Viruses are Used for Gene Therapy

Viruses Are Used for Gene Therapy

Although viruses have usually been seen as problems for humans, there is one field now in which they are being used for good. Viruses can be used to make alterations in somatic cells, whereby a genetic disease is treated by the introduction of a gene for a missing protein. This is called gene therapy. The most successful form of gene therapy to date involves the gene for adenosinedeaminase (ADA), an enzyme involved in purine catabolism. If this enzyme is missing, dATP builds up in tissues, inhibiting the action of the enzyme ribonucleotide reductase. This results in a deficiency of the other three deoxyribonucleoside triphosphates (dNTPs). The dATP (in excess) and the other three dNTPs (deficient) are precursors for DNA synthesis. This imbalance particularly affects DNA synthesis in lymphocytes, on which much of the immune response depends. Individuals who are homozygous for adenosine deaminase deficiency develop severe combined immune deficiency (SCID), the “bubble-boy”syndrome. These individuals are highly prone to infection because of their highly compromised immune systems. The ultimate goal of the planned gene therapy is to take bone marrow cells from affected individuals; introduce the gene for adenosine deaminase into the cells using a virus as a vector; and then reintroduce the bone marrow cells in the body, where they will produce the desired enzyme. The first clinical trials for ADA-SCID were simple enzyme-replacement therapies begun in 1982. The patients were given injections of ADA. Later clinical trials focused on correction of the gene in mature T cells. In 1990, transformed T cells were given to recipients via transfusions.

In trials at the National Institutes of Health (NIH), two girls, ages 4 and 9 at the start of treatment, showed improvement to the extent that they could attend regular public schools and have no more than the average number of infections. Administration of bone marrow stem cells in addition to T cells was the next step; clinical trials of this procedure were undertaken with two infants, ages 4 months and 8 months, in 2000. After 10 months, the children were healthy and had restored immune systems.

There are two types of delivery methods in human gene ther-apy. The first, called ex vivo, is the type used to combat SCID. Ex vivo delivery means that somatic cells are removed from the patient, altered with the gene therapy, and then given back to the patient. The most common vector for this is the retrovirus Maloney murine leukemia virus (MMLV). The figure shows howthe virus is used for gene therapy. Some of the MMLV is altered to remove the gag, pol, and env genes, rendering the virus unable to replicate. These genes are replaced with an expression cas-sette, which contains the gene being administered, such as theADA gene, along with a suitable promoter. This mutated virus is used to infect a packaging cell line. Normal MMLV is also used to infect the packaging cell line, which is not susceptible to the MMLV. The normal MMLV does not replicate in the packaging cell line, but its gag, pol, and env genes restore the mutated virus’s ability to replicate, but only in this cell line. These controls are necessary to keep mutant viruses from escap-ing to other tissues. The mutated virus particles are collected from the packaging cell line and used to infect the target cells, such as bone marrow cells in the case of SCID. MMLV is a ret-rovirus, so it infects the target cell and produces DNA from its RNA genome, and this DNA can then incorporate into the host genome, along with the promoter and ADA gene. In this way, the target cells that were collected have been transformed and produce ADA. These cells are then put back into the patient.

The second delivery method, called in vivo, means that the virus is used to directly infect the patient’s tissues. The most common vector for this delivery is the adenovirus (which is a DNA virus). A particular vector can be chosen based on spe-cific receptors on the target tissue. Adenovirus has receptors in lung and liver cells, and it has been used in clinical trials for gene therapy of cystic fibrosis and ornithine transcarbamoylase deficiency.

Clinical trials using gene therapy to combat cystic fibrosis and certain tumors in humans are under way. In mice, gene therapy has been successful in fighting diabetes. The field of gene therapy is exciting and full of promise, but there are many obstacles to success in humans. There are also many risks, such as the risk of a dangerous immunological response to the vector carrying the gene or the danger of a gene becoming incorpo-rated into the host chromosome at a location that activates a cancer-causing gene.

The second delivery method, called in vivo, means that the virus is used to directly infect the patient’s tissues. The most common vector for this delivery is the adenovirus (which is a DNA virus). A particular vector can be chosen based on spe-cific receptors on the target tissue. Adenovirus has receptors in lung and liver cells, and it has been used in clinical trials for gene therapy of cystic fibrosis and ornithine transcarbamoylase deficiency.

Clinical trials using gene therapy to combat cystic fibrosis and certain tumors in humans are under way. In mice, gene therapy has been successful in fighting diabetes. The field of gene therapy is exciting and full of promise, but there are many obstacles to success in humans. There are also many risks, such as the risk of a dangerous immunological response to the vector carrying the gene or the danger of a gene becoming incorporated into the host chromosome at a location that activates a cancer-causing gene.

Gene therapy via retroviruses. The Maloney murine leukemia virus(MMLV) is used for ex vivo gene therapy. (a) Essential genes (gag, pol,env) are removed from the virus and (b) replaced with an expression cas-sette containing the gene being replaced with gene therapy. Removal of the essential viral genes renders the viruses unable to replicate. (c) The altered virus is then grown in a packaging cell line that allows replication.

(d) Viruses are collected and then used to infect cultured target cells from the patient needing the gene therapy. (e) The altered virus produces RNA, which then produces DNA via reverse transcriptase. The DNA then integrates in the patient’s cells’ genome, and then his or her cells produce the desired protein. The cultured cells are then given back to the patient. (Adapted from Figure 1 in Crystal, R. G., 1995. Transfer of genes to humans: Early lessons and obstacles to success. Science270, 404.)