Group 3 Fabiha Aziz Fariha Munir Fatima Tuz Zahra Fatima Tuz Zahra Kh Muhammad Ahmad Saneea Imran
Tatheer Fatima Contents 1.Introduction of the disease or disorder 2.Types of mutations involved 3.Conventional treatment 4.Type of vector 5.The procedure of gene therapy
6.History of clinical trials 7.Commercially available treatments Introduction What is hemophilia? What is does to our body? What causes hemophilia?
What is hemophilia? Hemophilia is an inherited bleeding disorder in which the blood fails to clot effectively. Hemophiliacs do not bleed more extensively or more quickly than the normal people but they do bleed for a longer time after an injury or surgery. What it does to our body?
The bleeding may be internal or external Internal bleeding occurs in knees ankles tissues and muscles. If left untreated the bleeding in the joints may lead to agonizing pain And can be fatal when the bleeding occurs in the vital organ like brain Hemophilia A
All races Affects men and women become carriers as gene is present on the X chromosome. Factor Replacement Therapy MUTATION By: Fatima Tuz Zahra
Mutation of Hemophilia A Coagulation factors are proteins that work together in the blood clotting process. F8 gene provides instructions for making a coagulation factor VIII Mutations in the F8 gene lead to the production of an abnormal version of coagulation factor VIII.
28 region of X Chromosome. Inversion and Crossing Over of Intron22 Conventional Treatments
By: Tatheer Fatima Conventional treatment The two main approaches to treatment are Preventive treatment : Refers to the regular medications that are taken to prevent episodes of bleeding and subsequent complications
Episodic treatment : Used to manage episodes of prolonged bleeding. Conventional treatment Treatment With Replacement Therapy : Concentrates of clotting factor VIII (hemophilia A) or clotting factor IX (hemophilia B) are slowly dripped or injected into a vein
Preparation of clotting factor concentrate : Human Blood Clotting factor concentrate Recombinant Clotting factors Conventional treatment Types of Replacement therapy : Preventive or prophylactic therapy: Replacement therapy on a regular basis to prevent
bleeding Demand therapy: only need replacement therapy to stop bleeding when it occurs Conventional treatment 1.
2. 3. 4. PROS Quicker treatment. Early treatment lowers the risk of
complications Fewer visits to the doctor or emergency room Costs less than treatment in a medical care setting Helps children accept
treatment and take responsibility for their own health CONS 1. Antibody(proteins) that attack the clotting factor
2. Viral infections from human clotting factors 3. Damage to joints, muscles, or other parts of the body resulting from delays
Conventional treatment Desmopressin Man made hormone Can treat mild hemophilia A Cannot treat hemophilia B or severe hemophilia A Stimulates the release of stored factor VIII Injection or as nasal spray Only in certain situations. e.g. you may take this
medicine prior to dental work or before playing certain sports to prevent or reduce bleeding Conventional treatment Anti fibrinolytic Medicines Tranexamic acid and epsilon aminocaproic acid Can be used with replacement therapy Given as a pill and they help keep blood clots from
breaking down. Most often are used before dental work or to treat bleeding from the mouth or nose or mild intestinal bleeding. Types of vector By: Muhammad Ahmed
Ex-vivo Cells transduced in culture, then returned to patient Greater control over transfection conditions Unchanged cells can be screened out Difficult to transpant In-vivo Cells modified within the body
Introduction of vector with genetic material in patients body No transplantation issues Cost-effective May provoke immune system Ultimate goal for gene therapy Viral Vectors
Retroviral vectors Moloney murine leukemia virus (MoMLV)-based Can infect wide variety, integrate into host genome, relatively non-immunogenic Only in actively dividing cells Problems: insertional mutagenesis, inactivation by complement
Adenoviral vectors Relatively large, double-stranded DNA viruses Infect non-dividing cells Can transfer multiple copies of gene, in vivo Problems: Many humans may be immune to virus, not integrated into host genome
Adeno-associated (AAV) vectors Relatively small, single stranded DNA parvovirus Viral coding sequence replaced by transgene Non-dividing cells Lentiviral Vectors Integrate into genome, infect non-dividing cells Problems: limited range of cell targets, pathogens!
