Gene Therapy What is gene therapy? Gene therapy is an experimental technique that uses genes to treat or prevent disease.

In the future, this technique may allow doctors to treat a disorder by inserting a gene into a patients cells instead of using drugs or surgery. Researchers are testing several approaches to gene therapy, including:

Replacing a mutated gene that causes disease with a healthy copy of the gene. Inactivating, or knocking out, a mutated gene that is functioning improperly. Introducing a new gene into the body to help fight a disease.

Although gene therapy is a promising treatment option for a number of diseases (including inherited disorders, some types of cancer, and certain viral infections), the technique remains risky and is still under study to make sure that it will be safe and effective. Gene therapy is currently only being tested for the treatment of diseases that have no other cures. How does gene therapy work? Gene therapy is designed to introduce genetic material into cells to compensate for abnormal genes or to make a beneficial protein. If a mutated gene causes a necessary protein to be faulty or missing, gene therapy may be able to introduce a normal copy of the gene to restore the function of the protein. A gene that is inserted directly into a cell usually does not function. Instead, a carrier called a vector is genetically engineered to deliver the gene. Certain viruses are often used as vectors because they can deliver the new gene by infecting the cell. The viruses are modified so they can't cause disease when used in people. Some types of virus, such as retroviruses, integrate their genetic material (including the new gene) into a chromosome in the human cell. Other viruses, such as adenoviruses, introduce their DNA into the nucleus of the cell, but the DNA is not integrated into a chromosome. The vector can be injected or given intravenously directly into a specific tissue in the body, where it is taken up by individual cells. Alternately, a sample of the patient's cells can be removed and exposed to the vector in a laboratory setting. The cells containing the vector are then returned to the patient. If the treatment is successful, the new gene delivered by the vector will make a functioning protein. Researchers must overcome many technical challenges before gene therapy will be a practical approach to treating disease. For example, scientists must find better ways to deliver genes and target them to particular cells. They must also ensure that new genes are precisely controlled by the body. Is gene therapy safe? Gene therapy is under study to determine whether it could be used to treat disease. Current research is evaluating the safety of gene therapy; future studies will test whether it is an effective treatment option. Several studies have already shown that this approach can have very serious health risks, such as toxicity, inflammation, and cancer. Because the techniques are relatively new, some of the risks may be unpredictable; however, medical researchers, institutions, and regulatory agencies are working to ensure that gene therapy research is as safe as possible.

A new gene is injected into an adenovirus vector, which is used to introduce the modified DNA into a human cell. If the treatment is successful, the new gene will make a functional protein.

What are the ethical issues surrounding gene therapy? Because gene therapy involves making changes to the bodys set of basic instructions, it raises many unique ethical concerns. The ethical questions surrounding gene therapy include:
How can good and bad uses of gene therapy be distinguished? Who decides which traits are normal and which constitute a disability or disorder? Will the high costs of gene therapy make it available only to the wealthy? Could the widespread use of gene therapy make society less accepting of people who are

different?
Should people be allowed to use gene therapy to enhance basic human traits such as height,

intelligence, or athletic ability? Current gene therapy research has focused on treating individuals by targeting the therapy to body cells such as bone marrow or blood cells. This type of gene therapy cannot be passed on to a persons children. Gene therapy could be targeted to egg and sperm cells (germ cells), which would allow the inserted gene to be passed on to future generations. This approach is known as germline gene therapy. The idea of germline gene therapy is controversial. While it could spare future generations in a family

from having a particular genetic disorder, it might affect the development of a fetus in unexpected ways or have long-term side effects that are not yet known. Because people who would be affected by germline gene therapy are not yet born, they cant choose whether to have the treatment. Germ-line gene therapy is technically more difficult, and raises more ethical challenges. The two main methods of performing germ-line gene therapy would be: 1) to treat a pre-embryo that carries a serious genetic defect before implantation in the mother (this necessitates the use of IVF techniques); or 2) to treat the germ cells of afflicted adults so that their genetic defects would not be passed on to their offspring. This approach requires the technical expertise to delete the defective gene and insert a properly functioning replacement.

