The advent of recombinant DNA technology allowed researchers to create transgenic organisms. From this discovery the possibility of modifying the human genome to cure inherited disorders became theoretically possible. By transecting a host with a modified virus containing a copy of therapeutic DNA it is theoretically possible for the body of the host to produce the corresponding protein of that gene. However, this process is not without its issues and human trials have been shut down several times due to adverse reactions to treatment and even death. The promises of gene therapy are great but there still lies many unaddressed issues inhibit its efficiency.
Before I get into gene therapy and genetic engineering I feel it is essential to discuss the concept of recombinant DNA technology; the technology that makes gene therapy possible. The procedure is to extract and cut up DNA from a donor genome into fragments and insert the needed fragments (fragments containing desired genes) into autonomously replicating DNA molecules such as bacterial plasmids. The plasmids act as vectors, or carriers for the inserted gene. Both the vector DNA and the Donor DNA are digested with restriction enzyme (same enzyme for each). Restriction enzymes are endonucleases, enzymes that cleave the phosphodiester bond in within a polynucleotide chain. Digestion of the plasmids with the restriction enzyme converts the circular plasmid DNA into a linear molecule with sticky ends. Each DNA sequence is then mixed in a test tube to allow the sticky ends of vector and donor DNA to bind together and form what are called recombinant molecules. The spliced DNA recombinants can be sealed back into plasmids by the addition of the enzyme DNA ligase, which binds the sugar phosphate backbone of DNA back together. The recombinant plasmid DNA is then taken up by bacterial cells, and these cells in turn divide into millions of other bacterial cells creating many clones of that gene. These transgenic bacterial cells are then able produce the proteins that correspond to the cloned gene. (source)
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Gene therapy uses Recombinant DNA technology to replace a mutant allele with a functioning one within the human genome to correct a genetic disorder. This acts genotypically and theoretically allows us to permanently cure a disease through a single therapeutic application. When the gene is replaced with a functional one the patients body should theoretically be able to produce its corresponding protein, thereby correcting the disorder. The process of gene therapy can be divided into four steps.
First, the wildtype gene of interest is isolated and cloned. Second, the DNA for the desired functional gene is inserted into an appropriate vector. This is accomplished using restriction enzymes to produce a gene/vector hybrid. A vector is a DNA carrier that is used to deliver the gene to the patient's genome. Commonly used vectors are viruses that are modified to be harmless to humans. These vectors include modified retroviruses, adenoviruses etc. Other vectors have been used but the idea is to exploit the natural ability of a virus to seek out certain cells and insert their genes. One of the limitations of this method is that every current vector has at least some problems, and may be the reason for unsuccessful gene therapy trials (Marshall 2000a). The third step is a process called transfection where the gene/vector hybrid is inserted into the new host. This can be accomplished in two ways "In vivo" or "ex vivo." In vivo or "in the living tissue" involves injection of our vector hybrid directly into the patient's body where needed. Alternatively, ex vivo or "outside the living body" means the removal of human cells from the host, transfection of those cells and then reinsertion back into the host (Crystal 1995). This could be done on the host adult somatic cells and the results would only be temporary until the cell dies or it could be done to embryonic cells and the genes would be distributed throughout the entire body of the adult. If done on germ cells, the host would pass the gene on to subsequent generations. In the last step, the DNA must be integrated successfully by the host, transcribed and translated so that the host produces the new gene's product protein. This last step, however, involves issues with gene regulation, differentiation of cell types, and developmental stages. Because of this the inserted genes only expresses their proteins for a matter of weeks and then cease to function. (Human Gene Therapy)
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Initially hypothesized as gene surgery, gene thearapy was proposed in 1983 for the treatment of inherited diseases caused by defective genes. It had been suggested and hypothesized since 1970 when the first restriction endonucleases (restriction enzymes) were isolated and this led to the onset of recombinant DNA technology. This new found technology allowed genetic engineering and transgenic animals that, in turn, led to many medical advancements. However human genetic engineering, or what has now come to be known as gene therapy was not attempted till the 1990's. In 1990, the first human trial of gene therapy ever attempted was on a four-year-old girl. She was treated for severe combined immunodeficiency syndrome (SCID), a fatal inherited disease that that prevented her body from producing a crucial enzyme called adenosine deanimase (ADA). Researchers took the girl's white blood cells which were deficient in ADA and infused them with ADA producing genes. These modified white blood cells were then transferred back to the patient. The young girl continued to show increased stability but debate arose over whether this was the result of the gene therapy or the other drug treatments she had been receiving. She is alive today and is cured from the more adverse effects of her illness but requires periodic conventional treatment to stay healthy.(Human Gene THERAPY)
Gene therapy trials continued on several inherited diseases including Familial hypercholesterolemia (FH) and Cystic fibrosis (CF). Treatment of familial hypercholesterolemia, an autosomal dominant mutation that causes elevated blood cholesterol levels which leads to heart attacks at a very young age, promised some degree of success but variation in patient's responses to the treatment precluded continuation of trials. Research continued on Cystic fibrosis, though, a recessive mutation in the gene for cystic fibrosis transmembrane conductance regulator (CFTR). Defective proteins from this mutation result in the accumulation of mucus around the epithelial tissues of the lungs. This environment is ideal for infectious germs that lead to chronic lung disease an early death (Zabner et al. 1993). Cystic Fibrosis can only be treated symptomatically and is the most common fatal genetic disorder among Caucasians. This makes it one of the most researched human diseases in gene therapy. There have been over a dozen trials, using a variety of vectors, in an attempt to deliver a wildtype CFTR gene to respiratory tract tissues but there have been limited results. Research continues as they attempt to find a cure for this genetic disaster. (Human Gene THERAPY)
On September 17, 1999, for the first time, a patient died during a human gene therapy trial. An 18-year-old male volunteer, died while being treated for partial ornithine transcarbamylase deficiency, a condition caused by a missing or defective X-linked gene responsible for producing the liver enzyme ornithine transcarbamylase (OTC) (Marshall 2000a). A deficiency of this enzyme means the inability to break down nitrogenous compounds. This results in a toxic buildup of ammonia in the bloodstream and could be fatal. The young male however had a less severe case of the disease and was reasonably healthy before the trial. The OTC was administered in an adenovirus chimera to the livers of 18 volunteers through their hepatic arteries. The 18 -year- old died four days after transfection due to an immune response to the adenovirus vector, which caused multiple organ failure. In other words, the boy was killed by the vector and ultimately gene therapy. This revelation caused a huge setback for researchers and was a great impediment for the advancement of human genetic engineering as the FDA temporarily halted all research.
