gene therapy

Also from Answers.com... Cancer Treatments Learn about current and promising cancer treatments.

More about Gene Therapy: The biological basis of gene therapy Viral vectors The history of gene therapy Diseases targeted for treatment by gene therapy The Human Genome Project The future of gene therapy The ethics of gene therapy Resources

Definition

Gene therapy is a rapidly growing field of medicine in which genes are introduced into the body to treat diseases. Genes control heredity and provide the basic biological code for determining a cell's specific functions. Gene therapy seeks to provide genes that correct or supplant the disease-controlling functions of cells that are not, in essence, doing their job. Somatic gene therapy introduces therapeutic genes at the tissue or cellular level to treat a specific individual. Germ-line gene therapy inserts genes into reproductive cells or possibly into embryos to correct genetic defects that could be passed on to future generations. Initially conceived as an approach for treating inherited diseases, like cystic fibrosis and Huntington's 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 advanced rapidly, many questions surround its use. For example, some scientists are concerned that the therapeutic genes themselves may cause disease. Others fear that germ-line gene therapy may be used to control human development in ways not connected with disease, like intelligence or appearance.

— Katherine Hunt, MS

The treatment of certain disorders, especially those caused by genetic anomalies or deficiencies, by introducing specific engineered genes into a patient's cells.

Definition

Classic gene therapy is the direct use of genetic material in the treatment of disease. This usually involves inserting a functional gene or DNA fragment into key cells to mitigate, or cure, a disease. A broader definition of gene therapy includes all applications of DNA technology to treat disease. For people with certain neurological conditions such as Parkinson's disease and Canavan disease, initial gene therapy trials have shown promise. Developing gene therapies for treating disorders of the nervous system poses unique challenges, such as how to introduce the therapeutic gene across the blood-brain barrier or how to target the therapeutic gene to one specific area of the brain.

Purpose

Genes play a role in every function of the human body. Defects or mutations within a gene can lead to malfunction or disease of cells, tissues, and/or organs. Although standard drug therapy is usually effective in treating the symptoms of a disorder, a patient may be required to take the drugs for an extended time and there may be serious or unpleasant side effects. However, a patient may be cured with few negative consequences if treatment can be targeted directly at the specific cause of the disease (the gene defect), or if that cause can be neutralized or reversed. Therefore, gene therapy provides an attractive alternative to drug therapy as it seeks to provide treatment strategies that will be more complete and less toxic to the patient. Furthermore, gene therapy may provide a way of treating diseases that cannot be managed by standard therapies.

Description

There are many diverse approaches to gene therapy since the biological basis of each disease is unique, presenting a different set of parameters and challenges. However, in each case, a basic set of criteria must be met. First, it is essential to fully understand the disease to be treated. The cells or tissues associated with the disease must be well defined and accessible. The gene and the specific mutation or mutations causing the disease must be known, and it must be possible to isolate or synthesize a normal, functional copy of that gene and to incorporate it into a vector. The vector then transfers the new gene to the target cells where, hopefully, the gene will become fully active. The most common roles for the expressed gene include replacing a defective gene, inhibiting or degrading a deleterious DNA, RNA, or protein, or directly or indirectly killing the cell.

Single gene disorders resulting in a loss of gene function in one specific target tissue provide the easiest options for gene therapy, though strategies for many types of mutations have been investigated. A broad spectrum of diseases has been considered for gene therapy, including:

• neurological disorders, e.g., Parkinson disease, Huntington disease
• muscular dystrophies
• immunological disorders, e.g., severe combined immunodeficiency syndrome (SCIDS)
• blood abnormalities, e.g., thalassemias, hemophilia
• cancer

Unfortunately, many of the more commonly occurring disorders, including heart disease, diabetes, and high blood pressure, result from defects in multiple genes making them unlikely candidates for gene therapy using existing technologies.

For each disease, it must be determined if ex vivo or in vitro technology is the best approach. In ex vivo technology, patient cell samples are collected and cultured in the laboratory. The new gene is incorporated into the growing cells, and these are subsequently transferred back into the patient. Not all of the cultured cells will include the new gene, and not all will survive the transfer. The hope is that a sufficient number of the modified cells will be functional in the patient such that the therapy will reverse the disease. In vitro therapy involves injecting the new gene directly into the target tissue where the individual cells must pick it up. Of the two, this method is technically easier and cheaper, but it is harder to determine how many of the target cells actually acquire the new gene. Ex vivo therapy is more expensive and time consuming, but allows greater control of the conditions.

Both processes require the use of a vector to get the new gene across the cell membrane and into a cell. Viruses have proven to be highly effective as vectors since these are biological entities with a natural function of infecting host cells. DNA technology allows viruses to be manipulated to replace the normal payload of disease-causing genetic material with therapeutic genes. The virus will retain its ability to infect a host cell but, instead of causing a disease, it will deposit the new gene into the cell.

Other mechanisms of gene transfer have also been investigated. Artificial chromosomes have been developed, but these are often too large to move across cell membranes. Liposomes, structures with lipid membranes, that encompass genetic material can be successfully used as vectors if the liposome is absorbed by the cell or if its membrane fuses with the cell membrane releasing the new gene inside the cell.

Once the gene enters the cell, one of two things occurs. It may be degraded and lost, which is an unfavorable outcome. Preferably, the gene will stably incorporate into the DNA of the target cell so that it can be processed as a normal part of that genome. If the gene therapy is designed to replace a defective gene, the best-case scenario is for the new gene to integrate into a completely renewable cell such as a stem cell. Theoretically, in this situation, the gene will be permanently incorporated into the patient's body and no further therapy will be required. Alternatively, if the gene integrates into a genome of a cell with a finite lifespan, the beneficial effects of the gene will only exist while that cell lives, requiring the gene therapy to be repeated at a later time.

