Gene Therapy Understanding Basics
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Gene Therapy Understanding Basics



Gene Therapy Understanding Basics

Gene Therapy Understanding Basics



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Gene Therapy Understanding Basics Gene Therapy Understanding Basics Presentation Transcript

  • GENE THERAPY Basics Dr.T.V.Rao MD Dr.T.V.Rao MD 1
  • A Tribute to Gregory Mendel Dr.T.V.Rao MD 2
  • Gregory Mendel - GENETICS – He used pollen from some plants to carefully fertilize other plants – He was able to observe that characteristics are inherited in a predictable manner. – He determined that alleles of a single trait segregate (separate) independently. – He observed that the alleles for different traits assort independently. – This gave rise to a new discipline that we now call Dr.T.V.Rao MD Genetics 3
  • What Genes can do Genes, which are carried on chromosomes, are the basic physical and functional units of heredity. Genes are specific sequences of bases that encode instructions on how to make proteins. it‟s the proteins that perform most life functions and even make up the majority of cellular structures. Dr.T.V.Rao MD 4
  • Why Genetic Disorders When genes are altered so that the encoded proteins are unable to carry out their normal functions, genetic disorders can result. Dr.T.V.Rao MD 5
  • All of us carry some defective Genes,some are apparent and many in apparent Each of us carries about half a dozen defective genes. We remain blissfully unaware of this fact unless we, or one of our close relatives, are amongst the many millions who suffer from a genetic disease. About one in ten people has, or will develop at some later stage, an inherited genetic disorder, and approximately 2,800 specific conditions are known to be caused by defects (mutations) in just one of the patients genes. Dr.T.V.Rao MD 6
  • What we Inherit from Parents?  Most of us do not suffer any harmful effects from our defective genes because we carry two copies of nearly all genes, one derived from our mother and the other from our father. Dr.T.V.Rao MD 7
  • Why Males differ from Females• The only exceptions to this rule are the genes found on the male sex chromosomes. Males have one X and one Y chromosome, the former XX from the mother and the latter from the father, so each cell has only one copy of the genes on these chromosomes XY Dr.T.V.Rao MD 8
  • Law of Inheritance In the majority of cases, one normal gene is sufficient to avoid all the symptoms of disease. If the potentially harmful gene is recessive, then its normal counterpart will carry out all the tasks assigned to both. Only if we inherit from our parents two copies of the same recessive gene will a disease develop. Dr.T.V.Rao MD 9
  • Gene therapy is …. Gene therapy is the insertion of genes into an individuals cells and tissues to treat a disease, such as a hereditary disease in which a deleterious mutant allele is replaced with a functional one. Although the technology is still in its infancy, it has been used with some success. Dr.T.V.Rao MD 10
  • What is gene therapy?• 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. Dr.T.V.Rao MD 11
  • How It Works A vector delivers the therapeutic gene into a patient‟s target cell The target cells become infected with the viral vector The vector‟s genetic material is inserted into the target cell Functional proteins are created from the therapeutic gene causing the cell to return to a normal state Dr.T.V.Rao MD 12
  • Gene Therapy is Experimental Advances in understanding and manipulating genes have set the stage for scientists to alter a persons genetic material to fight or prevent disease. Gene therapy is an experimental treatment that involves introducing genetic material (DNA or RNA) into a persons cells to fight disease. Dr.T.V.Rao MD 13
  • Majority are Trails Gene therapy is being studied in clinical trials (research studies with people) for many different types of cancer and for other diseases. It is not currently available outside a clinical trials Dr.T.V.Rao MD 14
  • Vivo to Vitro Dr.T.V.Rao MD 15
  • What Gene therapy can Achieve 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 Dr.T.V.Rao MD to 16 help fight a disease.
