viral_vectors_II27[1] - Genetherapy Genetherapy (OTCdeficiency Hemophilia( SCID(severecombinedim

viral_vectors_II27[1] - Genetherapy Genetherapy...

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Unformatted text preview: Gene therapy Gene therapy: to correct a genetic defect by transferring of a functional normal copy of the gene into cells Examples of diseases caused by genetic defect Ornithine transcarbamylase (OTC deficiency) Hemophilia (blood coagulation factors VIII or IX) SCID( severe combined immunodeficiency) Muscular dystrophy Cystic fibrosis Sickle cell anemia Application of gene therapy Genetic disorder (deficiency): OTC Cancer Autoimmunity diseases: rheumatoid arthritis Genetic predisposition Mutation in oncogene or tumor suppressor gene Delivery of counteracting gene Diseases involve several genes and the environmental interact: diabetes Factors to be considered in Gene therapy How to deliver genes to specific cells, tissue and whole animals? (methods of delivery) How much and how long the introduced gene will be expressed? The site and dose of gene delivery Is there any adverse immunological consequence of both delivery vehicle (Virus) and the gene in animals? Is there any toxic effects? Methods of gene delivery Viral Vectors: Non­viral vector based Adenovirus Retrovirus Lentivirus Adeno­associated virus (AAV) Herpes simplex virus (HSV) Naked DNA (plasmid DNA): injection or genegun Liposomes (cationic lipids): mix with genes Ex­vivo In vivo Why use viral vectors Virus are obligate intracellular parasites Very efficient at transferring viral DNA into host cells Specific target cells: depending on the viral attachment proteins (capsid or glycoproteins) Gene replacement: non­essential genes of virus are deleted and exogenous genes are inserted Generation of viral vector for gene therapy Replication­competent virus Replication­defective virus Amplicon: doesn’t encode structural proteins Can’t replicate beyond the first cycle of infection Elements needed to generate amplicon Transfer Vector: plasmid (promoter, gene of interest, ori, packaging signal) Packaging vector (cosmid or cell lines): provide the viral structural proteins for packaging of transfer vector Helper virus (packaging of transfer vector): deleted Packaging signal sequence pac Amplicon Plasmid ori Amplicon Vector System Cotransfection with HSV­1 bacmid lacking packaging sequences ("helper genome") Plasmid is replicated into concatemers, cut into genome­unit length molecules & packaged into viral particles by the helper genome; the helper itself cannot be packaged Helper­free amplicons Adenoviral vectors Non­enveloped ds DNA, 36 kilobases Early proteins (E1A, E1B, E2,E3 and E4), late proteins (L1­L5) Causes a benign respiratory infections in human Serotypes 2 and 5 are commonly used as vectors Early generations of adenoviral vectors (replication defective) Death of 18­year old Jesse Gelsinger • Liver disease: OTC deficiency (genetic disease) • University of Pennsylvania • High dose of adenoviral vector (E1 and E4 genes deleted ) carrying the normal copy of OTC gene was administered • Suspected cause of death • Toxicity of high titer adenoviral vector • High immunogenicity of adenoviral vector (an immune revolt) Gutless Adenoviral vector (Amplicon) Modification of the tropism of adenovirus vector Adenovirus fiber binds to CAR (coxsakie and adenovirus receptor, CAR), receptor which is ubiquitous Modify the fiber protein Characteristics of adenoviral vector Advantages High titers Both dividing and non­dividing cells Wide tissue tropism Easily modify tissue tropism Disadvantages Transient expression ( not good for genetic diseases) Highly immunogenic High titers of virus can be toxic More suitable for cancer immunotherapy Retroviral vector Moloney murine leukemia virus (MuLV) Generation of replication defective retroviral vector Transfer plasmid vector: Gene of interest Long terminal repeats(LTR): promoter, polyA, integration, replication and reverse transcription Primer binding site (PBS) ( origin of replication) RNA packaging signal Poly purine tract (important for replication) Packaging vector Cell line stably transfected with plasmid constructs containing Gag/pol and Env Generation of retroviral vector Pseudotyped retroviral vector Characteristics of retroviral vector Advantages Integration: permanent expression Pseudotyped virus Disadvantages Only infecting dividing cells Insertional mutagenesis (tumor formation) Activate oncogenes Inhibit tumor suppressor genes A case of leukemia in a SCID child treated with a retroviral vector SCID disease or ‘Bubble boy disease’ ( T cell deficiency) or Overall quite successful, over 1000 peoples received retroviral gene therapy A French baby’s treated with retroviral vector 3 years ago A leukemia-like illness developed this summer. Nine other children treated same time show no sign of leukemia But the side effect isn’t a big enough risk yet that genetic experiments for children with an often fatal immune disease should stop People receiving retroviral gene therapy should be warned about the risk of developing leukemia Lentiviral vectors Infection of non­dividing cells (hepatocytes, neurons) HIV, a human lethal pathogen Delete accessory genes Provide an envelope from a non­retrovirus (VSV) Develop vectors from lentiviruses of non­ human pathogens SIV, FIV, EIAV etc Herpesvirus vectors Herpes simplex virus 1, mild disease in human, no risk Linear ds DNA, 152 kb, about half of the total 81 genes are non­essential for virus replication 40­50 kb of foreign DNA can be accommodated Neurotropic virus, target to nervous system Replication defective amplicon particles Adeno­associated virus vectors Non­pathogenic human parvovirus, non­ enveloped ss DNA virus, 4.6 kilobases Dependent on a helper virus ( adenovirus or herpesvirus) for replication (dependovirus) AAV­2 mostly used for vector Generation of adeno­associated virus vector Characteristics of AAV vector Advantages Integration and persistent expression No insertional mutagenesis Infecting dividing and nondividing cells Safe Disadvantages Size limitation, 4.9 kb Low titer of virus, low level of gene expression Comparison of different viral vectors Viral vector titers manupilation of immunogenicity tropism infecting of non­dividing cells Adenovirus 1011 terrific very high yes Retrovirus 107 good low only lentivirus Herpesvirus 107 not so good low yes AAV 107 not so good low yes ...
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