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Gene Therapy

In research facilities all around the world scientist are attempting to stop diseases at their very roots. Instead of trying to find drugs to cure illnesses they are trying to change the genes that cause the diseases. The process by which this is done is called gene therapy. Gene therapy is the deliberate alteration of the human genome for alleviation of disease. The studies of gene therapy began in the mid 1980’s to early 1990’s. The focus then was “treating diseases caused by such single-gene defects as hemophilia, Duchene’s muscular dystrophy, and sickle-cell anemia.

As time passed new technologies, techniques, strategies and ideas for transferring genes have been presented. William French Anderson, Michael Blaise, and Ken Culver performed the first successful gene therapy on a human in 1990. They developed a protocol for treating Adenosine deaminase (ADA) deficiency, a severe combined immune deficiency, also known as the “Boy in the Bubble disease. ” ADA deficiency is a result of inheriting two copies of the defective ADA gene. Possession of a normal gene leads to the continuous, regular production of ADA in cells throughout the body.

Without at least one properly functioning gene, children have no way of converting deoxyadenosine (a waste product) into inosine. This leads to the rapid build-up of deoxyadenosine in the system, which becomes phosphorlyzed into a toxic triphosphate, which kills T-cells. The result is an almost complete failure of the immune system and early death. Previous treatment options included bone marrow transplants, which worked well with matched donors. A major breakthrough occurred with the development of polyethylene glycol coated ADA (PEG-ADA).

This treatment introduces coated ADA into the blood stream, although not into the cells. It requires expensive, painful shots on a weekly basis, but it succeeded in giving children with ADA deficiency a new lease on life. While their immune systems were far from normal, PEG-ADA allows some semblance of a normal life and a much-increased life span. The first patient to undergo federally approved gene therapy was a young girl named Ashanti DeSilva, in 1990. Ashanti’s success immediately sparked a torrent of gene therapy investment and research. There are two main types of gene therapy.

There is somatic gene therapy, which encompasses all of the cells of the body excluding sperm and egg cells. The second type is germline gene therapy. The difference between the two is that changes made in somatic gene therapy are not passed on to offspring, whereas in germline gene therapy the changes are passed onto the next generation. Not much research is being done in germline gene therapy because of technical and ethical reasons. The first step of gene therapy is to find the location of the problem gene or genes. DNA probes are used to find the problem DNA.

The technique relies upon the fact that complimentary pieces of DNA stick together. “5 The Human Genome Project is helping to piece together the location of all of the human genome. The U. S. Human Genome Project began officially in 1990 as a $3-billion, 15-year program to find the estimated 80,000 human genes and determine the sequence of the 3 billion DNA building blocks that underlie all of human biology and its diversity. The early phase of the Human Genome Project was characterized by efforts to create the biological, instrumentation, and computing resources necessary for efficient production-scale DNA sequencing.

The first 5-year plan was revised in 1993 due to remarkable technological progress, and the second plan projected goals through 1998. Observers have predicted that the 21st century will be the “biology century. ” The analytical power arising from the reference DNA sequences of several entire genomes and other genomic resources is anticipated to help jump-start the new millennium. The HGP’s continued emphasis is on obtaining a complete and highly accurate reference sequence (1 error in 10,000 bases) that is largely continuous across each human chromosome.

Scientists believe that knowing this sequence is critically important for understanding human biology and for applications to other fields. The next step to gene therapy is to find a vehicle to transport the new genes. This is done through the use of vectors. A vector is a DNA molecule into which a DNA fragment can be cloned and which can replicate in a suitable host organism. The majority of vectors used today are attenuated or modified viruses. “The modified viruses can not replicate in the patient but do retain the ability to efficiently deliver genetic material.

There are two main modes used to deliver the genes of interest to the patient: non-viral and viral delivery vectors have been used. Both of them have advantages, but also a not-so-short list of disadvantages. “Non-viral vectors represent basically the approach of direct injection of the genetic material (the DNA) into the tissue. This is very straight forward, methodically not very difficult and has proven good results in animal experiments for a transient (non-stable) expression of these genes. However, it is difficult to precisely locate the area of action and the efficiency is not very good.