  • Uses of gene therapy Replace missing or defective genes; Deliver genes that speed the destruction of cancer cells; Supply genes that cause cancer cells to revert back to normal cells; Deliver bacterial or viral genes as a form of vaccination; Provide genes that promote or impede the growth of new tissue; and; Deliver genes that stimulate the healing of damaged tissue. Dr.T.V.Rao MD 17
  • Genes are Medicine ? Gene therapy is „the use of genes as medicine‟. It involves the transfer of a therapeutic or working gene copy into specific cells of an individual in order to repair a faulty gene copy. Thus it maybe used to replace a faulty gene, or to introduce a new gene whose function is to cure or to favourably modify the clinical course of a condition. Dr.T.V.Rao MD 18
  • Delivering desired Genes Dr.T.V.Rao MD 19
  • Gene Therapy Corrects Gene therapy is a technique for correcting defective genes responsible for disease development. Researchers may use one of several approaches for correcting faulty genes: Dr.T.V.Rao MD 20
  • Goal of Gene therapy A normal gene may be inserted into a non-specific location within the genome to replace a non-functional 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. Dr.T.V.Rao MD 21
  • Steps in Gene Therapy Dr.T.V.Rao MD 22
  • Dr.T.V.Rao MD 23
  • Manipulation corrects the Defective Genes Dr.T.V.Rao MD 24
  • Gene Therapy delivers Proteins Today, gene therapy is the ultimate method of protein delivery, in which the delivered gene enters the bodys cells and turns them into small "factories" that produce a therapeutic protein for a specific disease Dr.T.V.Rao MD 25 over a prolonged
  • Antisense therapy Antisense therapy is a form of treatment for genetic disorders or infections. When the genetic sequence of a particular gene is known to be causative of a particular disease, it is possible to synthesize a strand of nucleic acid (DNA, RNA or a chemical analogue) that will bind to the messenger RNA (mRNA) produced by that gene and inactivate it, effectively turning that gene "off". Dr.T.V.Rao MD 26
  • Antisense Therapy Antisense therapy is not strictly a form of gene therapy, but is a genetically- mediated therapy and is often considered together with other methods Dr.T.V.Rao MD 27
  • Dr.T.V.Rao MD 28
  • First Approved Gene Therapy On September 14, 1990 at the U.S. National Institutes of Health, W. French Anderson M.D. and his colleagues R. Michael Blaese, M.D., C. Bouzaid, M.D., and Kenneth Culver, M.D., performed the first approved gene therapy procedure on four-year old Ashanthi DeSilva. Born with a rare genetic disease called severe combined immunodeficiency (SCID), Dr.T.V.Rao MD 29
  • What did they do In Ashanthis gene therapy procedure, doctors removed white blood cells from the childs body, let the cells grow in the laboratory, inserted the missing gene into the cells, and then infused the genetically modified blood cells back into the patients bloodstream. Dr.T.V.Rao MD 30
  • A success story As of early 2007, she was still in good health, and she was attending college. Some would state that the study is of great importance despite its indefinite results, if only because it demonstrated that gene therapy could be practically attempted without adverse consequences. Dr.T.V.Rao MD 31
  • Gene Therapy Depends on Delivery of Corrective Genes Viral vectors are a tool commonly used by molecular biologists to deliver genetic material into cells. This process can be performed inside a living organism (in vivo) or in cell culture (in vitro). Viruses have evolved specialized molecular mechanisms to efficiently transport their genomes inside the cells they infect. Dr.T.V.Rao MD 32
  • Viruses are used as Delivery Tolls Viruses are used as vectors to introduce the genetic material inside the bodies. These viruses are inactivated, they are not able to reproduce Adenoviruses Herpes viruses DNA tumor viruses Retroviruses RNA tumor viruses Dr.T.V.Rao MD 33
  • Making the new Genetic Material Functional Gene that is inserted directly into a cell usually does not function. Instead, a carrier called a vector is used to introduce the therapeutic gene into the patients target cells. The most common vector that is used is a virus that has been genetically altered to carry normal human DNA. Viruses cause diseases in humans by encapsulating and delivering the genes into cells. Dr.T.V.Rao MD 34
  • Somatic and Germ Line Gene Therapy Gene therapy can target somatic (body) or germ (egg and sperm) cells. In somatic gene therapy the recipients genome is changed, but the change is not passed on to the next generation; whereas with germ line gene therapy the newly introduced gene is passed on to the offspring. Dr.T.V.Rao MD 35
  • Safety Safety: Although viral vectors are occasionally created from pathogenic viruses, they are modified in such a way as to minimize the risk of handling them. MD Dr.T.V.Rao 36
  • Making safe Protocols Low toxicity: The viral vector should have a minimal effect on the physiology of the cell it infects. Stability: Some viruses are genetically unstable and can rapidly rearrange their genomes. This is detrimental to predictability and reproducibility of the work conducted using a viral vector and is avoided in their design. Dr.T.V.Rao MD 37
  • Cell type specificity Cell type specificity: Most viral vectors are engineered to infect as wide a range of cell types as possible. However, sometimes the opposite is preferred. The viral receptor can be modified to target the virus to a specific kind Dr.T.V.Rao MD 38 of cell.
