腺病毒伴随病毒载体的研究进展

腺病毒伴随病毒载体的研究进展姚二梅冯泽华侯云德随着对人类基因愈加深入的了解，以及对真核基因改造和转移技术的发展，人类基因治疗已成为现实。基因治疗的关键问题就是要寻找有效的基因转移系统。在病毒介导和非病毒介导的两类基因转移系统中，感染性的重组病毒载体是...     

腺病毒载体,腺病毒相关病毒载体与基因治疗

近年来，腺病毒载体（ＡＶ），腺病毒相关病毒载体（ＡＡＶ）已经成为继反转录病毒载体（ＲＶ）以来最重要的病毒载体。与反转录病毒载体相比较，ＡＶ有其独特的优点，构建带有目的基因的腺病毒载体通过２９３辅助细胞产生重组腺病毒颗粒，用于活体基因转移及靶细胞感染。目前已利用ＡＶ研究了ｎｅｏ，ｌａｃＺ，ＨＢｓＡｇ及ＣＦＴＲ等基因在不用组织中的表达，其中人们已利用ＣＦＴＲ基因通过ＡＶ介导对囊性纤维化进行基因治疗临床     

携带eGFP及人endostatin-K5的嵌合腺病毒载体Ad5/11的构建及其体外实验研究

目的:构建携带报告基因eGFP及人内皮他丁en-dostatin-K5的嵌合型腺病毒载体ADS/11.方法:采用重叠PCR及在细菌E coil.BJ5183中同源重组的方法,获得表达eGFP报告基因的嵌合腺病毒骨架载体,然后以经Pac Ⅰ线性化的嵌合腺病毒载体骨架与Pac Ⅰ线性化的表达endostatin-K5的腺病毒E1穿梭载体共转染真核细胞HEK 293细胞中获得携带报告基因eGFP及人endostatin.K5的嵌合型腺病毒载体Ad5/11-E1-CMV-endo-K5/E3-CMV-eGFP.以获得的嵌合型腺病毒载体感染U87MG细胞,在荧光显微镜观察eGFP的表达,采用RT-PCR检测endostatin-K5的表达;将所构建的嵌合病毒载体ADS/11-E1-CMV-endo-KS/E3-CMV-eGFP与未经修饰的腺病毒载体Ad5-E1-CMV-eGFP在体外感染人胶质瘤细胞系A172及乳腺癌细胞系MDA-MB-231;通过荧光蛋白eG-FP的表达,比较它们对以上肿瘤细胞的感染效率.结果:嵌合病毒载体Ad5/11-E1-CMV-endo-K5/E3-CMV-eGFP可成功地表达eGFP及endostafin-K5.以经过修饰的嵌合型腺病毒载体AdS/11-E1-CMV-endo-KS/E3-CMV-eGFP感染人脑胶质瘤细胞系A172及人乳腺癌细胞MDA-MB-231的感染效率,明显高于对照未经修饰的腺病毒载体Ad5-E1-CMV-eGFP.结论:携带eGFP及人endostatin-K5的嵌合型腺病毒载体ADS/11,可明显提高对人脑胶质瘤细胞系A172及人乳腺癌细胞系MDA-MB-231的感染效率.     

腺病毒相关病毒载体的研究进展

腺病毒相关病毒(AAV)是一种缺陷型的单链DNA病毒,它能感染造血干细胞、脑细胞等非分裂细胞是其显著特征之一。现有资料显示AAV载体能以特异定向整合的方式存在。AAV重组体在细胞内能长期稳定表达,且在体内未引起明显的病理改变,表明AAV作为基因治疗的载体是安全、有效和可行的。     

腺病毒相关病毒载体的研究进展

实现基因治疗的关键在于目的基因的高效转移并适度表达 ,这将直接影响其治疗的效率和安全性。因此 ,探寻理想的基因转移载体显得尤为重要。腺病毒相关病毒 (adeno- associated virus,AAV)是一种缺陷型的单链DNA病毒 ,既可以转染分裂细胞又可以转染非分裂细胞。 AAV在宿主体内以定向整合的方式存在。 AAV重组体在细胞内能长期稳定地表达 ,且在体内不引起明显的病理变化 ,表明 AAV作为基因治疗的载体是安全、有效和可行的     

腺病毒及腺病毒相关病毒载体与基因治疗

腺病毒及腺病毒相关病毒能感染增殖与静止的细胞,宿主范围广。其稳定、安全滴度高、转染效率高。腺病毒载体能承载较大的外源基因,腺病毒相关病毒载体能定向整合入靶细胞基因组内等特点,使腺病毒及腺病毒相关病毒载体成为体内用于基因治疗的理想运载系统。     

腺病毒相关病毒载体的安全性评价

腺病毒相关病毒（AAV）作为基因治疗的病毒载体之一，已经受到广泛地关注。与其它载体相比，重组AAV具有宿主范围广、免疫原性小和安全性较好等优点，然而对重组AAV的靶向整合、毒性、在体内分布情况、产生免疫应答反应以及是否诱发肿瘤等问题仍还不十分清楚。本文就腺病毒相关病毒载体的安全性作一较全面而系统地评价。     

