分子病毒学-8病毒载体.ppt

1、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 Ex-vivo In vivo,Why use viral vectors,Viru

2、s 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

3、vector for gene therapy,Replication-competent virus Replication-defective virus Amplicon: doesnt encode structural proteins Cant replicate beyond the first cycle of infection Elements needed to generate amplicon Transfer Vector: plasmid (promoter, gene of interest, ori, packaging signal) Packaging v

4、ector (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

5、 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 V

6、irus 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 Saf

7、e 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

8、,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 adenov

9、iral 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,M

10、oloney 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 P

11、oly 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 vi

12、rus 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-ret

13、rovirus (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 accommod

14、ated Neurotropic virus, target to nervous system Replication defective amplicon particles,Comparison of different viral vectors,Viral vectortitersmanupilation of immunogenicityinfecting of tropism non-dividing cells Adenovirus1011terrificvery highyes Retrovirus107goodlowonly lentivirus Herpesvirus10

15、7not so goodlowyes AAV107not 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 coagulation factors VIII or

16、 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 counte

17、racting 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

18、 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 (