One of the early successes of gene therapy was for a four-year-old girl with adenine deaminase (ADA) deficiency. This is a form of SCIDS that results in malfunction of the immune system and can lead to death as a result of severe infection. Conventional treatment had failed for this patient, making her a candidate for gene therapy. A normal ADA gene was incorporated into a retroviral vector that transferred the gene into the patient's lymphocytes in vitro. The modified cells were returned to her circulation by transfusion. After five months, her levels of ADA activity had risen from less than 1% to 50%. With additional therapies over the next two years, her health improved as the enzyme activity stabilized, and she was able to begin a normal life. Twelve years later, she still demonstrates reasonable levels of ADA activity, but the gene therapy was not a cure as she must continue to receive the standard enzyme replacement therapy to maintain her health.

Acquired diseases can also be treated with gene therapy as demonstrated by a novel strategy for treating brain cancer. The thymidine kinase (TK) gene from the herpes simplex virus (HSV) has an enzymatic property that converts the drug ganciclovir into a toxic substance that can kill human cells. It was postulated that this could be used as a targeted killing tool. To investigate, cloned HSV TK genes were injected into brain tumors. In the brain, only the tumor cells are dividing, so these are the only cells that will be infected by the viral vector, and are thus the only cells that will receive the HSV TK gene. When the patient is subsequently treated with ganciclovir, the tumor cells that have incorporated the HSV TK gene will be selectively killed. Clinical trials proved that tumor cells could be selectively eliminated by demonstrating a reduction in the size of the brain tumors in seven of nine patients.

A completely different set of therapies is possible if the idea of gene therapy includes the use of DNA for patient treatment in ways other than inserting new genes into cells. One example is the drug Gleevec that was approved in 2001 for use in patients with chronic myelogenous leukemia (CML). Gleevec is a substance that binds to the defective protein produced in CML, blocking that protein's activity and alleviating the symptoms of the disease. This is a targeted therapy that affects only the cells with the CML mutation, so there are very few side effects. Recombinant DNA technology has also been utilized to generate genetically engineered copies of vaccines (Recombivax HB), antibodies, and normal gene products (insulin).

Aftercare

If the new DNA can be stably incorporated into the proper regenerative target cells, the patient may be cured of disease. No additional care should be required, although periodic monitoring of the patient is appropriate.

For gene therapies in which the new DNA is inserted into cells with a finite lifespan, the therapeutic effect will be lost when those cells die. In these situations, the patient will require continuing treatments. Monitoring of patients who receive drugs and substances arising from recombinant DNA technology is the same as standard drug therapy.

Precautions

Currently classic gene therapy is still experimental. Although many patients have shown significant improvement following their treatment, at least two individuals have died as a result of this type of therapy. Therefore, experts carefully review all protocols before any studies are undertaken. Initial research is done in an animal model system, and any problems detected are carefully evaluated before the same treatments are attempted in humans.

Risks

A patient who is receiving gene therapy may face a number of potential problems. The viral vectors used may cause infection and/or inflammation of tissues, and artificial introduction of viruses into the body may initiate other disease processes. Functional gene therapy relies on stable incorporation of a new gene into an individual's own DNA. As the integration is random, occasionally the new gene may insert within another normally functioning gene, causing its damage or inactivation. This, in turn, could lead to cancer or other disease. It is also critical that the new gene have the proper regulatory controls so that the gene product is produced in the proper amount. Over-expression of certain genes can have deleterious results. Any of these problems could render the gene therapy ineffective, or, at worst, cause the death of the subject.

Normal results

Classic gene therapy seeks to treat or cure a defined disease by incorporating a functional gene or gene product into target cells of an affected individual.

Resources

BOOKS

George, Linda. Gene Therapy. Woodbridge, CT: Blackbirch Marketing, 2003.

Nussbaum, Robert L., Roderick R. McInnes, and Huntington F. Willard. Thompson and Thompson Genetics in Medicine, 6th edition. Philadelphia, PA: W. B. Saunders Company, 2001.

Strachan, T., and Andrew P. Read. Human Molecular Genetics, 2nd edition. New York, NY: John Wiley and Sons, 1999.

OTHER

National Cancer Institute. Questions and Answers about Gene Therapy. Cited January 4, 2004 (March 23, 2004). http://cis.nci.nih.gov/fact/7_18.htm.

Constance K. Stein

Gene therapy is the treatment of human disease by gene transfer. Many, or maybe most, diseases have a genetic component — asthma, cancer, Alzheimer's disease, for example. However, most diseases are polygenic, i.e. a subtle interplay of many genes determines the likelihood of developing a disease condition, whereas, so far, gene therapy can only be contemplated for monogenic diseases, in which there is a single gene defect. Even in these cases only treatment of recessive diseases can be considered, where the correct gene is added in the continued presence of the faulty one. Dominant mutations cannot be approached in this way, as it would be necessary to knock out the existing faulty genes in the cells where they are expressed (i.e. where their presence shows an effect), as well as adding the correct genetic information.

Gene therapy for recessive monogenic diseases involves introducing correct genetic material into the patient. This can be approached in two different ways. Cells can be taken from the patient, modified in the laboratory (‘in vitro’), and then re-introduced, or a carrier (‘vector’) of the correct genetic material can be delivered directly into the patient. Examples are adenosine deaminase (ADA) deficiency and cystic fibrosis, respectively.

ADA deficiency is a lethal monogenic disease in which adenosine is not normally metabolized, leading to high levels of 2′-deoxyadenosine, which is selectively toxic to cells of the immune system (T and B cells) and leads to immunodeficiency. The gene therapy for this disease is to isolate T cells from patients and treat these cells in vitro with a retroviral vector — a virus, normally capable of causing disease, which has been modified to carry the required genetic information. Cells that have incorporated the new genetic information into their genomes are selected and reinfused back into the patient, allowing the appropriate metabolism of adenosine to occur.

Cystic fibrosis (CF) is also a lethal disease resulting from a single gene mutation. It disables normal cell membrane function. The gene codes for a protein, the cystic fibrosis ‘transmembrane conductance regulator’ (CFTR), which acts as a chloride ion channel in the epithelia of the airways, alimentary canal, and numerous other hollow organs. The correct genetic sequence can be incorporated into a type of virus which ordinarily can infect the respiratory tract (adenoviruses), or into genetic particles (plasmids) linked to lipids to form lipocomplexes. Either of these can be delivered directly into the patient's lungs.

