GENE THERAPY
Background :
In the 1980s, advances in molecular biology had already enabled human genes to be sequenced and cloned. Scientists looking for a method of easily producing proteins, such as the protein deficient in diabetics — insulin, investigated introducing human genes to bacterial DNA. The modified bacteria then produce the corresponding protein, which can be harvested and injected in people who cannot produce it naturally.
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 it to the right site on the genome.
What is Gene therapy ?
Genes, which are carried on chromosomes, are the basic physical and functional units of heredity. Genes are specific sequences of bases that encode instructions on how to make proteins. Although genes get a lot of attention, it’s the proteins that perform most life functions and even make up the majority of cellular structures. When genes are altered so that the encoded proteins are unable to carry out their normal functions, genetic disorders can result.
Gene therapy is a technique for correcting defective genes responsible for disease development. Researchers may use one of several approaches for correcting faulty genes:
- 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.
Gene therapy is an experimental technique that uses genes to treat or prevent disease. In the future, this technique may allow doctors to treat a disorder by inserting a gene into a patient’s cells instead of using drugs or surgery. Although gene therapy is a promising treatment option for a number of diseases (including inherited disorders, some types of cancer, and certain viral infections), the technique remains risky and is still under study to make sure that it will be safe and effective. Gene therapy is currently only being tested for the treatment of diseases that have no other cures
Much attention has been focused on the so-called genetic metabolic diseases in which a defective gene causes an enzyme to be either absent or ineffective in catalyzing a particular metabolic reaction effectively. A potential approach to the treatment of genetic disorders in man is gene therapy. This is a technique whereby the absent or faulty gene is replaced by a working gene, so that the body can make the correct enzyme or protein and consequently eliminate the root cause of the disease.
The most likely candidates for future gene therapy trials will be rare diseases such as Lesch-Nyhan syndrome, a distressing disease in which the patients are unable to manufacture a particular enzyme. This leads to a bizarre impulse for self-mutilation, including very severe biting of the lips and fingers. The normal version of the defective gene in this disease has now been cloned.
If gene therapy does become practicable, the biggest impact would be on the treatment of diseases where the normal gene needs to be introduced into only one organ. One such disease is phenylketonuria (PKU). PKU affects about one in 12,000 white children, and if not treated early can result in severe mental retardation. The disease is caused by a defect in a gene producing a liver enzyme. If detected early enough, the child can be placed on a special diet for their first few years, but this is very unpleasant and can lead to many problems within the family.
The types of gene therapy described thus far all have one factor in common: that is, that the tissues being treated are somatic (somatic cells include all the cells of the body, excluding sperm cells and egg cells). In contrast to this is the replacement of defective genes in the germline cells (which contribute to the genetic heritage of the offspring). Gene therapy in germline cells has the potential to affect not only the individual being treated, but also his or her children as well. Germline therapy would change the genetic pool of the entire human species, and future generations would have to live with that change. In addition to these ethical problems, a number of technical difficulties would make it unlikely that germline therapy would be tried on humans in the near future.
Before treatment for a genetic disease can begin, an accurate diagnosis of the genetic defect needs to be made. It is here that biotechnology is also likely to have a great impact in the near future. Genetic engineering research has produced a powerful tool for pinpointing specific diseases rapidly and accurately. Short pieces of DNA called DNA probes can be designed to stick very specifically to certain other pieces of DNA. The technique relies upon the fact that complementary pieces of DNA stick together. DNA probes are more specific and have the potential to be more sensitive than conventional diagnostic methods, and it should be possible in the near future to distinguish between defective genes and their normal counterparts, an important development.
The Human Genome Program in the U.S. will provide about $200 million each year to scientists in multidisciplinary research centers who are attempting to determine the makeup of all human genes. Together with similar programs in Europe, it is hoped that in 15 years time we shall be able to identify and treat all diseases to which humans are susceptible. This will revolutionize modern medicine, and hopefully improve the quality of life of all men, women, and children. Already, the genes for Duchenne muscular dystrophy, cystic fibrosis, and retinoblastoma have been identified, and more such information is emerging all the time.
Example to study “GENE THERAPY”
Imagine that you accidentally broke one of your neighbor's windows. What would you do? You could:
1. Stay silent: no one will ever find out that you are guilty, but the window doesn't get fixed.
2. Try to repair the cracked window with some tape: not the best long-term solution.
3. Put in a new window: not only do you solve the problem, but also you do the honorable thing.
What does this have to do with gene therapy?
You can think of a medical condition or illness as a "broken window." Many medical conditions result from flaws, or mutations, in one or more of a person's genes. Mutations cause the protein encoded by that gene to malfunction. When a protein malfunctions, cells that rely on that protein's function can't behave normally, causing problems for whole tissues or organs. Medical conditions related to gene mutations are called genetic disorders.
So, if a flawed gene caused our "broken window," can you "fix" it? What are your options?
1. Stay silent: ignore the genetic disorder and nothing gets fixed.
2. Try to treat the disorder with drugs or other approaches: depending on the disorder, treatment may or may not be a good long-term solution.
