重组腺病毒理论课件

Protocols for Recombinant Adenovirus 腺病毒基因工程的实验方法学 范雄林, Ph.D, Prof. Email: Tel:I. Introduction of Gene Transfer 基因转移的简介 Gene transfer (基因转转移) is an invaluable tool routinely used in vitro or in animal models to investigate the molecular mechanisms underlying diverse biological functions lin vivo gene transfer(体内基因转移): is to insert a gene into an organism using a vector (that will transfer only the required gene to the desired target cells). lgene therapy(基因治疗): which aims to use genes as therapeutic entities in humans (is the ultimate goal of gene transfer). The goal is to deliver genetic information to a target cell, either to replace a defective function (monogenic disease), or to introduce an additional function to treat (as in cancer) or to prevent (as in a vaccine) disease Aims of Gene transfer lex vivo gene therapy(体外基因治疗): target cells are first extracted from the patient. The desired gene is then inserted into these cells, and once the transfer is completed, the cells are returned to the patient. This technique has had promising results, but is restricted to a limited number of target cell types and diseases (Aiuti et al., 2002; Hacein-Bey-Abina et al., 2002). lin vivo gene therapy(体内基因治疗): the vector has to be able to deliver the selected gene directly into the target cells within the whole organism. The introduction of the therapeutic gene into the target cell can be achieved in two ways: ex vivo or in vivo II. Overview of gene therapy clinical trials worldwide 基因治疗的临床现状 Number of trials initiated per year Since 1990, 1500 clinical trials Someday people will look back on the era before gene therapy in the same way we look back on the era before antibiotics and vaccines. It is now possible to think about treating a whole series of diseases with a one-shot therapy that would last a lifetime. Dr. Rochelle Hirschhorn (Professor of Medicine, New York University) Ashanti was the first patient to be treated with gene therapy. She received infusions of T cells that had been transduced with a gene for ADA (an enzyme that she lacks), resulting in an amelioration of the symptoms of her severe combined immunodeficiency(SCID). Ashanti de Silva, a 4-year-old girl received 11 infusions from September 1990 to August 1992 Ex vivo gene transfer to bone marrow-derived CD34+ hematopoietic stem cells (HSCs) of a patient with inherited severe immune deficiency. Autologous HSCs are cultured and infected with a recombinant retroviral or lentiviral vector carrying a functional copy of the defective gene (for example, ADA, gamma-c, or ABCD1). The gene-corrected cells are then injected back into the patient. Eight of ten children have essentially been cured in an Italian trial. For the past 8 years, these pediatric patients no longer required enzyme replacement therapy, they respond well to vaccination, and they live normal lives, attending school instead of living as “bubble” boys or girls Retroviral transduction of HSC continues to show high therapeutic efficacy in ADA-SCID without the development of leukemia (Aiuti et al., 2009; Aiuti et al., 2002). In vivo gene transfer for ocular diseases. A. An adeno-associated viral (AAV) vector is injected into the subretinal space using a surgical procedure. B. Gene transfer of RPE-65 (色素表皮细胞特异的65kDa蛋白)to the retina restored light sensitivity in patients with Lebers congenital amaurosis (LCA) (from Proc Natl Acad Sci USA 105(39):15112-7,2008. C. A similar protocol for AAV-mediated L -opsin corrected color blindness in squirrel monkeys. Adeno-associated Viral (AAV) Vectors for the Treatment of Inherited Blindness four children (8-11 years old) are now able to play sports and attend school without the use of learning aids Indications addressed （基因治疗疗主治） l肿瘤 l心血管疾病 l单基因遗传 病 l感染性疾病 l神经系统疾 病 l眼科疾病 Vectors used 基因转移技术 l腺病毒 l反转录病毒 l裸DNA/质粒 DNA l脂质体 l痘病毒 l腺相关病毒 l单纯疱疹病 毒 lRNA转移 病毒类载体占主导地位 基因治疗的策略 依据对病变细胞基因采取的措施不同,分为 : l基因置换 l基因修正 l基因修饰 l基因失活 l基因疫苗 基因治疗的步骤 l目的基因准备 l受体细胞培养 l载体的选择 l目的基因导入靶细胞 l 转导细胞的选择和鉴定 基因治疗对基因转移 载体的要求 l高滴度的生产能力 l外源基因的长期表达 l对载体转导的细胞无免疫反应 l载体转移后对人体无毒性 l 转导细胞的选择和鉴定 III An efficient, non-toxic, gene delivery system（基因转移方式） is the major challenge of in vivo gene therapy 基因转移方式 Functions of Cell membrane: l Separating the cell from environments; l Forming the boundaries of different organelles inside cells l Allowing only controlled exchange of materials among different parts and surroundings DNA: l large molecular