Both the examples given must still be regarded as at the experimental stage. In ADA deficiency the transformed T cells persist for only 6 months, while in CF the new genetic material is not incorporated into the genome and is expressed outside of it (episomally) for only a few weeks. The major problems with gene therapy relate to the delivery of new genetic instructions to the appropriate body targets. Ideally, delivery should be to stem cells — the progenitor cells which give rise to replacement cells as adult, mature cells die. Furthermore, the material should be incorporated into the genome so that all future generations of cells carry the correct instructions. Retroviral vectors (viruses carrying the genetic material) are incorporated in the genome, but this can only be used in the in vitro type of procedure, when the treated cells are outside the body. Incorporation of the new material into an inappropriate position may switch on an oncogene which can lead to tumour formation. Thus it is very necessary to be sure that cells transformed in vitro behave normally before reintroducing them to the patient. These problems do not occur with adenoviral vectors, but expression persists for only a short time, thus requiring repeated administration, leading to immune reactions. Use of lipoplexes avoids this latter problem, but the efficiency of gene delivery by this route is very low.

Gene therapy will undoubtedly have much to offer for the future. Unlike many conventional therapies, it aims to cure disease rather than simply treat the symptoms. The principles of gene therapy are established, but technical problems, primarily related to efficient and safe ways of delivery, have still to be overcome.

— Alan W. Cuthbert

See also genetics, human; immune system.

A procedure that involves injection of “health genes” into the bloodstream of a patient to cure or treat a hereditary disease or similar illness.

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. Hauser

For more information on gene therapy, visit Britannica.com.

Treatment of disorders by correcting faulty genes by, for example, swapping an abnormal gene for a normal gene, or by repairing an abnormal gene. Although directed at combating disease, gene therapy has the potential to enhance sport performance. For example, a gene that, when defective is responsible for muscular dystrophy, when normal increases the production of a protein called mechano growth factor (MGF). MGF boosts muscle mass and improves a muscle's ability to repair itself. Research indicates that gene therapy could be used to either treat muscular dystrophy or improve the performance of a healthy athlete. See also gene doping.

Gene therapy involves replacement of an "abnormal" disease-causing gene with a "normal" gene. The normal gene is delivered to target cells using a vector, which is usually a virus that has been genetically engineered to carry human DNA. The virus genome is altered to remove disease-causing genes and insert therapeutic genes. Target cells are infected with the virus. The virus then integrates its genetically altered material containing the human gene into the target cell, where it should produce a functional protein product.

A promising technology that involves replacing a defective gene in the body with a healthy one. This can be done by removing cells from the body, using genetic engineering techniques to change defective sequences in the DNA, and then reinserting the cells. This technique has been carried out successfully, for example, on bone marrow cells, in which defective cells were successfully replaced with healthy, genetically engineered cells. Scientists hope to find an agent, such as a therapeutic virus, that will be able to correct defective DNA in situ. (See cloning vector.)

This article may be inaccurate in or unbalanced towards certain viewpoints. Please improve the article by adding information on neglected viewpoints, or discuss the issue on the talk page. (June 2009)

Gene therapy is the insertion of genes into an individual's cells and tissues to treat a disease, such as a hereditary disease in which a deleterious mutant allele is replaced with a functional one. Although the technology is still in its infancy, it has been used with some success. Antisense therapy is not strictly a form of gene therapy, but is a genetically-mediated therapy and is often considered together with other methods.

Gene therapy using an Adenovirus vector. A new gene is inserted 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.

Contents 1 Background 2 Types of gene therapy 2.1 Germ line gene therapy 2.2 Somatic gene therapy 3 Broad methods 4 Vectors in gene therapy 4.1 Viruses 4.1.1 Retroviruses 4.1.2 Adenoviruses 4.1.3 Adeno-associated viruses 4.1.4 Envelope protein pseudotyping of viral vectors 4.2 Non-viral methods 4.2.1 Naked DNA 4.2.2 Oligonucleotides 4.2.3 Lipoplexes and polyplexes 4.3 Hybrid methods 4.4 Dendrimers 5 Major developments in gene therapy 5.1 2002 and earlier 5.2 2003 5.3 2006 5.4 2007 6 Problems and ethics 7 In popular culture 8 See also 9 References 10 External links

Background

On September 14, 1990 at the U.S. National Institutes of Health, W. French Anderson M.D. and his colleagues R. Michael Blaese, M.D., C. Bouzaid, M.D., and Kenneth Culver, M.D., performed the first approved gene therapy procedure on four-year old Ashanthi DeSilva. Born with a rare genetic disease called severe combined immunodeficiency (SCID), she lacked a healthy immune system, and was vulnerable to every passing germ or infection. Children with this illness usually develop overwhelming infections and rarely survive to adulthood; a common childhood illness like chickenpox is life-threatening. Ashanthi led a cloistered existence — avoiding contact with people outside her family, remaining in the sterile environment of her home, and battling frequent illnesses with massive amounts of antibiotics.

In Ashanthi's gene therapy procedure, doctors removed white blood cells from the child's body, let the cells grow in the laboratory, inserted the missing gene into the cells, and then infused the genetically modified blood cells back into the patient's bloodstream. Whether the therapy strengthened Ashanthi's immune system is unclear, because the study did not have a control arm and she continued to receive standard therapy for her condition during the study. Moreover, the white blood cells treated genetically only work for a few months, after which the process must be repeated (VII, Thompson [First] 1993). As of early 2007, she was still in good health, and she was attending college. Some would state that the study is of great importance despite its indefinite results, if only because it demonstrated that gene therapy could be practically attempted without adverse consequences.^[1]

The road to the first approved gene therapy procedure was rocky and fraught with controversy. The biology of human gene therapy is very complex, and there are many techniques that still need to be developed and diseases that need to be understood more fully before gene therapy can be used appropriately. The public policy debate surrounding the possible use of genetically engineered material in human subjects has been equally complex. Major participants in the debate have come from the fields of biology, government, law, medicine, philosophy, politics, and religion, each bringing different views to the discussion.^{[citation needed]}

Scientists took the logical step of trying to introduce genes straight into human cells, focusing on diseases caused by single-gene defects, such as cystic fibrosis, hemophilia, muscular dystrophy and sickle cell anemia. However, this has been much harder than modifying simple bacteria, primarily because of the problems involved in carrying large sections of DNA and delivering them to the correct site on the comparatively large human genome^{[citation needed]}. Today, most gene therapy studies are aimed at cancer.