HIV big risk of recombination infectious virus Non-Viral Vectors Through plasmids Problem: Transient expression of genes (several days) Direct injection of naked DNA
Problems: Low efficiency of transduction, not integrated into genome Procedure of Gene Therapy for Hemophilia A By: Saneea Imran
Gene therapy for hemophilia A Hemophilia is an interesting target for gene therapy as the genetics of the disease are well understood. Introducing a functional copy of the mutated gene through a vector can provide an effective cure. Phenotype
Clotting factor level = Clinical Gene therapy for Hemophilia A Vector: Both viral and non-viral vectors can be used Target Cell: Hematopoietic stem cells, hepatocytes, skeletal muscle cells, endothelial cells.
Approach: Both in-vivo and ex-vivo gene therapy approach can be used. Non-Viral vector for gene therapy of Hemophilia A Year: 2001 Non-viral somatic cell gene therapy for hemophilia A Vector used: Plasmid
Cells targeted: Dermal fibroblasts Procedure Desired gene that produces Factor VIII Restriction enzymes
Preparing the vector Gene of interest Transfection
Antibioti c gene Dermal fibroblasts of patient ( skin biopsy ) Cells that
successfully produce FVIII are selected and inserted into patient
Patient with Hemophilia Viral Vectors for gene therapy of Hemophilia A Vectors that have been used are: 1. Retroviral and Lentiviral Vectors 2. Adeno Associated Viral Vectors
Using Retroviral Vectors Producing recombinant Retroviral vectors Targeting cells with Retrovirus Clinical Trials
By: Fatima Tuz Zahra Khan Stem cell-based gene transfer Implantation of Genetically Modified Stem Cells Genetic modification and transplantation of hemopoietic stem cells (HSCs) Use of Retroviral and lentiviral vectors Trials remain un-successful due to insufficient levels
of FVIII In Vivo methods Retroviral vectors Retroviral vectors transduce replicating cells by stably integrating into the host genome. Preclinical studies involving rabbits and dogs did show possible benefits.
Routes? Subjects received a peripheral intravenous infusion of retroviral vector carrying a B domaindeleted human FVIII gene. Efficiency? 9 of 12 subjects showed a FVIII level above 1% Insertional mutagenesis and the risk of subsequent
LENTIVIRAL VECTORS Lentiviruses efficiently transduce nondividing cells. Overcoming a hurdle faced by retroviral vector systems. Routes? In murine models, intraperitoneal administration of lentivirus transduced liver, spleen, blood and bone marrow Efficieny? four- to sixfold rise in expression
Concerns over genomic integration and subsequent malignancy. ADENOVIRAL VECTORS One of the most efficient vectors Transducing dividing and nondividing cells, with a tropism for hepatocytes Genetic payload capacity making them ideal vectors for
the FVIII gene Routes? Intramuscular Injection. Liver-directed administration Efficiency? Single Human Trail Adeno associated Virus:
Success in mouse and other large animal models. Limitations due to small genome size. Routes? Intramuscular Injection. Liver-directed administration Efficiency? Two trials have examined the use of AAV-2 vectors in human subjects
Limited efficacy Commercially available Treatments Treatment Regular infusions of DDAVP or clotting factor Clot-preserving medications (antifibrinolytics)
Fibrin sealants Physical therapy First aid for minor cuts Vaccinations Clinical Trials Currently are for hemophilia B AAV vector encoding factor IX (FIX) under the
control of a liver-restricted promoter Promising results Problems First clinical trial of hepatic artery infusion of an AAV2-FIX vector quickly encountered obstacles that had not been identified in the preclinical studies Risk of vertical transmission
Risk for potential offspring if the spermatocytes are transduced References Haemophilia A: from mutation analysis to new therapies Jochen Graw, Hans-Hermann Brackmann, Johannes Oldenburg, Reinhard Schneppenheim, Michael Spannagl & Rainer Schwaab Genetics Home Reference
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