Contents ii. Introduction iii. History of gene therapy iv. Types of gene therapy y. Somatic gene therapy vi. Germ line gene therapy vii. Outcomes of gene therapy viii. Functional classification ix. Genetic disorders Targets for gene therapy Technologies used in gene therapy x. Gene Targeting xi. Success cases of gene therapy xii. Advantages & disadvantages xiii. Challenges xiv. Recent upcoming xv. Conclusion xvi. ReferencesABSTRACT Gene therapy is the introduction of genes into existing cells to prevent or cure a wide range of diseases. It is a technique for correcting defective genes responsible for disease development. The first approved gene therapy experiment occurred on September 14, 1990 in US when Ashanti DeSilva was treated for ADA-SCID. Gene therapy is designed to introduce genetic material into cells to compensate for abnormal genes or to make a beneficial protein. If a mutated gene causes a necessary protein to be faulty or missing, gene therapy may be able to introduce a normal copy of the gene to restore the function of the protein. A gene that is inserted directly into a cell usually does not function. Instead, a carrier called a vector is genetically engineered to deliver the gene. Certain viruses are often used as vectors because they can deliver the new gene by infecting the cell. The viruses are modified so they can’t cause disease when used in people. Some types of virus, such as retroviruses, integrate their genetic material (including the new gene) into a chromosome in the human cell. Other viruses, such as adenoviruses, introduce their DNA into the nucleus of the cell, but the DNA is not integrated into a chromosome,INTRODUCTION Gene therapy is an experimental technique that uses genes to treat or prevent disease. In the future, this technique may allow doctors to treat a disorder by inserting a gene into a patient's cells instead of using drugs or surgery. Researchers are testing several approaches to gene therapy, including: + Replacing a mutated gene that causes disease with a healthy copy of the gene, + Inactivating, or “knocking out,” a mutated gene that is functioning improperly. + Introducing a new gene into the body to help fight a disease. Although gene therapy is a promising treatment option for a number of diseases (including inherited disorders, some types of cancer, and certain viral infections), the technique remains risky and is still under study to make sure that it will be safe and effective, Gene therapy is currently being tested only for diseases that have no other cures. An experimental technique for correcting defective genes that are responsible for disease development. The most common form of gene therapy involves inserting a normal gene to replace an abnormal gene. It is intracellular delivery of genes to generate a therapeutic effect by correcting an existing abnormality.History and Development of Gene Therapy 1960 : The concepts of gene therapy was introduced. 1970: Friedmann and Roblin author of paper in science tittled “Gene therapy for human genetic disease?” cite the first attempt to perform gene therapy. 1990: The first approved gene therapy case at the National institute of Health, U.K. it was performed on four year old girl named Ashanti DaSilva. It was a treatment fora genetic defect that left her with an immune system deficiency. New gene therapy approach repairs errors in messenger RNA derived from defective gene.This technique has the potential to treat the blood disorder Thalassaemia, Cystic fibrosis, and some cancers. Sickle cell disease is successfully treated in mice. 1992: Doctor Claudio Bordignon working at the Vita-Salute San Raffaele University, Milan, Italy performed the first procedure of gene therapy using hematopoietic stem cells as vectors to deliver gene intended to correct hereditary diseases. 1999: Death of Jesse Gelsinger in a gene therapy experiment resulted in a significant setback to gene therapy research in the United States. 2006: Scientists at the National Institute of Health (Bethesda, Maryland) have successfully treated metastatic melanoma in two patients. This study constitutes one of the first demonstrations that gene therapy ean be effective in treating cancer. 2007-200: Research is still ongoing and the number of diseases that has been treated successfully by gene therapy increases. ¥ Retinal disase ¥ Colour blindness ¥ Adrenoleukodystrophy 20u: Medical community accepted that it can cure HIV as in 2008, Gero Hutter has cured a man from HIV using gene therapy.TYPES OF GENE THERAPY 1) Germ Line gene therapy 2) Somatic gene therapy Germ line gene therapy Therapeutic genes transferred into the germ cells. It is heritable and passed on to later generations. Result is permanent changes. Potential for offering a permanent therapeutic effect for all who inherit the target gene. Possibility of eliminating some diseases from a particular family. E.g. Genes introduced into eggs. and sperms. Somatic Cell Gene Therapy Therapeutic genes transferred into the somatic cells. Affects only the target cells in the patient, and is not passed to future generations. Short-lived because the cells of most tissues ultimately die and are replaced by new cells. Transporting the gene to the target cells or tissueis also problematic. E.g. Introduction of genes into bone marrow cells, blood cells, skin cells ete. Outcome of gene therapy > Replaces a mutated gene with a healthy one. Deactivates a gene that isn't functioning properly & Introduces a new gene in the body to help fight the disease > Enhances the effect of a normally functioning gene. > Activates the gene that was shut down during fetal