18-Gene Therapy-2.ppt

Gene Therapy 1 * Learning objectives • • • • What is Gene therapy? Approaches used in Gene therapy. Applications. Case reports of Gene therapy. 2 * Gene therapy: • The main goal of gene therapy is to treat loss-of-function genetic disorders by delivering correcting therapeutic DNA sequences i...

Gene Therapy 1 * Learning objectives • • • • What is Gene therapy? Approaches used in Gene therapy. Applications. Case reports of Gene therapy. 2 * Gene therapy: • The main goal of gene therapy is to treat loss-of-function genetic disorders by delivering correcting therapeutic DNA sequences into the nucleus of a cell, allowing its expression at physiologically relevant levels. 3 • Gene therapy may be divided into: 1) Germ-line gene therapy: it aims for the introduction of therapeutic genes into germ-cells or specific type of embryonal cells (at the 4-8 cellular stage). As a result, all the cells of the individual derived from these cells will carry the therapeutic gene, including his or her germ cells. Further offspring will also carry the therapeutic gene (Slide 5). 4 2) Somatic gene therapy: it is the introduction of gene(s) into somatic cells. The ethical considerations of somatic gene therapy have been widely discussed and a consensus has emerged which allows genetic manipulation of a patient’s somatic cells for the purpose of correcting severe disorders. 5 Principles of Gene Therapy Isolation and amplification of -1 .normal gene Inserting gene into diseased -2 .cell The gene either integrate itself -3 into the chromosome or remain as an extra-chromosomal .genetic element (episome) .Gene expression -4 6 Ex vivo Gene Therapy 7 In vivo Gene Therapy 8 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 period of treatment – Would have to have multiple rounds of therapy • Immune Response – new things introduced into the body 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 • Limitation with multigene disorders – Heart disease, high blood pressure, Alzheimer’s, arthritis and diabetes are difficult 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 9 Disease targets for gene therapy 10 First case of treatment • The first gene therapy was performed on September 14th, 1990 – Ashanti DeSilva was treated for SCID • Sever combined immunodeficiency – Doctors removed her white blood cells, inserted the missing gene into the WBC, and then put them back into her blood stream. – This strengthened her immune system – Only worked for a few months – Must have repeated intake of the therapy 11 First gene therapy product Gendicine is a recombinant adenovirus • engineered to express wildtype-p53. This virus is designed to treat patients with tumors which .have mutated p53 genes 12 Requirements of Gene Therapy 13 Viral Vectors 14 Episome system 15 Non-viral Vectors 16 • Direct injection: – Simple. – Safe. – Poor efficiency. – Low level of stable integration of DNA. 17 • Particle bombardment: – Repeated injections. – Large number of DNA. – Doesn’t work with target tissue that is difficult to access directly. 18 • Liposomes 19 20 Clinical Applications of Gene Therapy 21 Treatment approaches for “Cancer” 22 Cancer Gene Therapy (Common approaches) Suicide gene therapy* 23 Suicide gene therapy • Herpes simplex virus Thymidine kinase (HSV-tk) gene coupled with the Ganciclovir (GCV) • Cytosine deaminase (CD) gene coupled with the pro-drug 5fluorocytosine (5-FC) • It is also known as V/GDEPT 24 Treatment approaches for “Monogenetic inherited disorders” 25 Ex 1: Cystic Fibrosis • Occurs with 2 defective copies of gene encoding the protein called cystic fibrosis transmembrane conductance regulator (CFTR). • Normally it serves as a pump at the cell membrane to move chloride ions out of the cells. • If cells can’t move chloride out, they absorb water trying to dilute the chloride in the cell. • This leads to the production of THICK sticky mucus that clogs airways; ideal environment for infections (leading to pneumonia, 26 etc…). CF Gene Therapy • Uses viruses (adenoassociated viral vector) and liposomes sprayed into nose & mouth. • In vivo gene therapy • Expensive treatment. • NOT a reliable cure yet. • Requires multiple applications. • May not produce adequate protein. 27 Ex 2: Duchenne Muscular Dystrophy (DMD): • a genetic disorder where dystrophin gene is mutated which codes for the protein dystrophin. It affects muscles causing muscular degeneration and premature death. • Gene therapy approach: DMD naked plasmid DNA-dystrophin 28