Gene Therapy

David Petechuk & Brian Cobb. The Gale Encyclopedia of Science. Editor: K Lee Lerner & Brenda Wilmoth Lerner. Volume 3. Detroit: Gale, 2008. 

Gene therapy refers to the deliberate introduction of genes into an organism. The intent of gene therapy is to correct a genetic defect or alleviate the symptoms of a genetically-determined disease when the introduced gene is expressed and its product is produced.

Gene therapy is the name applied to the treatment of inherited diseases by corrective genetic engineering of the dysfunctional genes. It is part of a broader field called genetic medicine, which involves the screening, diagnosis, prevention and treatment of hereditary conditions in humans. The results of genetic screening can pinpoint a potential problem to which gene therapy can sometimes offer a solution.

Genes represent the genetic material that organisms pass on from one generation to the next. Therefore, genes are responsible for controlling hereditary traits and provide the basic biological code or blueprint for living organisms. Genes produce protein such as hair and skin as well as proteins that are important for the proper functioning of organs. Mutated or defective genes often cause disease. The purpose of gene therapy is to replace a defective gene with a normal copy of the same gene in attempt to restore function.

Somatic gene therapy introduces a normal gene into tissues or cells to treat an individual that has an abnormal gene. Germline gene therapy inserts genes into reproductive cells (the egg or the sperm) or into embryos to correct genetic defects that could be passed on to future generations. Germline gene therapy differs from somatic gene therapy in that germline integration of a gene will ideally correct every progenitor cell that differentiates from the germ cell. Somatic gene therapy involves integrating corrected genes into cell and tissues that are fully differentiated or mature. An example of the latter is the efforts to treat cystic fibrosis in children or adults by the introduction of the normal gene that specifies a protein that forms a chloride transport channel through the epithelial cell membrane (in cystic fibrosis, the impaired transport of chloride causes the buildup of sticky mucus in the lungs, which can lead to bacterial infection).

Initially conceived as an approach for treating inherited diseases like cystic fibrosis and Huntington disease, the scope of potential gene therapies has grown to include treatments for cancers, arthritis, and infectious diseases. Although gene therapy testing in humans has rapidly advanced, in general, the field of gene therapy has proven to be problematic and complicated by a variety of ethical issues. For example, some scientists are concerned that the integrating genes into the human genome may cause disease. This concern has arisen because of evidence that randomly integrating corrected genes might disrupt other genes in the genome and if the disrupted gene is a tumor suppressor gene, cancer may develop. Others fear that germline gene therapy may be used to control human development in ways not connected with disease, like intelligence or appearance.

The Biological Basis of Gene Therapy

Gene therapy has grown out of the field of molecular biology. Life begins with a single cell, the basic building block of all multicellular organisms. Humans, for instance, are made up of trillions of cells, that make up tissues that form into organs. Each cell type can perform a specific function. Within the cells nucleus (the center part of a cell that regulates its chemical functions) are pairs of chromosomes. These threadlike structures are made up of DNA (deoxyribonucleic acid), which carries the blueprint of life in the form of codes, or genes, that are interspersed throughout the DNA sequence.

A DNA molecule looks like a twisted ladder. The rungs of these represent bonds between each letter of the DNA sequence called base pairs. Base pairs are made up of nitrogenous molecules. Thousands of these base pairs, or DNA sequences, can make up a single gene, specifically defined as a segment of the chromosome. The gene, or combination of genes formed by these base pairs ultimately direct an organisms growth and characteristics through the production of certain proteins, which are important for many biochemical functions.

Scientists have long known that defects in genes present within cells can cause inherited diseases such as cystic fibrosis, sickle-cell anemia, and hemophilia. Similarly, a gain or a loss of an entire chromosome can cause diseases such as Down syndrome or Turners syndrome. As the study of genetics advanced, however, scientists learned that an altered genetic sequence can also make people more susceptible to develop diseases making these individuals predisposed to having atherosclerosis, cancer, or schizophrenia. These diseases have a genetic component, but are also influenced by environmental factors (like diet and lifestyle). The objective of gene therapy is to treat diseases by introducing corrected genes into the body to replace a missing or dysfunctional protein. The inserted genes can be naturally-occurring genes that produce the desired effect or may be genetically engineered (or altered) genes.

Scientists have known how to manipulate the structure of a gene in the laboratory since the early 1970’s through a process called gene splicing. The process involves cutting a sequence of the genome with restriction enzymes, or proteins that act like molecular scissors. The ends where the DNA has been cut are sticky in the sense that they will easily bind to another sequence of DNA that was cut with the same enzyme. A DNA sequence and a gene sequence to be integrated in the DNA sequence can both be cut with the same type of enzyme and their ends will stick together. The new DNA sequence will now have the gene inserted into it. The resulting product is called genetic engineered recombinant DNA.

