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Gene Therapy Gene therapy is a technique for correcting defective genes responsible for disease development. There are 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.

Principle involved in Gene Therapy. 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 viral 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.. Some of the different types of viruses used as gene therapy vectors:


 * Retroviruses - A class of viruses that can create double-stranded DNA copies of their RNA genomes. These copies of its genome can be integrated into the chromosomes of host cells. Human immunodeficiency virus (HIV) is a retrovirus.

·Adenoviruses - A class of viruses with double-stranded DNA genomes that cause respiratory, intestinal, and eye infections in humans. The virus that causes the common cold is an adenovirus. ·adeno-associated viruses - A class of small, single-stranded DNA viruses that can insert their genetic material at a specific site on chromosome 19. ·Herpes simplex viruses - A class of double-stranded DNA viruses that infect a particular cell type, neurons. Herpes simplex virus type 1 is a common human pathogen that causes cold sores.

Some of the problems that remain to be solved include:

·how to avoid an immune response in the patient, which can interfere with gene therapy in two ways:
 * the vector provokes inflammation(a problem with adenovirus vectors)
 * the vector elicits antibodies that destroy the vector when it is administered again

·how to get the gene into non-dividing cells like liver, muscle, and neurons; ·how to get the gene integrated into the DNA of the host cell so that it will be replicated (in dividing cells) and expressed indefinitely but ·minimize the risk that it inserts near a proto-oncogene which it could activate producing a cancer. (This occurred in three little boys treated with a retroviral vector based on the murine leukemia virus. ·how to get the gene to be expressed as needed; that is, how to bring the gene under normal physiological controls so that its product is produced when and in the amounts needed.

Strategey in Action In the 1 January 1999 issue of Science, James M. Wilson and his colleagues reported the results of using this strategy in both mice and rhesus monkeys. They injected their experimental animals with two vectors.

Vector 1.. This piece of DNA contained (among other things): ·the DNA of adeno-associated virus (AAV) ·a gene encoding a protein containing two domains: oa portion of the molecule ("p65") that is needed to activate gene transcription but that by itself cannot bind to DNA oa portion ("FRB") that binds the drug rapamycin. ·a gene encoding another protein with two domains: oa portion of molecule ("ZFHD1") that binds specifically to the DNA sequence in the promoter of the erythropoietin gene but that by itself cannot activate transcription of the gene; oa portion ("FKBP12") that also binds to rapamycin. ·Promoters (not shown) that allow continuous expression (transcription and translation) of the two genes. But note that, by themselves, the two gene products are inactive..

Vector 2. This piece of DNA contained (among other things): ·the DNA of adeno-associated virus (AAV); ·12 identical promoters (green boxes) of the erythropoietin gene; ·the structural gene for erythropoietin (EPO) itself.

The Experiment..

The experimental animals were injected (in their skeletal muscles) with many copies of both vectors. Skeletal muscle was chosen because muscle fibers are multinucleate. Once across the plasma membrane, there are many nuclei which the vectors can enter and hence many opportunities to integrate into the DNA of the host. Later the animals were injected with rapamycin. This small molecule is an immunosuppressant and is currently being tested in transplant recipients to help them avoid rejection of the transplant. It was used here because of its ability to simultaneously bind to the FRB and FKBP12 domains of the two gene products of vector 1. The resulting trimer is an active transcription factor for the erythropoietin gene.

The Results.

In mice.. ·injections of the two vectors had — by themselves — no effect on the production of EPO nor on the number of red blood cells (hematocrit), but ·every time these animals were given an injection of rapamycin, they oquickly began to produce EPO (with levels increasing as much as 100 fold) and the number of red blood cells rose (hematocrits increasing from 42% to 60%). ·The amount of EPO produced was directly related to the amount of rapamycin given. ·Even after 5 months, a single injection of rapamycin produced a sharp rise in the level of EPO in the blood.

Factors affecting for an effective Gene Therapy for Curing genetic disease..

·Short-lived nature of gene therapy - Before gene therapy can become a permanent cure for any condition, the therapeutic DNA introduced into target cells must remain functional and the cells containing the therapeutic DNA must be long-lived and stable. Problems with integrating therapeutic DNA into the genome and the rapidly dividing nature of many cells prevent gene therapy from achieving any long-term benefits. Patients will have to undergo multiple rounds of gene therapy. ·Immune response - Anytime a foreign object is introduced into human tissues, the immune system is designed to attack the invader. The risk of stimulating the immune system in a way that reduces gene therapy effectiveness is always a potential risk. Furthermore, the immune system's enhanced response to invaders it has seen before makes it difficult for gene therapy to be repeated in patients. ·Problems with viral vectors - Viruses, while the carrier of choice in most gene therapy studies, present a variety of potential problems to the patient --toxicity, immune and inflammatory responses, and gene control and targeting issues. In addition, there is always the fear that the viral vector, once inside the patient, may recover its ability to cause disease. ·Multigene disorders - Conditions or disorders that arise from mutations in a single gene are the best candidates for gene therapy. Unfortunately, some the most commonly occurring disorders, such as heart disease, high blood pressure, Alzheimer's disease, arthritis, and diabetes, are caused by the combined effects of variations in many genes. Multigene or multifactorial disorders such as these would be especially difficult to treat effectively using gene therapy.