weight; l Sensitive to nucleases Relationship of DNA vs. Cell The key problem remains gene delivery lTransfer of DNA from the site of DNA administration to the surface of target cells; lTransfer of DNA across the plasma membrane to cytoplasm; lTransfer of DNA across the nuclear membrane to nucleus to initiate gene expression DNA entering to cell, three major obstacles lProtecting DNA against nuclease degradation; lTransporting DNA to the target cells; lFacilitating transport of DNA across the plasma membrane; lPromoting the import of DNA into the nucleus. An ideal gene delivery should include: In1928, Frederick Griffith described the phenomenon of “transformation” in Streptococcus pneumoniae ; In 1951, Joshua Lederberg and Norton Zinder discovered a new type of gene transfer mediated by viruses. During the lytic infection, the virus picked up some host genes that were then inserted into another host. They called this process “transduction”; The gene-mediated transfer of infectious virus was termed “transfection” to distinguish from infection by a natural route of viral entry. History of Gene Transfer Methods of Gene Transfer 基因转转移的方法 lNon-virus-dependent methods:(非病毒介导导 ） Chemical methods（化学方法） Physical methods（物理方法） lVirus-dependent methods（病毒介导导） 腺病毒载体 腺相关病毒载体 反转录病毒载体 单纯疱疹病毒载体 比较基因转移方式的优缺点 Chemical methods Calcium Phosphate DEAE-Dextran Cationic Lipids Cationic Polymers Combined Systems: liposomes mixed with polylysine, peptides Commercial sources for Chemical transfection reagents Mechanisms of DNA Delivery by Chemical methods Calcium phosphate method: forming calcium -phosphate-DNA precipitates; Others: forming complexes with DNA through electrostatic interaction between DNA and the synthetic vectors Methods Techniques Purification of DNA Cell activty Affecting factors of transfection Liposome-mediated transfection Use of cationic lipids chemical and physical similarities to biological lipids spontaneous formation of complexes with DNA, called lipoplexes High efficiency for in vitro transfections Can carry larger DNA than viruses Safer than viruses Low in vivo efficiency Physical methods Injection Bombardment Electroporation Encapsulated microspheres Untrasound Comparison of different physical methods for gene delivery I. DELIVERY USING ADENOVIRUSES II. DELIVERY USING RETROVIRUSES and /or LENTIVIRUSES III. DELIVERY USING ADENO-ASSOCIATED VIRUSES IV. DELIVERY USING HERPES SIMPLEX VIRUSES V. DELIVERY USING VACCINIA VRIUS or POXVIRUS Virus-dependent methods IV. 病毒介导的基因转移方式的总原则 回顾病毒的基本特性 lViruses are naturally very efficient at transducing their own genetic information into host cells for their own replication lBy replacing non-essential viral genes with foreign genes of therapeutic interest, recombinant viral vectors can be used to transduce the cell type that they would normally infect Principle：Trojan horses of the target cells acellular organisms whose genomes consist of nucleic acid (RNA or DNA but not both), and which obligately replicate inside host cells using host metabolic machinery and ribosomes to form a pool of components which assemble into particles called VIRIONS （病毒体）, which are released and infect other cells. VIRUS S.aureus (1000nm） Cowpox 300250nm A B C D E F G Rickettsia 450nm Chlamydia 390nm A、Bacteriophage ( 65 95nm ) B、Adenovirus (70nm ) C、Poliovirus (30nm ) D、JEV ( 40nm ) E、Protein (10nm ) F、Influenza virus ( 100nm ) G、TMV VIRION（病毒体） the complete infectious unit of virus particle structurally mature, extracellular virus particles core（nucleic acid ） capsid（protein） envelope 核衣壳 包膜病毒 Naked viruses 裸病毒 Neucleocapsid Enveloped viruses STRUCTURE OF VIRUSES Viral core viral nucleic acid genome control the viral heredity and variation Genome either DNA or RNA double-stranded or single stranded linear or circular segmented or non-segmented open reading frame (ORF) overlapping gene Viral Capsid The protein shell, or coat, that encloses the nucleic acid genome. Functions: a. Protect the viral nucleic acid b. Participate in the viral infection c. Share the antigenicity Nucleocapsid The core of a virus particle consisting of the genome plus a complex of proteins. Envelope A lipid-containing membrane that surrounds some viral particles. It is acquired during viral maturation by a budding process through a cellular membrane. enveloped virus and naked virus Spike or Peplomere 刺突/包膜子粒 Glycoproteins enchased in envelope Functions of envelope