病毒载体-2014-10
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Βιβλιοθήκη Baidu
病毒载体
病毒载体
Gene therapy
• Recent years for several types of vectors, particularly viral vectors, which have been used in 70% of gene therapy clinical trials as of January 2007
interest on four types • retroviruses (lentiviruses) • adenoviruses • adeno-associated viruses • herpes simplex virus type
Retrovirus Vectors
• • • • a class of enveloped viruses ssRNA molecule as the genome,10kb integrates into the host genome three genes: gag (coding for core proteins), pol (coding for reverse transcriptase) & env (coding for the viral envelope protein) • long terminal repeats (LTRs) • packaging sequences
Cloning Gene of Interest Homologous Recombination in vivo in Bacteria Virus Production in AD-293 Cells
Recombinant Adenovirus Construction
Step 1:Cloning a Gene of Interest (GOI) into Shuttle Vectors: • Sub-cloning the GOI into the appropriate intermediary, shuttle vector. • Confirmation through restriction digestion. Step 2: Transfer the expression cassette into the Adenovirus Vector: • Transferring the entire expression cassette into the large, adenovirus genome vector. • Confirmation through restriction mapping.
TRE: Tet-Response Element. A regulatory sequence consisting of seven direct repeats of a 42-bp sequence that contains the tetO.
靶向性优化
Viruses as Vectors
• non-enveloped viruses • a linear double stranded DNA genome • over 40 serotype strains of adenovirus( subgroup C serotypes 2 or 5 for the vectors) • not involve integration into the host genome
• produced at high titres (>1011/ml) • very efficient at transducing target cells in vitro & vivo • intravenous injection, 90% of the vector is degraded in the liver • the majority of problems for clinical trials will be degraded & presented to the immune system
将病毒cDNA经适当改造的载体： 5′LTR / 3′LTR 包装信号（ψ序列） Neo基因/ Ampr 酶切位点（多克隆位点）
包装细胞(packaging cell)
– 将缺失了包装信号及相关序列的缺陷型逆转录病毒导 入哺乳动物细胞，产生大量病毒包装蛋白。NIH/3T3， ψ—2，PA317，ψCRIP – 逆转录病毒载体导入包装细胞后，载体转录出病毒基 因组的部分序列、标记基因、外源基因，被包装成病 毒颗粒。 – 缺陷型的逆转录病毒颗粒 – 一过性感染 – 病毒滴度106CFU/ml以上
定义 临床应用 基本过程 载体优化 常用载体
病毒载体
载体(vector)
• 是携带外源DNA 进入宿主细胞进行 扩增和表达的DNA,它们一般是通过 改造质粒、噬菌体或病毒等构建的。
载体应具备以下条件：
• １、 能在适当的宿主细胞中复制； • ２、 具有多种限制酶的单一切点（即所谓多克隆 位点）以便外源DNA 插入； • ３、 具有筛选标志以区别阳性与阴性重组分子； • ４、 载体分子较小，以便体外基因操作，同时载 体DNA 与宿主DNA 便于分离； • ５、 对于表达型载体还应具有与宿主细胞相适应 的启动子、增强子、加尾信号等基因表达元件。
Viral Vectors 病毒载体
Institute of Molecular Biology, CTGU 刘朝奇
2014-10
病毒学基本概念
病毒基因组
基因组（genome） 1个生殖细胞（精子或卵子），1 个单倍体细胞或1个病毒所包含的全套基因。病毒核酸或为 DNA或为RNA，可以统称为病毒染色体。 完整的病毒颗粒具有蛋白质外壳，以保护病毒核酸不 受核酸酶的破坏，并能识别和侵袭特定的宿主。类病毒例 外，它是一类植物病毒，为很小的单链闭环RNA（250-400 碱基）。
• selectable markers, such as neomycin & beta galactosidase, can be included • The available carrying capacity for retroviral vectors is approximately 7.5 kb • Altering the env gene or its product has proved a successful means of manipulating the cell range
• Tissue specific expression was by using cellular promoter/enhancers e.g. the myosin light chain 1 promoter • fibre coat protein in the adenovirus binding to MHC class I molecule • Increased expression of integrin, heparin sulphate receptors can increase viral uptake
www.clontech.com
Tet-Off and Tet-On Gene Expression Systems
