Gene therapy
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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.
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. Antisense therapy is not strictly a form of gene therapy, but is often lumped together with them.
Contents
[hide]· 1 Background
· 2 Basic process
· 3 Types of gene therapy
· 4 Broad methods
· 5 Vectors in gene therapy
o 5.1 Viruses
§ 5.1.1 Retroviruses
§ 5.1.2 Adenoviruses
§ 5.1.3 Adeno-associated viruses
§ 5.1.4 Envelope protein pseudotyping of viral vectors
o 5.2 Non-viral methods
§ 5.2.1 Naked DNA
§ 5.2.2 Oligodeoxynucleotides
§ 5.2.3 Lipoplexes and polyplexes
o 5.3 Hybrid methods
· 6 Recent developments in gene therapy
· 7 Problems and ethics
· 8 In popular culture
· 9 Publications
· 10 See also
· 11 External links
· 12 References
· 13 Further reading
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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.
On September 14, 1990 researchers at the U.S. National Institutes of Health performed the first (approved) gene therapy procedure on four-year old Ashanti DeSilva. Born with a rare genetic disease called severe combined immune deficiency (SCID), she lacked a healthy immune system, and was vulnerable to every passing germ. Children with this illness usually develop overwhelming infections and rarely survive to adulthood; a common childhood illness like chickenpox is life-threatening. Ashanti 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 Ashanti's gene therapy procedure, doctors removed white blood cells from the child's body, let the cells grow in the lab, inserted the missing gene into the cells, and then infused the genetically modified blood cells back into the patient's bloodstream. Laboratory tests have shown that the therapy strengthened Ashanti's immune system; she no longer has recurrent colds, she has been allowed to attend school, and she was immunized against whooping cough. This procedure was not a cure; the white blood cells treated genetically only work for a few months, and the process must be repeated every few months. (VII, Thompson [First] 1993).
Although this simplified explanation of a gene therapy procedure sounds like a happy ending, it is little more than an optimistic first chapter in a long story; 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.
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.
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Basic process
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 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.
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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.
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Broad methods
There are a variety of different methods to replace or repair the genes targeted in gene therapy.
· A normal gene may be inserted into a nonspecific location within the genome to replace a nonfunctional gene. This approach is most common.[citationneeded]
· 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.
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Vectors in gene therapy
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Viruses
Main article: Viral vector
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. Some types of viruses actually physically insert their genes into the host's genome (it is the defining feature of retroviruses, the family of viruses that includes HIV, the virus that causes AIDS). 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 actually 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 your 'good 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.
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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 (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, we can say 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.
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 (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[1] or by including certain sequences such as the beta-globin locus control region[6] to direct the site of integration to specific chromosomal sites.
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. Ten 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 Adenosine Deaminase (ADA) enzyme continue with relative success in the USA, Italy and Japan.
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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 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 absense of integration into the host cell's genome should prevent the type of cancer seen in the SCID trials. This vector system has shown real promise in treating cancer and indeed the first gene therapy product to be licenced is an adenovirus to treat cancer.
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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, 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 be expressed for a long time. In recent human trials, CD8+ immune cells have recognised the AAV infected cells as compromised and killed these cells accordingly. This action appears to be triggered by part of the capsid or outer coat of the type 2 virus. Recent studies have shown that humans will likley react in the same way against the new serotype 8 AAV as well.