GENE THERAPY

Genetics Encyclopedia: Gene Therapy

Did You Know?

In April 2002 researchers announced that ex vivo gene therapy for severe combined immunodeficiency had been successful in five boys for up to 2.5 years.

Gene therapy is a new and largely experimental branch of medicine that uses genetic material (DNA) to treat patients. Researchers hope one day to use this therapy to treat several different kinds of diseases. While rapid progress has been made in this field in recent years, very few patients have been successfully treated by gene therapy, and a great deal of additional research remains to be done to bring these techniques into common use.

Disease Targets

Humans possess two copies of most of their genes. In a recessive genetic disease, both copies of a given gene are defective. Many such illnesses are called loss-of-function genetic diseases, and they represent the most straightforward application of gene therapy: If a functional copy of the defective gene can be delivered to the correct tissue and if it makes ("expresses") its normal protein there, the patient could be cured. Other patients suffer from dominant genetic diseases. In this case, the patient has one defective copy and one normal copy of a given gene. Some of these disorders are called gain-of-function diseases because the defective gene actively disrupts the normal functioning of their cells and tissues (some recessive diseases are also gain-of-function diseases). This defective copy would have to be removed or inactivated in order to cure these patients.

Gene therapy may also be effective in treating cancer or viral infections such as HIV-AIDS. It can even be used to modify the body's responses to injury. These approaches could be used to reduce scarring after surgery or to reduce restenosis, which is the reclosure of coronary arteries after balloon angioplasty. Each of these cases will be discussed in more detail below, but first we will deal with two technical issues of gene transfer: gene delivery and longevity of gene expression.

Gene Delivery

Whether given as pills or injections, most conventional drugs simply need to reach a minimal level in the bloodstream in order to be effective. In gene therapy, the drug (DNA) must be delivered to the nucleus of a cell in order to function, and a huge number of individual cells must each receive the DNA in order for the treatment to be effective. The situation is further complicated by the fact that a given gene may normally function in only a small portion of the cells in the body, and ectopic expression may be toxic. Thus, successful gene therapy often requires highly efficient delivery of DNA to a very restricted population of cells within the body.

To achieve these goals, many researchers have turned to viruses. Viruses are parasites that normally reproduce by infecting individual cells in the human body, delivering their DNA to the nucleus of those cells. Once there, the viral DNA takes over the cell, converting it to a factory to make more viruses. The cell eventually dies, releasing more viruses to continue the cycle. Scientists can remove or disable some of the genetic material of the virus, making it unable to reproduce outside of the laboratory. This genetic material can then be replaced by the gene needed to treat a patient. The modified (or recombinant) virus can then be administered to the patient, where it will carry the therapeutic gene into the target cells. In this way, scientists can take advantage of the virus's ability, gained over millions of years of evolution, to deliver DNA to cells with tremendous efficiency. One of the most commonly used is a cold virus called adenovirus. Recombinant adenoviruses have been used in experimental gene therapy for muscle diseases, and can deliver genes to almost all of the cells in a small region surrounding the site of injection. Unfortunately, while adenoviruses excel at gene delivery, evolution is a double-edged sword, and the many mechanisms our own bodies have evolved to combat harmful viral infections are also used against therapeutic viruses, as will be discussed in more detail below.

Recombinant adenoviruses cannot be used to transfer DNA to all cell types, because they cannot reproduce themselves outside of the laboratory. When a cell with a recombinant adenovirus in it divides, only one of the two resulting cells contains the virus and the therapeutic gene it bears. The treatment of some diseases requires gene transfer to a stem cell, a cell that actively divides to create many new cells. For example, white blood cells live for only a short time, and must be constantly replenished by the division of precursor cells called hematopoietic stem cells. Gene therapy to treat an immune disease affecting white blood cells would thus require targeting these rapidly dividing cells. Researchers use a different kind of virus to accomplish this: retroviruses, so called because they contain RNA (a different kind of genetic material) rather than DNA.

When a retrovirus infects a cell, it converts its RNA to DNA and inserts it into the chromosome of the target cell. As the cell subsequently copies its own DNA during cell division, it copies the viral DNA as well, so that all of the progeny cells contain the retroviral DNA. At some later time, the viral DNA can liberate itself from the chromosome, direct the manufacture of many new viruses, and go on to repeat its life cycle. Recombinant retro-viruses are engineered so that they can enter the target cell's chromosome, but become trapped there, unable to liberate themselves and continue their life cycle. Because all progeny cells still carry the recombinant retrovirus, they will also carry the therapeutic gene.

This is a great advantage over adenoviruses as a tool for gene delivery to dividing cells, but retroviruses have some drawbacks as well. They can only infect cells that are dividing quickly, and in most cases this infection must be carried out in the laboratory. Cells must be removed from the patient, infected with the recombinant retrovirus, grown for several weeks in the lab, and then reintroduced to the patient's body. This process, called ex vivo gene transfer, is extremely expensive and labor intensive. Nonetheless, this form of gene therapy has been used in one of the most successful clinical applications to date, the treatment of two patients with severe combined immune deficiency (SCID) caused by a defect in the adenosine deaminase gene.