原核生物体内的四环素阻碍蛋白(Tet repressor，Tet R) / 真核细胞内转录因子的 转录激活结构域 (单纯疱疹病毒编码的VPl6 的酸性结构域) 两部分构成的融合蛋白(tTA或rtTA)可以受 四环素或者其衍生物的调节而发生构象变 化，从而改变与相应的DNA序列(四环素反 应元件， Tet-Response Element ，TRE) 的结合能力
Moloney murine leukaemia virus (Mo-MLV)
• amphotrophic virus • The essential regions include the 5' & 3' LTRs & the packaging sequence lying downstream of the 5' LTR. • Transgene expression can either be driven by the promoter/enhancer region in the 5' LTR, or by alternative viral (e.g. cytomegalovirus, Rous sarcoma virus) or cellular (e.g. beta actin, tyrosine) promoters
病毒核酸与所有的原、真核生物的核酸组比较，最为 突出的特点是每种病毒颗粒只含1种核酸，或DNA或RNA， 两者不共存于1种病毒颗粒中，据此，病毒可以分成两组， 即DNA病毒和RNA病毒。 病毒核酸分子量大小：RNA病毒小106-107D，DNA病毒 大；107-108D 基因数：3-几百个
• • • • •
逆转录病毒载体的特点
• 缺陷型的逆转录病毒感染细胞 • RNA进入靶细胞后，变成前病毒并整合到 宿主细胞的染色体上，可稳定表达 • 只能感染处于增殖期的细胞
安全性问题
• • • • • • 逆转录病毒感染的可能性 污染的可能性 逆转录病毒在靶细胞基因组上的整合 破坏细胞正常生长的必须基因/抑癌基因 LTR激活原癌基因 染色体重排激活原癌基因
Advantages
High Transduction Effciency Insert Size up to 8kB Integrates into host genome resulting in sustained expression of vector Extremely well studied system Vector proteins not expressed in host
优点
• Broad range of infectivity and high titer • Infection does not require an actively dividing host cell • Expressed human proteins are properly folded and modified • Large insert size • AdEasy vector is non-insertional
Obstacles to systemic targeting
• The first: Reactions with the complement system and pre-existing antibodies • The next: the endothelial cell layer • Additional: the stroma and the basalmembrane-like structure to reach the tumor
柯萨奇腺病毒受体(coxsackie adenovirus receptor, CAR)
• • • •
The wild type approximately 35 kb up to 30 kb replaced with foreign DNA four early transcriptional units (E1, E2, E3 & E4) only the inverted terminal repeats (ITRs) & a packaging sequence around the transgene • all the necessary viral genes being provided in trans by a helper virus
Disadvantages
Low Titers (106- 107) Integration is random In vivo delivery remains poor. Effective only when infecting helper cell lines
Adenovirus Vectors
病毒载体
病毒载体
Gene therapy
• Recent years for several types of vectors, particularly viral vectors, which have been used in 70% of gene therapy clinical trials as of January 2007
interest on four types • retroviruses (lentiviruses) • adenoviruses • adeno-associated viruses • herpes simplex virus type
Retrovirus Vectors
• • • • a class of enveloped viruses ssRNA molecule as the genome,10kb integrates into the host genome three genes: gag (coding for core proteins), pol (coding for reverse transcriptase) & env (coding for the viral envelope protein) • long terminal repeats (LTRs) • packaging sequences
Cloning Gene of Interest Homologous Recombination in vivo in Bacteria Virus Production in AD-293 Cells
Recombinant Adenovirus Construction
Step 1:Cloning a Gene of Interest (GOI) into Shuttle Vectors: • Sub-cloning the GOI into the appropriate intermediary, shuttle vector. • Confirmation through restriction digestion. Step 2: Transfer the expression cassette into the Adenovirus Vector: • Transferring the entire expression cassette into the large, adenovirus genome vector. • Confirmation through restriction mapping.
TRE: Tet-Response Element. A regulatory sequence consisting of seven direct repeats of a 42-bp sequence that contains the tetO.