How does gene therapy work? In most gene therapy studies, a "normal" gene is inserted into the genome to replace an "abnormal," disease-causing gene. A carrier molecule called a vector must be used to deliver the therapeutic gene to the patient's target cells. Currently, the most common vector is a virus that has been genetically altered to carry normal human DNA. Viruses have evolved a way of encapsulating and delivering their genes to human cells in a pathogenic manner. Scientists have tried to take advantage of this capability and manipulate the virus genome to remove disease-causing genes and insert therapeutic genes. Target cells such as the patient's liver or lung cells are infected with the viral vector. The vector then unloads its genetic material containing the therapeutic human gene into the target cell. The generation of a functional protein product from the therapeutic gene restores the target cell to a normal state. Some of the different types of viruses used as gene therapy vectors:
Retroviruses - A class of viruses that can create double-stranded DNA copies of their RNA

genomes. These copies of its genome can be integrated into the chromosomes of host cells.
Adenoviruses - A class of viruses with double-stranded DNA genomes that cause respiratory,

intestinal, and eye infections in humans.

Adeno-associated viruses - A class of small, single-stranded DNA viruses that can insert their

genetic material at a specific site on chromosome 19.

Herpes simplex viruses - A class of double-stranded DNA viruses that infect a particular cell

type, neurons. Besides virus-mediated gene-delivery systems, there are several non-viral options for gene delivery. * The simplest method is the direct introduction of therapeutic DNA into target cells. This approach is limited in its application because it can be used only with certain tissues and requires large amounts of DNA. * Another non-viral approach involves the creation of an artificial lipid sphere with an aqueous core. This liposome, which carries the therapeutic DNA, is capable of passing the DNA through the target cell's membrane. * Therapeutic DNA also can get inside target cells by chemically linking the DNA to a molecule that will bind to special cell receptors. Once bound to these receptors, the therapeutic DNA

constructs are engulfed by the cell membrane and passed into the interior of the target cell. This delivery system tends to be less effective than other options. * Researchers also are experimenting with introducing a 47th (artificial human) chromosome into target cells. This chromosome would exist autonomously alongside the standard 46 --not affecting their workings or causing any mutations. It would be a large vector capable of carrying substantial amounts of genetic code, and scientists anticipate that, because of its construction and autonomy, the body's immune systems would not attack it. A problem with this potential method is the difficulty in delivering such a large molecule to the nucleus of a target cell. What is the current status of gene therapy research? Current gene therapy is experimental and has not proven very successful in clinical trials. Little progress has been made since the first gene therapy clinical trial began in 1990, gene therapy suffered a major setback with the death of 18-year-old Jesse Gelsinger. Jesse was participating in a gene therapy trial for ornithine transcarboxylase deficiency (OTCD). He died from multiple organ failures 4 days after starting the treatment. His death is believed to have been triggered by a severe immune response to the adenovirus carrier. Another major blow came in January 2003, when the FDA placed a temporary halt on all gene therapy trials using retroviral vectors in blood stem cells. FDA took this action after it learned that a second child treated in a French gene therapy trial had developed a leukemia-like condition. Both this child and another who had developed a similar condition in August 2002 had been successfully treated by gene therapy for X-linked severe combined immunodeficiency disease (X-SCID), also known as "bubble baby syndrome." What factors have kept gene therapy from becoming an effective treatment for genetic disease?
Short-lived nature of gene therapy - Before gene therapy can become a permanent cure for

any condition, the therapeutic DNA introduced into target cells must remain functional and the cells containing the therapeutic DNA must be long-lived and stable. Problems with integrating therapeutic DNA into the genome and the rapidly dividing nature of many cells prevent gene therapy from achieving any long-term benefits. Patients will have to undergo multiple rounds of gene therapy.
Immune response - Anytime a foreign object is introduced into human tissues, the immune

system is designed to attack the invader. The risk of stimulating the immune system in a way that reduces gene therapy effectiveness is always a potential risk.

