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Russo [Vic]Enzo; Cove David 1995 Genetic engineering : dreams and nightmares
Oxford ; W.H. Freeman/Spektrum, New York :

FROM GENETIC DIAGNOSIS TO GENE THERAPY ...........................................................................................................................................

To know or not to know, that is the question! That is the dramatic question that more and more people win have to ask themselves in the future. We know that many common diseases are due to defective genes, often to a defect in a single gene. A number of the genes concemed have already been cloned. As a result, a person can today check if she or he has a mutant gene that causes cystic fibrosis, Huntington's disease, fragile-X syndrome* or many other diseases. We only need to give a few drops of our blood to a laboratory, which will carry out tests using information obtained from cloned genes, and a few days later we will have the answer. The children of a person suffen'ng from Huntington's disease have a 50 percent chance of developing the disease themselves. Do they want to know that they may develop a disease for which there is at present no cure? Do they want to plan their life accordingly or would they prefer to remain ignorant, with the n'sk that they in tum will pass on the disease to their children? One out of every 2000 people of North European origin will face this dilemma. The sister of a mentally retarded brother with the fragile-X syndrome will face the same choice. Although she will not suffer from the condition herself, there is a 50 percent chance that she will be a carrier of the mutant gene. If she is a carrier, on average half her sons will be mentally retarded. If she knows that she has a mutant gene, she can either decide not to have children, or if her conscience allows it, she could allow the genetic diagnosis of this disease for each male foetus, aborting those which are affected.

* Fragile-X syndrome is so called because the X chromosome is altered so that it tends to break when chromosomes are prepared for microscopic observation. The alteration often leads to mental retardation and is, next to Down's Syndrome, the most common cause of mental retardation that can be specifically diagnosed.

How many mutant genes which give rise to genetic disease have been cloned? As we have seen, there are at least 2000 different diseases which are caused by a defect in one or more genes. We called these defective genes mutant genes. All of these mutant genes have arisen by mutation of normal genes. When one of these genes is cloned, it is the normal gene which is cloned. The number of such normal genes that have been cloned is growing rapidly, and some are listed in Table 11.1. This list also gives some details of the basis of the inherited condition and of the type of mutation which occurs to give rise to it. A puzzling discovery made in 1993, was that the mutant gene causing Huntington's disease is longer than the normal gene. The DNA sequence CAG is repeated a few times in the non-nal gene but in the mutant gene this sequence is repeated hundreds of times. No one yet knows how this expan- sion can happen or why it is deleterious.

Some cloned human genes causing genetic diseases when defective

  1. Disease Gene Year affected cloned Type of defect
  2. Thalassaemia HBB 1975 Point mutation or deletion
  3. Sickle-cell anaemia HBB 1975 Point mutation
  4. Phenylketonuria PH 1983 Point mutation or deletion
  5. Familial hypercholesterolaemia LDL receptor 1985 Point mutation or deletion
  6. Severe combined immune ADA 1986 Point mutation or deletion
  7. Duchenne Muscular dystrophy DMD 1986 Deletion
  8. Cystic fibrosis CFTR 1989 Point mutation or deletion
  9. Fragile-X syndrome FMR-1 1991 Long repeat of the triplet CGG"
  10. Huntington's disease HD 1993 Long repeat of the triplet CAG"

Genetic disease is caused by a gene that does not work any more because it is defective, as a result of mutation, e.g. point mutation or deletion (see ipter 4). Therefore radioactivity, UV irradiation, and contact with mutagenic substances, such as aflatoxin, should be avoided. A chemical is called mutagen if it causes mutations in the DNA. There are hundreds if not thousands of chemicals which cause mutation in our world. Chemicals are mutagenic also cause cancer. How much of any mutagen is harmful and what is the acceptable limit to release the environment is the subject of long debate and argument between ntists, environmentalists, industrialists and politicians. Many mutant genes present in populations today are thought to be due to mistakes made during the duplication of DNA. We can probably do very little about this, we should do our best to exclude mutagens from the environment. There are a number of distinct strategies for dealing with genetic disease. potential parents can be screened for mutant genes and counselled if there is risk that offspring may be genetically abnormal. Foetuses may be genetically screened and abortion of affected foetuses offered. This approach causes problems as abortion is unacceptable to many persons. An altemative fertilize eggs in vitro, screen them for genetic abnormalities and then slant only fertilized eggs which are genetically normal. A fourth possibility is the emerging technology of gene therapy, which will be discussed later. Cystic fibrosis is a devastating genetic disease common in people of north- European descent. This disease is due to the defect in a gene called rR. This gene is not located on the X chromosome, but is on an autosome. The mutant gene is recessive and so to be affected, a child must receive a copy of the mutant gene from both parents. Five percent of people of them European descent are carriers of the mutant gene. If a man ws that he has one mutant gene, he has to consider if it is wise to be with a woman who is a carrier, and vice versa. On average, 1 out of 2 children from such matings will have cystic fibrosis. Nowadays it is easy to check if a person has a mutant CFTR gene. If a couple find they are both carriers, they then have the choice of either not having children or having children but making a prenatal diagnosis and aborted foetuses. Screening for a defective gene is however complicated by the fact that there may be many different kinds of mutation (e.g. point mutations and deletions) that cause a normal gene to become defective. Screening can often only detect one type of mutant gene. Screening is usually for the most common mutant type of the gene in the population and cannot exclude with 100 percent certainty that an individual has a different mutant gene that is undetected.