靶向性优化
Viruses as Vectors
• non-enveloped viruses • a linear double stranded DNA genome • over 40 serotype strains of adenovirus( subgroup C serotypes 2 or 5 for the vectors) • not involve integration into the host genome
• produced at high titres (>1011/ml) • very efficient at transducing target cells in vitro & vivo • intravenous injection, 90% of the vector is degraded in the liver • the majority of problems for clinical trials will be degraded & presented to the immune system
将病毒cDNA经适当改造的载体： 5′LTR / 3′LTR 包装信号（ψ序列） Neo基因/ Ampr 酶切位点（多克隆位点）
包装细胞(packaging cell)
– 将缺失了包装信号及相关序列的缺陷型逆转录病毒导 入哺乳动物细胞，产生大量病毒包装蛋白。NIH/3T3， ψ—2，PA317，ψCRIP – 逆转录病毒载体导入包装细胞后，载体转录出病毒基 因组的部分序列、标记基因、外源基因，被包装成病 毒颗粒。 – 缺陷型的逆转录病毒颗粒 – 一过性感染 – 病毒滴度106CFU/ml以上
定义 临床应用 基本过程 载体优化 常用载体
病毒载体
载体(vector)
• 是携带外源DNA 进入宿主细胞进行 扩增和表达的DNA,它们一般是通过 改造质粒、噬菌体或病毒等构建的。
载体应具备以下条件：
• １、 能在适当的宿主细胞中复制； • ２、 具有多种限制酶的单一切点（即所谓多克隆 位点）以便外源DNA 插入； • ３、 具有筛选标志以区别阳性与阴性重组分子； • ４、 载体分子较小，以便体外基因操作，同时载 体DNA 与宿主DNA 便于分离； • ５、 对于表达型载体还应具有与宿主细胞相适应 的启动子、增强子、加尾信号等基因表达元件。
Viral Vectors 病毒载体
Institute of Molecular Biology, CTGU 刘朝奇
2014-10
病毒学基本概念
病毒基因组
基因组（genome） 1个生殖细胞（精子或卵子），1 个单倍体细胞或1个病毒所包含的全套基因。病毒核酸或为 DNA或为RNA，可以统称为病毒染色体。 完整的病毒颗粒具有蛋白质外壳，以保护病毒核酸不 受核酸酶的破坏，并能识别和侵袭特定的宿主。类病毒例 外，它是一类植物病毒，为很小的单链闭环RNA（250-400 碱基）。
• selectable markers, such as neomycin & beta galactosidase, can be included • The available carrying capacity for retroviral vectors is approximately 7.5 kb • Altering the env gene or its product has proved a successful means of manipulating the cell range
• Tissue specific expression was by using cellular promoter/enhancers e.g. the myosin light chain 1 promoter • fibre coat protein in the adenovirus binding to MHC class I molecule • Increased expression of integrin, heparin sulphate receptors can increase viral uptake
www.clontech.com
Tet-Off and Tet-On Gene Expression Systems
原核生物体内的四环素阻碍蛋白(Tet repressor，Tet R) / 真核细胞内转录因子的 转录激活结构域 (单纯疱疹病毒编码的VPl6 的酸性结构域) 两部分构成的融合蛋白(tTA或rtTA)可以受 四环素或者其衍生物的调节而发生构象变 化，从而改变与相应的DNA序列(四环素反 应元件， Tet-Response Element ，TRE) 的结合能力
Moloney murine leukaemia virus (Mo-MLV)
• amphotrophic virus • The essential regions include the 5' & 3' LTRs & the packaging sequence lying downstream of the 5' LTR. • Transgene expression can either be driven by the promoter/enhancer region in the 5' LTR, or by alternative viral (e.g. cytomegalovirus, Rous sarcoma virus) or cellular (e.g. beta actin, tyrosine) promoters
病毒核酸与所有的原、真核生物的核酸组比较，最为 突出的特点是每种病毒颗粒只含1种核酸，或DNA或RNA， 两者不共存于1种病毒颗粒中，据此，病毒可以分成两组， 即DNA病毒和RNA病毒。 病毒核酸分子量大小：RNA病毒小106-107D，DNA病毒 大；107-108D 基因数：3-几百个
• • • • •
逆转录病毒载体的特点
• 缺陷型的逆转录病毒感染细胞 • RNA进入靶细胞后，变成前病毒并整合到 宿主细胞的染色体上，可稳定表达 • 只能感染处于增殖期的细胞
安全性问题
• • • • • • 逆转录病毒感染的可能性 污染的可能性 逆转录病毒在靶细胞基因组上的整合 破坏细胞正常生长的必须基因/抑癌基因 LTR激活原癌基因 染色体重排激活原癌基因
Advantages
High Transduction Effciency Insert Size up to 8kB Integrates into host genome resulting in sustained expression of vector Extremely well studied system Vector proteins not expressed in host
优点
• Broad range of infectivity and high titer • Infection does not require an actively dividing host cell • Expressed human proteins are properly folded and modified • Large insert size • AdEasy vector is non-insertional
Obstacles to systemic targeting
• The first: Reactions with the complement system and pre-existing antibodies • The next: the endothelial cell layer • Additional: the stroma and the basalmembrane-like structure to reach the tumor
柯萨奇腺病毒受体(coxsackie adenovirus receptor, CAR)
• • • •
The wild type approximately 35 kb up to 30 kb replaced with foreign DNA four early transcriptional units (E1, E2, E3 & E4) only the inverted terminal repeats (ITRs) & a packaging sequence around the transgene • all the necessary viral genes being provided in trans by a helper virus
Disadvantages
Low Titers (106- 107) Integration is random In vivo delivery remains poor. Effective only when infecting helper cell lines
Adenovirus Vectors