Medicine is transforming rapidly. During this era of human history, scientists are forever changing lives, because for the first time humans are not powerless against their genes! We now have the power to not only predict, but also alter our very future. The Genetic Revolution is here!
With the power of gene therapy, scientists now have a new tool that enables them to change a patient's DNA. Medical treatments usually counteract the symptoms of a disease. But with gene therapy, doctors can cure diseases. Click to learn more
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Medicine is transforming rapidly. During this era of human history, scientists are forever changing lives, because for the first time humans are not powerless against their genes! We now have the power to not only predict, but also alter our very future. The Genetic Revolution is here!
Gene Therapy Basics
Genes contain instructions on how to make proteins. If the genes are altered, it causes the proteins to be altered as well. This can cause genetic diseases or it might make a person more susceptible to diseases. The genes might become oncogenes, or defective genes. Gene therapy is a process that enables scientists to modify the genes inside a person's cells in order to stop or limit a disease. If scientists can replace the genes with "healthy" ones, it is possible that the disease can be treated. Furthermore, scientists can also turn off the mutated genes in order to prevent it from being harmful, or scientists can turn on "healthy" genes already in the patient's genome to inhibit the disease.
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Scientists have developed several approaches for fixing abnormal genes and dealing with diseased cells:
- Swap an abnormal gene with a normal gene (using homologous recombination)
- Repair the abnormal gene (using selective reverse mutation)
- Change the degree in which a gene is turned on or off (the regulation)
- Insert a normal gene into a specific location in the genome in order to replace the nonfunctional gene (this is the most common method)
- Insert a gene into the diseased cell that would cause its death (toxins)
- Insert a gene that could assists the immune system in killing the diseased cells
One of the key concept of gene therapy is the insertion of the "healthy" DNA into the targeted or affected cell. To perform this task, scientists use something called a vector. Although there are many types of vectors, the most common one is a genetically altered virus carrying normal human DNA.
First scientists delete all of the viral DNA of the virus to prevent it from being harmful. They then insert human genetic materials into the cell. The targeted cell is then infected with the virus, which uploads its genetic materials, causing the abnormal cell to start making normal proteins. This will restore the cell back to its normal state. In addition, scientists also use nonviral vectors as well.
Viruses, through time, have gained the ability to take over a cell's DNA in order to use its "protein-maker" to create new viruses. Each viruses have an outer "coat" with proteins that determine the cells it will infect. Viruses have specific proteins that can only bind with specific cells. Scientists have had some success with manipulating these outer proteins.
Scientists use a variety of different viruses in therapeutic gene therapy. Here are the viruses and their descriptions:
- Retroviruses: This group of viruses carry their genetic material in RNA form, instead of the conventional DNA. The most known retrovirus is the HIV (Human Immunodeficiency Virus). This virus infects only dividing cells, and can target specific cell types if its surface proteins are specially engineered. When the virus infects a cell, its RNA travels into the nucleus of the cell, and then convert its RNA into DNA before activating genes. It then integrates its genes into the host cell's genome. It can cause an immune response.
- Adenoviruses: These viruses carry their genetic materials in the form of a double-stranded DNA. One of the most common adenovirus is the common cold. It can be engineered to target specific cells by changing its surface proteins. This virus can not integrate its genes into the host cell's genome, making its effects short-termed. It can cause an immune response.
- Adeno-associated Viruses: This virus carries its genetic material in a single-stranded DNA. It is not known to cause any illness or diseases in humans. Adeno-associated viruses can target dividing and non-diving cells, and can be engineered to target specific cells. The virus will infect a cell, and its DNA will then travels to the cell's nucleus, and activate its cells. The virus normally integrate its genes into the host cell genome, specifically in Chromosome 19 (95% of the time). It does not cause an immune response.
- Herpes Simplex Viruses: This virus is responsible for oral and genital herpes, carrying its genetic materials in the form of a single-stranded DNA. It normally affects cells of the nervous system, and does not integrate into the host cell's genome. Although it doesn't integrate, it can stay in the nucleus of the cell for a long time in the form of a separate circular piece of DNA that can replicate with the dividing cells. It can cause an immune response.
