腺相关病毒载体介导siRNA抑制HBV的表达与复制

目的：构建并制备能够有效抑制HBV mRNA表达的siRNA重组腺相关病毒。 方法：将筛选到的siRNA片段克隆入骨架质粒pAAVMCS中,与pAAVRC和pHelper质粒共转染人胚肾293T细胞,包装出重组腺相关病毒,将纯化后的重组病毒直接感染Hep G 2.2.15细胞,通过酶联免疫法和荧光定量PCR法检测重组腺相关病毒的抑制效果。结果：重组腺相关病毒显著降低HBV 分泌的相关抗原蛋白以及DNA和RNA水平。结论：重组腺相关病毒载体介导的siRNA能够有效抑制HBV的表达与复制。     

腺相关病毒载体基因治疗的研究进展

腺相关病毒 (ＡＡＶ)属于病毒中最小、也是最简单的一种———细小病毒属 ,它是一种有缺陷的非致病性的人类细小病毒 ,重组的腺相关病毒 (ｒＡＡＶ)作为基因治疗的载体 ,对长期基因矫正和基因治疗具有优越性。新的生产工艺提高了ｒＡＡＶ的滴度 ,去除了腺病毒的污染 ,并构建了适用于不同靶细胞的可诱导载体。这些改进有望加速进一步的动物实验研究 ,使临床治疗人类疾病早日成为可能。1 .ｒＡＡＶ的制备方法在 1 989年 ,首次产生了没有野生型ＡＡＶ帮助复制的重组的ＡＡＶ ,这以后 ,为了增加病毒颗粒数 ,人们制造了许多ＡＡＶ辅助质粒 ,其中…     

重组腺相关病毒：很有潜力的基因治疗载体

腺相关病毒(AAV)是细小病毒家族的一员,为无包膜的线性单链DNA病毒.由于AAV具有长期潜伏于人体而不具有任何明显致病性等优点,人们对AAV作为一种理想的基因治疗载体给予了很大期望.但是,近来发现,这类载体在应用上有许多明显的缺陷,包括某些细胞膜上病毒受体数量极少,重组AAV载体位点特异性整合不足,AAV衣壳成分和转基因产物引起宿主的免疫反应等等.这些缺陷促使人们加大对AAV生物学特性和转染过程的研究,从而更好地对AAV载体进行改进,使新一代重组AAV载体具备基因治疗所必需的安全性、高效性和靶向性,以期更广泛地应用于临床.     

重组腺相关病毒载体在肝疾病基因治疗中的应用及局限性

基因治疗(gene therapy)是近十年来随着现代分子生物学技术的发展而诞生的新的生物医学治疗技术.重组腺病毒伴随病毒是一种具有开发潜质的病毒载体,因此近年来对它的研究很是受人关注.肝炎、肝硬化、肝癌在我国乃至全世界是严重损害人们的健康,据此,国内外的研究人员对rAAV在肝疾病基因治疗方面做了大量的工作,本文对rAAV在肝疾病基因治疗中的应用和局限性研究进行综述.     

腺相关病毒介导的小鼠IX因子在血友病B小鼠体内表达研究

通过重组HSV/AAV辅助病毒方法制备出高滴度(>10~(12))的重组AAV/mFⅨ病毒颗粒,并注射到血友病B小鼠的后肢骨骼肌中,获得了稳定的高水平小鼠Ⅸ因子表达(>370ng/mL),持续时间超过350d。治疗组小鼠体内的Ⅸ因子活性达到正常值的30％,而且出血症状得到显著改善。在小鼠血浆中未发现抗Ⅸ因子的中和抗体,第1次注射后抗AAV抗体的水平也非常低。重复注射AAV/mFⅨ病毒后,不同剂量组的小鼠体内的抗AAV抗体出现了不同的结果。组织分析结果证实了血浆中有功能的FⅨ因子确实来源于骨骼肌。以上结果显示,AAV介导的基因转移是血友病B基因治疗的极有前途的方法。     

