain viral infections such as Acquired Immune Deficiency Syndrome (AIDS). At the present time however, gene therapy remains an experimental discipline and much research remains to be done before this approach to the treatment of disease will realize its full potential.

There are over 600 clinical gene therapy trials initiated or approved worldwide. Of these, the majorities are being conducted in the United States and Europe, with only a modest number initiated in other countries, including Australia. Perhaps surprisingly, the majority of trials focuses on treating acquired diseases, such as cancer and AIDS, although an increasing number of inherited conditions are being targeted.

A form of immune deficiency called adenosine deaminase (ADA) deficiency was the first disease to be treated with a gene therapy approach in humans in the early 1990’s, and was also the first condition for which therapeutic gene transfer into stem cells has been attempted in the clinical arena.

Another form of immune deficiency is due to a mutation in a gene located on the X chromosome and is called Severe Combined Immune Deficiency (SCID). This “X-linked condition” only affects boys. The use of gene therapy in the treatment of this condition was started by a French research group Cavazzana-Calvo and co-workers (2000). It was hailed as the first example of a genetic condition being successfully treated by gene therapy and is a milestone in medical history.

The US Food and Drug Administration (FDA) have not yet approved any human gene therapy product for sale. Current gene therapy is experimental and has not proven very successful in clinical trials. Little progress has been made since the first gene therapy clinical trial began in 1990.

In 1999, gene therapy suffered a major setback with the death of 18-year old lesse Gelsinger lesse was participating in a gene therapy trial for ornithine trans-carboxylase deficiency (OTCD). He died from multiple organ failures 4 days after starting the treatment. His death is believed to have been triggered by a severe immune response to the adenovirus carrier.

The most active research being done in gene therapy for children has been for genetic disorders such as cystic fibrosis. Other gene therapy trials involve children with severe immune-deficiencies, such as adenosine deaminase (ADA) deficiency (a rare genetic disease that makes children prone to serious infection), and those with familial hypercholesterolemia (extremely high levels of serum cholesterol).

Advances in understanding and manipulating genes have set the stage for scientists to alter a person’s genetic material to fight or prevent disease. Gene therapy is an experimental treatment that involves introducing genetic material (DNA or RNA) into a person’s cells to fight disease. Gene therapy is being studied in clinical trials (research studies with people) for many different types of cancer and for other diseases. It is not currently available outside a clinical trial.

Researchers are studying several ways to treat cancer using gene therapy. Some approaches target healthy cells to enhance their ability to fight cancer. Other approaches target cancer cells, to destroy them or prevent their growth.

Some gene therapy techniques under study are described below:

1. In one approach, researchers replace missing or altered genes with healthy genes. Because some missing or altered genes (e.g., p53) may cause cancer, substituting “working” copies of these genes may be used to treat cancer.

2. Researchers are also studying ways to improve a patient’s immune response to cancer. In this approach, gene therapy is used to stimulate the body’s natural ability to attack cancer cells. In one method under investigation, researchers take a small blood sample from a patient and insert genes that will cause each cell to produce a protein called a T-cell receptor (TCR).

The genes are transferred into the patient’s white blood cells (called T lymphocytes) and are then given back to the patient. In the body, the white blood cells produce TCRs, which attach to the outer surface of the white blood cells.

The TCRs then recognize and attach to certain molecules found on the surface of the tumor cells. Finally, the TCRs activate the white blood cells to attack and kill the tumor cells.

3. Scientists are investigating the insertion of genes into cancer cells to make them more sensitive to chemotherapy, radiation therapy, or other treatments. In other studies, researchers remove healthy blood-forming stem cells from the body, insert a gene that makes these cells more resistant to the side effects of high doses of anticancer drugs, and then inject the cells back into the patient.

4. In another approach, researchers introduce “suicide genes” into a patient’s cancer cells. A pro-drug (an inactive form of a toxic drug) is then given to the patient. The pro-drug is activated in cancer cells containing these “suicide genes,” which leads to the destruction of those cancer cells.

5. Other research is focused on the use of gene therapy to prevent cancer cells from developing new blood vessels (angiogenesis).

Some of the recent developments in gene therapy research include:

1. University of California, Los Angeles, research team gets genes into the brain using liposome’s coated in a polymer call polyethylene glycol (PEG). The transfer of genes into the brain is a significant achievement because viral vectors are too big to get across the “blood-brain barrier.” This method has potential for treating Parkinson’s disease.

2. RNA interference or gene silencing may be a new way to treat Huntington’s. Short pieces of double-stranded RNA (short, interfering RNAs or siRNAs) are used by cells to degrade RNA of a particular sequence. If a siRNA is designed to match the RNA copied from a faulty gene, then the abnormal protein product of that gene will not be produced.

3. New gene therapy approach repairs errors in messenger RNA derived from defective genes. Technique has potential to treat the blood disorder thalassemia, cystic fibrosis, and some cancers.