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(81) Retrovirus
(82) Adenovirus
(84) Adeno-associated Virus
(84) Herpes Simplex Virus
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Viral Vectors have shown extremely high levels of expressing and incorporating their DNA into the targeted cells. It is also very good at entering cells and can be engineered to specifically target certain types of cells.
Since our bodies are use to fighting off foreign invaders, it is possible that a systematic immune response could occur because of the vector. Modern viral vectors, however, had been engineered without most of its surface proteins, greatly decreasing the chance of an immune response. Another problem associated with this vector, is that some genes are too big for a virus to hold and carry.
There is also the risk factor associated with using this vector, for there is a good chance that the virus could mutate, or revert back to its disease causing form.
The following vectors are nonviral, and does not involved viruses at all.
Liposome: These lipid based spheres carries circular pieces of double-stranded DNA called plasmids. Liposomes are not cell specific, and do not integrate well into the host cell's genome. Liposomes do not cause an immune response, but can be toxic. There are no limit to the size of the transfer DNA. Furthermore these lipid complexes are easy to prepare.
Direct Injection: Sometimes DNA can be injected directly into a specific tissues using a syringe and needle. DNA can also be coated onto metal pellets and then safely fired into cells using a special gun. These direct injections are simple and sometimes safe, but it is poorly efficient in gene transfer and integration. When used in dividing cells, regular injections would be required.
Nonviral vectors are often more appeasing to use because it is often time easier to create and mass produced then viral vectors are, but viral vectors seem to show better integration based on performances.
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(88) A picture showing a plasmid
(89) A machine use to inject DNA
In this technique, cells are surgically removed from the affected tissue area. Scientists then inject these cells with new DNA, and allow them to divide in culture. These tissues are then placed back into the affected area of the patient.
The main advantage of this type of therapy is that are no immune response since autologous cells are used. This gene therapy can also allow scientists to increase the chance of success. Usually scientists will insert the "healthy" gene into the removed cells, and let them divide. Scientists can then examine the cells, and choose the cells that express the "healthy" genes the most in order to transplant them back into the patient. Scientists can see ahead of time if the gene therapy is successful in the cultured cells.
There are small disadvantages to this therapy though. This therapy is only usable with cells that can be safely removed from the patient, and grown in cultures. The cells must also be able to survive for a long period of time in the patient.
(90) Ex Vivo Gene Therapy
(91) Diagram comparing ex vivo and in vivo gene therapy
(92) Petri dish use to culture cells
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In this technique, the genes are transferred into the targeted cells directly, by use of a nonviral or viral vector.
This therapy is generally use for cells that can't be grown and cultured in large enough numbers for transplant. It is also use for cells that can't be safely re-implanted into the patient.
One major disadvantage to this technique, is that there are no way to choose or increase the number of cells with successful gene expression beforehand, therefore the success of this type of therapy depends extremely on the vector and correct expression of the gene.
Cells constantly divide in the human body. Sometimes the dividing cells will create "daughter cells" with the therapeutic gene in it, other times it does not. The "integration" of the gene(s) into cells depends on whether not it is in the chromosomes.
Cells with the wanted genes integrated into their chromosomes will carry the integrated genes with them during cell division. This creates a long-term and stable expression of the gene, which is a very promising solution to curing genetic disorders.
The long-termed nature of chromosomal integration means that if a mistake were to occur in the insertion of the gene, then a worser problem would harm the patient for a long time. Because insertion occurs randomly, the genes could vary in its location in each cell. The gene could be successful, but it could also deactivate other cells or cause cancer.
To counter this disadvantage, ex vivo gene therapy is specifically used to target cells and scan them for problems before transferring them back into the body for the greatest result.
Of course, all of the previously mentioned disadvantages of ex vivo gene therapy also apply.
