單擊此處編輯母版標(biāo)題樣式,單擊此處編輯母版文本樣式,第二級(jí),第三級(jí),第四級(jí),第五級(jí),,,*,,viral vector,韓 曉,,,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), late proteins (L1-L5),Causes a,benign respiratory infections,in human,Serotypes 2 and 5,are commonly used as vectors,,,,Early generations of adenoviral vector (replication defective),,,,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 non-dividing 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,,,,Lentiviral vectors,Infection of non-dividing cells (hepatocytes, neurons),HIV, a human lethal pathogen,Delete accessory genes,Provide an envelope from a non-retrovirus (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 non-essential for virus replication,40-50 kb of foreign DNA,can be accommodated,Neurotropic virus,, target to nervous system,Replication defective amplicon particles,,,Comparison of different viral vectors,Viral vector titers manupilation of immunogenicity infecting of,tropism non-dividing cells,Adenovirus 10,11,terrific very high yes,Retrovirus 10,7,good low only lentivirus,Herpesvirus 10,7,not so good low yes,AAV 10,7,not so good low yes,,,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,Genetic predisposition,Mutation in oncogene or tumor suppressor gene,Autoimmunity diseases: rheumatoid arthritis,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?,,,,Death of 18-year 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),,,,A case of leukemia in a SCID child treated with a retroviral vector,SCID disease,or,‘Bubble boy disease’ ( T cell deficiency),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,,,,謝謝,!,,,