To increase efficiency, the DNA is added to substances (e. g. polylysine) that allow the genes to cross cell membranes. But still, the expression is transient and injections have to be performed repeatedly to maintain a constant expression level. Viral vectors can be split in different groups: retroviral vectors, lentiviral vectors, and adenoviral vectors. A few more are under investigation. The principle of all these vectors is the packaging of the DNA into the viral capsule by replacing part (or all) of the viral genes.

By removing the crucial viral genes, the viruses also loose their pathogenic effect. After the manipulation of the viral genome, the virus will be used to deliver the genetic material by viral infection to the target cell. Retroviruses have a limitation because they are unable to infect non-dividing cells. This drawback was circumvented by the so-called ex-vivo gene therapy. In this procedure, cells are removed from the target tissue and grown in vitro. During this period the virus is added and the cells become infected. Subsequently, the cells are transplanted back to the target tissue.

Lentiviruses are a special type of retroviruses; the most prominent member is the human immunodeficiency virus (HIV). These viruses are able to infect non-dividing cells and can therefore be used for in vivo gene therapy. Results so far are promising, the system offers a targeted delivery and stable expression of the transgene (the genetic material that is delivered). Adenoviruses can infect both dividing and non-dividing cells. One major difference to the retroviruses is that the genetic material delivered by adenoviruses does not integrate into the host genome.

This resolves the problem of random integration at places that might be crucial for gene regulation or in the coding region of genes the target cells use as it is seen in retroviral vector systems. This is of some concern because it can lead to activation of oncogenes or the inactivation of tumor suppressor genes that ultimately leads to cancer. The disadvantage of the adenoviral vectors is the fact that the host is producing an immune response. This kills the infected cells and leads to the production of antibodies preventing a subsequent infection by the same virus.

But this system nevertheless is useful in situation where treatment for a short period of time is required, e. g. for fighting off cancer cells. ” Vectors like these can be found in places like the National Gene Vector Laboratories. There are currently three NGVL’s. They are located at the University of Michigan, University of Indiana, and University of Pennsylvania. The National Institute of Health funds the laboratories. The goal of the laboratories is to, “provide eligible investigators with clinical grade vectors for gene therapy applications.

The National Gene Vector laboratory at University of Michigan produces non-viral vectors for use in gene therapy phase 1 and 2 clinical trials. The National Gene Vector Laboratory at the Institute of Human Gene Therapy, University of Pennsylvania produces adenoviral vectors. The National Gene Vector Laboratory at Indiana University produces retroviral vectors, and is the coordinating center for the NGVL. Gene therapy solutions are currently being sought for diseases such as prostate cancer, cystic fibrosis, Parkinson’s disease and, retinitis pigmentosa. “Physicians perform gene therapy using an ex vivo or in vivo approach.

The most popular ex vivo way to deliver genes is through a retrovirus vector that targets rapidly dividing cells, such as cancer cells. Scientist start with a disarmed mouse-derived retrovirus, spiking it with therapeutic genes and injecting the modified virus into a patient. But ex vivo therapy is complicated and costly. Most researchers agree that as gene therapy evolves, in vivo use will emerge. Right now, the most publicized in vivo approach is the use of an adenovirus vector to shuttle a missing chloride ion-processing gene into the mucus-filled lungs of cystic fibrosis patients.

Although this method has yet to work, researchers say they are making significant progress. Producing effective viral vectors is tricky. They often generate poor gene expression, forcing researchers to administer repeat doses, which are expensive and can irritate tissue. ” 6 In 1989, the cystic fibrosis gene was isolated on chromosome 7. Current work focuses on the safe, efficient delivery of a functional cystic fibrosis transmembrane conductance gene to patients who are afflicted with cystic fibrosis and the expression of the gene in somatic cells to correct the genetic defect and restore chloride channel function.

It is hoped that successful gene therapy will be a feasible and cost-efficient approach that will lead to a cure. Following the cloning of the cystic fibrosis (CF) gene, in vitro studies rapidly established the feasibility of gene therapy for this disease. Unlike ex vivo approaches that have been utilized for other genetic diseases such as adenosine deaminase deficiency, gene therapy for CF will likely require direct in vivo delivery of gene transfer vectors to the airways of patients with CF.

Hence, major research efforts have been directed at the development of efficient gene transfer vectors that are safe for use in human subjects. Several vectors have now emerged from the laboratory for evaluation in clinical safety and efficacy trials in the United States and in the United Kingdom. Adenovirus-mediated gene transfer has been utilized for initial clinical safety and efficacy trials in the United States, while liposome-mediated gene transfer has been chosen for initial clinical safety and efficacy trials in the United Kingdom.