3. Put in a normal, functioning copy of the gene: if you can do this, it may solve the problem!
If it is successful, gene therapy provides a way to fix a problem at its source. Adding a corrected copy of the gene may help the affected cells, tissues and organs work properly. Gene therapy differs from traditional drug-based approaches, which may treat the problem, but which do not repair the underlying genetic flaw.
But gene therapy is not a simple solution - it's not a molecular bandage that will automatically fix a disorder. Although scientists and physicians have made progress in gene therapy research, they have much more work to do before they can realize its full potential. In this module, you'll explore several approaches to gene therapy, try them out yourself, and figure out why creating successful gene-based therapies is so challenging.
How does gene therapy work ?
In most gene therapy studies, a "normal" gene is inserted into the genome to replace an "abnormal," disease-causing gene. A carrier molecule called a vector must be used to deliver the therapeutic gene to the patient's target cells. Currently, the most common vector is a virus that has been genetically altered to carry normal human DNA. Viruses have evolved a way of encapsulating and delivering their genes to human cells in a pathogenic manner. Scientists have tried to take advantage of this capability and manipulate the virus genome to remove disease-causing genes and insert therapeutic genes.
Target cells such as the patient's liver or lung cells are infected with the viral vector. The vector then unloads its genetic material containing the therapeutic human gene into the target cell. The generation of a functional protein product from the therapeutic gene restores the target cell to a normal state. See a diagram depicting this process.
One potential benefit of the Human Genome Project will be the further refinement of gene therapy. When all of our genes and their functions are known, we will have a blueprint that tells us what genes, and what mutations of genes, are responsible for a vast array of human diseases. Gene therapy is intended to stop many of those diseases in their tracks, at their source.
If a person carries a defective or mutated form of a particular gene, that gene's protein product will not do the job it is intended to do. This can lead to disease. Most of our current therapies for such diseases are aimed at treating the symptoms of the diseases produced by the defective genes. Gene therapy is intended to cure the disease by replacing the defective gene with one that produces the correct protein. Genes can be attached to modified versions of viruses or similar structures that have the ability to penetrate the nucleus of a cell and become incorporated into the cell's existing DNA.
Gene therapy typically involves three "steps":
- Administration: The introduction of the correct form of the gene into the body.
- Delivery: The transfer of that correct gene to the nucleus of the cells for which it is intended.
- Expression: The production of the proper protein by the cells that have received the corrected form of the gene.
Fundamentals of Gene Therapy :
You look a little like your mother and a little like your father because of the genes they gave to you. Genes, those conceptual units composed of deoxyribonucleic acid—DNA, carry the information needed to make proteins, the building blocks of our bodies. The body buries genes deep in the heart of every cell, the nucleus, and organizes them in the chromosomes that hold the DNA. But when your DNA is damaged, it no longer makes all the needed proteins and disease results.
To reverse disease caused by genetic damage, researchers isolate normal DNA and package it into a vector, a molecular delivery truck usually made from a disabled virus. Doctors then infect a target cell —usually from a tissue affected by the illness, such as liver or lung cells—with the vector. The vector unloads its DNA cargo, which then begins producing the missing protein and restores the cell to normal.
Recently, French researchers reported dramatic results in treating a disease called severe combined immune deficiency (SCID), the disorder suffered by David, The Boy in the Bubble. A broken gene eliminates the production of an enzyme essential for the development of a normal immune system. Scientists isolated the normal copy of the gene and packaged it into a vector. In the laboratory, they then used the vector to transport the gene into the patient's own bone marrow cells. Bone marrow cells create the immune system. The treated bone marrow cells are then given back to the patient in a germ-free isolation room, where they reconstitute a normal, functioning immune system, freeing the patient from the need to remain in isolation.
Gene therapy is designed to introduce genetic material into cells to compensate for abnormal genes or to make a beneficial protein. If a mutated gene causes a necessary protein to be faulty or missing, gene therapy may be able to introduce a normal copy of the gene to restore the function of the protein.
A gene that is inserted directly into a cell usually does not function. Instead, a carrier called a vector is genetically engineered to deliver the gene. Certain viruses are often used as vectors because they can deliver the new gene by infecting the cell. The viruses are modified so they can't cause disease when used in people. Some types of virus, such as retroviruses, integrate their genetic material (including the new gene) into a chromosome in the human cell. Other viruses, such as adenoviruses, introduce their DNA into the nucleus of the cell, but the DNA is not integrated into a chromosome.
The vector can be injected or given intravenously (by IV) directly into a specific tissue in the body, where it is taken up by individual cells. Alternately, a sample of the patient's cells can be removed and exposed to the vector in a laboratory setting. The cells containing the vector are then returned to the patient. If the treatment is successful, the new gene delivered by the vector will make a functioning protein.
Researchers must overcome many technical challenges before gene therapy will be a practical approach to treating disease. For example, scientists must find better ways to deliver genes and target them to particular cells. They must also ensure that new genes are precisely controlled by the body.
A new gene is injected into an adenovirus vector, which is used to introduce the modified DNA into a human cell. If the treatment is successful, the new gene will make a functional protein.
