分子病毒学 8病毒载体
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• 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)
• Non-viral vector based
– Naked DNA (plasmid DNA): injection or genegun – Liposomes (cationic lipids): mix with genes
• Ex-vivo • In vivo
2
Why use viral vectors
– 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
Receptors –AAV2 Heparan Sulphate –AAV5 Sialic Acid
6
7
8
9
10
11
Generation of adeno-associated virus vector
12
13
14
15
Characteristics of AAV vector
• Advantages
17
Early generations of adenoviral vector (replication defective)
18
Gutless Adenoviral vector (Amplicon)
19
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
20
Characteristics of adenoviral vector
• Gene replacement: non-essential genes of
virus are deleted and exogenous genes are inserted
3
Generation of viral vector for gene therapy
• Replication-competent virus • Replication-defective virus
– 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
4
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
5
Adeno-Associated Virus
• Single Stranded DNA Virus
–Viral ITRs & Rep / Cap –~ 5kb Capacity
(AAV)
• Integrating / Concatameric
–Long Term Expression
• Complex Production &
Purification –Multi pDNA &/or Helper Virus –Lower Titre Than Adenovirus
• Serotype Differences In
– 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)
viral vector
1
Methods of gene delivery
• Viral Vectors:
– Adenovirus – Retrovirus – Lentivirus – Adeno-associated virus (AAV) – Herpes simplex virus (HSV)
16
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 Biblioteka Baiduommonly used as vectors
host cells
• Specific target cells: depending on the viral
attachment proteins (capsid or glycoproteins)
• Non-viral vector based
– Naked DNA (plasmid DNA): injection or genegun – Liposomes (cationic lipids): mix with genes
• Ex-vivo • In vivo
2
Why use viral vectors
– 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
Receptors –AAV2 Heparan Sulphate –AAV5 Sialic Acid
6
7
8
9
10
11
Generation of adeno-associated virus vector
12
13
14
15
Characteristics of AAV vector
• Advantages
17
Early generations of adenoviral vector (replication defective)
18
Gutless Adenoviral vector (Amplicon)
19
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
20
Characteristics of adenoviral vector
• Gene replacement: non-essential genes of
virus are deleted and exogenous genes are inserted
3
Generation of viral vector for gene therapy
• Replication-competent virus • Replication-defective virus
– 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
4
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
5
Adeno-Associated Virus
• Single Stranded DNA Virus
–Viral ITRs & Rep / Cap –~ 5kb Capacity
(AAV)
• Integrating / Concatameric
–Long Term Expression
• Complex Production &
Purification –Multi pDNA &/or Helper Virus –Lower Titre Than Adenovirus
• Serotype Differences In
– 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)
viral vector
1
Methods of gene delivery
• Viral Vectors:
– Adenovirus – Retrovirus – Lentivirus – Adeno-associated virus (AAV) – Herpes simplex virus (HSV)
16
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 Biblioteka Baiduommonly used as vectors