There are basically two types of gene therapy. Germ-line gene therapy introduces genes into reproductive cells (sperm and eggs) or into embryos in order to correct genetic defects that could be passed on to future generations. Most of the current research, however, has been in the applications of somatic cell gene therapy. In this type of gene therapy, therapeutic genes are inserted into tissue or cells to produce a naturally occurring protein or substance that is lacking or not functioning correctly in an individual patient. The main downside to this approach is that as each corrected cell dies, the therapeutic effects from gene therapy are lessened.

Viral Vectors

In both types of therapy, scientists need something to transport either the entire gene or a recombinant DNA to the cells nucleus, where the DNA is located. In essence, vectors are molecular delivery trucks. One of the first and most popular vectors developed was viral vectors, or vectors made of viruses because they invade cells as part of a natural infection process. Viruses were originally considered the most ideal vector because they have a specific relationship with the host in that they can infect specific cell types or tissues. As a result, vectors are chosen according to their affinity for certain cells and areas of the body.

One of the first viral vectors used was the retro-virus. Because these viruses are easily cloned (artificially reproduced) in the laboratory, scientists have studied them extensively and learned a great deal about their biological characteristics. They have also learned how to remove the genetic information that governs viral replication, thus reducing the chances of multiple rounds of infection. Additionally, many of the proteins from these viruses that can cause an immune response can be removed.

Retroviruses work best in actively dividing cells, but most of the cells in the body particularly those that are fully differentiated are relatively stable and do not divide often. As a result, these cells are used primarily for ex vivo (outside the body) manipulation. First, the cells are removed from the patient’s body, and the virus, or vector, carrying the gene is inserted into them. Next, the cells are placed into a nutrient culture where they grow and replicate. Once enough cells are gathered, they are returned to the body, usually by injection into the blood stream. Theoretically, as long as these cells survive, they can have therapeutic potential.

Another class of viruses, called the adenoviruses, have proven to be good gene vectors in certain cases. These cells can effectively infect nondividing cells in the body, where the desired gene product is then expressed. These viruses, which cause respiratory tract infections, are more easily purified and more stable than retroviruses, resulting in less chance of an unwanted viral infection. These viruses live for several days in the body and can have potentially life-threatening complications related to immune cell responses. Other viral vectors include influenza viruses (that causes the flu), Sindbis virus, and a herpes virus that infects nerve cells. Each of these vectors can be modified to minimize the risk of causing disease or immune cell responses.

Scientists have also developed nonviral vectors. These vectors rely on the natural biological process in which cells uptake (or gather) macromolecules (large molecules). One approach is to use liposomes, or globules of fat produced by the body and taken up by cells. Scientists are also investigating the introduction of recombinant DNA by directly injecting it into the bloodstream or placing it on microscopic beads of gold shot into the skin with a gene gun. Another possible vector under development is based on den-drimer molecules. This is a class of polymers or naturally occurring or artificial substances that have a high molecular weight and are formed by smaller molecules of the same or similar substances. They have been used in manufacturing Styrofoam, polyethylene cartons, and Plexiglass.

In the laboratory, dendrimers have shown the ability to transport genetic material into human cells. They can also be designed with a high affinity for the membrane of a cell by attaching sugars and protein groups to it. The use of liposomes (spherical bags whose membrane is comprise of lipids) as a carrier of DNA has shown promise, especially since other molecules can be incorporated into the lipid membrane that help target to liposome to a specific site in the body. As of 2006, liposome use is still at the experimental stage. However, the research has so far been very promising.

Another approach to gene therapy introduces DNA that can bind to a target stretch of DNA. The binding prevents the expression of the target sequence, which typically produces a product that contributes to the disease. This strategy is known as antisense therapy or gene silencing. As of 2006, this tact has shown great experimental promise.

The History of Gene Therapy

In the early 1970s, scientists proposed what they called “gene surgery” for treating inherited diseases caused by defective genes. In 1983, a group of scientists from Baylor College of Medicine in Houston, Texas, proposed that gene therapy could one day be a viable approach for treating Lesch-Nyhan disease, a rare neurological disorder. The scientists conducted experiments in which an enzyme-producing gene for correcting the disease was injected into a group of cells. The scientists theorized the cells could then be injected into people with Lesch-Nyhan disease.