Types of gene therapy :
In theory it is possible to transform either somatic cells (most cells of the body) or cells of the germline (such as stem cells, sperm cells and ova). All gene therapy so far in people has been directed at somatic cells, whereas germline engineering in humans remains only a highly controversial prospect. For the introduced gene to be transmitted normally to offspring, it needs not only to be inserted into the cell, but also to be incorporated into the chromosomes by genetic recombination.
Somatic gene therapy can be broadly split in to two categories: ex vivo (where cells are modified outside the body and then transplanted back in again) and in vivo (where genes are changed in cells still in the body.) Recombination-based approaches in vivo are especially uncommon, because for most DNA constructs recombination is a very low probability event.
Ex vivo :
The ex vivo approach was the first to be put in to practice. In 1990 trials were run designed to treat children with an inherited immune deficiency, as well as children or adults with high serum cholesterol. Cells were removed from the patients body and incubated with vectors that inserted copies of the genes. Most gene-therapy vectors are viruses, although there are techniques for delivering DNA directly as well. After modification, the cells are transplanted back in to the patient where they will hopefully replicate and produce functional descendants for the life of the transplanted individual.
This technique is best used for diseases where the desired cells can be extracted easily, such as the blood or liver.
When familiar viruses are detected in the bloodstream the body sends antibodies to bind to and consume them. In retroviral and other recombination-based approaches, a second problem arises in the unpredictability of where the new DNA inserts into the chromosomes of transfected cells. If the gene is inserted in a bad place — for example within the sequence of an important gene, or within non-coding (intron) regions that the cell will never translate to produce protein — then the new gene would not be properly expressed and the cell could be made worse or even cancerous.
Scientists are researching an interesting way of bypassing the DNA problems by actually introducing an extra chromosome into the body. Existing alongside existing DNA, this 47th chromosome would contain the genes needed. Introduced into the body as a large vector, it is not expected to be targeted by the immune system because of its construction.
Vectors in gene therapy :
Viruses attack their hosts to insert their genetic material into the genetic material of the host. This genetic material contains instructions to produce these viruses. The host cell will carry out these instructions and produce the viruses. This is how viruses spread, in general.
In addition to the instructions producing the components of the virus itself, viruses can carry additional genes containing instructions for creating other kinds of proteins. In theory, if we insert a gene that is missing from a patient in a virus, and infect that patient with the virus, the virus will spread the missing gene in all the cells of the patient. The missing gene is now replaced and the disease is cured. This technique is called gene therapy.
Three types of viruses are currently used as vectors in gene therapy: retroviruses, adenoviruses and adeno-associated viruses. They differ in their mechanisms of action and results.
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 into the cell. This RNA molecule from the retrovirus must produce a DNA copy from its RNA molecule before it can be considered part of 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, or the chromosomes of the cell. 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, we can say that the host cell is now modified to contain a new gene. When this host cell divides later, its descendants will all contain the new genes.
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. 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. If the gene happens to be one regulating cell division, uncontrolled cell division (i.e., cancer) can occur.
Gene therapy trials to treat severe combined immunodeficiency (SCID) were halted or restricted in the USA when leukemia was reported in three of eleven patients treated in the French Therapy X-linked SCID (XSCID) gene therapy trial. Five XSCID patients treated in England have not presented leukemia to date and have had similar success in immune reconstitution. Gene therapy trials to treat SCID due to deficiency of the Adenosin Deaminase (ADA) enzyme continue with relative success in the USA, Italy and Japan.
Adenoviruses :
Adenoviruses are viruses that carry their genetic material in the form of DNA. When these viruses infect a host cell, they introduce their DNA molecule into the host. The genetic material of the adenoviruses is not incorporated into the host cells 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. This means that treatment with the adenovirus will require regular doses to add the missing gene every time
Adeno-associated viruses :
Adeno-associated viruses, from the parvovirus family, are small viruses with a genome of single stranded DNA. There are a few disadvantages to using AAV, mainly the small amount of DNA it can carry 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
Herpes simplex viruses :
A class of double-stranded DNA viruses that infect a particular cell type, neurons. Herpes simplex virus type 1 is a common human pathogen that causes cold sores.
Besides virus-mediated gene-delivery systems, there are several nonviral options for gene delivery. The simplest method is the direct introduction of therapeutic DNA into target cells. This approach is limited in its application because it can be used only with certain tissues and requires large amounts of DNA.
Another nonviral approach involves the creation of an artificial lipid sphere with an aqueous core. This liposome, which carries the therapeutic DNA, is capable of passing the DNA through the target cell's membrane.
Therapeutic DNA also can get inside target cells by chemically linking the DNA to a molecule that will bind to special cell receptors. Once bound to these receptors, the therapeutic DNA constructs are engulfed by the cell membrane and passed into the interior of the target cell. This delivery system tends to be less effective than other options.
Researchers also are experimenting with introducing a 47th (artificial human) chromosome into target cells. This chromosome would exist autonomously alongside the standard 46 --not affecting their workings or causing any mutations. It would be a large vector capable of carrying substantial amounts of genetic code, and scientists anticipate that, because of its construction and autonomy, the body's immune systems would not attack it. A problem with this potential method is the difficulty in delivering such a large molecule to the nucleus of a target cell.