腺病毒载体研究新进展

近年来,病毒载体尤其是腺病毒载体的研究成为基因治疗研究中的一个热点。本文综述了腺病毒载体的构建和应用方面的最新进展,表明腺病毒载体是基因转移的重要手段,具有众多的优点,在基因治疗领域具有广阔的应用前景。     

腺病毒相关病毒载体的安全性评价

腺病毒相关病毒(AAV)作为基因治疗的病毒载体之一,已经受到广泛地关注。与其它载体相比,重组AAV具有宿主范围广、免疫原性小和安全性较好等优点,然而对重组AAV的靶向整合、毒性、在体内分布情况、产生免疫应答反应以及是否诱发肿瘤等问题仍还不十分清楚。本文就腺病毒相关病毒载体的安全性作一较全面而系统地评价。     

腺病毒载体介导肿瘤基因治疗的安全性分析

随着分子生物学和基因工程技术的发展,腺病毒载体在肿瘤基因治疗领域发挥了不可小觑的作用,同时其安全性也成了最为关键的问题之一。本文从腺病毒在体内的组织分布、复制型腺病毒的随机出现、腺病毒载体对宿主的免疫原性、转基因表达时间和水平、重组腺病毒载体对宿主器官的损伤作用等方面就腺病毒载体介导肿瘤基因治疗的安全性作一综述。     

Gene transfer by viral vectors for gene therapy

Conclusions The search for useful virus vectors and for improvements in currently available retrovectors which may have the capability of transportation by natural transport systems in the human body will open effective ways for targeting human genes to specific cells in tissues in situ. Genetic engineering of virus vectors is a subject of prime importance to the developing gene therapy protocols in humans.Abbreviations HSV Herpes simplex virus     

Adenoviral protein V promotes a process of viral assembly through nucleophosmin 1

Adenoviral infection induces nucleoplasmic redistribution of a nucleolar nucleophosmin 1/NPM1/B23.1. NPM1 is preferentially localized in the nucleoli of normal cells, whereas it is also present at the nuclear matrix in cancer cells. However, the biological roles of NPM1 during infection are unknown. Here, by analyzing a pV-deletion mutant, Ad5-dV/TSB, we demonstrate that pV promotes the NPM1 translocation from the nucleoli to the nucleoplasm in normal cells, and the NPM1 translocation is correlated with adenoviral replication. Lack of pV causes a dramatic reduction of adenoviral replication in normal cells, but not cancer cells, and Ad5-dV/TSB was defective in viral assembly in normal cells. NPM1 knockdown inhibits adenoviral replication, suggesting an involvement of NPM1 in adenoviral biology. Further, we show that NPM1 interacts with empty adenovirus particles which are an intermediate during virion maturation by immunoelectron microscopy. Collectively, these data implicate that pV participates in a process of viral assembly through NPM1.     

表达自杀基因的减毒伤寒沙门氏菌对肿瘤的抑制作用

目的 研究表达自杀基因的减毒鼠伤寒沙门氏菌对肿瘤的抑制作用。方法 把表达自杀基因的质粒（pc-CD)导入减毒的鼠伤寒沙门氏菌,以荷瘤鼠为模型研究沙门氏菌／自杀基因与5－FC系统对肿瘤的抑制作用。结果 该系统能抑制肿瘤生长,延长荷瘤鼠的存活期。结论 鼠伤寒沙门氏菌可以作为基因治疗的候选载体。     

Enhancing the therapeutic efficacy of adenovirus in combination with biomaterials

With the reason that systemically administered adenovirus (Ad) is rapidly extinguished by innate/adaptive immune responses and accumulation in liver, in vivo application of the Ad vector is strictly restricted. For achieving to develop successful Ad vector systems for cancer therapy, the chemical or physical modification of Ad vectors with polymers has been generally used as a promising strategy to overcome the obstacles. With polyethylene glycol (PEG) first in order, a variety of polymers have been developed to shield the surface of therapeutic Ad vectors and well accomplished to extend circulation time in blood and reduce liver toxicity. However, although polymer-coated Ads can successfully evacuate from a series of guarding systems in vivo and locate within tumors by enhanced permeability and retention (EPR) effect, the possibility to entering into the target cell is few and far between. To endow targeting moiety to polymer-coated Ad vectors, a diversity of ligands such as tumor-homing peptides, growth factors or antibodies, have been introduced with avoiding unwanted transduction and enhancing therapeutic efficacy. Here, we will describe and classify the characteristics of the published polymers with respect to Ad vectors. Furthermore, we will also compare the properties of variable targeting ligands, which are being utilized for addressing polymer-coated Ad vectors actively.     

