IV.

Methods

In gene therapy, one or more genes are inserted into a cell, where they produce a missing protein or enzyme. Researchers have developed several methods for transporting genes into cells. The most common technique is to attach healthy genes to genetically modified viruses. These infectious agents, known as vectors, carry the genes into a cell’s nucleus and incorporate them into the genetic material of the infected cell. Another gene-delivery method still under development is chimeraplasty, in which segments of DNA are inserted into a cell’s nucleus. The DNA segment binds with a defective gene in a way that helps the cell’s repair mechanisms identify and fix the defective gene.

A.

Virus Vectors

About 15 years ago scientists demonstrated that viruses could be used as vectors for the delivery of healthy genes. Scientists use four types of viruses in gene therapy experiments—retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex viruses.

A.1.

Retroviruses

Retroviruses were the first viruses used as vectors in gene therapy experiments. They are unusual because instead of using DNA to carry their genetic information to the cell’s protein-making machinery, retroviruses use a related material called ribonucleic acid (RNA) as their primary carrier of genetic information. When retroviruses invade a cell, they use an enzyme called reverse transcriptase to make a DNA copy of their genes. Other enzymes then incorporate this DNA copy into the infected cell's DNA.

In one application, scientists use a retrovirus that causes leukemia in mice but no known disease in humans. The researchers remove the genes that cause disease and, in their place, insert an RNA copy of a healthy gene into the virus. They also add a piece of genetic information called a promoter. The promoter is, in effect, an “on/off switch” for a gene. When it is turned on, usually with a drug, it tells the cell's protein-making machinery to begin producing the inserted gene's protein.

Although retroviruses have been used in most gene therapy experiments so far, they still present many problems. Retroviruses can invade only cells that are actively dividing, limiting potential targets for therapy to blood cells, skin cells, stem cells, and other fast-growing tissues. In addition, the viruses have no specific targets in the infected cells' chromosomes. As a result, the genes they carry are inserted in a haphazard manner.

Ideally, retroviruses insert genes into the middle of a strand of DNA that does not contain other genes. The genes might, however, be inserted smack in the middle of a crucial gene, rendering it defective and blocking key cellular functions, causing more damage than repair. Retroviruses could also integrate new genes into a stretch of DNA where they could cause cancer. Despite the presence of promoters, moreover, the added genes typically do not produce sufficient amounts of proteins to effectively treat disease. In addition, the patient’s body generally recognizes retroviruses as foreign invaders, provoking adverse immune responses.

Researchers approached the use of retroviruses with caution because of concerns that they might attack inappropriate cells. To avoid this problem, researchers initially removed blood or other target cells from the patient's body before treatment with the retrovirus. They then monitored the cells to ensure the therapy was working properly before returning the cells to the patient’s body.

As researchers have grown more confident, however, they have begun injecting altered retroviruses directly into tissues where the corrected genes are needed and have, so far, observed few problems. In clinical trials of patients with cystic fibrosis, a disease in which a mutated gene impairs lung function, healthy genes are inserted directly into the lining of bronchial tubes. In studies of animals, researchers have used retroviruses to inject genes directly into muscle tissue to learn if the genes will produce normal muscle proteins. Researchers hope this treatment will one day help people with muscular dystrophy.

A.2.

Adenoviruses

To avoid the problem of inserting genes at the wrong sites, some researchers have turned to other types of viruses, such as the adenoviruses, which cause the common cold. Stripped of their disease-causing genes, adenoviruses take healthy genes into the nucleus of cells, where the DNA is located, but do not usually integrate them into a cell's DNA. Researchers thus trade safety for impermanence, because the genes persist in the cell’s DNA only for days to weeks. Adenoviruses can also infect a broader variety of cells than retroviruses do, including cells that divide more slowly, such as lung cells. However, adenoviruses are also more likely to be attacked by the patient's immune system, and the high levels of virus required for treatment often provoke an undesirable inflammatory response. Despite these drawbacks, adenoviruses have been used in attempts to treat cancers of the liver and ovaries.

A.3.

Adeno-Associated Virus

One of the most promising potential gene-delivery systems, or vectors, is a recently discovered virus called the adeno-associated virus, which infects a broad range of cells, including both dividing and nondividing cells. Researchers believe that most humans carry adeno-associated viruses, which do not cause disease and do not provoke an immune response. Scientists have demonstrated that the adeno-associated virus can be used to correct genetic defects in animals. It is now being used in preliminary studies to treat hemophilia, a hereditary blood disease, in humans.

The chief drawback of the adeno-associated virus is that it is small, carrying only two genes in its natural state. Its payload is therefore relatively limited. It can produce unintended genetic damage because the adeno-associated virus inserts its genes directly into the host cell's DNA. Researchers have also had difficulties manufacturing large quantities of the altered virus.

A.4.

Herpes Simplex Virus

Scientists have found that the herpes simplex virus, the cause of the common cold sore, has a very large genome compared to other virus vectors. This large genome enables scientists to insert more than one therapeutic gene into a single virus, paving the way for the treatment of disorders caused by more than one gene defect. The virus makes an ideal vector because it can infect a wide variety of tissues, including muscle, tumor, liver, pancreas, nerve, and lung cells.

One problem with using herpes simplex virus is that the virus is cytopathic—that is, it kills the cells that it infects. In addition, the virus can cause encephalitis (inflammation of the brain) if it replicates freely in the brain. Scientists are developing a form of herpes simplex virus in which the genes that direct the virus’s replication and cell-killing abilities have been removed.

B.

Chimeraplasty

Some researchers believe that in the near future a process called chimeraplasty may make it possible to fix defective genes within a cell directly, making it unnecessary to insert new genes into cells. Researchers have developed short segments of DNA called oligomers, whose nucleotide sequences complement those of a gene in which a defect occurs. When inserted into the cell's nucleus, oligomers bind to the defective gene where the sequences are correct, but they do not bind properly at defective sites. The cell's repair machinery sees this “bump” in the DNA and interprets it as a signal to repair the defective gene. Chimeraplasty has been successfully tested in animals, and investigators have recently begun to test it in humans.

C.

Clinical Trials

Once gene therapy methods have been developed in a laboratory and tested on animals, scientists need to prove that they work in humans. Scientists approach any experiments in humans with great care. Since gene therapy is a new technique that may have unforeseen risks, they develop a proposed experiment, known as a protocol, that incorporates strict safety guidelines.

In the United States, a gene therapy protocol must be reviewed and approved by the Recombinant DNA Advisory Committee of the National Institutes of Health (NIH). If approved, the protocol is then submitted to the Food and Drug Administration (FDA) for approval. After the FDA gives permission for human testing to begin, they continue to monitor the experiments. In the course of a clinical trial, researchers are required to report any harmful side effects resulting from the gene therapy treatment under study. The FDA has approved more than 400 clinical trials of gene therapy, although in early 2003 the FDA placed a temporary ban on 30 of the clinical trials that used retrovirus vectors. The FDA took this precautionary measure after the French gene therapy trial for SCID resulted in a life-threatening disorder that may have been caused by the procedure. The FDA expects to permit the U.S. trials to resume if safety measures are taken to minimize the risk of using retrovirus vectors.