分子病毒学8病毒载体课件

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1、单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,viral vector,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,E

2、x-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,exogeno

3、us genes,are inserted,Generation of viral vector for gene therapy,Replication-,competent,virus,Replication-,defective,virus,Amplicon,:,doesn,t encode structural proteins,Cant replicate beyond the first cycle of infection,Elements needed to generate,amplicon,Transfer Vector:,plasmid,(promoter,gene of

4、 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,

5、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&Pu

6、rification,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,Infectin

7、g 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,

8、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

9、,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,immunothe

10、rapy,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,),

11、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 expr

12、ession,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 enve

13、lope 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

14、 be accommodated,Neurotropic,virus,target to nervous system,Replication defective,amplicon,particles,Comparison of different viral vectors,Viral vectortiters,manupilation,of,immunogenicity,infecting of,tropism non-dividing cells,Adenovirus10,11,terrificvery highyes,Retrovirus10,7,goodlowonly,lentivi

15、rus,Herpesvirus,10,7,not so goodlowyes,AAV10,7,not so goodlowyes,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 coagul

16、ation 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 an