Introduction
What is a Gene? 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. So what is Gene Therapy? 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.
Basic process
In most gene therapy studies, a "normal" gene is inserted into the genome to replace an "abnormal," disease-causing gene. A carrier called a vector must be used to deliver the therapeutic gene to the patient's target cells. All viruses attack their hosts and introduce their genetic material into the host cell as part of their replication cycle. This genetic material helps them 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 the virus, leading to more and more cells becoming infected. Some types of viruses actually physically insert their genes into the host's genome (a defining feature of retroviruses, the family of viruses that includes HIV, is that the virus will introduce the enzyme reverse transcriptase into the host and thus use its RNA as the "instructions"). This incorporates the genes of that virus among the genes of the host cell for the life span of that cell. Scientists have tried to harness this ability by manipulating the viral genome to remove disease-causing genes and insert therapeutic ones.
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. 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 to date on humans has been directed at somatic cells, whereas germline engineering in humans remains controversial.[2] 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. Two methods exist for inserting genetic material into human chromosomes: The first, called the ex vivo technique, involves surgically removing cells from the affected tissue area, injecting or splicing the new DNA (the DNA that will correct the disease) into the cells and letting them divide in cultures. The new tissues are placed back into the affected area of the patient. Often, doctors need only culture the patient’s bone marrow because it produces the blood that will eventually travel throughout the body. This type of surgery, however, is especially painful, and patients usually have to undergo it twice--once to extract the marrow and then again to replace it--because the culturing time takes many hours to complete. The second method is called the in vivo technique and requires no surgery or even anesthesia. In this process, the therapeutic DNA is injected directly into the body cells, usually via one of two types of viruses. o The Perfect Retrovirus The most frequently used type is the very simple retrovirus. Dr. Richard Mulligan of MIT has synthetically created the perfect retrovirus: it has no reproduction sequence and exists solely to deliver therapeutic DNA during gene therapy. It has no viral DNA (DNA that would make the cell--and you-- sick) whatsoever and only carries the new DNA that has been spliced into it. After injecting the diseased cell with the new therapeutic DNA, it then dies. Using retroviruses is very safe and provides long-lasting effects. Unfortunately, the new DNA it injects will only help the new daughter cells and not those that already exist.
o Adenovirus The second type of virus used for the in vivo technique is called an adenovirus, the equivalent of the common cold virus. Although this virus will also die after injecting its spliced therapeutic DNA, it will be attacked by the immune system and the patient will suffer from a temporary sore throat and runny nose. The adenovirus works the same way the retrovirus does, but its effects are much more immediate--within 48 hours.
Unlike the retrovirus, though, the new DNA’s effects wear off within weeks. Scientists like the fact that only a few millimeters of altered adenovirus solution is needed to cure the patient, whereas several liters of retrovirus are needed to obtain a much slower result.
o 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 with near 100% certainty. As now known, Gene Therapy has a very much important step known as Gene Splicing..
So what is Gene Splicing? Gene Splicing
Gene splicing is just what it sounds like: cutting the DNA of a gene to add base pairs. Contrary to the immediate image, however, no sharp instruments are involved; rather, everything is done chemically.
Chemicals called restriction enzymes act as the scissors to cut the DNA. Thousands of varieties of restriction enzymes exist, each recognizing only a single nucleotide sequence. Once it finds that sequence in a strand of DNA, it attacks it and splits the base pairs apart, leaving single helix strands at the end of two double helixes. Scientists are then free to add any genetic sequences they wish into the broken chain and, afterwards, the chain is repaired (as a longer chain with the added DNA) with another enzyme called ligase
Non-viral methods
There are other gene therapy techniques, although they aren’t as frequently used. 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. One more method involves inserting therapeutic DNA into cultured endothelium tissue (endothelium is the membrane that lines all of the blood vessels) and then grafting it into the patient.
Another technique requires the patient to receive an electric shock while submerged in a bath of a therapeutic DNA solution. The shock opens the skin pores, allowing the DNA to enter. Still other options include skin grafts, connective tissue grafts, and injecting the liver with the therapeutic DNA. Oligonucleotides The use of synthetic oligonucleotides in gene therapy is to inactivate the genes involved in the disease process. There are several methods by which this is achieved. One strategy uses antisense specific to the target gene to disrupt the transcription of the faulty gene. Another uses small molecules of RNA called siRNA to signal the cell to cleave specific unique sequences in the mRNA transcript of the faulty gene, disrupting translation of the faulty mRNA, and therefore expression of the gene. A further strategy uses double stranded oligodeoxynucleotides as a decoy for the transcription factors that are required to activate the transcription of the target gene. The transcription factors bind to the decoys instead of the promoter of the faulty gene, which reduces the transcription of the target gene, lowering expression
Gene Therapy-It Works!
A Background to some important developments and breakthrough research work o
18 May 2007
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Researchers at London’s Moorfields Eye Hospital have made the world’s first attempt at using gene therapy to treat a visual disorder. The team operated on Robert Johnson, who lives in the UK and has a sight disorder that deteriorates with age. Johnson’s disorder is due to a defect in a gene called RPE65, which stops the photoreceptor cells in the retina at the back of the eye from detecting light. In people with healthy vision, these cells usually detect light. However, as they are damaged in Johnson’s case, he is unable to see properly. The gene therapy administered involved the injecting of normal copies of the defective gene into the back of one of Johnson’s eye. The normal gene is injected between two layers of cells that form the retina. If successful, the normal genes will restore the cells in the pigment layer and help the photoreceptor cells in the retina detect light again. The cells can then send nerve impulses to the optic nerve and eventually to the brain and improve Johnson’s sight. After the operation, Johnson can currently see outlines during the day but little at night.
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(October 2006).
Scientists at the National Institutes of Health (Bethesda, Maryland) have successfully treated metastatic melanoma in two patients using killer T cells genetically retargeted to attack the cancer cells. This study constitutes the first demonstration that gene therapy can be effective in treating cancer. The study results have been published in Science
Researchers at the National Cancer Institute (NCI), part of the National Institutes of Health, successfully reengineer immune cells, called lymphocytes, to target and attack cancer cells in patients with advanced metastatic melanoma. This is the first time that gene therapy is used to successfully treat cancer in humans. See New Method of Gene Therapy Alters Immune Cells for Treatment of Advanced Melanoma (August 30, 2006).
o (March 2006) An international group of scientists announced the successful use of gene therapy to treat two adult patients for a disease affecting myeloid cells. The study, published in Nature Medicine, is believed to be the first to show that gene therapy can cure diseases of the myeloid system o (May 2006)
A team of scientists led by Dr. Luigi Naldini and Dr. Brian Brown from the San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET) in Milan, Italy reported a breakthrough for gene therapy in which they developed a way to prevent the immune system from rejecting a newly delivered gene. Similar to organ transplantation, gene therapy has been plagued by the problem of immune rejection. So far, delivery of the 'normal' gene has been difficult because the immune system does not recognize the new gene and rejects the cells carrying it. To overcome this problem, the HSR-TIGET group utilized a newly uncovered network of genes regulated by molecules known as microRNAs. Dr. Naldini's group reasoned that they could use this natural function of microRNA to selectively turn off the identity of their therapeutic gene in cells of the immune system and prevent the gene from being found and destroyed. This work will have important implications for the treatment of hemophilia and other genetic diseases by gene therapy [5].