2. Gene therapy definition: is the insertion of genes into an individual's cell and biological tissues to treat disease, such as cancer where deleterious mutant alleles are replaced with functional ones. wikipedia

3. It is now possible to treat genetic disorders by the addition of functional copies of the gene responsible for the disorder. This is, at present, limited to treating somatic cells. (any cells forming the body of an organism)cells The major problem in gene therapies is finding the optimal vector to transfer the therapeutic gene into the recipient's cells.

4. This limits their use to ex vivo application. Lentiviruses may prove a more useful alternative. Adenoviruses can also be used but their DNA does not integrate into the target cell's chromosomes and their effect is transient. They also may induce immune response in the patient. An alternative to the use of viral vectors involves encapsulating DNA in liposomes. This causes no side effects, but is much less efficient in transferring DNA to target cells. ex vivo means that which takes place outside an organism. In science, ex vivo refers to experimentation or measurements done in or on tissue in an artificial environment outside the organism with the minimum alteration of natural conditions

5. The major technical advances in molecular genetics made in the past 20 years have enabled us to isolate, clone and sequence genes from all species including humans. This has raised the question of whether it would be possible to use genetic methods to cure such individuals. This would require the necessary gene to be isolated and targeted into the cells of the affected individual- gene therapy.

6. In the case of inherited syndromes there is a choice of approaches: a normal copy of a gene can be introduced into either the # germ line # somatic cells of the affected individual. germline of a mature or developing individual is the line (sequence) of germ cells that have genetic material that may be passed to a child.developinggerm cells For example, sex cells such as the sperm or the egg, are part of the germlinesex cellsspermegg

7. in proposed gene therapies for cancer it is often the aim to introduce genes into tumor cells that will induce cell death. ex the major obstacle to the development of a gene therapy lies in finding suitable vectors to introduce genes into target cells efficiently while not causing serious side- effects. funnily enough the most promising candidate vectors for gene therapy are derived from viruses that cause disease in humans Cancer:

8. Gene therapy can be carried out by introducing genes into the patient and hoping they will find an appropriate target cell - in vivo therapy. This is useless process, and in some cases it is possible to remove cells from the patient, manipulate them in cell culture, and then return them to the patient. This is known as ex vivo therapy.

9. It is important to realize that targets for therapeutic genes will differ. In certain cases the gene must be delivered to a specific cell type, ex: replacement of globin genes in inherited hemoglobinopathies in which the gene must be delivered to erythroid cells. Cell that will give rise to erythrocytes

10. Ex: In other cases it is only necessary to increase the level of a specific protein, for example a hormone, in the body. Here any cell type that will allow the product to be secreted into the blood system will be a suitable target.

11. Before a virus can be used as a vector to carry therapeutic genes those genes necessary for viral functions must be removed. This prevents spread of the virus within the body, and reduces the response of the patient's immune system. Viruses deleted in this way are referred to as gutted viruses. The desired gene is inserted into the remaining virus genome along with DNA sequences to promote and regulate its transcription, and this recombinant DNA molecule is packaged into the appropriate viral coat so that it can infect human cells. Several different virus vectors can be used.

12. Retroviruses are easily adapted to carry a human gene insert. These have the advantage that they will infect most, if not all, cell types and are efficient at integrating their genome into the host cell. 1- Their main disadvantage is that they will only infect actively dividing cells. For this reason their use is largely limited to ex vivo use with cells grown in culture. 2- Another problem is that the inserted gene is often only expressed for a short period, usually less than 2 weeks.

13. Non viral options for gene delivery 1. Direct introduction of therapeutic cDNA into target cells. This approach can be used only with certain tissues and requires large amount of DNA. 2. Artificial liposome which carry therapeutic cDNA. It is capable of passing the DNA through the target cells membrane Retno Dwi Wulandari 10 Pebruari 2006

14. Non viral options continued......... 3. Chemically linking therapeutic cDNA to a molecule that will bind to special cell receptors. Once bound to these receptors the cDNA construct are engulfed by the cell membrane and passed into the interior of the target cell. This methode is not very effective 4. Introducing 47th artificial human chromosome into target cells. This chromosome would exist autonomously alongside the standard.However, a method for inserting the chromosome into the cell, or the nucleus is yet to be found Retno Dwi Wulandari 10 Pebruari 2006

15. What diseases could be treated with gene therapy? About 4000 diseases have been traced to gene disorders. Current and possible candidates for gene therapy include cancer, AIDS, cystic fibrosis, Parkinsons, Alzheimer’s disease, cardiovascular disease and arthritis. (AMA gene therapy) Retno Dwi Wulandari 10 Pebruari 2006

16. Has gene therapy been successfullyused in humans? Gene therapy is likely to be most successful with diseases caused by single gene defects. The 1st successful gene therapy on humans was performed in 1990 by researchers at the National Institute of Health for treatment patient with adenosine deaminase (ADA) deficiency, a rare genetic disease. Patients with this condition do not have normal ADA genes and do not produce ADA enzyme, causes severe immunodeficiency and prone to repeated serious infections. Retno Dwi Wulandari 10 Pebruari 2006

17. In 2003, more than 600 gene therapy clinical trials were under way in the US but only a handful of these are in advanced stages. (AMA gene therapy) Retno Dwi Wulandari 10 Pebruari 2006

18. What factors have kept gene therapy from becoming an effective treatment for genetic disease ? Short-lived nature of gene therapy. Immune response Problem with viral vector : viruses may present a variety of potential problems to the patient – toxicity, immune and inflammatory response and also cause disease Multigene disorder : multigene or multifactorial disorders would be difficult to treat effectively using gene therapy

19. Cystic fibrosis, is the result of recessive mutation of a single gene, however the frequency of mutant alleles of this gene is much higher, particularly in Caucasian populations. The defect is in a gene responsible for the transport of chloride ions across the cell membrane. Lack of this function has major effects on the cells that line the lungs and gut. The gene therapy approach was initially similar to that for ADA in that the gene was cloned and engineered into a vector. Europe, North Africa, the Horn of Africa, West Asia, Central Asia, and South Asia.

20. The majority of clinical trials involving gene therapy have been directed at tumor cells. Given that tumors arise as a result of gene mutation it is theoretically possible to prevent further growth of tumor cells by replacing defective tumor suppressor genes. Another approach is to use exogenous genes either to induce death in tumor cells or to render them more sensitive to the normal defence mechanisms of the body.

21. One potentially useful approach to specific killing of tumor cells is transfection with the thymidine kinase gene of the herpes simplex virus. This enzyme differs from its human equivalent, and can phosphorylate a wider range of substrates. One substrate that cannot be utilized by human thymidine kinase is gancyclovir. This compound is nontoxic to human cells but its phosphorylated derivatives are highly toxic. antiviral medication

22. Transfer of the herpes thymidine kinase gene to tumor cells thus make them specifically sensitive to gancyclovir treatment. There are two other advantages in this methodology: (i) thymidine kinase is active only in cells that are replicating DNA and hence nontumor cells are less likely to be affected; and (ii) the phosphorylated gancyclovir molecules, which are produced only in those tumor cells that have taken up the gene, are exported to surrounding tumor cells through gap junctions. The result is that the proportion of tumor cells killed by gancyclovir is greatly increased. This approach has been used with brain and ovarian tumors, and is set out schematic ally in

24. As yet no gene therapy approach has been successful in eradicating all tumor cells from a patient. It may be that gene therapy will never be totally successful on its own but will become an extra treatment regime to be used in conjunction with surgery and conventional drug and radiation therapies.