Types of gene therapy

Gene therapy may be classified into the following types:

Germ line gene therapy

In the case of germ line gene therapy, germ cells, i.e., sperm or eggs, are modified by the introduction of functional genes, which are ordinarily integrated into their genomes. Therefore, the change due to therapy would be heritable and would be passed on to later generations. This new approach, theoretically, should be highly effective in counteracting genetic disorders. However, many jurisdictions prohibit this for application in human beings, at least for the present, for a variety of technical and ethical reasons.

Somatic gene therapy

In the case of somatic gene therapy, therapeutic genes are transferred into the somatic cells of a patient. Any modifications and effects will be restricted to the individual patient only, and will not be inherited by the patient's offspring.

Broad methods

There are a variety of different methods to replace or repair the genes targeted in gene therapy.^[2]

• A normal gene may be inserted into a nonspecific location within the genome to replace a nonfunctional gene. This approach is most common.
• An abnormal gene could be swapped for a normal gene through homologous recombination.
• The abnormal gene could be repaired through selective reverse mutation, which returns the gene to its normal function.
• The regulation (the degree to which a gene is turned on or off) of a particular gene could be altered.

Vectors in gene therapy

Viruses

Main article: Viral vector

All viruses bind to their hosts and introduce their genetic material into the host cell as part of their replication cycle. This genetic material contains basic 'instructions' of how to produce more copies of these viruses, hijacking the body's normal production machinery to serve the needs of the virus. The host cell will carry out these instructions and produce additional copies of the virus, leading to more and more cells becoming infected. Some types of viruses physically insert their genes into the host's genome (a defining feature of retroviruses, the family of viruses that includes HIV, is that the virus will introduce the enzyme reverse transcriptase into the host and thus use its RNA as the "instructions"). This incorporates the genes of that virus among the genes of the host cell for the life span of that cell.

Doctors and molecular biologists realized that viruses like this could be used as vehicles to carry 'good' genes into a human cell. First, a scientist would remove the genes in the virus that cause disease. Then they would replace those genes with genes encoding the desired effect (for instance, insulin production in the case of diabetics). This procedure must be done in such a way that the genes which allow the virus to insert its genome into its host's genome are left intact. This can be confusing, and requires significant research and understanding of the virus' genes in order to know the function of each. An example: A virus is found which replicates by inserting its genes into the host cell's genome. This virus has two genes- A and B. Gene A encodes a protein which allows this virus to insert itself into the host's genome. Gene B causes the disease this virus is associated with. Gene C is the "normal" or "desirable" gene we want in the place of gene B. Thus, by re-engineering the virus so that gene B is replaced by gene C, while allowing gene A to properly function, this virus could introduce the required gene - gene C into the host cell's genome without causing any disease.

All this is clearly an oversimplification, and numerous problems exist that prevent gene therapy using viral vectors, such as: trouble preventing undesired effects, ensuring the virus will infect the correct target cell in the body, and ensuring that the inserted gene doesn't disrupt any vital genes already in the genome. However, this basic mode of gene introduction currently shows much promise and doctors and scientists are working hard to fix any potential problems that could exist.

Retroviruses

The genetic material in retroviruses is in the form of RNA molecules, while the genetic material of their hosts is in the form of DNA. When a retrovirus infects a host cell, it will introduce its RNA together with some enzymes, namely reverse transcriptase and integrase, into the cell. This RNA molecule from the retrovirus must produce a DNA copy from its RNA molecule before it can be integrated into the genetic material of the host cell. The process of producing a DNA copy from an RNA molecule is termed reverse transcription. It is carried out by one of the enzymes carried in the virus, called reverse transcriptase. After this DNA copy is produced and is free in the nucleus of the host cell, it must be incorporated into the genome of the host cell. That is, it must be inserted into the large DNA molecules in the cell (the chromosomes). This process is done by another enzyme carried in the virus called integrase.

Now that the genetic material of the virus is incorporated and has become part of the genetic material of the host cell, it can be said that the host cell is now modified to contain a new gene. If this host cell divides later, its descendants will all contain the new genes. Sometimes the genes of the retrovirus do not express their information immediately.

One of the problems of gene therapy using retroviruses is that the integrase enzyme can insert the genetic material of the virus in any arbitrary position in the genome of the host- it randomly shoves the genetic material into a chromosome. If genetic material happens to be inserted in the middle of one of the original genes of the host cell, this gene will be disrupted (insertional mutagenesis). If the gene happens to be one regulating cell division, uncontrolled cell division (i.e., cancer) can occur. This problem has recently begun to be addressed by utilizing zinc finger nucleases^[3] or by including certain sequences such as the beta-globin locus control region to direct the site of integration to specific chromosomal sites.

Gene therapy trials using retroviral vectors to treat X-linked severe combined immunodeficiency (X-SCID) represent the most successful application of gene therapy to date. More than twenty patients have been treated in France and Britain, with a high rate of immune system reconstitution observed. Similar trials were halted or restricted in the USA when leukemia was reported in patients treated in the French X-SCID gene therapy trial. To date, four children in the French trial and one in the British trial have developed leukemia as a result of insertional mutagenesis by the retroviral vector. All but one of these children responded well to conventional anti-leukemia treatment. Gene therapy trials to treat SCID due to deficiency of the Adenosine Deaminase (ADA) enzyme continue with relative success in the USA, Britain, Italy and Japan.