A paclitaxel-conjugated adenovirus vector for targeted drug delivery for tumor therapy

Tumor-targeted drug delivery is an attractive strategy in cancer treatment. Our previous study demonstrated that modified adenovirus has strong tumor targeting ability and less toxicity to surrounding normal tissue. In this study, Paclitaxel (PTX), a widely used clinical anticancer drug, was conjugated to folate-modified adenovirus (Ad) nanoparticles by using succinic anhydride and Fmoc-Glu(OtBu)-OH linkers to form two prodrugs, FA-Ad-Suc-PTX and FA-Ad-ICG02-Glu-PTX. Near-infrared (NIR) fluorescent dye ICG-Der-02 was attached to -NH₂-Glu(OtBu)-PTX for in vivo optical imaging. In vitro and acute toxicity study demonstrates the low toxicity of the prodrug FA-Ad-Suc-PTX and FA-Ad-ICG02-Glu-PTX compared to the free drug. The dynamic behaviors and targeting ability of FA-Ad-ICG02-Glu-PTX on MDA-MB-231 tumor-bearing mice were investigated by NIR fluorescence imaging. The result show that PTX-conjugated Ad vector could enhance the targeting and residence time in tumor site. In vitro and in vivo studies demonstrate that Coxsackie adenovirus receptor (CAR) or foliate receptor (FR)-mediated uptake of FA-Ad-loaded PTX induced highly anti-tumor activity. The results support the potential of using chemically modified Ad vector as drug-loaded tumor-targeting delivery system.     

视网膜母细胞瘤基因治疗的实验研究

目的观察外源性Ｒｂ基因在视网膜母细胞瘤（ｒｅｔｉｎｏｂｌａｓｔｏｍａ,ＲＢ）移植瘤内的表达情况,及其对ＲＢ移植瘤生长的影响,为进行ＲＢ体内基因治疗提供实验依据。方法建立裸鼠眼玻璃体腔ＲＢ移植瘤模型,构建Ｒｂ基因的逆转录病毒表达载体,用脂质体将Ｒｂ基因导入裸鼠ＲＢ移植瘤,免疫组织化学染色及流式细胞仪间接免疫荧光法检测Ｒｂ基因表达,裸鼠ＲＢ移植瘤生长的活体观察、分级、组织病理形态观察。结果脂质体Ｄｏｓｐｅｒ介导的Ｒｂ基因转染可在ＲＢ移植瘤内表达至少７天,并部分抑制ＲＢ移植瘤的生长,抑制效果与Ｒｂ蛋白表达量、治疗时机有关。结论外源性Ｒｂ基因可在ＲＢ移植瘤内表达并部分抑制ＲＢ移植瘤的生长。     

腺病毒载体在肿瘤基因治疗和基因疫苗领域的应用研究进展

腺病毒载体是目前应用最为广泛的基因治疗病毒载体之一。相较其他病毒载体,腺病毒具有较高的基因转导效率,广泛的组织亲和性,不整合入宿主基因组等优点。腺病毒可以被改造为复制缺陷病毒载体和选择性复制的溶瘤病毒载体。腺病毒载体较强的免疫原性使其可以被应用于基因疫苗和抗癌治疗中。目前众多的临床试验都证实了腺病毒载体在上述治疗试验中的安全性和有效性。     

An improved Tet-On regulatable FasL-adenovirus vector system for lung cancer therapy

Gene therapy is a new therapeutic approach for the treatment of human cancers. Gene expression systems that can be regulated by drugs have been developed to improve the safety and efficacy of therapeutic transgene delivery. One of the most promising systems is the tetracycline (Tet)-responsive system in the Tet-On configuration. A major problem of the Tet-On system if used in viral vectors is the high basal activity of the Tet response element (TRE) promoter leading to leaky expression of transgenes under uninduced conditions. We therefore evaluated novel TRE promoters for controlling gene expression in an adenovirus vector (AdV) Tet-On system and further investigated them for expression of the pro-apoptotic CD95/Fas ligand (FasL) in human epithelial carcinoma cell line (HeLa) and lung cancer cells. Plasmid-based reporter gene assays showed that modifications within the tetO ₇ and minimal immediate early cytomegalovirus promoter (CMV)_min sequence of the TRE promoter reduced its leakiness and led to a markedly improved regulatability by doxycycline. Among several TRE promoters tested, a new construct (TRE-Tight1) containing modifications of both the tetO ₇ sequence and the CMV_min showed 11-fold reduced leakiness and 1.5-fold increased absolute transgene expression levels after induction, as compared to the original TRE. Under induced conditions, a TRE-Tight1 promoter-dependent AdV expressing the pro-apoptotic CD95L/FasL induced apoptosis and cell lysis in HeLa cells as efficiently as an AdV containing the original TRE promoter. In contrast to the latter, however, the vector with the modified TRE promoter left cells totally unaffected in the absence of the inducer. Stringently regulated induction of apoptosis and cell death by TRE-Tight1-AdV was also demonstrated in three human lung cancer cell lines. These data show that the novel TRE-Tight1 promoter has a high potential for closely controlled and efficient expression of cytotoxic genes in AdV-based anti-cancer approaches. Isaac Sipo, Almudena Hurtado Picó, Wolfgang Poller and Henry Fechner have equally contributed to this work.