Somatic vs. germ-line therapy :
Gene therapy can be somatic, or directed at existing mutated cells in the body. Somatic therapy cannot be passed on to future generations because only the mature cells with the defective gene are affected. Another form of gene therapy is germ-line therapy, directed at egg or sperm cells and intended to be inherited for generations. For ethical and technical reasons, gene therapy research efforts are largely aimed at somatic treatment.
The proper forms of the genes are either introduced in the laboratory or directly into the body. For laboratory procedures, the cells with the defective gene are removed from the body, the genes are introduced in culture dishes, and the altered cells returned to the body.
Alternatively, the corrected genes, attached to the vectors (often harmless viruses) that carry them into the nuclei, can be injected into the body. Current technology requires that such genes be injected quite near or directly into the tissue that needs them. Scientists are now investigating new techniques that would permit the injection to take place at any site.
Altered Genes :
Each of us carries about half a dozen defective genes. We remain blissfully unaware of this fact unless we, or one of our close relatives, are amongst the many millions who suffer from a genetic disease. About one in ten people has, or will develop at some later stage, an inherited genetic disorder, and approximately 2,800 specific conditions are known to be caused by defects (mutations) in just one of the patient's genes. Some single gene disorders are quite common - cystic fibrosis is found in one out of every 2,500 babies born in the Western World - and in total, diseases that can be traced to single gene defects account for about 5% of all admissions to children's hospitals.
In the U.S. and Europe, there are exciting new programs to 'map' the entire human genome - all of our genes. This work will enable scientists and doctors to understand the genes that control all diseases to which the human race is prone, and hopefully develop new therapies to treat and predict diseases.
Diseases of Genetic Origin :
Most of us do not suffer any harmful effects from our defective genes because we carry two copies of nearly all genes, one derived from our mother and the other from our father. The only exceptions to this rule are the genes found on the male sex chromosomes. Males have one X and one Y chromosome, the former from the mother and the latter from the father, so each cell has only one copy of the genes on these chromosomes. In the majority of cases, one normal gene is sufficient to avoid all the symptoms of disease. If the potentially harmful gene is recessive, then its normal counterpart will carry out all the tasks assigned to both. Only if we inherit from our parents two copies of the same recessive gene will a disease develop.
On the other hand, if the gene is dominant, it alone can produce the disease, even if its counterpart is normal. Clearly only the children of a parent with the disease can be affected, and then on average only half the children will be affected. Huntington's chorea, a severe disease of the nervous system, which becomes apparent only in adulthood, is an example of a dominant genetic disease.
Finally, there are the X chromosome-linked genetic diseases. As males have only one copy of the genes from this chromosome, there are no others available to fulfill the defective gene's function. Examples of such diseases are Duchenne muscular dystrophy and, perhaps most well known of all, hemophilia.
Queen Victoria was a carrier of the defective gene responsible for hemophilia, and through her it was transmitted to the royal families of Russia, Spain, and Prussia. Minor cuts and bruises, which would do little harm to most people, can prove fatal to hemophiliacs, who lack the proteins (Factors VIII and IX) involved in the clotting of blood, which are coded for by the defective genes. Sadly, before these proteins were made available through genetic engineering, hemophiliacs were treated with proteins isolated from human blood. Some of this blood was contaminated with the AIDS virus, and has resulted in tragic consequences for many hemophiliacs. Use of genetically engineered proteins in therapeutic applications, rather than blood products, will avoid these problems in the future.
Not all defective genes necessarily produce detrimental effects, since the environment in which the gene operates is also of importance. A classic example of a genetic disease having a beneficial effect on survival is illustrated by the relationship between sickle-cell anemia and malaria. Only individuals having two copies of the sickle-cell gene, which produces a defective blood protein, suffer from the disease. Those with one sickle-cell gene and one normal gene are unaffected and, more importantly, are able to resist infection by malarial parasites. The clear advantage, in this case, of having one defective gene explains why this gene is common in populations in those areas of the world where malaria is endemic.
What is the current status of gene therapy research?
The Food and Drug Administration (FDA) has not yet approved any human gene therapy product for sale. Current gene therapy is experimental and has not proven very successful in clinical trials. Little progress has been made since the first gene therapy clinical trial began in 1990. In 1999, gene therapy suffered a major setback with the death of 18-year-old Jesse Gelsinger. Jesse was participating in a gene therapy trial for ornithine transcarboxylase deficiency (OTCD). He died from multiple organ failures 4 days after starting the treatment. His death is believed to have been triggered by a severe immune response to the adenovirus carrier.
Another major blow came in January 2003, when the FDA placed a temporary halt on all gene therapy trials using retroviral vectors in blood stem cells. FDA took this action after it learned that a second child treated in a French gene therapy trial had developed a leukemia-like condition. Both this child and another who had developed a similar condition in August 2002 had been successfully treated by gene therapy for X-linked severe combined immunodeficiency disease (X-SCID), also known as "bubble baby syndrome."