Adenoviruses

Adenoviruses are viruses that carry their genetic material in the form of double-stranded DNA. They cause respiratory, intestinal, and eye infections in humans (especially the common cold). When these viruses infect a host cell, they introduce their DNA molecule into the host. The genetic material of the adenoviruses is not incorporated (transient) into the host cell's genetic material. The DNA molecule is left free in the nucleus of the host cell, and the instructions in this extra DNA molecule are transcribed just like any other gene. The only difference is that these extra genes are not replicated when the cell is about to undergo cell division so the descendants of that cell will not have the extra gene. As a result, treatment with the adenovirus will require readministration in a growing cell population although the absence of integration into the host cell's genome should prevent the type of cancer seen in the SCID trials. This vector system has been promoted for treating cancer and indeed the first gene therapy product to be licensed to treat cancer, Gendicine, is an adenovirus. Gendicine, an adenoviral p53-based gene therapy was approved by the Chinese FDA in 2003 for treatment of head and neck cancer. Advexin, a similar gene therapy approach from Introgen, was turned down by the US FDA in 2008.

Concerns about the safety of Adenovirus vectors were raised after the 1999 death of an Arizona teenager participating in a gene therapy trial. Since then, work using adenovirus vectors has focused on genetically crippled versions of the virus.

Adeno-associated viruses

Adeno-associated viruses, from the parvovirus family, are small viruses with a genome of single stranded DNA. The wild type AAV can insert genetic material at a specific site on chromosome 19 with near 100% certainty. But the recombinant AAV, which does not contain any viral genes and only the therapeutic gene, does not integrate into the genome. Instead the recombinant viral genome fuses at its ends via the ITR (inverted terminal repeats) recombination to form circular, episomal forms which are predicted to be the primary cause of the long term gene expression. There are a few disadvantages to using AAV, including the small amount of DNA it can carry (low capacity) and the difficulty in producing it. This type of virus is being used, however, because it is non-pathogenic (most people carry this harmless virus). In contrast to adenoviruses, most people treated with AAV will not build an immune response to remove the virus and the cells that have been successfully treated with it. Several trials with AAV are on-going or in preparation, mainly trying to treat muscle and eye diseases; the two tissues where the virus seems particularly useful. However, clinical trials have also been initiated where AAV vectors are used to deliver genes to the brain. This is possible because AAV viruses can infect non-dividing (quiescent) cells, such as neurons in which their genomes are expressed for a long time.

Envelope protein pseudotyping of viral vectors

The viral vectors described above have natural host cell populations that they infect most efficiently. Retroviruses have limited natural host cell ranges, and although adenovirus and adeno-associated virus are able to infect a relatively broader range of cells efficiently, some cell types are refractory to infection by these viruses as well. Attachment to and entry into a susceptible cell is mediated by the protein envelope on the surface of a virus. Retroviruses and adeno-associated viruses have a single protein coating their membrane, while adenoviruses are coated with both an envelope protein and fibers that extend away from the surface of the virus. The envelope proteins on each of these viruses bind to cell-surface molecules such as heparin sulfate, which localizes them upon the surface of the potential host, as well as with the specific protein receptor that either induces entry-promoting structural changes in the viral protein, or localizes the virus in endosomes wherein acidification of the lumen induces this refolding of the viral coat. In either case, entry into potential host cells requires a favorable interaction between a protein on the surface of the virus and a protein on the surface of the cell. For the purposes of gene therapy, one might either want to limit or expand the range of cells susceptible to transduction by a gene therapy vector. To this end, many vectors have been developed in which the endogenous viral envelope proteins have been replaced by either envelope proteins from other viruses, or by chimeric proteins. Such chimera would consist of those parts of the viral protein necessary for incorporation into the virion as well as sequences meant to interact with specific host cell proteins. Viruses in which the envelope proteins have been replaced as described are referred to as pseudotyped viruses. For example, the most popular retroviral vector for use in gene therapy trials has been the lentivirus Simian immunodeficiency virus coated with the envelope proteins, G-protein, from Vesicular stomatitis virus. This vector is referred to as VSV G-pseudotyped lentivirus, and infects an almost universal set of cells. This tropism is characteristic of the VSV G-protein with which this vector is coated. Many attempts have been made to limit the tropism of viral vectors to one or a few host cell populations. This advance would allow for the systemic administration of a relatively small amount of vector. The potential for off-target cell modification would be limited, and many concerns from the medical community would be alleviated. Most attempts to limit tropism have used chimeric envelope proteins bearing antibody fragments. These vectors show great promise for the development of "magic bullet" gene therapies.

Non-viral methods

Non-viral methods present certain advantages over viral methods, with simple large scale production and low host immunogenicity being just two. Previously, low levels of transfection and expression of the gene held non-viral methods at a disadvantage; however, recent advances in vector technology have yielded molecules and techniques with transfection efficiencies similar to those of viruses.

Naked DNA

This is the simplest method of non-viral transfection. Clinical trials carried out of intramuscular injection of a naked DNA plasmid have occurred with some success; however, the expression has been very low in comparison to other methods of transfection. In addition to trials with plasmids, there have been trials with naked PCR product, which have had similar or greater success. This success, however, does not compare to that of the other methods, leading to research into more efficient methods for delivery of the naked DNA such as electroporation, sonoporation, and the use of a "gene gun", which shoots DNA coated gold particles into the cell using high pressure gas.

Oligonucleotides

The use of synthetic oligonucleotides in gene therapy is to inactivate the genes involved in the disease process. There are several methods by which this is achieved. One strategy uses antisense specific to the target gene to disrupt the transcription of the faulty gene. Another uses small molecules of RNA called siRNA to signal the cell to cleave specific unique sequences in the mRNA transcript of the faulty gene, disrupting translation of the faulty mRNA, and therefore expression of the gene. A further strategy uses double stranded oligodeoxynucleotides as a decoy for the transcription factors that are required to activate the transcription of the target gene. The transcription factors bind to the decoys instead of the promoter of the faulty gene, which reduces the transcription of the target gene, lowering expression. Additionally, single stranded DNA oligonucleotides have been used to direct a single base change within a mutant gene. The oligonucleotide is designed to anneal with complementarity to the target gene with the exception of a central base, the target base, which serves as the template base for repair. This technique is referred to as oligonucleotide mediated gene repair, targeted gene repair, or targeted nucleotide alteration.

