分子病毒学病毒载体优秀课件
合集下载
相关主题
- 1、下载文档前请自行甄别文档内容的完整性，平台不提供额外的编辑、内容补充、找答案等附加服务。
- 2、"仅部分预览"的文档,不可在线预览部分如存在完整性等问题,可反馈申请退款(可完整预览的文档不适用该条件!)。
- 3、如文档侵犯您的权益，请联系客服反馈,我们会尽快为您处理(人工客服工作时间：9:00-18:30)。
– 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
.
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 commonly used as vectors
• 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
.
17
Early generations of adenoviral vector (replication defective)
.
18
Gutless Adenoviral vector (Amplicon)
.
19
Modification of the tropism of adenovirus vector
viral vector
韩晓
.
1
Methods of gene delivery
• Viral Vectors:
– Adenovirus – Retrovirus – Lentivirus – Adeno-associated virus (AAV) – Herpes simplex virus (HSV)
(AAV)
• Integrating / Concatameric
–Long Term Expression
• Complex Production &
Purification –Multi pDNA &/or Helper Virus –Lower Titre Than Adenovirus
• Serotype Differences In
herpesvirus) for replication (dependovirus)
• AAV-2 mostly used for vector
.
5
Adeno-Associated Virus
• Single Stranded DNA Virus
–Viral ITRs & Rep / Cap –~ 5kb Capacity
• Advantages
– 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
.
4
Biblioteka Baidu
Adeno-associated virus vectors
• Non-pathogenic human parvovirus, non-
enveloped ss DNA virus, 4.6 kilobases
• Dependent on a helper virus ( adenovirus or
• Adenovirus fiber binds to CAR (coxsakie and
adenovirus receptor, CAR), receptor which is ubiquitous
• Modify the fiber protein
.
20
Characteristics of adenoviral vector
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
• 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
– 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)
– Helper virus (packaging of transfer vector): deleted Packaging signal sequence
.
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 commonly used as vectors
• 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
.
17
Early generations of adenoviral vector (replication defective)
.
18
Gutless Adenoviral vector (Amplicon)
.
19
Modification of the tropism of adenovirus vector
viral vector
韩晓
.
1
Methods of gene delivery
• Viral Vectors:
– Adenovirus – Retrovirus – Lentivirus – Adeno-associated virus (AAV) – Herpes simplex virus (HSV)
(AAV)
• Integrating / Concatameric
–Long Term Expression
• Complex Production &
Purification –Multi pDNA &/or Helper Virus –Lower Titre Than Adenovirus
• Serotype Differences In
herpesvirus) for replication (dependovirus)
• AAV-2 mostly used for vector
.
5
Adeno-Associated Virus
• Single Stranded DNA Virus
–Viral ITRs & Rep / Cap –~ 5kb Capacity
• Advantages
– 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
.
4
Biblioteka Baidu
Adeno-associated virus vectors
• Non-pathogenic human parvovirus, non-
enveloped ss DNA virus, 4.6 kilobases
• Dependent on a helper virus ( adenovirus or
• Adenovirus fiber binds to CAR (coxsakie and
adenovirus receptor, CAR), receptor which is ubiquitous
• Modify the fiber protein
.
20
Characteristics of adenoviral vector
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
• 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
– 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)