FDA's Biological Response Modifiers Advisory Committee (BRMAC) met at the end of February 2003 to discuss possible measures that could allow a number of retroviral gene therapy trials for treatment of life-threatening diseases to proceed with appropriate safeguards. FDA has yet to make a decision based on the discussions and advice of the BRMAC meeting.
What factors have kept gene therapy from becoming an effective treatment for genetic disease?
- Short-lived nature of gene therapy - Before gene therapy can become a permanent cure for any condition, the therapeutic DNA introduced into target cells must remain functional and the cells containing the therapeutic DNA must be long-lived and stable. Problems with integrating therapeutic DNA into the genome and the rapidly dividing nature of many cells prevent gene therapy from achieving any long-term benefits. Patients will have to undergo multiple rounds of gene therapy.
- Immune response - Anytime a foreign object is introduced into human tissues, the immune system is designed to attack the invader. The risk of stimulating the immune system in a way that reduces gene therapy effectiveness is always a potential risk. 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, while the carrier of choice in most gene therapy studies, present a variety of potential problems to the patient --toxicity, immune and inflammatory responses, and gene control and targeting issues. In addition, there is always the fear that the viral vector, once inside the patient, may recover its ability to cause disease.
- Multigene disorders - Conditions or disorders that arise from mutations in a single gene are the best candidates for gene therapy. Unfortunately, some the most commonly occurring disorders, such as heart disease, high blood pressure, Alzheimer's disease, arthritis, and diabetes, are caused by the combined effects of variations in many genes. Multigene or multifactorial disorders such as these would be especially difficult to treat effectively using gene therapy. For more information on different types of genetic disease, see Genetic Disease Information.
What are some recent developments in gene therapy research?
- University of California, Los Angeles, research team gets genes into the brain using liposomes coated in a polymer call polyethylene glycol (PEG). The transfer of genes into the brain is a significant achievement because viral vectors are too big to get across the "blood-brain barrier." This method has potential for treating Parkinson's disease. 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).
- New gene therapy approach repairs errors in messenger RNA derived from defective genes. Technique has potential to treat the blood disorder thalassaemia, cystic fibrosis, and some cancers. See Subtle gene therapy tackles blood disorder at NewScientist.com (October 11, 2002).
- Gene therapy for treating children with X-SCID (sever combined immunodeficiency) or the "bubble boy" disease is stopped in France when the treatment causes leukemia in one of the patients. See 'Miracle' gene therapy trial halted at NewScientist.com (October 3, 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 is successfully treated in mice. See Murine Gene Therapy Corrects Symptoms of Sickle Cell Disease from March 18, 2002, issue of The Scientist.
How is gene therapy being studied in the treatment of cancer?
Researchers are studying several ways to treat cancer using gene therapy. Some approaches target healthy cells to enhance their ability to fight cancer. Other approaches target cancer cells, to destroy them or prevent their growth. Some gene therapy techniques under study are described below.
· In one approach, researchers replace missing or altered genes with healthy genes. Because some missing or altered genes may lead to cancer, substituting “working” copies of these genes may keep cancer from developing.
· Researchers are also studying ways to improve a patient’s immune response to cancer. In this approach, gene therapy is used to stimulate the body’s natural ability to attack cancer cells.
· In some studies, scientists inject cancer cells with genes that make them more sensitive to chemotherapy, radiation therapy, or other treatments. In other studies, researchers place a gene into healthy blood-forming stem cells to make these cells more resistant to the side effects of high doses of anticancer drugs.
· In another approach, researchers inject cancer cells with genes that can be used to destroy the cells. In this technique, “suicide genes” are introduced into cancer cells. Later, a pro-drug (an inactive form of a toxic drug) is given to the patient. The pro-drug is activated in cancer cells containing these “suicide genes,” which leads to the destruction of those cancer cells.
· Other research is focused on the use of gene therapy to prevent cancer cells from developing new blood vessels (angiogenesis).
How are genes transferred into cells so that gene therapy can take place?
In general, a gene cannot be directly inserted into a person’s cell. It must be delivered to the cell using a carrier, or “vector.” The vectors most commonly used in gene therapy are viruses. Viruses have a unique ability to recognize certain cells and insert their DNA into the cells.
In some gene therapy clinical trials, cells from the patient’s blood or bone marrow are removed and grown in the laboratory. The cells are exposed to the virus that is carrying the desired gene. The virus enters the cells and inserts the desired gene into the cells’ DNA. The cells grow in the laboratory and are then returned to the patient by injection into a vein. This type of gene therapy is called ex vivo because the cells are grown outside the body. The gene is transferred into the patient’s cells while the cells are outside the patient’s body.
In other studies, vectors (often viruses) or liposomes (fatty particles) are used to deliver the desired gene to cells in the patient’s body. This form of gene therapy is called in vivo, because the gene is transferred to cells inside the patient’s body
What types of viruses are used in gene therapy, and how can they be used safely?