Antigenicity some viruses possess neuraminidase Infectivity Resistance Enveloped Virus Naked Virus Cubic Helical Virion Capsid Viral core Envelope REPLICATION OF VIRUSES Replicative cycle vself-replication vAs obligate intracellular parasites, virus must enter and replicate in living cells in order to “reproduce” themselves. This “growth cycle” involves specific attachment of virus, penetration and uncoating, nucleic acid transcription, protein synthesis, maturation and assembly of the virions and their subsequent release from the cell by budding or lysis. Attachment/Adsorption Virus attaches to the cell surface via specific receptors. Cells without the appropriate receptors are not susceptible to the virus. 病毒受体感染细胞 HIVCD4分子CD4T细胞 EB病毒CR2（补体受 体2） B细胞 流感病毒糖蛋白末端神 经氨酸 许多细胞 丙型肝炎病毒CD81肝细胞等 adsorption Penetration (Virus enters the cell) Enveloped viruses the virus envelope fuses with the plasma membrane to facilitate entry Non-enveloped viruses engulfed into vacuoles by “endocytosis” penetration herpesviruses, paramyxoviruses, HIV 直接穿入 penetration 直接穿入 penetration Uncoating Nucleic acid has to be sufficiently uncoated that virus replication can begin at this stage. When the nucleic acid is uncoated, infectious virus particles cannot be recovered from the cell - this is the start of the ECLIPSE phase - which lasts until new infectious virions are made. Uncoating is usually achieved by cellular proteases “opening up” the capsid. Biosynthesis genome synthesis mRNA production protein synthesis Maturation and Assembly The stage of viral replication at which a virus particle becomes infectious; nucleic acids and capsids are assembled together. Release Disintegration: naked virus cause the host cell lysis Budding: enveloped viruses Budding viruses do not necessarily kill the cell. Thus, some budding viruses may be able to set up persistence. 病毒复制周期 Products of viral replication Virion Defective virus（缺陷病毒） Abortive infection（流产感染） Integration（整合） DEFECTIVE VIRUS deficiency in some aspects of replication, but interfering the replication of normal viruses HELPER VIRUS ABORTIVE INFECTION When a virus infects a cell (or host), but cannot complete the full replication cycle ( not biosynthesize their components or not assemble virions.), i.e. a non-productive infection. Cytopathic effect (CPE) The presence of the virus often gives rise to morphological changes in the host cell. Any detectable changes in the host cell due to infection are known as a cytopathic effect. cell rounding, swelling or shrinking, death, detachment from the surface, etc. Many viruses induce apoptosis (programmed cell death) in infected cells. The cytopathic effects produced by different viruses depend on the virus and the cells on which it is grown. This can be used in the clinical virology laboratory to aid in identification of a virus isolate. cytocidal infection溶细胞感染 mostly naked viruses X. From wild-type virus to replication-defective viral vectors （复制缺陷的病毒载体） 如何建立病毒载体 Outline of the possible modifications of viral properties required to transform a virus into a gene transfer vector ITR inverted terminal repeat; L left; R right; encapsidation signal; oriS replication origin. The four main viral vectors: plasmids and viral particles AAV vector Retroviral vector Gutless adenoviral vector Amplicon vector derived from HSV-1 常用病毒载载体的特性和适用范围围 病毒载体 生物学特性 适用范围 腺病毒载体 可感染分裂细胞或非分裂细胞 In vivo基因治疗 不整合到靶细胞染色体上 肿瘤基因治疗 基因表达水平高 表达时间短 免疫原性强 反转录病毒载体 可感染分裂细胞 ex vivo基因治疗 整合到靶细胞染色体上，有致癌危险 肿瘤基因治疗 表达时间长 腺相关病毒载体 可感染分裂细胞或非分裂细胞 In vivo基因治疗 整合到靶细胞染色体上，无致病性 ex vivo基因治疗 表达时间长 遗传病、慢性病基因治疗 免疫原性弱 单纯疱疹病毒载体可感染分裂细胞或非分裂细胞 神经系统疾病、肿瘤 具有嗜神经性，可潜伏感染 基因治疗 容量大，免疫原性强 History Biology XI Adenoviruses as virus 腺病毒 History 哺乳动物腺病毒属 禽腺病毒属 10-30nm纤维突起 无包膜; 二十面体对称; 252个壳粒; (12个五邻体:种特 异性 240个六邻体：属 和亚属特异性) 直径80-90nm; 基因组:双链线性 DNA, 36kb ITR（insert terminal region):与病毒复制有关； E1:调节所有早期功能; E2:编码与DNA复制有关的蛋白质; E3:编码与细胞相互作用的病毒机制的多肽; E4:编码关闭细胞基因表达,有利于病毒复制蛋白质 ITRs ITRs l 复制和装配均在被感染细细胞核内进进行；复 制和转录转录 机制复杂杂。 