Problems with viral vectors - Viruses, while the carrier of choice in most gene therapy studies, present a variety of potential problems to the patient --toxicity, immune and inflammatory responses, and gene control and targeting issues. In addition, there is always the fear that the viral vector, once inside the patient, may recover its ability to cause disease. Multigene disorders - Conditions or disorders that arise from mutations in a single gene are the best candidates for gene therapy. Unfortunately, some the most commonly occurring disorders, such as heart disease, high blood pressure, Alzheimer's disease, arthritis, and diabetes, are caused by the combined effects of variations in many genes.

Multigene or multifactorial disorders such as these would be especially difficult to treat effectively using gene therapy.
Some recent developments in gene therapy research: Research team in University of California (2003), gets genes into the brain using liposomes

coated in a polymer call polyethylene glycol (PEG). The transfer of genes into the brain is a significant achievement because viral vectors are too big to get across the "blood-brain barrier." This method has potential for treating Parkinson's disease.
RNA interference or gene silencing may be a new way to treat Huntington's (2003). Short

pieces of double-stranded RNA (short, interfering RNAs or siRNAs) are used by cells to degrade RNA of a particular sequence. If a siRNA is designed to match the RNA copied from a faulty gene, then the abnormal protein product of that gene will not be produced.
New gene therapy approach repairs errors in messenger RNA derived from defective genes.

Technique has potential to treat the blood disorder thalassaemia, cystic fibrosis, and some cancers. (2002).
Gene therapy for treating children with X-SCID (sever combined immunodeficiency) or the

"bubble boy" disease is stopped in France when the treatment causes leukemia in one of the patients (2002).
Researchers created tiny liposomes 25 nanometers across that can carry therapeutic DNA

through pores in the nuclear membrane (2002).

Sickle cell is successfully treated in mice (2002).

Techniques The first somatic cell gene therapy procedure inserted a normal gene into the DNA of cells in order to compensate for the nonfunctioning defective gene. This technique involves obtaining blood cells from a person afflicted with a genetic disease and then introducing a normal gene into the defective cell. The normal gene is delivered using a domesticated retrovirus that infects the cell, introducing the properly functioning gene. Retroviruses can infect many types of cells, so it is important to develop gene transfer techniques that allow only retroviruses to deliver genes to a cell and then remain there. Furthermore, the new gene must not get to the wrong place in the genome of the cell. For cystic fibrosis, another kind of virus called an adenovirus has been used as the vector for the new gene. In still other studies, the new DNA is introduced directly into skin cells or even tumor cells. Candidate Diseases for Gene Therapy Gene therapy is likely to have the greatest success with diseases that are cause by single gene defects. By the end of 1993, gene therapy had been approved for use on such diseases as severe combined immune deficiency, familial hypercholesterolemia, cystic fibrosis, and Gaucher's disease. Most protocols to date are aimed toward the treatment of cancer; a few are also targeted toward AIDS. Numerous disorders are discussed as candidates for gene therapy: Parkinson's and Alzheimer's

diseases, arthritis, and heart disease. The Human Genome Project, an ongoing effort to identify the location of all the genes in the human genome, continues to identify genetic diseases. Eve Nichols describes the criteria for selection of disease candidates for human gene therapy: 1) the disease is an incurable, life-threatening disease; 2) organ, tissue and cell types affected by the disease have been identified; 3) the normal counterpart of the defective gene has been isolated and cloned; 4) the normal gene can be introduced into a substantial subfraction of the cells from the affected tissue; or that introduction of the gene into the available target tissue, such as bone marrow, will somehow alter the disease process in the tissue affected by the disease; 5) the gene can be expressed adequately (it will direct the production of enough normal protein to make a difference); and 6) techniques are available to verify the safety of the procedure.