Sever Combine Immunodeficiency Syndrome comes in several forms. The first trials in of gene therapy in 1990 tried an autosomal disease. SCID- XIis, a more lethal form of SCID, is caused by an X-linked mutation in a receptor for cytokines, hormones that signal inflammatory response in cells. People who suffer from this disease have severe immune system failures that force them into quarantine as they cannot handle being exposed to even the slightest infection without it being fatal.(Marshall 2000a). In May 2000 two infant girls, aged 8 and 11 months, with SCID-X1, underwent treatment by gene therapy. They extracted each patient's bone marrow cells and incubated them with a Maloney- derived retrovirus vector carrying the cytokine receptor cDNA. After incubation was complete specialists inserted the modified cells back into the bone marrow. Ten months following this transfection, the two girls showed enough improvement in their immune systems that they could be taken out of protective isolation and moved home (Cavazzana-Calvo et al. 2000). This was the first known documentable success of gene therapy that cured a genetic condition. Unfortunately the two girls, although cured of the genetic disease, later developed leukemia like symptoms that caused the FDA to temporarily shut down all gene therapy trials in 2003. (source)
Recent Developments and unaddressed issues
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There have been countless developments in the applications of gene therapy and much success in transgenic mice, however human trials are said to still be years away. As discussed above a "perfect" carrier or vector has not yet been identified. But, scientists are beginning to look into options other than virus-mediated delivery systems. Recent findings show many non-viral options in gene delivery. The simplest non-viral delivery system is directly introducing new DNA into target cells. This simple method however is limited because it requires large amounts of DNA and can only be used with certain types of tissue. Another approach is to have a synthesized liposome, an artificial lipid sphere with an aqueous core, deliver the therapeutic DNA through the cell membrane of the target cell. Researchers are also experimenting with an artificial human chromosome. It is a 47th chromosome that would exist independently of the other 46.This would ensure that its presence would not disrupt their natural workings and would theoretically help us avoid negative auto-immune responses to the genetic alterations. The extra chromosome could store substantial amounts of genetic information but such large molecule would be difficult to deliver into the cell's nucleus. Recent findings show promise but are still relatively premature for human trials. (source)
A recent step toward the use of gene therapy for treatment of cancer also shows promise. Scientists have attempted to combine both nanotechnologies with gene therapy to create something called "tumor bursting" genes. Theses genes are wrapped in nanoparticles that are only absorbed by cancer cells. Once absorbed these genes produce proteins that kill the cancerous cells. It has only been tested on mice is predicted to be ready for human trials in several years. (BBC article) In 2008 Researchers announced successful results for the first clinical trials of gene therapy on a type of inherited blindness. The treatment has been approved as safe and represents a huge landmark for gene therapy in improving eyesight. Gene therapy shows more and more development everyday for infinitely many genetic conditions and diseases. (source)
Important Unaddressed Issues
There remain many problems that have not been properly addressed in gene therapy and by addressing them we could greatly improve efficiency of the technology. Researchers have not yet developed a method to properly integrate the therapeutic DNA into the genome and the genes are not passed onto offspring. This means that patients must undergo multiple gene therapy treatments. Many viruses are recognized by the immune system as invaders and by using a viral delivery system to transfect cells there is a high risk of triggering the immune system in a way that negates the effectiveness of the therapy. This also creates problems for subsequent treatments because it is even more difficult to administer the therapy when the vector virus has already been detected as an invader. Problems with viral vectors also include gene control, specific tissue targeting, and toxicity and inflammatory responses. Researchers also fear the theoretical possibility of the virus regaining its invasive ability to cause disease after transfection. Currently gene therapy is only a suitable theoretical solution for mutations in a single gene that causes disease. Unfortunately multifactor disorders (diseases cause by multiple genes) would be very difficult to treat using the methods in gene therapy.