4. Researchers at Case Western Reserve University and Copernicus Therapeutics are able to create tiny liposomes 25 nanometers across that can carry therapeutic DNA through pores in the nuclear membrane.

5. Sickle cell is successfully treated in mice.

Limitations of Gene Therapy:

1. Short-lived Nature of Gene Therapy:

Before gene therapy can become a permanent cure for any condition, the therapeutic DNA introduced into target cells must remain functional and the cells containing the therapeutic DNA must be long-lived and stable. Problems with integrating therapeutic DNA into the genome and the rapidly dividing nature of many cells prevent gene therapy from achieving any long-term benefits. Patients will have to undergo multiple rounds of gene therapy.

2. Immune Response:

Anytime a foreign object is introduced into human tissues, the immune system is designed to attack the invader. The risk of stimulating the immune system in a way that reduces gene therapy effectiveness is always a potential risk. Furthermore, the immune system’s enhanced response to invaders makes it difficult for gene therapy to be repeated in patients.

3. Problems with Viral Vectors:

Viruses, while the carrier of choice in most gene therapy studies, present a variety of potential problems to the patient toxicity, immune and inflammatory responses, and gene control and targeting issues. In addition, there is always the fear that the viral vector, once inside the patient, may recover its ability to cause disease.

4. Multi-gene Disorders:

Conditions or disorders that arise from mutations in a single gene are the best candidates for gene therapy. Unfortunately, some the most commonly occurring disorders, such as heart disease, high blood pressure, Alzheimer’s disease, arthritis, and diabetes, are caused by the combined effects of variations in many genes. Multi-gene or multi-factorial disorders such as these would be especially difficult to treat effectively using gene therapy.

Ethical Considerations for Gene Therapy:

While the body has many billions of cells, only a very small proportion of these cells are involved in reproduction, the process by which our genes are handed on to future generations. In males these cells are located in the testes and in females, in the ovaries. These special reproductive cells are called “germ cells”. All other cells in the body, irrespective of whether they are brain, lung, skin or bone cells, are known as “somatic cells”.

In gene therapy, only somatic cells are targeted for treatment. So any changes to the genes of a person by gene therapy will only impact on the cells of their body and cannot be passed on to their children. Changes to the somatic cells cannot be passed on to future generations (inherited). Gene therapy treats the individual and has no impact on future generations.

The possible genetic manipulation of the egg or sperm cells (germ cells) remains the subject of intense ethical and philosophical discussion. The strong consensus view at present is that the risks of germ-line manipulation far exceed any potential benefit and should not be attempted.

Gene therapy does have risks and limitations. The viruses and other agents used to deliver the “good” genes can affect more than the cells for which they are intended. If a gene is added to DNA, it could be put in the wrong place, which could potentially cause cancer or other damage.

Genes can also be “over-expressed,” meaning they can drive the production of so much of a protein that they can be harmful. Another risk is that a virus introduced into one person could be transmitted to other people or into the environment.

Gene therapy trials with some children present an ethical question. We don’t test children to see if they are carriers of genetic diseases, because at this point, there’s nothing we can do about it. If a child knows he has a problem gene, there’s no guarantee he will be affected by it, but he has to live with the knowledge for the rest of his life. For example, the gene for some types of breast cancer has been identified. But if we test a young girl to see if she’s a carrier for it, what will she do with that information?

The principle objections are summarized as:

(i) The technology is imperfect:

The effects of gene transfer are unpredictable and, even if the target disease was cured, further defects could be introduced into the embryo.

(ii) Denial of human rights:

Individuals resulting from germ line gene therapy would have no say in whether their genetic material should have been modified.

(iii) Potential abuse:

Germ line gene therapy could be used not only to eliminate disease, but also to enhance favourable characteristics and suppress unfavourable ones. On a small scale, this would result in a generation of ‘designer children’, with traits chosen by their parents. On a large scale, gene therapy could result in eugenics manipulation of the genetic properties of a population.

Some questions to be considered by the society:

(i) What is normal and what is a disability or disorder, and who decides?

(ii) Are disabilities diseases? Do they need to be cured or prevented?

(iii) Does searching for a cure demean the lives of individuals presently affected by disabilities?

(iv) Is somatic gene therapy (which is done in the adult cells of persons known to have the disease) more or less ethical than germ-line gene therapy (which is done in egg and sperm cells and prevents the trait from being passed on to further generations)? In cases of somatic gene therapy, the procedure may have to be repeated in future generations.

(v) Preliminary attempts at gene therapy are exorbitantly expensive. Who will have access to these therapies? Who will pay for their use?

Application # 4. Recombinant DNA Technology in the Synthesis of Human Insulin:

Since Banting and Best discovered the hormone, insulin in 1921, diabetic patients, whose elevated sugar levels are due to impaired insulin production, have been treated with insulin derived from the pancreas glands of killed animals. The hormone, produced and secreted by the beta cells of the pancreas’ islets of Langerhans, regulates the use and storage of food, particularly carbohydrates.