Recent developments in Gene Therapy Research... ·University of California, Los Angeles, research team gets genes into the brain using liposomes coated in a polymer call polyethylene glycol (PEG). The transfer of genes into the brain is a significant achievement because viral vectors are too big to get across the "blood-brain barrier." This method has potential for treating Parkinson's disease. ·RNA interference or gene silencing may be a new way to treat Huntington's. Short pieces of double-stranded RNA (short, interfering RNAs or siRNAs) are used by cells to degrade RNA of a particular sequence. If a siRNA is designed to match the RNA copied from a faulty gene, then the abnormal protein product of that gene will not be produced. ·RNA interference or gene silencing may be a new way to treat Huntington's. Short pieces of double-stranded RNA (short, interfering RNAs or siRNAs) are used by cells to degrade RNA of a particular sequence. If a siRNA is designed to match the RNA copied from a faulty gene, then the abnormal protein product of that gene will not be produced. ·Gene therapy for treating children with X-SCID (sever combined immunodeficiency) or the "bubble boy" disease is stopped in France when the treatment causes leukemia in one of the patients. ·Researchers at Case Western Reserve University and Copernicus Therapeutics are able to create tiny liposomes 25 nanometers across that can carry therapeutic DNA through pores in the nuclear membrane. ·Sickle cell is successfully treated in mice.

In monkeys..

The results were similar to those in mice, but the effect wore off after 4 months. So here is a system where a gene introduced into an animal can then be ·switched on by giving the animal a small molecule. (In humans, rapamycin can be given by mouth as a pill.) ·can have its output regulated by the amount of the small molecule administered..

Curing Insulin-Dependent Diabetes Mellitus (IDDM) in mice and rats..... Researchers in Seoul, Korea reported in the 23 November 2000 issue of Nature that they have used an AAV-type vector to cure ·mice with inherited IDDM ·rats with IDDM induced by chemical destruction of their insulin-secreting beta cells.

Both groups of animals were injected (in their hepatic portal vein) with billions of copies of a complex vector containing:

·AAV ·the complementary DNA (cDNA) encoding a synthetic version of insulin ·a promoter that is active only in liver cells and is turned on by the presence of glucose ·the DNA encoding a signal sequence so that the insulin can be secreted. ·an enhancer to elevate expression of this artificial gene.

The results:

Both groups of animals gained control over their blood sugar level and kept this control for over 8 months. When given glucose, they proceeded to synthesize the synthetic insulin which then brought their blood glucose back down to normal levels.

Curing hemophilia B in mice....

Researchers at the Salk Institute reported (in the 30 March 1999 issue of the Proceedings of the National Academy of Sciences) work with mice.. ·whose genes for clotting factor IX had been "knocked out and ·thus were subject to uncontrolled bleeding like human patients with hemophilia B These mice were injected (also in the hepatic portal vein) with DNA containing ·AAV ·cDNA for factor IX (the dog gene) ·liver-specific promoter and enhancer sequences

The mice proceeded to make factor IX and were no longer susceptible to uncontrolled bleeding. More recently (2005), injection of embryonic stem cells with functioning factor IX genes into the liver of mice without the genes cured them.

Ethical aspects of gene therapy.. Gene therapy consists of a wilful modification of the genetic material in cells of a patient in order to bring about a therapeutic effect. This modification usually occurs by introducing exogenous DNA using viral vectors or other means. Although gene therapy is still in its infancy as a clinically useful therapeutic modality, a discussion of the ethical issues is useful in several respects because it involves ethical principles of broad applicability in clinical medicine. Furthermore, many current applications of genetic engineering in medicine (DNA vaccines, therapeutic use of encapsulated genetically modified cells) are conceptually close to gene therapy, so that the border between gene therapy in the narrow sense and other gene-based therapies is getting fuzzier as time goes by. Two conceptual distinctions are central to an understanding of the ethical issues of gene therapy:

1 - Therapy vs. enhancement. There is a consensus that gene therapy should be therapy, i.e. the correction of bona fide disease conditions, rather than enhancement, which would mean "improving the human species" (whatever that means...) and therefore would entail the introduction in human subjects of novel characteristics going beyond the usual, medical, understanding of health (i.e. health as absence of serious disease). 2 - Somatic vs. germ line gene therapy. All current research on humans deals with somatic gene therapy. In these projects somatic cells such as bone-marrow, liver, lung or vascular epithelium etc. are genetically modified. Since the germ line is not affected, all effects of therapy end with the life of the patient, at the very latest. In fact, most somatic therapies will probably require repeated applications, much like ordinary pharmacological treatments.