  • Lentivirus Lentivirus (lenti-, Latin for "slow") is a genus of slow viruses of the Retroviridae family, characterized by a long incubation period. Lent viruses can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Dr.T.V.Rao MD 39
  • Retroviruses Retroviruses can infect only dividing cells. The viral genome in the form of RNA is reverse- transcribed when the virus enters the cell to produce DNA, which is then inserted into the genome at a random position by the viral integrase enzyme Dr.T.V.Rao MD 40
  • Vectors deliver the Genetic Materials The vector, now called a provirus, remains in the genome and is passed on to the progeny of the cell when it divides. Dr.T.V.Rao MD 41
  • Adenoviruses As opposed to lenti viruses, adenoviral DNA does not integrate into the genome and is not replicated during cell division. Adenoviral vectors are occasionally used in in vitro experiments. Dr.T.V.Rao MD 42
  • Choosing non infective Adenovirus Their primary applications are in gene therapy and vaccination. Since humans commonly come in contact with adenoviruses, which cause respiratory, gastrointestinal and eye infections, they trigger a rapid immune response with potentially dangerous consequences To overcome this problem scientists are currently investigating adenoviruses to which humans do not have immunity. Dr.T.V.Rao MD 43
  • Adeno-associated viruses Adeno-associated virus (AAV) is a small virus which infects humans and some other primate species. AAV is not currently known to cause disease and consequently the virus causes a very mild immune response. AAV can infect both dividing and non-dividing cells and may incorporate its genome into that of the host cell. These features make AAV a very attractive candidate for creating viral vectors for gene therapy Dr.T.V.Rao MD 44
  • Limitation of Direct Gene Induction The simplest method is the direct introduction of therapeutic DNA into target cells. This approach is limited in its application because it can be used only with certain tissues and requires large amounts of Dr.T.V.Rao MD 45 DNA.
  • Nonviral approach  Nonviral approach involves the creation of an artificial lipid sphere with an aqueous core. This liposome, which carries the therapeutic DNA, is capable of passing the DNA through the target cells membrane Dr.T.V.Rao MD 46
  • Nonviral Vectors: Liposomes less Immunogenic DNA/lipid complexes are easy to prepare and there is no limit to the size of genes that can be delivered. Because carrier systems lack proteins, they may evoke much less immunogenic responses. More importantly, the cationic lipid systems have much less risk of generating the infectious form or inducing tumorigenic mutations because genes delivered have low integration frequency and cannot replicate or recombine. Dr.T.V.Rao MD 47
  • Nanotechnology in Gene therapy• Nanotechnology", is the study of the control of matter on an atomic and molecular scale. Generally nanotechnology deals with structures of the size 100 nanometres or smaller, and involves developing materials or devices within that size• The nanotechnology- based approach used by the researchers has minimal toxic side effects to normal cells Dr.T.V.Rao MD 48
  • Nano engineered substances Nonviral substances such as Ormosil have been used as DNA vectors and can deliver DNA loads to specifically targeted cells in living animals. (Ormosil stands for organically modified silica or silicate) Dr.T.V.Rao MD 49
  • Gene Therapy should not Interfere Germ LineThe germ line of a mature or developingindividual is the line (sequence) of germcells that have genetic material that may bepassed to a childGerm line cells are immortal, in the sensethat they can reproduce indefinitely. This islargely due to the activity of the enzymeknown as telomerase. This enzyme extendsthe telomeres of the chromosome,preventing chromosome fusions and othernegative effects of shortened telomeres. Dr.T.V.Rao MD 50
  • Creating New Chromosome Researchers are also experimenting with introducing a 47th artificial chromosome to the body. It would exist autonomously along side of the other 46, not affecting their workings or causing any mutations. It would be a large vector capable of carrying substantial amounts of genetic information and the body‟s immune system would not attack it. Dr.T.V.Rao MD 51
  • ADA deficiency was selected for the first approved human gene therapy trial for several reasons The disease is caused by a defect in a single gene, which increases the likelihood that gene therapy will succeed. The gene is regulated in a simple, “always-on” fashion, unlike many genes whose regulation is complex. The amount of ADA present does not need to be precisely regulated. Even small amounts of the enzyme are known to be beneficial, while larger amounts are also tolerated well Dr.T.V.Rao MD 52
  • Problems of Large Gene It would be a large vector capable of carrying substantial amounts of genetic code, and scientists anticipate that, because of its construction and autonomy, the bodys immune systems would not attack it. A problem with this potential method is the difficulty in delivering such a large molecule to the nucleus of a target cells. Dr.T.V.Rao MD 53