Before treatment, these patients had essentially no immune system at all, and would have been required to live as "bubble children," completely isolated in a sterile environment. While their treatment did not completely cure their genetic disorder, it restored their immune systems enough to allow them to leave their sterile isolation chambers and live essentially normal lives. Many other viruses are being engineered for application to gene delivery, including adeno-associated virus, herpes simplex virus, and even extensively modified forms of the human immunodeficiency virus (HIV), to name just a few.

Many researchers are also exploring nonviral methods for gene delivery. One of the most successful of these methods consists of coating the therapeutic DNA with specialized fat molecules called lipids. The resulting small fatty drops called vesicles can then be injected or inhaled to deliver the DNA to the target tissue. Many different lipid formulations have been tested and different formulations work better in different tissues. These approaches have the great advantage that they do not stimulate the serious immune response that some viral vectors do. However, in general, these nonviral methods are not as efficient as viruses at transferring DNA to the target cells. No clearly superior method for gene delivery has yet emerged, and scientists are still actively developing both viral and nonviral methods. It is likely that many different methods will eventually be used, with each method specifically tailored to work best in a specific tissue or organ of the body.

Longevity of Gene Expression

One of the most challenging problems in gene therapy is to achieve long-lasting expression of the therapeutic gene, also called the transgene. Often the virus used to deliver the transgene causes the patient's body to produce an immune response that destroys the very cells that have been treated. This is especially true when an adenovirus is used to deliver genes. The human body raises a potent immune response to prevent or limit infections by adenovirus, completely clearing it from the body within several weeks. This immune response is frequently directed at proteins made by the adenovirus itself.

To combat this problem, researchers have deleted more and more of the virus's own genetic material. These modifications make the viruses safer and less likely to raise an immune response, but also make them more and more difficult to grow in the quantities necessary for use in the clinic. Expression of therapeutic transgenes can also be lost when the regulatory sequences that control a gene and turn it on and off (called promoters and enhancers) are shut down. Although inflammation has been found to play a role in this process, it is not well understood, and much additional research remains to be done.

Examples of Gene Therapy Applications

There are many conditions that must be met in order to allow gene therapy to be possible. First, the details of the disease process must be understood. Of course, scientists must know exactly what gene is defective, but also when and at what level that gene would normally be expressed, how it functions, and what the regenerative possibilities are for the affected tissue. Not all diseases can be treated by gene therapy. It must be clear that replacement of the defective gene would benefit the patient. For example, a mutation that leads to a birth defect might be impossible to treat, because irreversible damage will have already occurred by the time the patient is identified. Similarly, diseases that cause death of brain cells are not well suited to gene therapy: Although gene therapy might be able to halt further progression of disease, existing damage cannot be reversed because brain cells cannot regenerate. Additionally, the cells to which DNA needs to be delivered must be accessible. Finally, great caution is warranted as gene therapy is pursued, as the body's response to high doses of viral vectors can be unpredictable. On September 12, 1999, Jesse Gelsinger, an eighteen-yearold participant in a clinical trial in Philadelphia, became unexpectedly ill and died from side effects of liver administration of adenovirus. This tragedy illustrates the importance of careful attention to safety regulations and extensive experiments in animal model systems before moving to human clinical trials.

Muscular Dystrophies

Duchenne and other recessive muscular dystrophies are well suited in many ways for gene therapy. These are loss-of-function recessive genetic diseases caused by mutations in the dystrophin gene or in genes for other structural muscle proteins. The normal levels of these proteins are known, as are many of the ways that they function in the muscle cell. There is ample evidence in animal model systems that these diseases can be cured by delivery of functional copies of the gene. This is true in large part because muscle tissue has a tremendous capacity for repair and regeneration, so one could imagine that the heavily damaged muscle could repair itself after successful gene transfer. Muscle tissue is also an excellent target for gene transfer.

Several different approaches have been used to transfer DNA to muscle. The most straightforward approach is the direct intramuscular injection of DNA in a circular form called a plasmid. The advantage to this approach is that it induces little to no immune response, although the overall number of cells expressing the gene is fairly low. In contrast, recombinant adenoviruses are extremely efficient at transferring genes to muscle, but give rise to a potent immune response that results in only short-term expression of the transferred genes. Because the efficiency of adenoviral transfer is so great, huge efforts are underway to reduce the immunogenicity of these vectors. These efforts have produced some significantly improved vectors, and research is now focusing on developing methods to prepare the large quantities necessary for clinical use. Adeno-associated virus combines the extremely high efficiency of adenoviral transfer with the very low immunogenicity of direct DNA transfer. However, this virus has a rather small capacity to carry DNA, so small that it cannot carry the dystrophin gene (one of the largest genes known), which is needed to treat Duchenne muscular dystrophy.