The rationale and laboratory studies are reviewed leading to initial clinical safety and efficacy trials. Also reviewed are the currently available vectors for potential use in clinical studies, their advantages and disadvantages, and the promises and pitfalls of current gene therapy efforts for CF in the United States focusing on adenovirus vectors in current clinical trials. Although the incidence and mortality rates of prostate cancer have decreased slightly in the past few years, prostate cancer remains a major health threat to American men.

This disease remains the most commonly diagnosed internal malignancy in men and the second leading cause of cancer death among men in the United States. Biologically, prostate cancer represents a heterogeneous disease entity that exhibits varying degrees of aggressiveness, patterns of metastasis, and response to therapy. Universal agreement has not been reached as to the best treatment for prostate cancer at any stage. Radical prostatectomy, external-beam radiation therapy, brachytherapy, and cryotherapy can affect local tumor control and are potentially curative in patients with clinically localized disease.

In spite of the widespread use of prostate-specific antigen (PSA) in early detection and screening, many cases are not diagnosed until the disease has advanced or metastasized beyond the reach of these local treatment modalities. Hormonal therapy and chemotherapy are the only systemic treatments available at the present time. Unfortunately, progressive disease develops in many patients who undergo these treatments, thus proving them to be noncurative. Gene therapy has emerged as an exciting new treatment that may affect both local and systemic control of prostate cancer.

With respect to cancer, the goal of gene therapy is to prevent or treat disease by using the therapeutic information encoded in the DNA sequences. Evidence suggests that tumor formation is caused by the overexpression of oncogenes or by mutations in suppressor genes in the presence or absence of cancer-causing environmental events. The future of gene therapy approaches to prostate cancer or other cancers will depend on further development of vector systems at the basic science level as well as a better understanding of the genes involved in tumor induction and proliferation.

Parkinson’s disease is a chronic degenerative neurologic condition affecting control of voluntary movement. The disease is most commonly treated with pharmaceutical products containing L-dopa and then in more advanced cases, with neurosurgical procedures. Cell Genesys, Inc. announced that company scientists have demonstrated long term production of L-dopa, a drug currently used to treat Parkinson’s disease, following just a single gene therapy treatment in Parkinson’s disease animal models.

An adeno-associated viral (AAV) gene delivery system was used to deliver human genes responsible for the production of L-dopa to the appropriate region of the brain, resulting in stable production of the enzyme responsible for L-dopa synthesis for one year, the duration of the study. “The potential for gene therapy to provide long term correction of Parkinson’s disease after just a single injection may represent a new approach to treating this disease. We have used AAV vectors to deliver genes to several types of tissue including brain, muscle, liver and cardiovascular tissues, and have observed long term gene expression in each.

This broad applicability allows for a wide variety of potential disease targets including hemophilia, cardiovascular disorders and neurologic disorders. “12 Cell Genesys scientists utilized AAV vectors to deliver two human genes to the specific area of the brain affected by Parkinson’s disease in Parkinson’s disease rat models. Following gene transfer, the chemical synthesis of L-dopa was demonstrated and the expression of the L-dopa producing enzyme was stable for one year, the duration of the study. There were no observable toxicities after treatment, and importantly, there were no other regions of the brain affected by the gene delivery.

The Fred Hutchinson Cancer Research Center and Cornell University reported that a genetic defect is responsible for progressive rod-cone degeneration (PRCD) in dogs. PRCD is known to cause blindness in at least four breeds of dogs: Portuguese water dogs, Labrador retrievers, poodles, and cocker spaniels. It is possible that many other breeds may also be affected. If so, PRCD would be caused by a relatively old mutation in the evolutionary history of dogs. PRCD starts with the deterioration of the retina and results in blindness. PRCD seems to be the dog version of retinitis pigmentosa.

RP17 appears to be the closest human equivalent. Prior to this study, the general scientific consensus was that a link between the two was unlikely. Since the study, scientists now believe that both diseases come from similar mutations to the underlying gene. Scientists hope that by identifying the gene responsible for PRCD, a diagnostic test could be developed for the disease in dogs and, eventually, in humans. Further treatment options could be developed using gene therapy techniques. Scientists would insert the normal gene into a vector.