life.Functional Classification Based on the purpose of gene therapy it can be - 1) Gene replacement therapy 2) Gene deactivation therapy 3) Transgenesis 4) Gene Enhancement therapy 5) Gene activation therapy Genetic Disorders A genetic disorder is an illness caused by one or more abnormalities in the genome, especially a condition that is present from birth (congenital). They are medical disorders related to gene mutation. Genetic disorders are heritable, and are passed down from the parents’ genes, Other defects may be caused by new mutations or changes to the DNA, In such cases, the defect will only be heritable if it occurs in the germ line. The same disease, such as some forms of cancer, may be caused by an inherited genetic condition in some people, by new mutations in other people, and by non-genetic causes in still other people. These diseases are totally random and difficult to prevent as they are not caused by external agents. Also as their root cause lies in the genome of the organism their cure was thought to be impossible until the breakthrough research unlocking the secrets of DNA leading to the development of biotechnology and hence gene therapy. Gene Therapy We can think of a medical condition or illness as a "broken window.” Many medical conditions result from flaws, or mutations, in one or more of a person's genes. Mutations cause the protein encoded by that gene to malfunction, When a protein malfunctions, cells that rely on that protein's function can'thehave normally, causing problems for whole tissues or organs. Medical conditions related to gene mutations are called genetic disorders,So, if a flawed gene caused our "broken window,” can we “fix” it? What are our options? 1. Stay silent: ignore the genetic disorder and nothing gets fixed. 2. Try to treat the disorder with drugs or other approaches: depending on the disorder, treatment may or may not be a good long-term solution. 3.Put in a normal, functioning copy of the gene: if you can do this, it may solve the problem! If it is suecessful, gene therapy provides a way to fix a problem at its source. Adding a corrected copy of the gene may help the affected cells, tissues and organs work properly. Gene therapy differs from traditional drug-based approaches, which may treat the problem, but which do not repair the underlying genetic flaw. Targets for Gene Therapy But now a question arises, which disorders or diseases can we target using gene therapy? Many disorders or medical conditions might be treated using gene therapy, but others may not be suitable for this approach. For a disease to be targeted by gene therapy it must satisfy the following conditions: 1. The condition must result from mutations in one or more genes 2. To treat a genetic flaw, the knowledge of which gene(s) to pursue is absolutely necessary. Also a DNA copy of that gene available in the laboratory. The best candidates for gene therapy are the so-called “single-gene” disorders - which are caused by mutations in only one gene. 3-To design the best possible approach, knowledge about how the gene factors into the disorder is required. For example: Which tissuesare affected? + What role does the protein encoded by the gene play within the cells of that tissue?> Exactly how do mutations in the gene affect the protein's function? 4. Adding a normal copy of the gene should fix the problem in the affected tissue. This may seem like obvious, but it's not. What if the mutated gene encodes a protein that prevents the normal protein from doing its job? Mutated genes that function this way are called dominant negative and adding back the normal protein won't fix the problem. 5. The gene delivery to cells of the affected tissue must be possible, It depends on: How accessible is the tissue? Is it fairly easy (skin, blood or lungs), or more difficult to reach (internal organs)? > What is the best mode of delivery? Techniques used in gene therapy The techniques of biotechnology have made it possible to isolate the required gene in the laboratory and also deliver the gene. Isolation of Gene of Interest The first step is to find and isolate the gene that will be inserted into the genetically modified organism. Finding the right gene to insert usually draws on years of scientific research into the identity and function of useful genes. Once that is known the DNA needs to be cut at specific locations to isolate the gene of interest. This can be done by using restriction enzymes also known as molecular scissors which cut DNA at specific sites containing palindromic DNA sequences. But in order to cut the DNA with restriction enzymes, it needs to be in pure form, free from other macro-molecules. Isolation of DNA Since the DNA is enclosed within the membranes, we have to break the cell open to release DNA along with other macromolecules such as RNA, proteins, polysaccharides and also lipids. This can be achieved by treating the bacterial cells/plant or animal tissue with enzymes such as lysozyme(bacteria), cellulase (plant cells), chitinase (fungus). Genes are located on long molecules of DNA intertwined with proteins such as histones, The RNA can be removed by treatment with ribonuclease whereas proteins can be removed by treatment with protease. Other molecules can be removed by appropriate treatments and purified DNA ultimately precipitates out after the addition of chilled ethanol. This can be seen as collection of fine threads in the suspension. Cutting of DNA Restriction enzyme digestions are performed by incubating purified DNA molecules with the restriction enzyme, at the optimal conditions for that specific enzyme. The cutting of DNA by