The prospects for gene therapy

There are an estimated 100,000 genes in the human body. By replacing defective genes with healthy ones, it will be possible to treat many hereditary diseases such as haemophilia, cystic fibrosis and, eventually, some forms of muscular dystrophy and cancer.

Rapid progress has been made in recent years in characterizing hereditary diseases and in understanding their causes. These diseases are due to alterations of genes, which encode the information for building the body and making it work. Genes are the carriers of hereditary traits and are contained in filaments of DNA (deoxyribonucleic acid) that are present in the cells of all organisms. The precise number of genes in the human body is unknown, but it is estimated to be around 100,000.

Every cell has two copies of each gene, one deriving from the father, the other from the mother. The information in the genes is used for making proteins, the agents for building the body and making it work. A disease appears when a gene is altered in such a way that either it does not make its corresponding protein, or else makes a protein that is incapable of functioning. Since the same protein is encoded in both genes of each pair, if only one fails enough protein continues to be made to satisfy the needs of the organisms, and there is no disease. A disease appears when both fail and no functional protein is made. This generally happens when a child is born from two healthy parents, each of whom carries an altered gene of the same pair. Such parents have a one-in-four chance of generating a defective child.

Hereditary genetic diseases can vary greatly in severity. At one extreme are diseases such as phenylketonuria, which, after detection at birth, can be completely prevented by adopting a suitable diet. At the other are those like Lesch-Nyhan syndrome, which affects only boys, causing a terrible form of mental retardation in which they tend to eat themselves and must be kept bound to their beds to prevent them from eating their fingers. Even so, they may eat their lips or their tongues. All hereditary diseases generate a degree of handicap, and for the majority of them there is not as yet any effective therapy.

Vectors and receptors

During the last ten years many of the genes responsible for hereditary diseases have been discovered, leading to the possibility of a new form of therapy known as "gene therapy" that is based on the introduction into the patient's cells of a good copy of the defective gene so as to perform the missing function. The concept is straightforward, but the implementation presents many problems, which so far have limited its use to only a few cases.

To treat a hereditary disease with gene therapy several conditions must be met. In the first place the gene responsible for the disease must be known and must be isolated in a form that contains all the information needed to make the protein. Many copies of the gene must be available, to maximize the number of cells into which it can be introduced. The fresh genetic material is usually inserted into the malfunctioning cells by means of a "vector" or carrier. Such vectors are often obtained from viruses; but many viruses may either kill cells or change them to cancer cells. To prevent these adverse effects the genes responsible for causing them are removed from the viruses.

The first vectors used in gene therapy were made from viruses known as "retro-viruses". However, retro-viral vectors have important drawbacks: they only enter into some types of cell, and then the gene they carry acts weakly. The major disadvantage is that they cannot become established in cells that do not multiply. More recently viruses of another kind, known as "adeno-viruses", have been used extensively because they can become established in cells that do not multiply. Today most gene therapy is being carried out using as a vector either adeno-viruses or other viruses with similar properties.

Many attempts have been made to use other ways of delivering genes into cells. One of them takes advantage of the ability of certain "factors" present in the blood or other body fluids to enter cells by interacting with "receptors" present at the surface of cells. For this method to work, the cell into which the gene must be introduced needs to have a suitable receptor at its surface, and this limits the types of cells that can be used.

A method suitable for any cell has thus been developed, which consists in embedding the gene into a microscopic vesicle called a "liposome", made up of an artificial membrane similar to those that surround the cells. When the vesicle touches the cell, its membrane fuses with the cellular membrane, and the gene it contains enters the cell. This method seems to have few drawbacks but it has not yet been used widely enough for its efficacy to be proved. Most recently it has been shown that naked DNA can enter cells, opening up the possibility of an even simpler method for gene delivery.

Inserting the gene into a suitable vector is the first step in gene therapy; the next step is to introduce the vector into the cells. The type of cell to be targeted depends on the nature of the disease produced by the genetic defect. In many of the attempts using viral vectors the cells are taken from the body, grown in vitro, and returned to the body after introduction of the gene. To carry out this procedure the cells must be accessible, like those of muscle, skin and blood, but unlike those of internal organs. With the newer approaches using receptor binding, liposomes, or naked DNA, the handicap is not so great, and genes have been introduced into the liver, lungs and muscle.

The first attempt to use gene therapy on a human being was carried out in 1990 on a girl affected by adenosine deaminase (ADA) deficiency, that is, the cells of the immune system lacked an essential enzyme and were unable to defend her body against infections. The child had to live under a plastic bell. She could not play with other children and could not go to school. The cells lacking the enzyme are accessible because they are made in the bone marrow and then enter the blood. The therapy was carried out by taking the cells from the blood, introducing the ADA gene into them, and then returning them to the patient's blood. The result has been good. The defence functions of the girl's immune system were re-established and she was able to leave her abode and go to school.