  • Gene Therapy Uses AIDS Virus to Fight AIDS In the study, immune cells were removed from the patients bodies, modified with a disabled AIDS virus known as a lentivirus, and then intravenously returned. The genetically altered cells disseminated anti- HIV material and prevented HIV from reproducing( 07 November, 2006) Dr.T.V.Rao MD 54
  • Cystic Fibrosis needs Correction-Gene therapy can be best option Dr.T.V.Rao MD 55
  • Technical Difficulties in Gene Therapy Gene delivery: Successful gene delivery is not easy or predictable, even in single-gene disorders. For example, although the genetic basis of cystic fibrosis is well known, the presence of mucus in the lungs makes it physically difficult to deliver genes to the target lung cells. Delivery of genes for cancer therapy may also be complicated by the disease being present at several sites. Gene-therapy trials for X-linked severe combined immunodeficiency (X-SCID), however, have been more successful Dr.T.V.Rao MD 56
  • Problems with Gene Therapy Short Lived  Hard to rapidly integrate therapeutic DNA into genome and rapidly dividing nature of cells prevent gene therapy from long time  Would have to have multiple rounds of therapy Immune Response  new things introduced leads to immune response  increased response when a repeat offender enters Viral Vectors  patient could have toxic, immune, inflammatory response  also may cause disease once inside Multigene Disorders  Heart disease, high blood pressure, Alzheimer‟s, arthritis and diabetes are hard to treat because you need to introduce more than one gene May induce a tumor if integrated in a tumor suppressor gene because insertional mutagenesis Dr.T.V.Rao MD 57
  • Law interferes in Gene Therapy Dr.T.V.Rao MD 58
  • Aldo Leopold The First Bioethicist  A thing is right when it tends to preserve the integrity, stability, and beauty of the biotic community. It is wrong when it tends otherwise." - Aldo Leopold, 1949, A Sand County Almanac Dr.T.V.Rao MD 59
  • What are the ethical issues in Gene therapy? How can “good” and “bad” uses of gene therapy be distinguished? Who decides which traits are normal and which constitute a disability or disorder? Will the high costs of gene therapy make it available only to the wealthy? Could the widespread use of gene therapy make society less accepting of people who are different? Should people be allowed to use gene therapy to enhance basic human traits such as height, intelligence, or athletic ability? Dr.T.V.Rao MD 60
  • The Future of Gene Therapy Current uses of gene therapy focus on treating or curing existing conditions. In the future, the focus could shift to prevention. As more of the human genome is understood, medicine will know more about which genes contribute to or cause disease. With that knowledge in hand, gene therapy could be used to head off problems before they occur. Dr.T.V.Rao MD 61
  • Creating 47th Chromosome Researchers are also experimenting with introducing a 47th artificial chromosome to the body. It would exist autonomously along side of the other 46, not affecting their workings or causing any mutations. It would be a large vector capable of carrying substantial amounts of genetic information and the body‟s immune system would not attack it. Dr.T.V.Rao MD 62
  • Several Diseases have Genetic basis Gene mutations probably play a role in many of todays most common diseases, such as heart disease, diabetes, immune system disorders, and birth defects. These diseases are believed to result from complex interactions between genes and environmental factors. When genes for diseases have been identified, scientists can study how specific environmental factors, such as food, drugs, or pollutants interact with those genes. Dr.T.V.Rao MD 63
  • Last two decades made rapid progress Over the last 20 years, the initial thoughts of gene therapy have been transformed into reality with more than 175 clinical trials and 2,000 patients already treated . Yet with all the trials, there is still no conclusive evidence for efficacy. Dr.T.V.Rao MD 64
  • Attempting on MostDisabling Diseases Dr.T.V.Rao MD 65
  • Lesch-Nyhan syndrome, The most likely candidates for future gene therapy trials will be rare diseases such as Lesch-Nyhan syndrome, a distressing disease in which the patients are unable to manufacture a particular enzyme. This leads to a bizarre impulse for self- mutilation, including very severe biting of the lips and fingers. The normal version of the defective gene in this disease has now been cloned. Dr.T.V.Rao MD 66
  • X-linked recessive Disease LNS is transmitted as and X-linked recessive trait. Female carriers do not show the symptoms. LNS is characterized by self-mutilating behaviours such as lip and finger biting and/or head banging. The deficiency of HPRT activity leads to accumulation of phosphoribosylpyrophos Dr.T.V.Rao MD 67 phate.