restriction endonucleases results in the fragments of DNA. These fragments can be separated by a technique known as gel electrophoresis. Since DNA fragments are negatively charged molecules they can be separated by forcing them to move towards the anode under an electric field through a medium/matrix. The separated bands of DNA are analysed for the required gene and then it is cut out from the agarose gel and extracted from the gel piece. This step is known as elution. Multiplication of Gene (PCR) PCR or polymerase chain reaction is then used to create multiple copies the gene of interest. In this reaction, multiple copies of the gene (or DNA) of interest is synthesised in vitro using two sets of primers (small chemically synthesised oligonucleotides that are complementary to the regions of DNA) and the enzyme DNA polymerase. The enzyme extends the primers using the nucleotides provided in the reaction and the genomic DNA as template. If the process of replication of DNA is repeated many times, the segment of DNA can be amplified to approximately billion times, i.e., 1 billion copies are made. Gene Targeting Gene delivery is one of the biggest challenges in the field of gene therapy. Gene Delivery includes:1. TARGETING the right cells, 2. ACTIVATING the gene. A gene's journey is not over when it enters the cell. it must go to the cell's nucleus and be “turned on,” meaning that its transcription and translation are activated to produce the protein product encoded by the gene. For gene delivery to be successful, the protein that is produced must function properly. 3. INTEGRATING the gene in the cells. The gene must stay put and continue working in the target cells. If so, it must be ensured that the gene integrates into, or becomes part of the host cell's genetic material, or that the gene finds another way to survive in the nucleus without being rejected. 4. AVOIDING harmful side effects. Anytime an unfamiliar biological substance is introduced into the body, there is a risk that it will be toxic or that the body will mount an immune response against it. If the body develops immunity against a specific gene delivery vehicle, future rounds of the therapy will be ineffective. Choosing the Best Vector There is no “perfect vector" that can treat every disorder. Like any type of medical treatment, a gene therapy vector must be customized to address the unique features of the disorder. We have learnt the lesson, of transferring genes into plants and animals from bacteria and viruses, which have known this for ages - how to deliver genes to transform eukaryotic cells and force them to do what the bacteria or viruses want. Part of the challenge in gene therapy is choosing the most suitable vector for treating the disorder. Some vectors commonly used are: Viruses: Usually when we think of viruses, we think of them causing diseases such as the commen cold, the flu, and HIV/AIDS. When faced with the problem of gene delivery, scientists looked to viruses. Why reinvent the wheel if there's a perfectly good one out there? If we can modify viruses todeliver genes without making people sick, we may have a good set of gene therapy tools. General advantages of viral vectors: They're very good at targeting and entering cells. -Some viral vectors might be engineered to target specific types of cells, -They can be modified so that they can't replicate and destroy the cell. General drawbacks of viral vectors: A virus can't “expand” to fit a piece of genetic material larger than it is naturally built to carry. Therefore, some genes may be too big to fit into a certain type of virus. Viruses can cause immune responses in patients, resulting in two potential outcomes: ~ Patients may get sick. A patient's immunity to a virus may prevent him from responding to repeated treatments. % However, modern viral vectors have been engineered without most of the proteins that would cause an immune response Non-Viral Vectors Although viruses can effectively deliver genetic material into a patient's cells, they do have some limitations. It is sometimes more efficient to deliver a gene using a non-viral vector, which has fewer size constraints and which won't generate an immune response. Non-viral vectors are typically circular DNA molecules, also known as plasmids. In nature, bacteria use plasmids to transfer genes from cell to cell. Scientists use bacteria and plasmids to easily and efficiently store and replicate genes of interest from any organism,Vectors used at present, are engineered in such a way that they help easy linking of foreign DNA and selection of recombinants from non-recombinants. These are not the only way to introduce alien DNA into host cells, Ina method known as micro-injection, recombinant DNA is directly injected into the nucleus of an animal cell. In another method, suitable for plants, cells are bombarded with high velocity micro-particles of gold or tungsten coated with DNA in a method known as biolistics or gene gun. Cystic Fibrosis Cystic fibrosis (CF), also known as mucoviscidosis, is an autosomal recessive genetic disorder that affects most critically the lungs, and also the pancreas, liver, and intestine. It is characterized by abnormal transport of chloride and sodium across an epithelium, leading to thick, viscous secretions, preventing the cilia from clearing debris which cause symptoms such as coughing, poor digestion and increased vulnerability to infection. CF is caused by a mutation in the gene for the protein cystic fibrosis transmembrane conductance regulator (CFTR) gene on chromosome 7. Most commonly, the mutation in the CFTR gene is a three-base-pair deletion, This