分子病毒学病毒载体优秀课件

2、DNA (plasmid DNA): injection or genegunNaked DNA (plasmid DNA): injection or genegun Liposomes (cationic lipids): mix with genesLiposomes (cationic lipids): mix with genesEx-vivoEx-vivoIn vivoIn vivo2 2. .Why use viral vectorsVirus are Virus are obligate intracellular parasitesobligate intracellular

3、 parasitesVery Very efficient at transferring viral DNA into efficient at transferring viral DNA into host cellshost cellsSpecific target cellsSpecific target cells: depending on the viral : depending on the viral attachment proteins (capsid or glycoproteins)attachment proteins (capsid or glycoprote

4、ins)Gene replacementGene replacement: non-essential genes of : non-essential genes of virus are deleted and virus are deleted and exogenous genesexogenous genes are are insertedinserted3 3. .Generation of viral vector for gene therapyReplication-Replication-competentcompetent virus virus Replication

5、-Replication-defective defective virus virus Amplicon: doesnt encode structural proteins Amplicon: doesnt encode structural proteins Cant replicate beyond the first cycle of infectionCant replicate beyond the first cycle of infectionElements needed to generate amplicon Elements needed to generate am

6、plicon Transfer Vector: plasmid (promoter, gene of interest, Transfer Vector: plasmid (promoter, gene of interest, oriori, , packaging signal)packaging signal) Packaging vector (cosmid or cell lines): Packaging vector (cosmid or cell lines): provide the provide the viral structural proteins for pack

7、aging of transfer viral structural proteins for packaging of transfer vectorvector Helper virus (packaging of transfer vector): Helper virus (packaging of transfer vector): deleted deleted Packaging signal sequencePackaging signal sequence 4 4. .Adeno-associated virus vectorsNon-pathogenic human par

8、vovirusNon-pathogenic human parvovirus, non-, non-enveloped ss DNA virus, 4.6 kilobasesenveloped ss DNA virus, 4.6 kilobasesDependent on a helper virus ( adenovirus or Dependent on a helper virus ( adenovirus or herpesvirus) for replication (dependovirus)herpesvirus) for replication (dependovirus)AA

9、V-2 mostly used for vectorAAV-2 mostly used for vector5 5. .Adeno-Associated Virus (AAV)Single Stranded DNA VirusSingle Stranded DNA Virus Viral ITRs & Rep / CapViral ITRs & Rep / Cap 5kb Capacity 5kb CapacityIntegrating / ConcatamericIntegrating / Concatameric Long Term ExpressionLong Term Expressi

10、onComplex Production & Complex Production & PurificationPurification Multi pDNA &/or Helper VirusMulti pDNA &/or Helper Virus Lower Titre Than AdenovirusLower Titre Than AdenovirusSerotype Differences In Serotype Differences In ReceptorsReceptors AAV2 Heparan SulphateAAV2 Heparan Sulphate AAV5 Siali

11、c Acid AAV5 Sialic Acid 6 6. .7 7. .8 8. .9 9. .1010. .1111. .Generation of adeno-associated virus vector1212. .1313. .1414. .1515. .Characteristics of AAV vectorAdvantagesAdvantages Integration and persistent expressionIntegration and persistent expression No insertional mutagenesisNo insertional m

12、utagenesis Infecting dividing and nondividing cellsInfecting dividing and nondividing cells SafeSafeDisadvantagesDisadvantages Size limitation, 4.9 kbSize limitation, 4.9 kb Low titer of virus, low level of gene expressionLow titer of virus, low level of gene expression1616. .Adenoviral vectorsNon-e

13、nveloped ds DNA, 36 kilobasesNon-enveloped ds DNA, 36 kilobasesEarly proteins (E1A, E1B, E2,E3 and E4), late proteins Early proteins (E1A, E1B, E2,E3 and E4), late proteins (L1-L5)(L1-L5)Causes a Causes a benign respiratory infectionsbenign respiratory infections in human in humanSerotypes 2 and 5Se

14、rotypes 2 and 5 are commonly used as vectors are commonly used as vectors1717. .Early generations of adenoviral vector (replication defective) 1818. .Gutless Adenoviral vector (Amplicon)1919. .Modification of the tropism of adenovirus vectorAdenovirus Adenovirus fiberfiber binds to CAR (coxsakie and

15、 binds to CAR (coxsakie and adenovirus receptor, CAR), receptor which is adenovirus receptor, CAR), receptor which is ubiquitousubiquitousModify the fiber proteinModify the fiber protein2020. .Characteristics of adenoviral vectorAdvantages Advantages High titersHigh titers Both dividing and non-divi

16、ding cellsBoth dividing and non-dividing cells Wide tissue tropismWide tissue tropism Easily modify tissue tropismEasily modify tissue tropismDisadvantagesDisadvantages Transient expression ( not good for genetic diseases)Transient expression ( not good for genetic diseases) Highly immunogenicHighly

17、 immunogenic High titers of virus can be toxicHigh titers of virus can be toxic More suitable for cancer immunotherapyMore suitable for cancer immunotherapy2121. .Retroviral vectorMoloney murine leukemia virus (MuLV)Moloney murine leukemia virus (MuLV)Generation of replication defective retroviral v

18、ectorGeneration of replication defective retroviral vector Transfer plasmid vector:Transfer plasmid vector: Gene of interestGene of interest Long terminal repeats(LTR): Long terminal repeats(LTR): promoter, polyA, integrationpromoter, polyA, integration, , replication and reverse transcriptionreplic

19、ation and reverse transcription Primer binding site (PBS) (Primer binding site (PBS) (origin of replicationorigin of replication) ) RNA packaging signalRNA packaging signal Poly purine tract (important for replication)Poly purine tract (important for replication) Packaging vectorPackaging vector Cel