Many gene therapy clinical trials rely on retroviruses to deliver the desired gene. Other viruses used as vectors include adenoviruses, adeno-associated viruses, lentiviruses, poxviruses, and herpes viruses. These viruses differ in how well they transfer the genes to cells, which cells they can recognize and infect, and whether they alter the cell’s DNA permanently or temporarily. Thus, researchers may use different vectors, depending on the specific characteristics and requirements of the study.
Scientists alter the viruses used in gene therapy to make them safe for humans and to increase their ability to deliver specific genes to a patient’s cells. Depending on the type of virus and the goals of the research study, scientists may inactivate certain genes in the viruses to prevent them from reproducing or causing disease. Researchers may also alter the virus so that it better recognizes and enters the target cell.
What risks are associated with current gene therapy trials?
Viruses can usually infect more than one type of cell. Thus, when viral vectors are used to carry genes into the body, they might infect healthy cells as well as cancer cells. Another danger is that the new gene might be inserted in the wrong location in the DNA, possibly causing cancer or other harmful mutations to the DNA.
In addition, when viruses or liposomes are used to deliver DNA to cells inside the patient’s body, there is a slight chance that this DNA could unintentionally be introduced into the patient’s reproductive cells. If this happens, it could produce changes that may be passed on if a patient has children after treatment.
Other concerns include the possibility that transferred genes could be “overexpressed,” producing so much of the missing protein as to be harmful; that the viral vector could cause inflammation or an immune reaction; and that the virus could be transmitted from the patient to other individuals or into the environment.
Scientists use animal testing and other precautions to identify and avoid these risks before any clinical trials are conducted in humans.
What major problems must scientists overcome before gene therapy becomes a common technique for treating disease?
Scientists need to identify more efficient ways to deliver genes to the body. To treat cancer and other diseases effectively with gene therapy, researchers must develop vectors that can be injected into the patient and specifically focus on the target cells located throughout the body. More work is also needed to ensure that the vectors will successfully insert the desired genes into each of these target cells.
Researchers also need to be able to deliver genes consistently to a precise location in the patient’s DNA, and ensure that transplanted genes are precisely controlled by the body’s normal physiologic signals.
Although scientists are working hard on these problems, it is impossible to predict when they will have effective solutions.
2. The first disease approved for treatment with gene therapy was adenosine deaminase (ADA) deficiency. What is this disease and why was it selected?
ADA deficiency is a rare genetic disease. The normal ADA gene produces an enzyme called adenosine deaminase, which is essential to the body’s immune system. Patients with this condition do not have normal ADA genes and do not produce functional ADA enzyme. ADA-deficient children are born with severe immunodeficiency and are prone to repeated serious infections, which may be life-threatening. Although ADA deficiency can be treated with a drug called PEG-ADA, the drug is expensive (more than $100,000 a year) and must be taken for life by injection into a vein.
ADA deficiency was selected for the first approved human gene therapy trial for several reasons:
· The disease is caused by a defect in a single gene, which increases the likelihood that gene therapy will succeed.
· The gene is regulated in a simple, “always-on” fashion, unlike many genes whose regulation is complex.
· The amount of ADA present does not need to be precisely regulated. Even small amounts of the enzyme are known to be beneficial, while larger amounts are also tolerated well.
How do gene therapy trials receive approval?
A proposed gene therapy trial, or protocol, must be approved by at least two review boards at the scientists’ institution. Gene therapy protocols must also be approved by the U.S. Food and Drug Administration (FDA), which regulates all gene therapy products. In addition, trials that are funded by the National Institutes of Health (NIH) must be registered with the NIH Recombinant DNA Advisory Committee (RAC). The NIH, which includes more than 20 institutes and offices, is the Federal focal point for biomedical research in the United States.
Why are there so many steps in this process?
Any studies involving humans must be reviewed with great care. Gene therapy in particular is a potentially very powerful technique, is relatively new, and could have profound implications. These factors make it necessary for scientists to take special precautions with gene therapy.
What are some of the social and ethical issues surrounding human gene therapy?
In large measure, the issues are the same as those faced whenever a powerful new technology is developed. Such technologies can accomplish great good, but they can also result in great harm if applied unwisely.
Gene therapy is currently focused on correcting genetic flaws and curing life-threatening disease, and regulations are in place for conducting these types of studies. But in the future, when the techniques of gene therapy have become simpler and more accessible, society will need to deal with more complex questions.
One such question is related to the possibility of genetically altering human eggs or sperm, the reproductive cells that pass genes on to future generations. (Because reproductive cells are also called germ cells, this type of gene therapy is referred to as germ-line therapy.) Another question is related to the potential for enhancing human capabilities—for example, improving memory and intelligence—by genetic intervention. Although both germ-line gene therapy and genetic enhancement have the potential to produce benefits, possible problems with these procedures worry many scientists.
Germ-line gene therapy would forever change the genetic make-up of an individual’s descendants. Thus, the human gene pool would be permanently affected. Although these changes would presumably be for the better, an error in technology or judgment could have far-reaching consequences. The NIH does not approve germ-line gene therapy in humans.
In the case of genetic enhancement, there is concern that such manipulation could become a luxury available only to the rich and powerful. Some also fear that widespread use of this technology could lead to new definitions of “normal” that would exclude individuals who are, for example, of merely average intelligence. And, justly or not, some people associate all genetic manipulation with past abuses of the concept of “eugenics,” or the study of methods of improving genetic qualities through selective breeding.