Lipoplexes and polyplexes

To improve the delivery of the new DNA into the cell, the DNA must be protected from damage and its entry into the cell must be facilitated. To this end new molecules, lipoplexes and polyplexes, have been created that have the ability to protect the DNA from undesirable degradation during the transfection process.

Plasmid DNA can be covered with lipids in an organized structure like a micelle or a liposome. When the organized structure is complexed with DNA it is called a lipoplex. There are three types of lipids, anionic (negatively charged), neutral, or cationic (positively charged). Initially, anionic and neutral lipids were used for the construction of lipoplexes for synthetic vectors. However, in spite of the facts that there is little toxicity associated with them, that they are compatible with body fluids and that there was a possibility of adapting them to be tissue specific; they are complicated and time consuming to produce so attention was turned to the cationic versions.

Cationic lipids, due to their positive charge, were first used to condense negatively charged DNA molecules so as to facilitate the encapsulation of DNA into liposomes. Later it was found that the use of cationic lipids significantly enhanced the stability of lipoplexes. Also as a result of their charge, cationic liposomes interact with the cell membrane, endocytosis was widely believed as the major route by which cells uptake lipoplexes. Endosomes are formed as the results of endocytosis, however, if genes can not be released into cytoplasm by breaking the membrane of endosome, they will be sent to lysosomes where all DNA will be destroyed before they could achieve their functions. It was also found that although cationic lipids themselves could condense and encapsulate DNA into liposomes, the transfection efficiency is very low due to the lack of ability in terms of “endosomal escaping”. However, when helper lipids (usually electroneutral lipids, such as DOPE) were added to form lipoplexes, much higher transfection efficiency was observed. Later on, it was figured out that certain lipids have the ability to destabilize endosomal membranes so as to facilitate the escape of DNA from endosome, therefore those lipids are called fusogenic lipids. Although cationic liposomes have been widely used as an alternative for gene delivery vectors, a dose dependent toxicity of cationic lipids were also observed which could limit their therapeutic usages.

The most common use of lipoplexes has been in gene transfer into cancer cells, where the supplied genes have activated tumor suppressor control genes in the cell and decrease the activity of oncogenes. Recent studies have shown lipoplexes to be useful in transfecting respiratory epithelial cells, so they may be used for treatment of genetic respiratory diseases such as cystic fibrosis.

Complexes of polymers with DNA are called polyplexes. Most polyplexes consist of cationic polymers and their production is regulated by ionic interactions. One large difference between the methods of action of polyplexes and lipoplexes is that polyplexes cannot release their DNA load into the cytoplasm, so to this end, co-transfection with endosome-lytic agents (to lyse the endosome that is made during endocytosis, the process by which the polyplex enters the cell) such as inactivated adenovirus must occur. However, this isn't always the case, polymers such as polyethylenimine have their own method of endosome disruption as does chitosan and trimethylchitosan.

Hybrid methods

Due to every method of gene transfer having shortcomings, there have been some hybrid methods developed that combine two or more techniques. Virosomes are one example; they combine liposomes with an inactivated HIV or influenza virus. This has been shown to have more efficient gene transfer in respiratory epithelial cells than either viral or liposomal methods alone. Other methods involve mixing other viral vectors with cationic lipids or hybridising viruses.

Dendrimers

A dendrimer is a highly branched macromolecule with a spherical shape. The surface of the particle may be functionalized in many ways and many of the properties of the resulting construct are determined by its surface.

In particular it is possible to construct a cationic dendrimer, i.e. one with a positive surface charge. When in the presence of genetic material such as DNA or RNA, charge complimentarity leads to a temporary association of the nucleic acid with the cationic dendrimer. On reaching its destination the dendrimer-nucleic acid complex is then taken into the cell via endocytosis.

In recent years the benchmark for transfection agents has been cationic lipids. Limitations of these competing reagents have been reported to include: the lack of ability to transfect a number of cell types, the lack of robust active targeting capabilities, incompatibility with animal models, and toxicity. Dendrimers offer robust covalent construction and extreme control over molecule structure, and therefore size. Together these give compelling advantages compared to existing approaches.

Producing dendrimers has historically been a slow and expensive process consisting of numerous slow reactions, an obstacle that severely curtailed their commercial development. The Michigan based company Dendritic Nanotechnologies discovered a method to produce dendrimers using kinetically driven chemistry, a process that not only reduced cost by a magnitude of three, but also cut reaction time from over a month to several days. These new "Priostar" dendrimers can be specifically constructed to carry a DNA or RNA payload that transfects cells at a high efficiency with little or no toxicity.

Major developments in gene therapy

This section may contain an inappropriate mixture of prose and timeline. Please help convert this timeline into prose or, if necessary, a list.

2002 and earlier

New gene therapy approach repairs errors in messenger RNA derived from defective genes. This technique has the potential to treat the blood disorder thalassaemia, cystic fibrosis, and some cancers. See Subtle gene therapy tackles blood disorder at NewScientist.com (October 11, 2002).

Researchers at Case Western Reserve University and Copernicus Therapeutics are able to create tiny liposomes 25 nanometers across that can carry therapeutic DNA through pores in the nuclear membrane. See DNA nanoballs boost gene therapy at NewScientist.com (May 12, 2002).

Sickle cell disease is successfully treated in mice. See Murine Gene Therapy Corrects Symptoms of Sickle Cell Disease from March 18, 2002, issue of The Scientist.