  • Lesch-Nyhan syndrome(an X-linked recessive disease) Dr.T.V.Rao MD 68
  • Successful One Year Gene Therapy Trial For Parkinsons Disease A successful Documentation  Neurologix a biotech company announced that they have successfully completed its landmark Phase I trial of gene therapy for Parkinsons Disease. Dr.T.V.Rao MD 69
  • Gene therapy for pain:emerging strategies and future directions Gene therapy to alleviate pain could appear surprising and perhaps not appropriate when opioids and other active molecules are available. However, the possibility of introducing a therapeutic protein into some targeted structures, where it would be continuously synthesised and exert its biological effect in the near vicinity of, or inside the cells, might avoid some drawbacks of "classical" drugs. Dr.T.V.Rao MD 70
  • Pain – Cancer a major research area Numerous other molecules involved in pain processing or associated with chronic pain have been identified and the gene-based techniques might be particularly adapted for the evaluation of the possible therapeutic interest of these new potential targets Dr.T.V.Rao MD 71
  • Correction of Maternal Inherited Mutation of Mitochondrial DNA• Mutationatioal disorders Dr.T.V.Rao MD 72
  • Risks associated with current gene therapy Viruses can infect more than one type of cells. Viral vectors may alter more than the intended cells. Or the new gene might be inserted into the wrong location in the DNA, causing cancer or other damage. When DNA is injected directly into a tumor there is a chance that some DNA could be introduced into germ cells, producing inheritable changes. Dr.T.V.Rao MD 73
  • Risks associated with current gene therapy• The gene might be over- expressed (toxicity); the viral vector could cause inflammation or immune reaction; the virus could be transmitted to other individuals or the environment Dr.T.V.Rao MD 74
  • Healthy Mitochondria fused with Oocyst Dr.T.V.Rao MD 75
  • A third Parent donates Mitochondria Dr.T.V.Rao MD 76
  • Cancer gene therapy in India• A cell-based gene therapy that can be used for to treat renal cell carcinoma, colon, breast, and lung cancer, has been given permission to be used in Indian patients• The Indian Department of Biotechnology will allow this innovative therapy to be given to patients Dr.T.V.Rao MD 77
  • Consent and Law• No research on or modification of the human genome, whether the modification has therapeutic or diagnostic aims, can be undertaken without the free and informed consent of the person concerned. In the case of minors and others legally incapacitated, parents or guardians should give such consent. Dr.T.V.Rao MD 78
  • What are some of the ethical considerations for using gene therapy?• -Some Questions to Consider...• What is normal and what is a disability or disorder, and who decides?• Are disabilities diseases? Do they need to be cured or prevented?• Does searching for a cure demean the lives of individuals presently affected by disabilities? Dr.T.V.Rao MD 79
  • Other ethical considerations for using Gene therapy?• Is somatic gene therapy (which is done in the adult cells of persons known to have the disease) more or less ethical than germ line gene therapy (which is done in egg and sperm cells and prevents the trait from being passed on to further generations)? In cases of somatic gene therapy, the procedure may have to be repeated in future generations. Preliminary attempts at gene therapy are exorbitantly expensive. Who will have access to these therapies? Who will pay for their use? Dr.T.V.Rao MD 80
  • Nature - UNESCO• Somatic cell gene therapy - encouraged for any disease - Somatic cell gene enhancement - not to be illegal - Germ-line gene therapy - not to be illegal - Germ-line gene enhancement - Dr.T.V.Rao MD 81 should not be done
  • Understanding Genome and Human Genome Project is a boost to Gene Therapy Dr.T.V.Rao MD 82
  • Dr.T.V.Rao MD 83
  • Dr.T.V.Rao MD 84
  • Changing from Mending the Bones to Patching the Genes Dr.T.V.Rao MD 85
  • Tomorrow’s imagination of life Dr.T.V.Rao MD 86
  • Dr.T.V.Rao MDCollins et al., Nature 4/24/03 87
  • Do not forget Genes can be Unpredictable ? Dr.T.V.Rao MD 88
  • Are we Distorting the Nature? Dr.T.V.Rao MD 89
  • Is Gene therapy totally safe ???• 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 only being tested for the treatment of diseases that have no other cures. Dr.T.V.Rao MD 90
  • • Created by Dr.T.V.Rao MD for Medical and ParamedicalProfessionals in the Developing World • Email • Dr.T.V.Rao MD 91