20、l line stably transfected with plasmid constructs Cell line stably transfected with plasmid constructs containing Gag/pol and Envcontaining Gag/pol and Env2222. .Generation of retroviral vector2323. .Pseudotyped retroviral vector2424. .2525. .Characteristics of retroviral vectorAdvantagesAdvantages

21、Integration: permanent expressionIntegration: permanent expression Pseudotyped virusPseudotyped virusDisadvantagesDisadvantages Only infecting dividing cellsOnly infecting dividing cells Insertional mutagenesis (tumor formation)Insertional mutagenesis (tumor formation)Activate oncogenesActivate onco

22、genesInhibit tumor suppressor genesInhibit tumor suppressor genes2626. .Lentiviral vectorsInfection of non-dividing Infection of non-dividing cells (hepatocytes, cells (hepatocytes, neurons)neurons)HIV, a human lethal HIV, a human lethal pathogenpathogen Delete accessory Delete accessory genesgenes

23、Provide an envelope Provide an envelope from a non-retrovirus from a non-retrovirus (VSV)(VSV) Develop vectors from Develop vectors from lentiviruses of non-lentiviruses of non-human pathogenshuman pathogensSIV, FIV, EIAV etcSIV, FIV, EIAV etc2727. .2828. .2929. .Herpesvirus vectorsHerpes simplex vi

24、rus 1, Herpes simplex virus 1, mild diseasemild disease in human, no in human, no riskriskLinear ds DNA, 152 kb, about half of the total 81 Linear ds DNA, 152 kb, about half of the total 81 genes are non-essential for virus replicationgenes are non-essential for virus replication40-50 kb of foreign

25、DNA40-50 kb of foreign DNA can be accommodated can be accommodatedNeurotropic virusNeurotropic virus, target to nervous system, target to nervous systemReplication defective amplicon particlesReplication defective amplicon particles3030. .Comparison of different viral vectorsViral vectortitersmanupi

26、lation of immunogenicityinfecting of tropism non-dividing cellsAdenovirus1011terrificvery highyesRetrovirus107goodlowonly lentivirusHerpesvirus107not so goodlowyesAAV107not so goodlowyes3131. .Gene therapy Gene therapy: Gene therapy: to correct a to correct a genetic defectgenetic defect by transfer

27、ring of a by transferring of a functional normal copyfunctional normal copy of the gene into cells of the gene into cellsExamples of diseases caused by genetic defectExamples of diseases caused by genetic defect Ornithine transcarbamylase (OTC deficiency)Ornithine transcarbamylase (OTC deficiency) H

28、emophilia (blood coagulation factors VIII or IX)Hemophilia (blood coagulation factors VIII or IX) SCID( severe combined immunodeficiency)SCID( severe combined immunodeficiency) Muscular dystrophy Muscular dystrophy Cystic fibrosisCystic fibrosis Sickle cell anemia Sickle cell anemia 3232. .Applicati

29、on of gene therapyGenetic disorder (deficiency): OTCGenetic disorder (deficiency): OTCCancer Cancer Genetic predispositionGenetic predisposition Mutation in oncogene or tumor suppressor geneMutation in oncogene or tumor suppressor geneAutoimmunity diseases: rheumatoid arthritisAutoimmunity diseases:

30、 rheumatoid arthritis Delivery of counteracting geneDelivery of counteracting geneDiseases involve several genes and the Diseases involve several genes and the environmental interact: diabetes environmental interact: diabetes3333. .Factors to be considered in Gene therapyHow to deliver genes to spec

31、ific cells, tissue and whole How to deliver genes to specific cells, tissue and whole animals? (animals? (methods of deliverymethods of delivery) )How much and how long the introduced gene will be How much and how long the introduced gene will be expressed?expressed?The site and dose of gene deliver

32、y The site and dose of gene delivery Is there any Is there any adverse immunological consequenceadverse immunological consequence of both of both delivery vehicle (Virus) and the gene in animals?delivery vehicle (Virus) and the gene in animals? Is there any toxic effects? Is there any toxic effects?

33、3434. . Death of 18-year old Jesse Gelsinger Liver disease: OTC deficiency (genetic disease)Liver disease: OTC deficiency (genetic disease) University of PennsylvaniaUniversity of Pennsylvania High dose of adenoviral vector (E1 and E4 genesHigh dose of adenoviral vector (E1 and E4 genesdeleted ) car

34、rying the normal copy of OTC gene was deleted ) carrying the normal copy of OTC gene was administeredadministered Suspected cause of deathSuspected cause of death-Toxicity of high titer adenoviral vector-Toxicity of high titer adenoviral vector-High -High immunogenicityimmunogenicity of adenoviral v

35、ector (an immune of adenoviral vector (an immune revolt)revolt)3535. .A case of leukemia in a SCID child treated with a retroviral vectorSCID diseaseSCID disease or or Bubble boy disease ( T cell deficiency)Bubble boy disease ( T cell deficiency)Overall quite successful, over 1000 peoples received r

36、etroviral Overall quite successful, over 1000 peoples received retroviral gene therapy gene therapy A French babys treated with retroviral vector 3 years agoA French babys treated with retroviral vector 3 years agoA leukemia-like illness developed this summer.A leukemia-like illness developed this s

37、ummer.Nine other children treated same time show no sign of Nine other children treated same time show no sign of leukemialeukemiaBut the side effect isnt a big enough risk yet that genetic But the side effect isnt a big enough risk yet that genetic experiments for children with an often fatal immun

38、e disease experiments for children with an often fatal immune disease should stopshould stopPeople receiving retroviral gene therapy should be warned People receiving retroviral gene therapy should be warned about the risk of developing leukemiaabout the risk of developing leukemia3636. .谢谢谢谢!3737. .

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