The success of a multi-center trial for treating children with SCID (severe combined immune deficiency or "bubble boy" disease) held from 2000 and 2002 was questioned when two of the ten children treated at the trial's Paris center developed a leukemia-like condition. Clinical trials were halted temporarily in 2002, but resumed after regulatory review of the protocol in the United States, the United Kingdom, France, Italy, and Germany.^[4]

In 1993 Andrew Gobea was born with severe combined immunodeficiency (SCID). Genetic screening before birth showed that he had SCID. Blood was removed from Andrew's placenta and umbilical cord immediately after birth, containing stem cells. The allele that codes for ADA was obtained and was inserted into a retrovirus. Retroviruses and stem cells were mixed, after which they entered and inserted the gene into the stem cells' chromosomes. Stem cells containing the working ADA gene were injected into Andrew's blood system via a vein. Injections of the ADA enzyme were also given weekly. For four years T-cells (white blood cells), produced by stem cells, made ADA enzymes using the ADA gene. After four years more treatment was needed.

2003

In 2003 a University of California, Los Angeles research team inserted genes into the brain using liposomes coated in a polymer called 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. See Undercover genes slip into the brain at NewScientist.com (March 20, 2003).

RNA interference or gene silencing may be a new way to treat Huntington's. 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. See Gene therapy may switch off Huntington's at NewScientist.com (March 13, 2003).

2006

Scientists at the National Institutes of Health (Bethesda, Maryland) have successfully treated metastatic melanoma in two patients using killer T cells genetically retargeted to attack the cancer cells. This study constitutes the first demonstration that gene therapy can be effective in treating cancer.^[5]

In March 2006 an international group of scientists announced the successful use of gene therapy to treat two adult patients for a disease affecting myeloid cells. The study, published in Nature Medicine, is believed to be the first to show that gene therapy can cure diseases of the myeloid system.^[6]

In May 2006 a team of scientists led by Dr. Luigi Naldini and Dr. Brian Brown from the San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET) in Milan, Italy reported a breakthrough for gene therapy in which they developed a way to prevent the immune system from rejecting a newly delivered gene.^[7] Similar to organ transplantation, gene therapy has been plagued by the problem of immune rejection. So far, delivery of the 'normal' gene has been difficult because the immune system recognizes the new gene as foreign and rejects the cells carrying it. To overcome this problem, the HSR-TIGET group utilized a newly uncovered network of genes regulated by molecules known as microRNAs. Dr. Naldini's group reasoned that they could use this natural function of microRNA to selectively turn off the identity of their therapeutic gene in cells of the immune system and prevent the gene from being found and destroyed. The researchers injected mice with the gene containing an immune-cell microRNA target sequence, and spectacularly, the mice did not reject the gene, as previously occurred when vectors without the microRNA target sequence were used. This work will have important implications for the treatment of hemophilia and other genetic diseases by gene therapy.

2007

On 1 May 2007 Moorfields Eye Hospital and University College London's Institute of Ophthalmology announced the world's first gene therapy trial for inherited retinal disease. The first operation was carried out on a 23 year-old British male, Robert Johnson, in early 2007.^[8] Leber's congenital amaurosis is an inherited blinding disease caused by mutations in the RPE65 gene. The results of the Moorfields/UCL trial were published in New England Journal of Medicine in April 2008. They researched the safety of the subretinal delivery of recombinant adeno associated virus (AAV) carrying RPE65 gene, and found it yielded positive results, with patients having modest increase in vision, and, perhaps more importantly, no apparent side-effects.^[9]

Problems and ethics

For the safety of gene therapy, the Weismann barrier is fundamental in the current thinking. Soma-to-germline feedback should therefore be impossible. However, there are indications^[10] that the Weissman barrier can be breached. One way it might possibly be breached is if the treatment were somehow misapplied and spread to the testes and therefore would infect the germline against the intentions of the therapy.

Some of the problems of gene therapy include:

• 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 has evolved to attack the invader. The risk of stimulating the immune system in a way that reduces gene therapy effectiveness is always a possibility. Furthermore, the immune system's enhanced response to invaders it has seen before makes it difficult for gene therapy to be repeated in patients.
• Problems with viral vectors – Viruses, 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 of 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.
• Chance of inducing a tumor (insertional mutagenesis) - If the DNA is integrated in the wrong place in the genome, for example in a tumor suppressor gene, it could induce a tumor. This has occurred in clinical trials for X-linked severe combined immunodeficiency (X-SCID) patients, in which hematopoietic stem cells were transduced with a corrective transgene using a retrovirus, and this led to the development of T cell leukemia in 3 of 20 patients.^[11]

Deaths have occurred due to gene therapy, including that of Jesse Gelsinger.^[12]

In popular culture

• In the TV series Dark Angel gene therapy is mentioned as one of the practices performed on transgenics and their surrogate mothers at Manticore, and in the episode Prodigy, Dr. Tanaka uses a groundbreaking new form of gene therapy to turn Jude, a premature, vegetative baby of a crack/cocaine addict, into a boy genius.

• Gene therapy is a crucial plot element in the video game Metal Gear Solid, where it has been used to enhance the battle capabilities of enemy soldiers.

• Gene therapy plays a major role in the sci-fi series Stargate Atlantis, as a certain type of alien technology can only be used if one has a certain gene which is given to the members of the team through gene therapy.

• Gene therapy also plays a major role in the plot of the James Bond movie Die Another Day.

• The Yellow Bastard from Frank Miller's Sin City was also apparently the recipient of gene therapy.

• In the The Dark Knight Strikes Again, Dick Grayson, the first Robin, becomes a victim of extensive gene therapy for years by Lex Luthor to become The Joker.

• Gene therapy plays a recurring role in the present-time sci-fi television program ReGenesis, where it is used to cure various diseases, enhance athletic performance and produce vast profits for bio-tech corporations. (e.g. an undetectable performance-enhancing gene therapy was used by one of the characters on himself, but to avoid copyright infringement, this gene therapy was modified from the tested-to-be-harmless original, which produced a fatal cardiovascular defect)

• Gene therapy is the basis for the plot line of the film I Am Legend.

• Gene therapy is an important plot key in the game Bioshock where the game contents refer to plasmids and [gene] splicers.

• The book Next by Michael Crichton unravels a story in which fictitious biotechnology companies which experiment with gene therapy are involved.

• In the television show Alias, a breakthrough in molecular gene therapy is discovered, whereby a patient's body is reshaped to identically resemble someone else. Protagonist Sydney Bristow's best friend was secretly killed and her "double" resumed her place.

