gene therapy PPT

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Gene therapy for hemophilia

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Hemophilia Hereditary bleeding disorder Lack of particular clotting factors bleeding from injury Hemophilia A Hemophilia B

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Occurrence 80% hemophilia A due to factor VIII deficiency 20% hemophilia B due to factor IX deficiency hemophilia A occurs in about 1 in every 5,000-10,000 male births 400 babies are born with hemophilia each year

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Differences between hemophilia A & B Size of DNA F VIII 4.4 KB F IX 1.5 kb Prevalence of inhibitary antibodies (lower in B) Levels of protein expression required F VIII 300 Nanograms F IX 5µg/ ml Hemophilia A gene – 7000 bp Hemophilia B gene – 1500bp

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Causes of Hemophilia Caused by Genetic error Females have two X chromosomes while Males have a Y and a X chromosomes Females are carriers Hemophilia only affects males

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Signs and Symptoms The major signs and symptoms are excessive bleeding and easy bruising Signs of excessive external bleeding: Bleeding in the mouth(cut, bite, losing a tooth) Nosebleeds for no reason Heavy bleeding from minor cut

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Treatment Main treatment- replacement therapy Replacement therapy- giving or replacing the clotting factor that’s too low or missing Replacement therapy on regular basis to prevent bleeding Take clotting factor concentrates that reduces the risks than use human blood.

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Gene Therapy Approaches for Hemophilia: Liver directed Muscle directed Bone marrow directed Transplantation of genitically - modified fibroblasts

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Ex Vivo vs. In Vivo Transfer into Liver Ex Vivo: Remove hepatocytes Modify in culture Reinject In vivo: Inject vector parenterally

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Summary of Success with Different Vectors in the Liver AAV (Adenovirus associated virus) Vectors Good expression for hemophilia B Safe Retroviral vectors Good expression for hemophilia A & B Safe Adenoviral vectors Great expression for hemophilia A or B Toxic No stable efficacy in large animals

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Adeno-associated virus (AAV) Vectors Small single-stranded DNA (4.5 kb) virus of the parvovirus family Does not require replicating cells for gene transfer

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Retroviral Vectors Single stranded RNA virus that gets copied into DNA that integrates into the chromosome Classes of retroviral vectors: Oncoretroviral vectors require replication for gene transfer Lentiviral vectors do not require replication for gene transfer

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Ways for Oncoretroviral Vectors to Transduce Hepatocytes : Adults: Inject hepatocyte growth factor (HGF) prior to injection of retroviral vectors Newborns: Replication is already sufficient for gene transfer due to the rapid rate of growth

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Clinical Trials in Patients IM injection of AAV for hemophilia B IV injection of RV for hemophilia A Implantation of genetically-modified fibroblasts for hemophilia A Hepatic artery injection of AAV for hemophilia A

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Transplantation of genetically-modified Fibroblasts

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Conclusions: No gene therapy approaches have evidence of long-term efficacy in patients IM injection of AAV is too inefficient Implantation of fibroblasts is very laborious and not very effective Liver delivery of AAV or RV should work

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Cystic Fibrosis

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Cystic Fibrosis monogenic disorder that presents as a multi system disease The first signs and symptoms occur in childhood Autosomal recessive disorder affecting 1 in 2500 live white births Inherited disease of secretory glands (which make mucous and sweat) This is characterised by abnormal salt and water transport That leads to abnormal airway secretions, impaired mucociliary clearance, chronic bacterial infection, bronchiectasis and pre mature death A variety of epithelial Tissues are affected in this disease , including airway, pancreatic, sweat ductal and gastrointestinal epithelia.

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Cystic Fibrosis Cause Mutation in CFTR (cystic fibrosis transmembrane conductance regulator) gene present in chromosome 7 Role of CFTR Protein : Protein moves chloride ions out of an epithelial cell to the covering mucus In CF No movement of cl This means water does not leave and it results in the mucus becoming thick

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Cystic Fibrosis Pathophysiology Mutation in the CFTR gene The protein created by this gene is anchored to the outer membrane of cells in the sweat glands, lungs, pancreas, and other affected organs The protein acts as a channel connecting the inner part of the cell (cytoplasm) to the surrounding fluid

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Cystic Fibrosis Pathophysiology This channel is responsible for controlling the movement of chloride from inside to outside of the cell however, in the sweat ducts it facilitates the movement of chloride from the sweat into the cytoplasm When the CFTR protein does not work, chloride is trapped inside the cells in the airway and outside in the skin

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Cystic Fibrosis Mucus becomes thick and sticky Mucus builds up in lungs and blocks airways Buildup of mucus makes it easy for bacteria to grow This leads to repeated, serious lung infections. Over time, these infections can severely damage lungs

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Cystic Fibrosis The thick, sticky mucus also can block tubes, or ducts in pancreas So that pancreas can’t pass the digestive enzymes to small intestine Intestines can't fully absorb fats and proteins This can cause vitamin deficiency and malnutrition It also can cause bulky stools, intestinal gas, a swollen belly from severe constipation, and pain or discomfort

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Cystic Fibrosis Also causes sweat to become very salty As a result body loses large amounts of salt during sweating This can upset the balance of minerals in blood CF causes increased risk for diabetes or osteoporosis Also causes infertility in men, and it can make it harder for women to get pregnant

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Cystic Fibrosis Hallmarks Salty tasting skin Normal appetite but poor growth Poor weight gain Excessive mucus production Coughing

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Cystic Fibrosis Males– infertile due to congenital B/L absence of vas deferens Meconium ileus – typical finding in NB babies with cystic fibrosis More common in western world

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Cystic Fibrosis Diagnosis Sweat testing Genetic testing Chorionic villous sampling amniocentesis

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Cystic Fibrosis Gene therapy Gene therapy at early age – to replace the defective gene Give active form of protein product that is deficient Lung transplantation in severe lung disease Pancreatic enzyme supplementation in severe pancreatic deficiency

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Gene therapy Introduction of functional genetic material into the target cell to replace the defective genes Gene transfer techniques Exvivo : Cells removed, genetically modified and transplanted back in to the patient Invivo : Direct transfer of genetic material into patient

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Gene transfer : Gene transfer Vectors viral non-viral Cells germ cells somatic cells

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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

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Problems with gene therapy : Short lived Immune response Viral vectors Multigene disorders

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