protein is required to regulate the components of sweat, digestive fluids, and mucus, CFTR regulates the movement of chloride and sodium ions across epithelial membranes, such as the alveolar epithelia located in the lungs. Since all of the cells of a CF patient have the defective protein, large quantities of thick, sticky mucus build up throughout the lungs and other organs, This results in the severity of symptoms seen in CF patients.Success Cases of gene therapy 1) Gene therapy cures blindness Cure blindness of inherited condition. Leber's conginetal amaurosis -Inherited disease caused by an abnormality in a gene called RPE65. -The condition appears at birth or in the first few months of life and causes progressive worse and loss of vision. How it work?? Used harmless viruses % Enable access to the cells beneath the retinas of patients. + By using a very fine needle - safe in an extremely fragile tissue and can improve vision in a condition previously considered wholly untreatable. 2)Gene therapy reduces Parkinson’s Disease Symptoms * It significantly improved the weakness of the symptoms such as tremors, motor skill problems, and rigidity. * Main- overactive brain region : the subthalamic nucleus should be introduced with gene. “ That would produce GABA-an inhibitory chemical- then they could potentially quiet that brain region and alleviate tremors, How it works?? % Done with local anesthesia, used a harmless, inactive virus [ AAV-2 GAD] ‘> Deliver the GAD gene into the pateinet’s subthalamic nucleus.The gene instructs cells to begin making GABA neurotransmitters to re-establish the normal chemical balance that becomes dysfunctional as the diseases progresses, Advantages “ Give a chance of normal life to baby born with genetic disease. “ Give hope of healthy life to cancer patients. * For certain disease that do not have any cure except gene therapy, it could save many lives. Disadvantages *? The genetic testing, screening and research in findind the availability of certain gene is very controversy. * May increase rate abortionif prenatal test regarding baby with genetic disease is done. “The cost is very and the patient might need an insurance to cover the treatment. “Cosmetic industry may monopolized this gene therapy if it is used in enhancing beauty and in vanishing the aging effect, rather than used for treatment of a disease.Is It A Good Target For Gene Therapy? To check this some questions must be answered:- 1. Does the condition result from mutation? Yes. 2. Is the biology of the disorder known? Yes. 3. Will adding a normal copy of the gene fix the problem in the affected tissue? Yes, While the mutated CFTR gene encodes a non-functional ion channel protein, it will not prevent a normal CFTR channel protein from working properly. Therefore, adding a normal copy of the CFTR gene should fix the problem Is it feasible to deliver the gene to the cells of the affected tissue? Yes, in part. Treating the lungs of patients with CF might be feasible, since the lung surfaces are exposed to the air and somewhat easy to reach, Because the digestive system is less accessible, however, it might be a more difficult region to treat. Hence we can conclude that it is a perfect disease to be treated by gene therapy.Challenges Some the factors that have kept gene therapy from becoming an effective treatment for genetic diseases are: + 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 anylong-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 Upcoming CRISPR, CRISPR stands for clustered regularly interspaced short palindromic repeats. These RNA sequences serve an immune function in archaea and bacteria, but in the last year or so, scientists have seized upon them to rewrite genes. The RNA sequence serves as a guide to target a DNA sequence in, say, a zygote or a stem cell. The guide sequence leads an enzyme, Casg, to the DNA of interest. Casg can cut the double strand, nick it, or even knock down gene expression. After Casg injures the DNA, repair systems fix the sequence - or new sequences canbe inserted, CRISPR, It isn’t the first or only method of gene repair therapy that's been developed, but the CRISPR technology, says Ramesar, is so special because, unlike previous methods which were more laborious and could only target one kind of cell in the body, it appears to be a “one size fits all delivery", adaptable for different tissues. The procedure also seems relatively simple to perform. Exciting as the development may be, CRISPR won't be delivering instant cures just yet. Ramesar says, from his initial impressions of the literature, that it would seem that localised, accessible abnormal tissue (as in the retina or skin) could be targeted more easily. Conditions affecting the body more systemically, however, such as certain developmental syndromes, or central nervous system disorders, might be problematic in terms of getting the repair technology into enough of the target cells in that tissue to make an effective difference.CONCLUSION Although early clinical failures led many to dismiss gene therapy as over-hyped, clinical successes since 2006 have bolstered new optimism in the promise of gene therapy. These include successful treatment of patients with the retinal disease Leber's congenital amaurosis, X-linked SCID, ADA-SCID, adrenoleukodystrophy, chronic lymphocytic leukaemia (CLL),acute lymphocytic leukaemia (ALL), multiple myeloma, haemophilia and Parkinson's disease. These recent clinical successes have led to a renewed interest in gene therapy, with several articles in scientific and popular publications calling for continued investmentin the field.