Gene therapy
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Gene therapy is the insertion of genes into an individual's cells and tissues to treat a disease, and hereditary diseases in particular. Gene therapy typically aims to supplement a defective mutant allele with a functional one. Although the technology is still in its infancy, it has been used with some success.
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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.
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 sperm cells, ova, and their stem cell precursors). 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 has a very low probability.
Vectors in gene therapy
All viruses attack 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 virus, leading to more and more cells becoming infected. Certain types of viruses actually physically insert their genes into the host's genome. This would incorporate the genes of that virus into the genes of the host cell for the life span of that cell. Most viruses do not do this, but some do (in fact, it is the defining feature of retroviruses, the family of viruses that includes HIV, the virus that causes AIDS).
Doctors and molecular biologists realized that viruses like this could potentially 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, he or she 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 as 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's genes in order to know which one has what function. An example:
A virus is found which replicates by inserting its genes into the host cell's genome. This virus has three genes - A, B, and C. Gene A encodes a protein which allows this virus to insert itself into the host's genome. Genes B and C actually cause the disease this virus is associated with. Thus, by re-engineering the virus so that genes B and C are removed and replaced by a beneficial gene, and leaving gene A alone, this virus could introduce your 'good gene' 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 into the cell. This RNA molecule from the retrovirus must produce a DNA copy from its RNA molecule before it can be considered for 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. This problem has recently begun to be addressed by utilizing Zinc finger nucleases to direct the site of integration to specific DNA sequences [1].
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 double-stranded DNA. They cause respiratory (especially the common cold), intestinal, and eye infections in humans. 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. These viruses can insert genetic material at a specific site on chromosome 19. 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 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 cells, such as neurons and can be expressed for a long time.
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 [1] 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.
In the Media
Gene therapy plays a major role in the sci-fi series Stargate SG1 and Stargate Atlantis.
Publications
Human Gene Therapy, published by Mary Ann Liebert, Inc., is a rapid-publication, peer-reviewed journal covering all aspects of human gene therapy.
See also
External links
- Gene Therapy: Molecular Bandage? University of Utah's Genetic Science Learning Center
- Latest Advances In Gene Research
- 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 an overcomming current hurdles to successful gene therapy
- Council for Responsible Genetics
References
- Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells. Nucleic Acids Res. 2005 Oct 26; 33(18): 5978-90 Full textca:Teràpia gènica
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