Gene Therapy

Sections

What is Gene Therapy?

Viral Gene Therapy

Non-Viral Gene Therapy

Practical Applications and Drawbacks

Activities

Additional Resources and Links

References

What is Gene Therapy?

Gene therapy is the act of manipulating an organism's genes, resulting in the alteration of cellular function. The manipulation is most often used to correct a harmful mutation in humans (Maugh II, 2002). It is most effective in correcting genetic disorders where the problem is derived from a single defective gene (NCI, 2004). There are different ways to correct a gene. The most common way is to insert a functional gene randomly. Three less common ways include: replacing a defective gene with a functional gene, correcting the gene, and altering how active a gene is within a cell (HGMIS, 2003).

Mutations corrected in body cells, or somatic cells, can correct inherited disorders. These corrections are not inherited by an individual's offspring. Germ-line gene therapy corrects glitches in egg and sperm cells of an individual. These corrections are passed down to offspring.

The most common method to insert new genetic material into a cell is by using a modified virus. These viruses are called vectors because they act as the mechanism for introducing the new DNA (Maugh II, 2002). Four types of virus vectors exist (HGMIS, 2003). Another way to change the genetic material is by chimeraplasty (Maugh II, 2002).

A basic overview of gene therapy involves a culture of cells, some sort of vector, and the newly created genetic material. Most often, cells from an organism are extracted. A vector is then prepared with the new genetic material, ensuring that the vector cannot create illness. The cells are injected with a vector. With the vector in the cell, the genetic material is inserted into the nucleus. The corrected cells are placed inside the organism again (Maugh II, 2002). This method is called ex vivo. The vectors can also be injected directly into the body. This method is called in vivo (NCI, 2004).

Viral Gene Therapy

Retrovirus Gene Therapy

Retroviruses are a type of virus used as a vector in gene therapy. It was the first vector to be used for such a purpose. Retroviruses use ribonucleic acid (RNA) to carry their genetic information instead of deoxyribonucleic acid (DNA). The RNA is manipulated to carry a copy of the desired gene as well as a promoter. The promoter determines whether the gene functions within the cell or not. A drug usually activates the promoter, which in turn, activates the gene. The activated gene is then translated into proteins (Maugh II, 2002).

Although retroviruses are the most popular vector in gene therapy, there are still many difficulties associated with their use. Retroviruses can only invade cells that divide often. Therefore, blood cells, skin cells, and many other tissues cannot be invaded by this vector. Also, they do not insert their genetic material in any specific place within the cell’s chromosomes. It might insert the genetic material in the middle of an important gene. The important gene could become defective or stop functioning and could do more harm than good. Another defect is that the genes are not always effective. Most of the time, they don't produce enough proteins to overcome the disease. The body also has its immune response, treating retroviruses as foreign invaders, thus destroying them (Maugh II, 2002).

Adenovirus Gene Therapy

Adenoviruses cause the common cold, and can effectively carry the new corrected DNA to the nucleus of the cell. In order to use these as vectors, their harmful genes are removed. They are safer to use than retroviruses because the genes are not integrated into the DNA. Therefore, adenoviruses are not permanent, lasting for a few days to a few weeks. Adenoviruses can also invade slower dividing cells, such as lung cells. Large amounts of adenovirus are required for treatment. Like retroviruses, this creates an immune response which may reduce its effectiveness (Maugh II, 2002).

Adeno-associated Virus Gene Therapy

Adeno-associated viruses are believed to occur naturally in humans, existing without causing disease or instigating an immune response from the body. These viruses were discovered fairly recently, and have many advantages (Maugh II, 2002). They insert their material specifically into chromosome 19, at a specific site (NCI, 2004). In addition to that, they can invade a variety of cells, both dividing and non-dividing (Maugh II, 2002).

The main disadvantage of this type of vector is that it can only carry two genes. It is hard to manufacture large amounts of altered adeno-associated virus as well (Maugh II, 2002).

Herpes Simplex Virus Gene Therapy

The herpes simplex virus method of gene therapy is usually used to target the nervous system because herpes simplex virus usually infects neurons (NCI, 2004).

Nonviral Gene Therapy

There are many other ways of introducing DNA into the cell. A simple way is to inject the DNA directly. However, it requires large amounts of DNA, and can only be done on certain tissues (HGMIS, 2003). Chimeraplasty is also used to correct genes. It uses oligomers, short DNA segments. These oligomers are synthesized to correspond to the corrected version of defective gene sequence. In the presence of the gene, the oligomer attaches to the gene only where the sequences match. The improperly matched parts are unable to attach. Mechanisms within the cell detect this and are able to correct the defective gene (Maugh II, 2002).

Other approaches use other vectors to transport DNA to the cell. Lipid spheres can be synthesized to carry DNA. These can pass through cell membranes. DNA can also be attached to chemicals. These chemicals are capable of binding to receptors on the cell membrane. Once this occurs, the DNA enters the cell by endocytosis. This method is not very effective (HGMIS, 2003).