From these examples, it should be clear that many different approaches to gene therapy for muscular dystrophy have been tried, but that each approach suffers from one or more key shortcomings. In addition, all of these approaches to treat muscular dystrophy face one common problem: Although it is easy to transfer genes to a small part of a single muscle, simultaneously delivering a gene to all parts of all the muscles of the body is impossible with today's technology.

Hemophilia and Sickle Cell Disease

Because of the difficulty in treating diseases such as muscular dystrophy, many researchers have chosen to focus on genetic diseases that may be easier to treat, particularly those resulting from the lack of proteins freely dissolved in the bloodstream. Hemophilia is one such disorder, caused by a lack of blood-clotting proteins. Such patients have long been treated by the infusion of the missing clotting proteins, but this treatment is extremely expensive and requires almost daily injections. Gene therapy holds great promise for these patients, because replacement of the gene that makes the missing protein could permanently eliminate the need for protein injections. It really does not matter what tissue produces these clotting factors as long as the protein is delivered to the bloodstream, so researchers have tried to deliver these genes to muscle and to the liver using several different vectors. Approaches using recombinant adenoviruses to deliver the clotting factor gene to the liver are especially promising, and tests have shown significant clinical improvement in a dog model of hemophilia.

Gain-of-function genetic diseases present a very different sort of challenge because the mutant gene or genes create a new biological activity that actively interferes with the normal functioning of the cell. An example of such a disorder is sickle cell disease. Patients suffering from this disease have a defective hemoglobin protein in their red blood cells. This defective protein can cause their red blood cells to be misshapen, clogging their blood vessels and causing extremely painful and dangerous blood clots. Most of our genes make an RNA transcript, which is then used as a blueprint to make protein. In sickle cell disease, the transcript of the mutant gene needs to be destroyed or repaired in order to prevent the synthesis of mutant hemoglobin.

The molecular repair of these transcripts is possible using special RNA molecules called ribozymes. There are several different kinds of ribozymes: some that destroy their targets, and others that modify and repair their target transcripts. The repair approach was tested in the laboratory on cells containing the sickle cell mutation, and was quite successful, repairing a significant fraction of the mutant transcripts. While patients cannot yet be treated using this technique, the approach illustrates how biologically damaging molecules can be inactivated. Similar approaches are being developed to treat HIV-AIDS infections, and these may one day be used along with other antiviral therapies to treat this dreaded disease.

Cancer

Very different strategies of gene therapy are used to treat cancer. When treating diseases such as muscular dystrophy, researchers try to deliver genes without detection by the patient's immune system. When treating cancer, the object is often precisely the opposite: to stimulate a patient's immune reaction to the tumor tissue and improve its ability to fight the disease. For this reason, tumor tissue is often transformed by the new gene to produce specific activators of the immune system, such as interleukins or GM-CSF (granulocyte monocyte colony stimulating factor).

Usually, cancer cells are not recognized by the immune system because they are in many ways identical to the patient's normal cells. These stimulating factors activate the immune system and help it recognize and attack the tumor tissue. In another approach, called "suicide therapy," a gene such as the herpes simplex virus thymidine kinase gene (HSV-TK) is transferred to the tumor. This gene normally does not occur in the human body, and it is not metabolically active. After several rounds of gene therapy have built up high levels of TK activity in the tumor, a drug called ganciclovir is given to the patient. This drug is inactive in normal cells, but the TK gene converts it into a potent toxin, killing the tumor cells. Even nearby tumor cells that do not have the TK gene can be killed by a phenomenon called the "bystander effect." This approach not only kills tumor cells directly, but also activates the immune system to further attack the tumor.

Anticancer gene therapy is a powerful adjunct to other more traditional forms of cancer treatment. Its advantages are that it can be beneficial even if only a portion of the tumor cells receive the transferred gene, there is no need for long-term gene expression, and it works with the immune system, rather than trying to defeat it. Anticancer gene therapy is already in significant use in the clinic, and is likely to become even more commonplace in the near future.

In summary, gene therapy covers several related areas of research and clinical treatment, all using the genetic material DNA as a drug. Gene therapy is currently being used, along with other techniques, to treat cancer. One day, gene therapy may also be used to treat a variety of hereditary and nonhereditary diseases, ranging from loss-of-function disorders such as muscular dystrophy and hemophilia, to gain-of-function disorders such as sickle cell disease, to viral diseases such as HIV-AIDS. Active areas of research include improvements in the methods of gene delivery to the individual tissues and cells of the body and the modulation of the immune response to gene delivery. Many challenges remain to the successful maturation of gene therapy from the laboratory to the clinical setting.

Bibliography

Beardsley, T. "Working under Pressure." Scientific American 282 (2000): 34.

Clark, William R. The New Healers: The Promise and Problems of Molecular Medicine in the Twenty-first Century. New York: Oxford University Press, 1999.

Vogel, G. "Gene Therapy: FDA Moves against Penn Scientist." Science 290 (2000):2049-2051.
Internet Resource

Institute for Human Gene Therapy. http://www.yshs.upenn.edu/ihgt/.

—Michael A. Haus