3. What is being done to address these social and ethical issues?
Scientists working on the Human Genome Project (HGP), which has completed mapping and sequencing all of the genes in humans, have recognized that the information gained from this work will have profound implications for individuals, families, and society. The Ethical, Legal, and Social Implications (ELSI) Program was established in 1990 to address these issues. The ELSI Program is designed to identify, analyze, and address the ethical, legal, and social implications of human genetics research at the same time that the basic scientific issues are being studied. In this way, problem areas can be identified and solutions developed before the scientific information becomes part of standard health care practice. More information about the HGP and the ELSI Program can be found on the National Human Genome Research Institute (NHGRI)
Obstacles to overcome :
Gene therapy is still in its infancy. In the laboratory, genes are successfully introduced into cells, and those cells produce the proper proteins. In the body, there are still obstacles to overcome. The genes must find the right tissue and get to the correct cells in those tissues. Once there, they must work into the nuclei of those cells, become incorporated properly into the existing DNA, get translated into the proper proteins, and be reproduced along with the rest of the cell's DNA. Other problems include side effects from the treatment. In 1999, 18-year-old Jesse Gelsinger died four days after undergoing gene therapy treatment for an inherited liver disease. His organs shut down, apparently due to a severe immune response to the adenovirus vector used for the therapy. And in January 2003, the FDA ordered a halt to all clinical gene therapy trials using retroviral vectors to alter blood stem cells after two children participating in a French trial developed leukemia. Despite these challenges, progress continues, and many believe effective gene therapy remains a realizable technology.
New Approaches to Gene Therapy
| In Tools of the Trade, we examined the viral and non-viral vectors commonly used for gene delivery. Each of those vectors is designed to deliver normal copies of a gene into cells that contain only a mutated copy. 
There are times, though, when adding a "good" copy of the gene won't solve the problem. For example, if the mutated gene encodes a protein that prevents the normal protein from doing its job, adding back the normal gene won't help. Mutated genes that function this way are called dominant negative. How to deal with a dominant negative? To address this situation, you could either repair the mutated gene's product, or you could get rid of it altogether. Here are some of the newest methods that scientists are developing as potential approaches to gene therapy. Each of these techniques also requires a specific and efficient means of delivering the gene to the target cell. A technique for repairing mutations: SMaRT™ The term SMaRT™ stands for "Spliceosome-Mediated RNA Trans-splicing." This technique targets and repairs the messenger RNA (mRNA) transcripts copied from the mutated gene. Instead of attempting to replace the entire gene, this technique repairs just the section of the mRNA transcript that contains the mutation. The sequence of a human gene contains regions that encode the protein (called exons) and regions that don't encode the protein (called introns). After a gene is copied into mRNA, the cell uses RNA-based machinery called spliceosomes (pronounced SPLICE-oh-zomes) to cut out the non-coding introns and splice the exons together. SMaRT™ involves three steps: 1. Delivery of an RNA strand that pairs specifically with the intron next to the mutated segment of mRNA. Once bound, this RNA strand prevents spliceosomes from including the mutated segment in the final, spliced RNA product. 2. Simultaneous delivery of a correct version of the segment to replace the mutated piece in the final mRNA product 3. Translation of the repaired mRNA to produce the normal, functional protein .SMaRT™ is a trademark of Intronn, Inc. Techniques to prevent the production of a mutated protein: · Triple-helix-forming oligonucleotides 
Triple-helix-forming oligonucleotide (pronounced AHL-ih-go-NOOK-leo-tide) gene therapy targets the DNA sequence of a mutated gene to prevent its transcription. This technique involves the delivery of short, single-stranded pieces of DNA, called oligonucleotides, that bind specifically in the groove between the double strands of the mutated gene's DNA. Binding produces a triple-helix structure that prevents that segment of DNA from being transcribed into mRNA. · Antisense 
Antisense gene therapy aims to turn off a mutated gene in a cell by targeting the mRNA transcripts copied from the gene. Genes are made up of two paired DNA strands. During transcription, the sequence of one strand is copied into a single strand of mRNA. This mRNA is called the "sense" strand because it contains the code that will be read by the cell as it makes a protein. The opposite strand is the "antisense" strand. Antisense gene therapy involves the following steps: 1. Delivery of an RNA strand containing the antisense code of a mutated gene 2. Binding of the antisense RNA strands to the mutated sense mRNA strands, preventing the mRNA from being translated into a mutated protein · Ribozymes Like antisense, ribozyme (pronounced RYE-bo-ZYME) gene therapy aims to turn off a mutated gene in a cell by targeting the mRNA transcripts copied from the gene. This approach prevents the production of the mutated protein. Ribozymes are RNA molecules that act as enzymes. Most often, they act as molecular scissors that cut RNA. For example, spliceosomes (described above) are believed to be a type of ribozyme. Read more about ribozymes and other forms of RNA in Bringing RNA into View. Ribozyme gene therapy involves the following steps: 1. Delivery of RNA strands engineered to function as ribozymes. 2. Specific binding of the ribozyme RNA to mRNA encoded by the mutated gene 3. Cleavage of the target mRNA, preventing it from being translated into a protein 
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Challenges in Gene Therapy
| Gene therapy is not a new field; it has been evolving for decades. Despite the best efforts of researchers around the world, however, gene therapy has seen only limited success. Why? The answer is that gene therapy poses one of the greatest technical challenges in modern medicine. It is very hard to introduce new genes into cells of the body. Let's look at some of the main technical issues in gene therapy.