The vector would then be injected into the organism in the hope that the normal gene would slow the genesis of the disease or even reverse its effects in the diseased cells. New gene therapy techniques are discovered everyday. Doctors at the University of Pennsylvania and ARIAD Pharmaceuticals have discovered a “molecular rheostat. “4 Researchers stripped two AAV’s of their viral genes and reloaded them with the gene for erythroprotein(Epo). Epo is a gene that stimulates red blood cell production and is used to treat anemia. It had been previously showed that Epo could be “switched on” by the drug rapamycin.

In mice and monkeys, rapamycin stimulated a rise in production of Epo and red blood cells. Conversely, when they stopped administering the drug Epo production was shut down. Epo was chosen because it action can easily be measured red blood cell counts. “The ability to achieve dosage control of a drug in the context of gene therapy is going to be critical to making a number of new treatments possibleMany of the proteins we would like to produce in the body using gene therapy are quite potent and can have side effects so being able to carefully adjust their levels within a therapeutic window is going to be key.

With the ever-increasing knowledge on gene therapy, many ethical and moral concerns have risen. Society as a whole shows both concern and apprehension on this contemporary issue. Many argue that the morality of gene therapy is misguided, mainly because of the effort private companies and the NIH are doing to have certain DNA sequences patented. With the research going into decoding the human genome, there is an unusual demand for the patenting of useless DNA sequences, undermining the sole purpose of patents.

Should DNA sequences should be patented at all or be treated as merchandise? Patenting any kind of DNA sequences is just like patenting life itself, and from history we know that the ownership of any human being is wrong. DNA or deoxyribonucleic acid sequences in an individual can thus be considered analogous to a piece of property. Another question about gene therapy involves every person’s right to true genetic inheritance instead of artificially manipulated ones. To many radicals, germ-line therapy serves essentially as an open door for another Hitler wanting a superhuman race.

Probably one of the largest debates over gene therapy has to deal with the use of fetal tissue to treat diseases such as Parkinson’s disease. Today, over 1. 5 million Americans suffer from Parkinson’s disease, which impairing speech, crippling gait, and eventually immobilizing the entire body, all because of the degeneration of a tiny portion in the brain called the substantia nigra. Some scientists are now using electively aborted six- to eight-week old fetal tissue obtained from abortion clinics in order to insert new brain tissue into the dying region of the brain.

Obviously, ethical concerns have been raised while inviting more controversy such as the abortion issue. Such therapy, however, has improved the well being of the patients. There are many other concerns dealing with gene therapy. For instance, once gene therapy has taken effect it cannot be stopped or in other words, it is irreversible. Unlike drugs, you can’t stop producing genes. In addition, the mixed results in delivering genes have raised some concern.

Some scientists fear, with retroviruses and their ability to introduce new genes in between other genes in the body, that the turning on of oncogenes will occur, causing cancer. In addition, retroviruses consist of RNA and only affect dividing cells; still, they may become dangerous if combined with other versions or helper viruses. Finally, although retroviruses are disabled from reproducing, they still posses potential problems because when they insert a new gene between an existing gene, they inevitably knock out the structural code for a protein.

Adenoviruses, like those associated with the common cold, are aimed for treatment of cystic fibrosis; yet, they may inflame “DNA containing mucus,” causing pneumonia, and even go through the rest of the body and into sex cells. 14 Furthermore, the lungs may not produce enough of the needed protein to correct cystic fibrosis and if the scientists try repeating the process, there might even be an immune response, making matters worse.

There is enormous money making potential in the future of gene therapy. It is estimated that by the year 2000 there will be at least 10 cancer gene therapies and that the gene therapy market will top $2 billion. “Over the past five years, estimated Michael Murphy, editor of the California Technology Stock Letter, companies have spent about $250 million on gene therapy research and development. When a product gets approved, he says the payoff in the annual sales will probably be ten times that. “6

With the current technological advances and world wide research efforts, the goal of eliminating all human disease through gene therapy may one day be possible. Worldwide about 200 clinical trials are currently carried out. Looking at the tremendous potential this technique offers, and considering the promising results obtained in animal experiments, chances are good that gene therapy will become standard practice soon. In summary, gene therapy far surpasses the power of vaccines and drugs, since it provides a means of eliminating, instead of alleviating, diseases or ailments.

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