分子病毒 8病毒载体.ppt

* 韩 晓 Methods of gene delivery Viral Vectors: Adenovirus Retrovirus Lentivirus Adeno-associated virus (AAV) Herpes simplex virus (HSV) Non-viral vector based Naked DNA (plasmid DNA): injection or genegun Liposomes (cationic lipids): mix with genes Ex-vivo 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: non-essential genes of virus are deleted and exogenous genes are inserted Generation of viral vector for gene therapy Replication-competent virus Replication-defective 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 Adeno-associated virus vectors Non-pathogenic human parvovirus, non-enveloped ss DNA virus, 4.6 kilobases Dependent on a helper virus ( adenovirus or herpesvirus) for replication (dependovirus) AAV-2 mostly used for vector  Adeno-Associated Virus (AAV) Single Stranded DNA Virus Viral ITRs Rep / Cap ~ 5kb Capacity Integrating / Concatameric Long Term Expression Complex Production Purification Multi pDNA /or Helper Virus Lower Titre Than Adenovirus Serotype Differences In Receptors AAV2 Heparan Sulphate AAV5 Sialic Acid Generation of adeno-associated 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 Adenoviral vectors Non-enveloped ds DNA, 36 kilobases Early proteins (E1A, E1B, E2,E3 and E4),