As the science of genetics advanced throughout the 1980s, gene therapy gained an established foothold in the minds of medical scientists as a viable approach to treatments for specific diseases. However, its promises were more than what it could deliver. One of the major impetuses in the growth of gene therapy was an increasing ability to identify the genetic abnormalities that cause inherited diseases. Interest grew as further studies showed that specific genetic defects in one or more genes occurred in successive generations of certain family members who suffered from diseases like intestinal cancer, manic-depression, Alzheimer disease, heart disease, diabetes, and many more. Although the genes may not be the sole cause of the disease in all cases, they may make certain individuals more susceptible to developing the disease because of environmental influences, such as smoking, pollution, and stress. In fact, many scientists believe that all diseases have a genetic component.

On September 14, 1990, a four-year old girl suffering from a genetic disorder that prevented her body from producing a crucial enzyme became the first person to undergo gene therapy in the United States. Since her body could not produce adenosine deaminase (ADA), she had a weakened immune system, making her extremely susceptible to severe, life-threatening infections. W. French Anderson and colleagues at the National Institutes of Health’s Clinical Center in Bethesda, Maryland, took white blood cells (which are crucial for proper immune system functioning) from the girl, inserted ADA producing genes into them, and then transferred the cells back into the patient. Although the young girl continued to show an increased ability to produce ADA, debate arose as to whether the improvement resulted from the gene therapy or from an additional drug treatment she received.

Nevertheless, a new era of gene therapy began as more and more scientists sought to conduct clinical trials in this area. In that same year, gene therapy was tested on patients suffering from melanoma (skin cancer). The goal was to help them produce antibodies (disease fighting substances in the immune system) to battle the cancer.

These experiments have spawned a growing number of attempts to refine develop new gene therapies. For example, gene therapy for cystic fibrosis, a disease that affects the airways, is being developed. However, due to the complications involved in penetrating the natural barriers that impedes viral entry into the airways, it is unlikely that currently used vectors for cystic fibrosis gene therapy represent a plausible approach. Modifications of these vectors by adding compounds that naturally bind to areas on the outermost membranes of the lung and gain entrance into these tissues are currently being investigated. Another approach was developed for treating brain cancer patients, in which the inserted gene was designed to make the cancer cells more likely to respond to drug treatment. Additionally, gene therapy for patients suffering from artery blockage, which can lead to strokes, that induces the growth of new blood vessels near clogged arteries improving normal blood circulation is also being investigated.

In the United States, both DNA-based (in vivo) treatments and cell-based (ex vivo) treatments are being investigated. DNA-based gene therapy uses vectors (like viruses) to deliver modified genes to target cells. Cell-based gene therapy techniques remove cells from the patient, which are genetically altered and then reintroduce them to the patient’s body. Presently, gene therapies for the following diseases are being developed: cystic fibrosis (using adeno-viral vector), HIV infection (cell-based), malignant melanoma (cell-based), kidney cancer (cell-based), Gaucher’s disease (retroviral vector), breast cancer (retroviral vector), and lung cancer (retroviral vector).

The medical has contributed to transgenic research that is supported by government funding. In 1991, the U.S. government provided $58 million for gene therapy research, with increases of $15-40 million dollars a year over the following four years. With fierce competition over the promise of major medical benefit in addition to huge profits, large pharmaceutical corporations moved to the forefront of transgenic research.

Diseases Targeted for Treatment by Gene Therapy

The potential scope of gene therapy is enormous. More than 4,200 diseases have been identified that result directly from defective genes. People suffering from cystic fibrosis lack a gene needed to produce a salt-regulating protein. This protein regulates the flow of chloride into epithelial cells, which cover the air passages of the nose and lungs. Without this regulation, cystic fibrosis patients suffer from a buildup of thick mucus, which can cause lung infections and respiratory problems, which usually leads to death within the first 30 years of life. A gene therapy technique to correct this defect might employ an adenovirus to transfer a normal copy of what scientists call the cystic fibrosis transmembrane conductance regulator, or CTRF, gene. The gene is introduced into the patient by spraying it into the nose or lungs.

Familial hypercholesterolemia is also an inherited disease, resulting in the inability to process cholesterol properly, which leads to high levels of artery-clogging fat in the blood stream. Patients often suffer heart attacks and strokes because of blocked arteries. A gene therapy approach that was still being investigated as of 2006 involves partially and surgically removing the patient’s liver (ex vivo transgene therapy). Corrected copies of a gene that serve to reduce cholesterol build-up are inserted into the liver sections, which are then transplanted back into the patient.