See also

• Antisense therapy
• DNA
• Genetic engineering
• Predictive Medicine
• Full Genome Sequencing
• Life extension
• List of life extension related topics
• Technology assessment
• Therapeutic gene modulation
• Pharmacological gene therapy

References

1. ^ Kahn J (2007-09-25). Wired (15.10): 3. http://www.wired.com/techbiz/people/magazine/15-10/ff_anderson?currentPage=3. 
2. ^ Human Genome Project Information: Gene Therapy
3. ^ Durai S, Mani M, Kandavelou K, Wu J, Porteus MH, Chandrasegaran S (2005). "Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells". Nucleic Acids Res. 33 (18): 5978–90. doi:10.1093/nar/gki912. PMID 16251401. 
4. ^ Cavazzana-Calvo M, Thrasher A, Mavilio F (Feb 2004). "The future of gene therapy". Nature 427 (6977): 779–81. doi:10.1038/427779a. PMID 14985734. 
5. ^ Morgan RA, Dudley ME, Wunderlich JR, et al. (Oct 2006). "Cancer regression in patients after transfer of genetically engineered lymphocytes". Science 314 (5796): 126–9. doi:10.1126/science.1129003. PMID 16946036. 
6. ^ Ott MG, Schmidt M, Schwarzwaelder K, et al. (Apr 2006). "Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1". Nat Med. 12 (4): 401–9. doi:10.1038/nm1393. PMID 16582916. 
7. ^ Brown BD, Venneri MA, Zingale A, Sergi Sergi L, Naldini L (May 2006). "Endogenous microRNA regulation suppresses transgene expression in hematopoietic lineages and enables stable gene transfer". Nat Med. 12 (5): 585–91. doi:10.1038/nm1398. PMID 16633348. 
8. ^ BBC NEWS | Health | Gene therapy first for poor sight
9. ^ Maguire AM, Simonelli F, Pierce EA, et al. (May 2008). "Safety and efficacy of gene transfer for Leber's congenital amaurosis". N Engl J Med. 358 (21): 2240–8. doi:10.1056/NEJMoa0802315. PMID 18441370. http://content.nejm.org/cgi/content/full/NEJMoa0802315. 
10. ^ Korthof G. "The implications of Steele's soma-to-germline feedback for human gene therapy". http://home.planet.nl/~gkorthof/kortho39a.htm. 
11. ^ Woods NB, Bottero V, Schmidt M, von Kalle C, Verma IM (Apr 2006). "Gene therapy: therapeutic gene causing lymphoma". Nature 440 (7088): 1123. doi:10.1038/4401123a. PMID 16641981. 
    Thrasher AJ, Gaspar HB, Baum C, et al. (Sep 2006). "Gene therapy: X-SCID transgene leukaemogenicity". Nature 443 (7109): E5–6; discussion E6–7. doi:10.1038/nature05219. PMID 16988659. 
12. ^ http://www.ornl.gov/sci/techresources/Human_Genome/medicine/genetherapy.shtml#status

• Tinkov, S., Bekeredjian, R., Winter, G., Coester, C., Polyplex-conjugated microbubbles for enhanced ultrasound targeted gene therapy,2008 AAPS Annual Meeting and Exposition, 16-20 November, Georgia World Congress Center, Atlanta, GA, USA, (http://www.aapsj.org/abstracts/AM_2008/AAPS2008-000838.PDF)
• Gardlík R, Pálffy R, Hodosy J, Lukács J, Turna J, Celec P (Apr 2005). "Vectors and delivery systems in gene therapy". Med Sci Monit. 11 (4): RA110–21. PMID 15795707. http://www.medscimonit.com/fulltxt.php?ICID=15907. 

• Staff (November 18, 2005). "Gene Therapy" (FAQ). Human Genome Project Information. Oak Ridge National Laboratory. http://www.ornl.gov/sci/techresources/Human_Genome/medicine/genetherapy.shtml. Retrieved on 2006-05-28. 

• Salmons B, Günzburg WH (Apr 1993). "Targeting of retroviral vectors for gene therapy". Hum Gene Ther. 4 (2): 129–41. doi:10.1089/hum.1993.4.2-129. PMID 8494923. 

• Baum C, Düllmann J, Li Z, et al. (Mar 2003). "Side effects of retroviral gene transfer into hematopoietic stem cells". Blood 101 (6): 2099–114. doi:10.1182/blood-2002-07-2314. PMID 12511419. 

• Horn PA, Morris JC, Neff T, Kiem HP (Sep 2004). "Stem cell gene transfer—efficacy and safety in large animal studies". Mol. Ther. 10 (3): 417–31. doi:10.1016/j.ymthe.2004.05.017. PMID 15336643. 

• Wang, Hongjie; Dmitry M. Shayakhmetov, Tobias Leege, Michael Harkey, Qiliang Li, Thalia Papayannopoulou, George Stamatoyannopolous, and André Lieber (September 2005). "A capsid-modified helper-dependent adenovirus vector containing the beta-globin locus control region displays a nonrandom integration pattern and allows stable, erythroid-specific gene expression". Journal of Virology 79 (17): 10999–1013. doi:10.1128/JVI.79.17.10999-11013.2005. PMID 16103151. 

External links

Wikibooks has a book on the topic of Genes, Technology and Policy

• Gene Therapy: Molecular Bandage? University of Utah's Genetic Science Learning Center
• The American Society of Gene Therapy
• The European Society of Gene Therapy
• 2003 news relating to gene therapy
• Research Group at Cambridge, UK working on overcoming current hurdles to successful gene therapy
• Council for Responsible Genetics
• Molecular Medicine and Gene Therapy at Lund University
• Dossier on gene therapy and clinical trials
• Gene Therapy Net The startpoint for all the information about gene therapy
• Cancer-genetherapy.com Site focusing on cancer gene therapy
• Gene Therapy Review.

This entry is from Wikipedia, the leading user-contributed encyclopedia. It may not have been reviewed by professional editors (see full disclaimer)