Initially, gene therapy was conceptualised mainly as a procedure to correct recessive monogenic defects by bringing a healthy copy of the deficient gene in the relevant cells. In fact, somatic gene therapy has a much broader potential if one thinks of it as a sophisticated means of bringing a therapeutic gene product to the right place in the body. The field has moved increasingly from a "gene correction" model to a "DNA as drug" model (ADN médicament, A. Kahn). This evolution towards an understanding of gene therapy as "DNA-based chemotherapy" underscores why the ethical considerations for somatic gene therapy are not basically different from the well-known ethical principles that apply in trials of any new experimental therapy..

·Favourable risk-benefit balance (principle of beneficence/non-maleficence); ·Informed consent (principle of respect for persons); ·Fairness in selecting research subjects (principle of justice).

Clearly, the mere fact that gene therapy has to do with genes and the genome does not, in itself, make it "special" or "suspicious". A further distinction ought to be made between in vivo and ex vivo somatic gene therapy. Ex vivo procedures entail the extraction of cells from the patient's body (for instance bone-marrow cells), genetic modification of the cells using appropriate vectors or other DNA-transfer methods and reimplantation of the cells in the patient. In vivo therapy uses a vector or DNA-transfer technique that can be applied directly to the patient. This is the case of current experiments aimed at correcting the gene defect of cystic fibrosis by exposing lung epithelium to adenovirus-derived vectors containing the CFTR gene. In the in vivo case, the potential for unintended dissemination of the vector is more of an issue. Therefore, biological safety considerations must also be subjected to ethical scrutiny in addition to the patient-regarding concerns already mentioned. In germ line therapy, the DNA of germ cells would be affected, the objective being to correct a genetic defect once and for all, in all descendants of the therapy recipient who will inherit the modified allele. Although germ line therapy is far more speculative than somatic gene therapy at this time, it is widely discussed because it raises important and difficult ethical questions that have relevance for other medical practices as well. The consensus against germ line therapy is broad, but not unanimous. The ethical debate on germ line therapy has usually revolved around two kinds of issues:

1 - Germ line therapy is "open-ended" therapy. Its effects extend indefinitely into the future. This basically fits the objective of germ line therapy (assuming that it becomes possible one day), namely to correct a genetic defect once and for all. But precisely there lies also an ethical problem: an experiment in germ line therapy would be tantamount to a clinical experiment on unconsenting subjects, which are the affected members of future generations. This raises a number of very complex questions and is, in my view, an important but not necessarily overriding argument. A recent symposium on germ line engineering has concluded with a cautious "yes-maybe" for germ line gene therapy.

2 - Germ line therapy may involve invasive experimentation on human embryos. Although there are other potential targets for germ-line interventions, much of the discussion revolves around the genetic modification of early embryos, where the germ line has not yet segregated from the precursors of the various somatic cell types. As a result, the ethical assessment of germ line gene therapy will hinge in part on the ethical standing accorded to the early human embryo and the moral (dis)approval of early embryo experimentation. Those who believe the early embryo to be the bearer of considerable intrinsic moral worth or even that it is "like" a human person in a morally-relevant sense will conclude that embryo experimentation is to be rejected and germ-line therapy as well. Others think that it is only later in development that humans acquire those features that make them ethically and legally protected human subjects to the fullest degree. For them, the use of early embryos is not objectionable and germ line therapy cannot be ruled out on these grounds alone. As might be expected in view of the moral pluralism of modern societies, the policies of European countries differ in this respect: some permit some invasive research on human embryos (UK, Spain, Denmark), others ban it (Germany, Norway), others are still undecided. More generally, embryo-centred controversies are expected to increase as the field of embryonic stem-cell research becomes ever more promising. It is expected that this field will catch much of the public attention that was devoted to gene therapy in the nineties. Clearly, the question of the ethical standing of the human embryo is also of major importance for other medical procedures in reproductive medicine such as in-vitro fertilisation, pre-implantation diagnosis, experimentation on human embryos in general and abortion. To go back to gene therapy, or rather to the therapeutic innovations due to genetic engineering such as DNA vaccines: some of these could potentially benefit a great number of people world-wide, contrary to early developments of genetic engineering in medicine, which where largely geared towards the health problems of rich countries. Although the course of biomedical progress is often unpredictable, the setting of research priorities does raise troubling issues of social ethics.