An artificial chromosome can also be introduced. This would not affect the other 46 chromosomes, functioning mainly as a vector that can carry huge amounts of corrected DNA. Like the adeno-associated virus, it would not cause an immune response. However, a method for inserting the chromosome into the cell, or the nucleus, is yet to be found (HGMIS, 2003).

Practical Applcations and Drawbacks

Practical Applications

Cancer research has been looking in using gene therapy for potential prevention methods or cures. There is strong belief that when certain genes are missing completely or mutated, they can lead to cancer or keep cancer from developing. Using viral vectors to insert these genes may prevent cancer. Another approach is to manipulate the immune system, promoting the attack on cancer cells (NCI, 2004).

Two other methods are dependent on drugs or treatment. The cancer cells have genes placed in them, increasing sensitivity to the radiation they are subjected to. Cancer cells can also be injected with a suicide gene, which contains something like a promoter. With a drug, the suicide gene is turned on and the cancer cell is destroyed (NCI, 2004). This method often utilizes the herpes simplex virus (Ramesh, Marrogi & Freeman, 1998) or the synthesized liposome as a vector (Martin & Boulikas, 1998).. Other times, normal cells are injected with genes that make them more resistant to the radiation (NCI, 2004).

There is also potential for a cancer vaccine. An in vivo method is used to immunize the body against tumor associated antigens. Antigens are a factor that can set off an immune response. Tumors produce particular proteins called tumor associated antigens. Should a tumor be present in the body, the immune system would be able to recognize these proteins and launch an attack. This would decrease the need for radiation therapy and tumors would be a smaller threat, more easily managed (Lee, Wang, Nottingham, Oh, Weiner & Kim, 1999).

Researchers are also looking for a way to prevent the damage that comes from a heart attack as well. They have located a gene in animals that is activated when their oxygen supplies are low. This gene synthesizes a protein and prevents cells from dying. Humans have the gene that synthesizes the protein as well, but they do not have the promoter. The promoter is activated by low oxygen supply. Injecting human cells with this gene could reduce the damage created from a heart attack or stroke, saving individuals from death. Testing of this method on rats has already been proven successful (Mann, Gibbons, Hutchinson, Poston, Hoyt, Robbins & Dzau, 1999).

There has been much success in gene silencing also. This technique uses a viral vector to insert RNA into the nucleus of cells, causing a defective gene to become inactive. Experiments with mice silenced a rare hereditary condition called ataxia. It is very similar to Huntington's disease. Success with silencing the gene is encouraging, and gene silencing has the potential to be applied to other hereditary neurological diseases, such as Alzheimer's and Huntington's disease (Mao, Eliason, Harper, Martins, Orr, Paulson, Yang, Kotin & Davidson, 2004).

In a clinical trial, people with Alzheimer's disease had modified neurons injected in the brain. These cells contained a gene that prevented neurons affected by Alzheimer's from dying and helped untreated cells work more successfully. This process included using the ex vivo method, extracting cells, manipulating them, allowing cell division, and injecting the cells back into the organism (Tuszynski, 2004).

Experiments, using both in vivo and ex vivo, are targeting hemophilia and searching for a way to produce the clotting factors the diseased individual lacks. Using the in vivo technique, the liver cells are exposed to vectors. The liver creates most of the needed clotting factor, and by modifying it, the hemophiliacs would no longer lack the clotting factors. With the ex vivo technique, a variety of different cells including skin cells and various blood cells are manipulated. With successful manipulation, the cells are then injected into the human, and continue manufacturing the needed clotting factors (Hoeben, 1998).

Drawbacks

Gene therapy has not been effective in treating genetic disorders for many reasons. A particularly common and widespread disadvantage is that the vectors used to carry genetic material to target cells often sets off an immune response. This greatly decreases success in curing any type of disease (HGMIS, 2003).

The therapy has also been proven to be very temporary. Any of the new DNA introduced into the cell must be stable. However, due to the fact that cells often divide quickly and new DNA does not become part of the chromosomes quickly, the new genetic information is lost. In order for the therapy to show any results, patients need to receive multiple rounds of the therapy (HGMIS, 2003).

As mentioned before, an immune response is instigated when a foreign organism enters the body. The immune system also can recognize invaders that had entered the body before. This defense mechanism prevents vectors from invading cells more than once. As a result, it is harder to have multiple rounds of gene therapy because each time the vectors are introduced, the immune system can quickly destroy them (HGMIS, 2003).

Another problem concerns the viral vectors themselves. There have been many problems using viruses as vectors. Most do not insert their genetic material anywhere specific. There are vast problems with the immune response. Furthermore, it is possible that the virus is capable of regaining genetic material enabling it to spread disease (HGMIS, 2003).

Lastly, most hereditary diseases are not confined to a single gene. Many disorders are caused by multiple genes which have defects. Alzheimer’s, diabetes, and heart disease are a few examples of disease caused by the multiple genes. These are called multigene disorders, or multifactorial disorders. It is hard to “find a cure” for diseases such as these, and gene therapy would not be extremely effective (HGMIS, 2003).