lTwo phases separated by the onset of viral DNA replication. lThe early phase 早期 lThe late phase 晚期 Replication cycle lentry into the cell（受体介导内吞）; ltransport of the viral genome to the nucleus;(附加子形 式） lfollowed by the transcription and translation of early viral genes. lThese events modulate the functions of the host cell to facilitate the replication of the virus DNA and the transcription and translation of the late genes. lIn permissive cells, the early phase takes 56 h, after which time viral DNA replication is first detected. The early phase lbegins concomitantly with the onset of DNA replication （DNA 半保留复制）. linvolves the expression of the late viral genes, leading to the assembly in the nucleus of the structural proteins and the maturation of infectious viruses. lthe host cells lyse to release progeny virions about 20 24 h postinfection. l 104-105 vp/cell The late phase 51个人腺病毒血清型的特点 依据纤维蛋白对各种血红细胞的凝集能力分A-F： Ultraviolet light 30min Adenoviruses as virus Vaccine prevention XII Adenoviruses as vectors 腺病毒载体的特点 l36kb基因组易于以重组技术操作； l感染静止和分裂细胞，并高水平表达蛋白质； l感染细胞类型多，包括成纤维、上皮、内皮、基质 细胞等，来源人、灵长类、狗、啮齿类 l在容许细胞可有效复制，104 vp/细胞 l 在非容许细胞，以附加子存在，表达时间较长 l常用血清型为2型，5型 l可用于基因治疗、重组疫苗、外源基因的真核高效 表达等 构建病毒载体常用元件 l真核表达质粒必须序列元件 l转录控制元件(启动子/增强子，polyA信号， 内含子剪接信号） l翻译控制元件（内部核糖体进入位点IRES) l病毒复制子和包装信号 Map of adenovirus serotype 5 genome and different generations of adenoviral vectors. Early transcripts are represented by E1E4 regions and late transcripts are represented by L1L5 regions. MLP: major late promoter; : packaging signal lcontaining the whole viral genome with the exception of the E1 Region; l several E1-expressing cell lines : 293, 911, N52.E6 and PER.C6. lcannot replicate in vivo; lresidual expression from adenoviral genes triggers a cytotoxic T lymphocyte (CTL) immune response towards infected cells, which finally leads to the elimination of transduced cells and, therefore, to the lost of therapeutic gene expression first-generation adenoviral vectors Design and Construction of Adenoviral Vectors “first-generation” replication-deficient vectors: lDeletion of the E1 region, while retaining the ITR and packaging signal, is designed to prevent expression of the E2 genes and thus block viral DNA replication and the synthesis of late structural proteins. lE1-deleted Ad vectors are therefore propagated in complementing human cell lines that provide the E1 proteins in trans. l E1-substituted Ad vectors lcombining deletion of different early regions (E1/E3 and E2/E4) lpermits to accommodate up to 14 kb and increase the vector cloning capacity lstill do not avoid in vivo associated immunogenicity and toxicity due to residual gene expression from remaining viral genes second-generation adenoviral vectors lgutless or gutted Ad: devoid of all coding viral regions lhelper-dependent adenoviruses: because of the need of a helper adenovirus that carries all coding regions lhigh-capacity adenoviruses: because they can accommodate up to 36 kb of DNA lonly keeps the 5 and 3 inverted terminal repeats (ITRs) and the packaging signal from the wild-type adenovirus. Third-generation adenoviral vectors Generation of gutless adenovirus using the Cre/loxP system. Gutless and helper genomes are cotransfected in permissive 293 Cre-expressing cells, where both genomes are amplified and viral proteins produced. Then, packaging signal of the helpers genome is excised by Cre recombinase, preventing its packaging into the viral capsid, while gutless genome is still packageable. Efficiency of the excision process allows 9099.9% purity of the gutless vector. Design and Construction of Adenoviral Vectors In order to provide additional cloning space in the vector, the E3 region, which is not necessary for viral replication in culture, is also commonly deleted. homologous recombination in an E1- complementing human cell line between two DNA molecules: one carrying sequences mapping to the left end of the Ad genome and the gene of interest; one carrying the Ad genome with the left end deleted but retaining some sequences that partially overlap the 3 end of the first molecule Homologous recombination linefficiency of homologous recombination in mammalian cells; lthe need for purification of individual viral plaques, which means it is both labor-intensive and time-consuming; lif no recombinants are generated, the researcher is unable to determine whether the problem is technical or biological. Problems lexploits the highly efficient homologous recombination machinery in bacteria to generate a recombinant Ad vector by homologous recombination in Escherichia coli between a large plasmid containing most of the Ad genome and a small shuttle plasmid containing the expression cassette flanked by sequences homologous to the region to be targeted in the viral genome; lThe recombinant Ad genome is then linearized by restriction digestion and used to transfect E1