Gene therapy has also been tested on AIDS patients. AIDS is caused by the human immunodeficiency virus (HIV), which weakens the body’s immune system to the point that sufferers are unable to fight off diseases such as pneumonia. An approach to treat AIDS is to insert genes into a patient’s bloodstream that have been genetically engineered to produce a receptor that would attract HIV and reduce its chances of replicating.

Several cancers also have the potential to be treated with gene therapy. A therapy that is currently being tested for the treatment of melanoma (a form of skin cancer), involves introducing a gene with an anticancer protein called tumor necrosis factor (TNF) into test tube samples of the patient’s own cancer cells, which are then reintroduced into the patient. In brain cancer, the approach is to insert a specific gene that increases the cancer cells susceptibility to a common drug used in fighting the disease. Gene therapy can also be used to treat diseases that involve dysfunctional enzymes. For example, Gaucher’s disease is an inherited disease caused by a mutant gene that inhibits the production of an enzyme called glucocerebrosi-dase. Gaucher patients have enlarged livers and spleens and eventually their bones fall apart. Clinical gene therapy trials focus on inserting the gene for producing this enzyme.

Gene therapy is also being considered as an approach to solving a problem associated with a surgical procedure known as angioplasty. In this procedure, a type of tubular scaffolding is used to open a clogged artery. However, in response to the trauma of the scaffold insertion, the body often initiates a natural healing process resulting in restenosis, or reclosing of the artery. The gene therapy approach to preventing this unwanted side effect is to cover the outside of the stents with a soluble gel. This gel is designed to contain vectors for genes that would reduce restenosis.

The Future of Gene Therapy

There are many obstacles and ethical questions concerning gene therapy. For example, some retrovirusal vectors, can also enter normal cells and interfere with the natural biological processes, possibly leading to other diseases. Other viral vectors, like the adeno-viruses, are often recognized and destroyed by the immune system so their therapeutic effects are shortlived. One of the primary limitations in gene therapy is that delivering a gene using a viral vector that can only undergo one round of infection (making it safer) may provide only temporary therapeutic value that lasts only as long as the corrected gene is expressed. As a result, some therapies need to be repeated often to provide long-lasting benefits.

One of the most pressing issues, however, involves gene regulation. Several genes may play a role in turning other genes on and off. For example, certain genes work together to stimulate cell division and growth, but if these are not regulated, the inserted genes could cause unregulated cell growth leading to the formation of a tumor. Another difficulty is learning how to make the gene be expressed in a regulated way. A specific gene should turn on, for example, when certain levels of a protein or enzyme are not sufficiently meeting cellular demands. This type of controlled regulation of gene expression for these delivered genes is very difficult to achieve.

Ethical Considerations in Gene Therapy

While gene therapy holds promise as a revolutionary approach to treating disease, ethical concerns over its use and ramifications have been expressed. For example, it is difficult to determine the long-term effect of exposure to viral vectors and the effects these engineered viruses have on the human genome.

As the technology develops and more mainstream applications become possible, it is likely that medically unrelated genetic traits might be the target of manipulation. For example, perhaps a gene could be introduced that prevents balding in males. Or what if genetic manipulation was used to alter skin color, prevent homosexuality, or to enhance physical attractiveness and intelligence? Will this only be available to the rich? Gene therapy has been surrounded by more controversy and scrutiny in both scientists and the general public than many other technologies.

As with every new medical technique, there are many potential dangers and unpredictable factors with gene therapy, which make its practical application risky. Even though every precaution is taken to prevent accidents, they sometimes do occur. Jesse Gelsinger, a 17 year-old boy suffering from the disease ornithine transcarbamylase (OTC) deficiency became the first tragic victim of gene therapy and died on September 17, 1999. He had volunteered to test the potential use of gene therapy in the treatment of OTC in young babies. His therapy consisted of an infusion of corrective genes, encased in a weakened adenovirus vector. Gelsinger suffered an unexpected chain reaction that resulted in his early death from multiple organ system failure. The reason for his extreme reaction to the treatment is suspected to have been an overwhelming inflammatory response to the viral vector, though the reason why is not known. Subsequent investigations revealed the deaths of six other gene therapy patients, some prior to Gelsinger, who were undergoing trials for the use of gene therapy in the treatment of heart conditions. Unlike Gelsinger, these latter six victims are thought to have died from complications stemming from their underlying illnesses rather than the gene therapy itself.

In 2006, Italian researchers were successful in preventing the immune system from rejecting introduced DNA, which had long been a problem in gene therapy. With further refinement, the advancement may enable gene therapy to become routinely successful.