Gene therapy will work only if we can deliver a normal gene to a large number of cells - say, several million - in a tissue. And they have to be the correct cells, in the correct tissue. Once the gene reaches its destination, it must be activated, or turned on to produce the protein encoded by the gene. Gene delivery and activation are the biggest obstacles facing gene therapy researchers. Tools of the Trade highlights some of the most common methods for addressing these challenges.
Introducing changes into the germline Targeting a gene to the correct cells is crucial to the success of any gene therapy treatment. Just as important, though, is making sure that the gene is not incorporated into the wrong cells. Delivering a gene to the wrong tissue would be inefficient and could cause health problems for the patient. For example, improper targeting could incorporate the therapeutic gene into a patient's germline, or reproductive cells, which ultimately produce sperm and eggs. Should this happen, the patient would pass the introduced gene on to his or her offspring. The consequences would vary, depending on the type of gene introduced. Immune response Our immune systems are very good at fighting off intruders such as bacteria, viruses and other biological substances. Gene delivery vectors must be able to escape the body's natural surveillance systems. Failure to do so can cause serious illness or even death. The story of Jesse Gelsinger illustrates this challenge well. Gelsinger, who had a rare liver disorder, participated in a 1999 gene therapy trial at the University of Pennsylvania. He died of complications from an inflammatory response shortly after receiving a dose of experimental adenovirus vector. His death halted all gene therapy trials in the United States for a time, sparking a much-needed discussion on how best to regulate experimental trials and report health problems in volunteer patients. Disrupting important genes in target cells The best gene therapy is the one that lasts. Ideally, we would want a gene that is introduced into a group of cells to remain there and continue working. For this to happen, the newly introduced gene must become a permanent part of each cell's genome, usually by integrating, or "stitching" itself, into the cell's existing DNA. But what happens if the gene stitches itself into an inappropriate location, disrupting another gene? This happened recently in a gene therapy trial to treat several children with X-linked Severe Combined Immune Deficiency (SCID). People with this disorder have virtually no immune protection against bacteria and viruses. To escape infections and illnesses, they must live in a completely germ-free environment. In the late 1990s, researchers tested a gene therapy treatment that would restore the function of a crucial gene, gamma c, to cells of the immune system. This treatment appeared very successful, restoring immune function to most of the children who received it. But later, two of these children developed leukemia. Researchers found that the leukemia occurred because the newly transferred gamma c gene had stitched itself into the wrong place, interrupting the function of a gene that normally helps regulate the rate at which cells divide. As a result, the cells began to divide out of control, causing the blood cancer leukemia. Although doctors have treated the children successfully with chemotherapy, the fact that they developed leukemia during treatment raises another important safety-related issue that gene therapy researchers must address. |
What Are Some Issues In Gene Therapy?
| We saw in Choosing Targets for Gene Therapy and Challenges in Gene Therapy that gene therapy research is complex and has many variables. Though several clinical trials have shown promising results, much more research is needed to guarantee the safety and efficiency of gene therapy procedures. As gene therapy comes closer to becoming a medical treatment for genetic diseases, other ethical, legal, and social issues must be kept in mind. What are the possible implications of gene therapy research to society? All of us - researchers, policymakers and the public - have a responsibility to explore the potential effects of gene therapy research on our lives so that we can make informed decisions. For each new application of gene therapy research, we must consider: - What are the benefits?
- What are the risks?
- Whom will the technology help? Whom will it potentially hurt?
- What does gene therapy mean for me? For my family? For the people in my community?
- Why might others not share my view?
Ethical, legal and social issues There are several types of issues to consider as we think about gene therapy:  Ethical issues ask us to consider the potential moral outcomes of gene therapy research.
 Legal issues require researchers and the public to help policymakers decide whether and how gene therapy research should be regulated by the government.
 Social issues involve the impact of gene therapy research on society as a whole.
Some questions to ponder - When should gene therapy be used? Should it be used to treat critically ill patients? Should it be used to treat babies and children?
- What effect would gene therapy have on future generations if germline (reproductive) cells were genetically altered? How might this alteration affect human variation?
- Who should decide what are "good" or "bad" uses of genetic modifications? How do you define "normal" with regard to human beings?
- What if we could alter human traits not associated with disease? Would it be okay to use gene therapy to improve or enhance a person's genetic profile?
- Who will have access to gene therapy, treatments and long-term follow-ups? Will gene therapy and genetic enhancements create an advantage for those who can afford it?
The questions raised here have no clear right or wrong answer. Your responses will depend on your values, as well as on the opinions of those around you. |