Why is gene therapy hard? The principle of gene therapy is simple -- put new genes into cells. In practice, however, it has been very difficult. One reason for the difficulty is that it takes many steps to get the gene to the right place:

needs The gene has to be designed. The gene needs to be manufactured with the same care as any other drug. The gene needs to be directed to the right cells in the body. The gene needs to go to a safe place in the chromosomes without disturbing the surrounding genes. The gene to be controlled, so the protein is produced only when it is needed. All of these challenges, and more, have to be overcome before anything can be given to a patient. Each of these problems has one or more solutions. Together, however, they have so far prevented success in all but a few single-gene diseases. Next, we will examine one of these problems in detail: How do scientists deliver the new genes to tissues in the body, and how do they make sure the genes get to the right parts of the body? Medical researchers have conceived two main ways of fixing gene defects: outside the body and inside it. Getting new genes into cells In this first approach, researchers remove certain cells from the body -- blood cells, for example -and modify their genes. The researchers then return these cells to the body. The hope is that the modified cells will produce enough of the needed protein. This approach has its own set of challenges, however:
Some proteins are needed in specific places in the body. These places cannot be removed

from the body. Treatment of a weak heart muscle is one example.

This is an expensive approach, requiring special lab equipment and time. Most cells, including blood cells, don't live forever. So, the procedure must be repeated.

One variant of this gene therapy has the promise to last a very long time, or even for life. In this approach, doctors treat special cells that can continue to divide in the body indefinitely. These cells are called stem cells. Scientists have only recently learned to isolate and grow stem cells, so they have little experience in using them for gene therapy. Physicians are accustomed to giving patients a

pill or an injection to treat disease. Researchers devising gene therapies have tried to follow that path. But how could pills or injections get genes into the patient's cells? Getting new genes into cells -- Method 2 "Gene vectors" carry genes from one location to another. Their slang name is "gene trucks." Currently, scientists are working with three major types of gene vectors.

Viruses

Viruses are the simplest organisms known. They consist of nothing more than genetic material encased in protein. But they do one thing well: inject their genetic material into cells they have evolved to infect. This property makes viruses seem ideal as gene vectors. Researchers reasoned that they could remove those parts of the virus's genetic material that make the virus dangerous, and replace it with a human gene that would treat disease. The viruses would infect cells, but instead of injecting virus DNA, they would inject the human gene. It was, and remains, a great solution, but there are challenges:
Even though the modified virus can't cause disease directly, the body may still recognize it as

a foe and attack it. A vigorous attack by the body's own immune system can be dangerous in itself. Some genes are too large for a single virus to carry. These gene vectors are very selective. They don't deliver to every cell. In some cases, the gene never arrives in the part of the cells where it can function. This method has its advantages, however. Some viruses inject their DNA only into certain types of cells. Suppose, for example, we had a harmless virus that only injected DNA into heart cells. We would then have a way of delivering genes to just the heart.

Liposomes

Another approach essentially consists of encasing genes in fat. Normal cells in the body are always enclosed by a thin layer of fat molecules, rather like a soap bubble. By enclosing genes in little bubbles of fat called liposomes, scientists found that the two fatty layers would melt together, like two soap bubbles merging into one. After merging, the gene will be inside the cell, as desired. Like virus delivery, this approach is only effective in a small percentage of the cells. But some researchers think this approach could even work to insert genes into skin cells, for example, to treat certain kinds of baldness.

Naked DNA Finally, sometimes cells can be induced to take up naked DNA that touches their surface. The advantage of this method is simplicity. DNA and little else is applied to the cells. This approach, however, works only on a few of the many cells treated. Most cells will not take up the DNA. This method may work in certain situations, such as in the treatment of skin cancers. Killing cancer cells The many diseases grouped under the name "cancer" pose special problems. In treating cancer, the goal has always been to kill cancer cells, but spare normal cells. This is difficult because they are, in truth, very similar. One approach to killing cancer cells is having them kill themselves. Most normal cells do precisely this -- they live their normal lifespan, and then quietly die in a process called "apoptosis." Many cancer cells, however, have lost the genes that cause their existence to end normally. If physicians could restore the genes controlling apoptosis, then cancer cells would be made to die. Since these would be normal genes, it's possible that this approach would not greatly harm normal cells.