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(97) Cancer Cells
Gene therapy involving nonintegrating genes are usually for short-termed effects. Because nonintegrated genes are "extrachromosomal elements", as the cells divide, its daughter cells may not be given the genes. This could results in extra and repeated treatment.
This therapy greatly reduce the risk of having a permanent problem caused by the random insertion of the gene into the patient's genome.
Because it is short-termed, this therapy is commonly studied for use in killing cancer cells. For example, a nonintegrating gene would be inserted into the cancer cells, causing them to die, or be more susceptible to death. Once the cancer cells are gone, the treatment would not be needed anymore.
All of the cells in the human body can be divided into two categories: somatic cells or germline cells.
Somatic cells involved all cells in the human body except for the egg or sperm cell. Gene therapy affecting somatic cells are considered to be safer and more conservative. This is because the changes made in the genome of a patient's somatic cells are not passable onto a patient's offspring.
This type of gene therapy does have some disadvantages though:
Repeated treatments are necessary since somatic cells generally die and become replaced with new cells quickly. Successfully transporting the cell can be difficult (although this is true of all types of gene therapy)
Gene therapy done on germ cells (eggs or sperm), may become permanent. If the therapy is done early in the embryologic development then the gene transfer could occur in all the cells of the developing embryo. This is especially useful and helpful for eliminating hereditary diseases that are passed down in family lines.
This type of therapy, however is VERY controversial. (See "Impact and Issue" section under Gene Therapy)
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(98) Dividing Cells in the Body
(99) Germline Gene Therapy Is Very Controversial
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Everyone knows that humans have a total of 46 chromosomes in each of their cells. Scientists, however, have been thinking of a new approach to gene therapy utilizing a 47th chromosome. Scientists hope to insert a synthetic made-in-a lab chromosome that will transfer with each dividing cell into their daughter cells. This chromosome would be able to carry and express many large genes. In addition, the chromosome would not be affected by the immune system. The chromosome would also be able to function and express its genes independently of the other chromosomes, not interfering them.
Chromos Molecular Systems in Burnaby, British Columbia, had been able to demonstrate that mother cells carrying the extra chromosomes transfer it to their daughter cells during cell division. Recent tests on mice had confirmed the therapeutic nature of this technology.
This technology aims to repair the messenger RNA (mRNA) transcripts that are used by the body to copy the mutated gene in order to make proteins. SMaRT is used to repair just the mRNA transcript, not the entire gene. First scientists will deliver an RNA strand that pairs with the intron (regions that don't encode the protein) part of the mutated RNA segment. The RNA strand will then bound to the targeted place, and prevent spliceosomes from including the mutated segment in the final RNA product.
Scientists will deliver the correct version of the segment at the same time to replace the mutated piece. The repaired mRNA should now create functional proteins.
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The goal of antisense gene therapy is to turn off a mutated gene and prevent its expression through targeting its mRNA transcripts. A DNA strand have two pair, but during transcription, the DNA unwinds and only one strand is copied into mRNA (called the "sense" strand). The opposite strand is called the "antisense" strand.
In antisense gene therapy, an RNA strand with the antisense code of a mutated gene is delivered. The antisense RNA strand will then bind to the mutated sense mRNA strand, preventing the translation of the mutated gene.
The target of this gene therapy technique is the mutated gene in order to prevent its transcription.
A short single stranded pieces of DNA (oligonucleotides) is delivered. It then binds in the groove between the mutated gene's DNA. This creates a structure that prevents the mRNA from transcribing the segment containing the mutation.
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Ribozymes are RNA molecules that cut RNA. In this therapy, RNA strands acting as ribozymes are delivered. These strands will then bind to the mRNA created by the mutated gene. The mutated mRNA is cut, preventing it from being translated.
(106) The picture on top depicts a ribozyme
In addition to all of the techniques above, scientists are also been developing newer therapeutic gene therapy techniques. Some of these new techniques are hybrids of two other techniques, while others are completely new and untested ideas.