One drawback of this therapy is that the modified cells do not last a long time, and so the treatment must be repeated at frequent intervals. This difficulty might be overcome by introducing the gene into large cells of the bone marrow known as stem cells, which have a much longer life span. However they do not multiply readily in vitro, and therefore the retro-viral vector would not work for them, and, moreover, they are very difficult to isolate. The therapy is therefore effective but still needs much additional work.

Hope for haemophiliacs

Another promising case for gene therapy is haemophilia, which causes a tendency to bleed due to lack of a coagulation factor which is made in the liver and then secreted into the blood stream. There are two forms of the disease: A and B, in each of which a different factor is lacking. Attention has been concentrated on haemophilia B because the gene responsible for the factor (called factor IX) is of manageable size; the other is too big for the available vectors.

Work has been carried out on dogs, which also suffer from this form of haemophilia, using a retro-viral vector. To replenish the blood with the factor two strategies were followed. In one case, cells were taken from the animal, either from subcutaneous tissue, muscle, or the epidermis, grown in vitro, and after introduction of the vector, were implanted into the animal, where they continued to produce the missing factor for many months, and perhaps indefinitely.

The other strategy was to introduce the same vector into the liver. To overcome the difficulty that the liver cells do not multiply, one third of the liver was surgically removed in order to cause a regeneration of the organ, and therefore cell multiplication. The result was a steady, although low, production of the factor, which again lasted for a long time, with a satisfactory correction of the disease. These results show that there is much hope for a long-term control of haemophilia through gene therapy.

A disease targeted for gene therapy since its gene was isolated a few years ago is cystic fibrosis, in which many types of secreting cells are affected, but especially those of the lung with accumulation of mucus and limitation of oxygen uptake. Results suggest that human gene therapy holds promise as a treatment for this disease, especially if it can be done by delivering the gene through nebulization, which is very simple.

Attempts to treat cancer

Attempts to use gene therapy have been made for a number of years in the treatment of cancer, which is a genetic disease, although not usually transmitted hereditarily. These attempts have taken two main directions. One, applied to brain tumours, is to introduce into the cancer cells a vector carrying a gene that converts an anti-herpes drug into a cell-killing substance. The gene, in a retro-virus vector, is injected into the cancer mass, where it is able to perform its action because the cells are multiplying, whereas it has no effect on normal brain cells, which do not multiply. Experiments with cancer transplanted into mice showed that the tumour was destroyed after the mice were injected with the anti-herpes drug. The problem in this experiment is that the vector only penetrates some of the cells so that the remaining cells may start a new growth (even though the toxic substance produced under the action of the gene can pass from one cell to another). Whether this approach will be useful in human patients remains to be seen; clinical trials are under way.

The other direction being taken by attempts to use gene therapy to treat cancer is to enhance the body's defences against the tumour. It is based on the idea that, at least in some cases, the body recognizes the cancer cells as foreign and should react immunologically, causing their destruction. This does not happen in the patient because the immunological defences are paralyzed. The approach is therefore to potentiate the immune response, which is attempted in two ways. One is to grow the immune cells in vitro in the presence of potentiating factors, and then introduce into them the gene for a cell-killing substance, after which they are injected back into the patient.

Preliminary trials in patients with melanoma--a malignant skin tumour--using the potentiated cells without the introduction of the gene have given encouraging results--a reduction of the skin tumours and of their metastases in the lungs. The other approach to potentiating the immune response consists in introducing into the cancer cells a gene that will cause them to activate the immune cells with which they come in contact; once these cells are activated they can attack cancer cells at other sites in the body. The modified cancer cells thus constitute a kind of vaccine against the cancer. Experiments in animals have yielded promising results.

Many approaches to gene therapy can be applied to hereditary human diseases, and some promising results have already been obtained. The future is bright for diseases such as adenosine deaminase (ADA) deficiency, haemophilia and cystic fibrosis, and the prospects are good for muscular dystrophy and for some cancers. Development of this field is obviously still at a very early stage, and improvements will certainly be made in areas such as the mode of delivery of the gene to the cells and the isolation and growth of suitable cells. There is every reason to expect that gene therapy will become a powerful weapon in the treatment of diseases caused by gene alterations.

RENATO DULBECCO is an Italian-born American virologist who was awarded the 1975 Nobel Prize for Physiology or Medicine, together with David Baltimore and Howard M. Temin, for their studies of the workings of cells, viral contamination and the genesis of cancer. He is the coordinator of the human genome project for Italy and Honorary President of the Salk institute for Biological Studies in La Jolla, California (U.S.A.). Among his published works is an 8-volume Encyclopaedia of Human Biology (1991).

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