Although bovine and porcine insulin are similar to human insulin, their composition is slightly different. Consequently, a number of patients’ immune systems produce antibodies against it, neutralizing its actions and resulting in inflammatory responses at injection sites.

These factors led researchers to consider synthesising Humulin (short form of human insulin) by inserting the insulin gene into a suitable vector, the Escherichia coli (E. coli) bacterial cell, to produce insulin that is chemically identical to its naturally produced insulin (Fig. 19.5, 6). This has been achieved using Recombinant DNA technology. This method is a more reliable and sustainable method than extracting and purifying the by-product from slaughtered animals.

Chemically, insulin is a small, simple protein. It consists of 51 amino acid, 30 of which constitute one polypeptide chain, and 21 of which comprise a second chain. The two chains are linked by a disulfide bond. The genetic code for insulin is found in the DNA at the top of the short arm of the eleventh chromosome.

It contains 153 nitrogen bases (63 in the A chain and 90 in the B chain). A weakened strain of the common bacterium, E. coli an inhabitant of the human digestive tract, is the ‘factory’ used in the genetic engineering of insulin. In E. coli, B-galactosidase is the enzyme that controls the transcription of the genes. To make the bacteria produce insulin, the insulin gene needs to be tied to this enzyme.

Some north-central Washington farmers have grown a genetically modified safflower plant the past two years for a Canadian biotechnical pharmaceutical company searching for a cheaper way to produce insulin. Eventually, SemBioSys Genetics Inc. of Calgary, Alberta, hopes to raise several thousand acres of genetically-modified-organism safflower. The company had developed the ability to produce insulin from GMO safflower seed for far less money than the traditional method of producing synthetic insulin genetic engineering of bacteria grown in large steel bioreactors.

The company could cut traditional production costs by up to 90 percent and meet the global demand for insulin on 10,000 to 20,000 acres of GMO safflower. SemBioSys is ambitiously hoping to have the safflower produced insulin on the market by 2010.

Application # 5. Hepatitis B Vaccine:

Hepatitis B (HepB) is a major public health problem worldwide. Approximately 30% of the world’s population, or about 2 billion persons, have serologic evidence of hepatitis B virus (HB V) infection. Of these, an estimated 350 million have chronic HBV infection and at least one million chronically infected persons die each year from liver cancer and cirrhosis.

HBV is second only to tobacco as a known human carcinogen. HepB vaccine is effective in preventing HBV infections when it is given either before exposure or shortly after exposure. At least 85-90% of HBV- associated deaths are vaccine-preventable.

Hepatitis B is a killer, taking the lives of 900,000 people each year. This disease is especially dangerous for infants, since those who are infected when young, may carry the infection for the rest of their lives, often without knowing it. Fortunately, hepatitis B vaccine, if provided to infants, helps protect them against these problems. In effect, it is the world’s first anticancer vaccine.

Due to the seriousness of hepatitis B disease, and because of the high effectiveness and safety of the vaccine, the World Health Organization (WHO) recommends that it be given to all children worldwide. The hepatitis B vaccine has been available for decades, but introduction into the developing world only began in the late 1980s. Currently, more than 100 countries routinely provide the vaccine, but many still cannot afford to do so.

Definition:

Hepatitis B vaccine (rDNA) is a preparation of hepatitis B surface antigen (HBsAg), a component protein of hepatitis B virus; the antigen may be adsorbed on a mineral carrier such as aluminum hydroxide or hydrated aluminum phosphate. The antigen is obtained by recombinant DNA technology.

Hepatitis B vaccine (rDNA) is produced by the expression of the viral gene coding for HBsAg in yeast (Saccharomyces cerevisiae) or mammalian cells (Chinese hamster ovary (CHO) cells or other suitable cell lines), purification of the resulting HBsAg and the rendering of this antigen into an immunogenic preparation. The suitability and safety of the cells are approved by the competent authority.

The vaccine may contain the product of the S gene (major protein), a combination of the S gene and pre-S2 gene products (middle protein) or a combination of the S gene, the pre-S2 gene and pre-S1 gene products (large protein).

The antigen must comply following standard quality criteria: Total protein value, Antigen content and identification using ELISA and SDS page electrophoresis, Antigenic purity by reaction, Composition in terms of the content of proteins, lipids, nucleic acids and carbohydrates, Host-cell- and vector-derived DNA presence, Caesium content (used for purity of antigen) and lastly sterility.

Recently, the transfer of hepatitis-B surface antigen gene in tobacco and expression of this recombinant gene in tobacco followed by partial purification of protein antigen from the plant showed possibility of producing this antigen through higher plants (Molecular Farming). When this protein was injected into mice, it elicited antibody response similar to that obtained with yeast derived commercially available vaccine.

This is clear that gene product obtained from two different organisms has same property and transgenic plants can be used as source of antibodies. Attempts are being made to produce many more molecules by cultivation of transgenic higher plants.