Counselling before starting a family cannot be of help for most causes of Down's Syndrome. This condition is due to the presence of an extra chromosome 21. It becomes increasingly more likely as the age of the mother increases. Only 0. I percent of offspning of mothers aged 20, are affected while two percent of offspring born to mothers aged 40 or more, are affected. This phenomenon is not due to the inheritance of a mutant gene but as far as we know, is caused by a random event. In this case only prenatal diagnosis and abortion can avoid the birth of a child with Down's Syndrome.

The problem of the carrier of a mutant CFTR gene is the problem of any carrier of a mutant gene. What should individuals do, what should govem- ments do? This is a complex ethical and social problem which we will return to later.

Current methods for gene therapy

Very few genetic diseases can be cured, and only the symptoms are treated. The mutant genes are not altered by the treatments. One of the best known treatments is the injection of insulin into diabetic patients. This therapy is very successful and not too expensive. Another example is the treatment of phenylketonuria by the use of a diet that controls the intake of phenylalanine. For blood diseases, bone marrow transplantation has been tried. Red blood cells have a limited life and are made continuously by special cells in the bone marrow. People with diseases affecting their red blood cells such as thalassaemia, could therefore be given a supply of normal bone marrow. The problem of bone marrow transplantation is that even transplants from close relatives are not always successful.

So the great hope for patients with a dominant mutant gene is to replace the mutant gene in the cells of the body with a normal one. In cases where the mutant gene is recessive, the hope is to give patients extra copies of the normal gene. The name of the game is gene replacement or to use medical terminology, gene therapy.

On the 14 September 1990 at the National Institutes of Health in Bethesda, USA, a 4-year old girl became the first known human being to be treated with gene therapy. She was suffering from Severe Combined immune Deficiency. This condition is caused by a defect in a single gene, whieh is known as ADA. As a result, the immune system does not work at all. Affected children are unable to defend themselves against infection, and have to live in a germ-free environment separated from everybody. Even so, their life is usually short. After two years of therapy, this young girl was able to attend school nominally, to swim, dance, ice skate with her family and friends. This scientific exploit was carried out by Kenneth Culver, Michael Blaese and French Anderson. What looks like a miracle is an example of how basic research in genetics and molecular biology can in the long run cure some of the plagues of mankind. Can we now cure any genetic disease? To answer this, we must as usual understand the principles involved in this success story. The ADA gene is a gene that is essential for the function of T-cells, white blood cells that are the cornerstone of our immune system. T-cells have a short life and, like red blood cells, are made continuously by the division of cells in the bone marrow. T-cells are easy to grow outside the body, in the laboratory and so Culver, Blaese and Anderson took some of these cells from the girl, grew them and then transformed them with DNA containing a normal copy of the ADA gene. They checked that the cells now contained the normal ADA gene and that the cells were using this gene to make mRNA. They then injected transformed T-cells back into the affected girl. This procedure has to be repeated periodically. The success of this strategy relied on at least three things. First, the gene concemed had been cloned. Second, the level of use of the ADA gene is not cn'tical. The immune system will work again as in a normal person, whether the normal gene is working at only one tenth of the normal level or at 50 times the normal level. Lastly, it is easy to remove T-cells and to cultivate them outside the human body. The combination of these three conditions made this first success possible. Unfortunately many genes must be active in the right tissues at the right level. It is not therefore enough just to supply the gene but it is essential to make sure that its use is regulated correctly too. For many genes, the DNA sequences needed for the correct regulation of their use are not yet known. Another major problem is that many different types of human cells, such as nerve cells, muscle cells and lung cells, cannot be cultivated in the laboratory. A successful gene therapy for cystic fibrosis would require the normal gene to be placed into the cells of the patient's lungs. For Alzheimer's disease, if the mutant genes can be found, the therapy would need to change the genes of the brain.

What strategies are there for achieving the supply of a normal gene to the correct tissue and getting it to be used at the correct time and tumed on at the right level? The main idea being explored at present is to put the DNA coding for a normal gene into the genome of a vector based on a virus. As we have seen earlier, some viruses show tissue specificity, infecting, for example, orily lung cells or brain cells. It would clearly be dangerous to use a pathogenic virus to transport the normal gene to the correct type of cell. For this reason, the viruses will need to be modified, again by genetic engineer- ing, so as to destroy their pathogenicity without affecting their ability to infect their target cells. This strategy sounds fine in principle, but would need to be introduced with extreme caution, trying it on animals first. The problem is that for many diseases, there are no animal models: no animal exists that has a disease similar to the human disease. It was only in 1992 that the first mouse with a mutant CFTR gene was obtained, and this was produced by genetic engineering.

How is it to be decided whether a new therapy is to be tried out? In the USA, there is a Recombinant DNA Advisory Committee of the National Institutes of Health, which gives permission before any gene therapy can be undertaken. Other countries are introducing similar structures. There are many biological and ethical problems still to be solved but there is also a huge potential to help millions of patients. In addition, gene therapy is also potentially very profitable.

The pharmaceutical industry has discovered a new Eldorado

Do you have cancer? Take the pill with the normal gene. Do you have Alzheimer's disease? Take the right medicine. Do you want to protect your- self from AIDS? Inject yourself with the genes for HIV immunity. All these are still dreams, but there are thousands of sen'ous scientists in dozens of new gene therapy companies who are working to make these dreams come true. For each of these diseases, there are millions of new patients every year. It is estimated that the sale of phan-naceutical and health products based on genetic engineering will increase from the few million dollars today to over one hundred billion dollars in ten years from now.