分子病毒学病毒载体.pptxVIP

Methods of gene delivery第1页/共37页Viral Vectors:AdenovirusRetrovirusLentivirusAdeno-associated virus (AAV)Herpes simplex virus (HSV)Non-viral vector based Naked DNA (plasmid DNA): injection or genegunLiposomes (cationic lipids): mix with genesEx-vivoIn vivo Why use viral vectors第2页/共37页Virus are obligate intracellular parasites Very efficient at transferring viral DNA into host cells Specific target cells: depending on the viral attachment proteins ( capsid orglycoproteins) Gene replacement: non- essential genes of virus are deleted and exogenous genes are inserted Generation of viral vector for gene therapyReplication-competent virusReplication-defective virus Amplicon: doesn’t encode structural proteins Can’t replicate beyond the first cycle of infectionElements 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 vectorHelper virus (pa第c3k页a/共g3i7页ng of transfer vector): Adeno-associated virus vectors第4页/共37页 Non- pathogenic human parvovirus, non- enveloped ss DNA virus, 4 . 6 kilobasesDependent on a helper virus ( adenovirus orherpesvirus) for replication ( dependovirus)AAV- 2 mostly used for vector Adeno-Associated Virus(AAV)Single Stranded DNA VirusViral ITRs Rep / Cap~ 5kb CapacityIntegrating /ConcatamericLong Term ExpressionComplex Production PurificationMulti pDNA /or Helper VirusLower Titre Than Adenovirus第5页/共37页 第6页/共37页 第7页/共37页 第8页/共37页 第9页/共37页 第10页/共37页 Generation of adeno-associated virus vector第11页/共37页 第12页/共37页 第13页/共37页 第14页/共37页 Characteristics of AAV vector第15页/共37页AdvantagesIntegration and persistent expressionNo insertional mutagenesisInfecting dividing and nondividing cellsSafeDisadvantagesSize limitation, 4.9 kb Low titer of virus, low level of gene expression Adenoviral vectors第16页/共37页Non-enveloped ds DNA, 36 kilobases Early proteins (E1A, E1B, E2,E3 and E4), late proteins (L1-L5) Cau