1、viral vector韓曉Methods of gene deliveryViral Vectors: AdenovirusRetrovirusLentivirusAdeno-associated virus (AAV)Herpes simplex virus (HSV)Non-viral vector basedNaked DNA (plasmid DNA): injection or genegunLiposomes (cationic lipids): mix with genesEx-vivoIn vivoWhy use viral vectorsVirus are obligate

2、 intracellular parasitesVery efficient at transferring viral DNA into host cellsSpecific target cells: depending on the viral attachment proteins (capsid or glycoproteins)Gene replacement: non-essential genes of virus are deleted and exogenous genes are insertedGeneration of viral vector for gene th

3、erapyReplication-competent virus Replication-defective virus Amplicon: doesnt encode structural proteinsCant 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

4、lines): provide the viral structural proteins for packaging of transfer vectorHelper virus (packaging of transfer vector): deleted Packaging signal sequence Adeno-associated virus vectorsNon-pathogenic human parvovirus, non-enveloped ss DNA virus, 4.6 kilobasesDependent on a helper virus ( adenoviru

5、s or herpesvirus) for replication (dependovirus)AAV-2 mostly used for vectorGeneration of adeno-associated virus vectorAdenoviral vectorsNon-enveloped ds DNA, 36 kilobasesEarly proteins (E1A, E1B, E2,E3 and E4), late proteins (L1-L5)Causes a benign respiratory infections in humanSerotypes 2 and 5 ar

6、e commonly used as vectorsEarly generations of adenoviral vector (replication defective) Gutless Adenoviral vector (Amplicon)Modification of the tropism of adenovirus vectorAdenovirus fiber binds to CAR (coxsakie and adenovirus receptor, CAR), receptor which is ubiquitousModify the fiber proteinChar

7、acteristics of adenoviral vectorAdvantages High titersBoth dividing and non-dividing cellsWide tissue tropismEasily modify tissue tropismDisadvantagesTransient expression ( not good for genetic diseases)Highly immunogenicHigh titers of virus can be toxicMore suitable for cancer immunotherapyRetrovir

8、al vectorMoloney murine leukemia virus (MuLV)Generation of replication defective retroviral vectorTransfer plasmid vector:Gene of interestLong terminal repeats(LTR): promoter, polyA, integration, replication and reverse transcriptionPrimer binding site (PBS) (origin of replication)RNA packaging sign

9、alPoly purine tract (important for replication)Packaging vectorCell line stably transfected with plasmid constructs containing Gag/pol and EnvPseudotyped retroviral vectorLentiviral vectorsInfection of non-dividing cells (hepatocytes, neurons)HIV, a human lethal pathogenDelete accessory genesProvide

10、 an envelope from a non-retrovirus (VSV)Develop vectors from lentiviruses of non-human pathogensSIV, FIV, EIAV etcHerpesvirus vectorsHerpes simplex virus 1, mild disease in human, no riskLinear ds DNA, 152 kb, about half of the total 81 genes are non-essential for virus replication40-50 kb of foreig

11、n DNA can be accommodatedNeurotropic virus, target to nervous systemReplication defective amplicon particlesComparison of different viral vectorsViral vectortitersmanupilation of immunogenicityinfecting of tropism non-dividing cellsAdenovirus1011terrificvery highyesRetrovirus107goodlowonly lentiviru

12、sHerpesvirus107not so goodlowyesAAV107not so goodlowyesGene therapy Gene therapy: to correct a genetic defect by transferring of a functional normal copy of the gene into cellsExamples of diseases caused by genetic defectOrnithine transcarbamylase (OTC deficiency)Hemophilia (blood coagulation factor

13、s VIII or IX)SCID( severe combined immunodeficiency)Muscular dystrophy Cystic fibrosisSickle cell anemia Application of gene therapyGenetic disorder (deficiency): OTCCancer Genetic predispositionMutation in oncogene or tumor suppressor geneAutoimmunity diseases: rheumatoid arthritisDelivery of count

14、eracting geneDiseases involve several genes and the environmental interact: diabetesFactors to be considered in Gene therapyHow 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 del

15、ivery 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 GelsingerLiver disease: OTC deficiency (genetic disease)University of PennsylvaniaHigh dose of adenoviral vector (E1 and E4 genesdelet

16、ed ) carrying the normal copy of OTC gene was administeredSuspected 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 vectorSCID disease or Bubble boy disease ( T cell deficiency)Overall quite successful, over 1000 peoples received retroviral gene therapy A French babys treated with retroviral vector 3 years agoA leukemia-like illness developed this summer.Nine other children treated