腺相关病毒介导的小鼠Ⅸ因子在血友病B小鼠体内表达研究

通过重组HSV/AAV辅助病毒方法制备出高滴度(>10¹²)的重组AAV/mFⅨ病毒颗粒, 并注射到血友病B小鼠的后肢骨骼肌中, 获得了稳定的高水平小鼠Ⅸ因子表达(>370 ng/mL), 持续时间超过350 d. 治疗组小鼠体内的Ⅸ因子活性达到正常值的30%, 而且出血症状得到显著改善. 在小鼠血浆中未发现抗Ⅸ因子的中和抗体, 第1次注射后抗AAV抗体的水平也非常低. 重复注射AAV/mFⅨ病毒后, 不同剂量组的小鼠体内的抗AAV抗体出现了不同的结果. 组织分析结果证实了血浆中有功能的FⅨ因子确实来源于骨骼肌. 以上结果显示, AAV介导的基因转移是血友病B基因治疗的极有前途的方法.     

腺相关病毒载体与中枢神经系统的基因治疗

本文对ＡＡＶ的分子生物学结构,重组和其对细胞的整合及ｒＡＡＶ载体在基因治疗中的作用,尤其是ｒＡＡ载体在中枢神经系统疾病基因方面的最新应用作了较全面阐述。     

重组腺相关病毒载体诱导的天然免疫反应及机制

重组腺相关病毒(rAAV)已成为基因治疗领域应用最广泛的载体之一。临床前研究显示其具有很高的安全性,但人体免疫毒性仍是制约其临床疗效的关键,因此有关rAAV免疫机制的研究成为近期热点。尽管天然免疫在获得性免疫反应中发挥重要作用,但与rAAV有关的天然免疫研究过去一直未被重视。直到最近,才确认有至少3种人体细胞(树突状细胞、巨噬细胞和内皮细胞)参与了rAAV的天然免疫,作用机制为可识别载体基因组的TLR9或病毒衣壳TLR2所介导,NF-κB或干扰素调节因子(IRFs)信号通路被激活,导致各种炎性因子及I型干扰素的大量表达。自身互补型rAAV诱导的TLR9依赖性天然免疫较单链rAAV更为强烈。本文重点对近期发现的激活天然免疫反应的宿主与rAAV的相互作用、涉及的信号通路、天然免疫对获得性免疫以及转基因表达的影响进行综述。     

表达siRNA的重组腺相关病毒载体的构建和鉴定

腺相关病毒(AAV)载体介导的RNA干扰(RNAi)可在哺乳动物细胞中长期特异性抑制同源基因表达。本研究以AAV Helper-Free System中的转移质粒pAAV-MCS为基础,引入增强型绿色荧光蛋白(EGFP)基因和H1启动子,将该质粒改造为可表达小干扰RNA(siRNA)和EGFP的质粒pAAV-EGFP-H1。该质粒与辅助质粒共转染包装细胞后,获得了具有感染性的重组AAV(rAAV)。以EGFP为靶基因的干扰实验证明:所得rAAV可以产生siRNA并能够特异性抑制EGFP靶基因表达;荧光显微镜观察、FACS分析和Real-time PCR方法均表明重组病毒rAAV-H1-sh EGFP引起EGFP的表达降低大于60%。     

重组腺相关病毒载体相关性杂质

可以稳定表达治疗基因而无明显不良反应的重组腺相关病毒 (rAAV) 载体被认为是最有发展前景的基因治疗载体。但如何建立可以有效去除rAAV载体内具有潜在致病危害的杂质、产品质量符合临床使用要求的纯化工艺是研究人员面临的巨大挑战。其中针对载体相关性杂质的纯化工艺尤为关键,因为该类杂质的性质与真正的rAAV载体极其相似,一旦存在便难以去除,且会引起严重不良反应。以下总结了该类杂质形成的过程及有别于rAAV载体的特点,并对可以防止其生成或将其与rAAV载体有效分离的技术手段进行了评价。     

重组腺相关病毒载体相关性杂质

可以稳定表达治疗基因而无明显不良反应的重组腺相关病毒(rAAV)载体被认为是最有发展前景的基因治疗载体。但如何建立可以有效去除rAAV载体内具有潜在致病危害的杂质、产品质量符合临床使用要求的纯化工艺是研究人员面临的巨大挑战。其中针对载体相关性杂质的纯化工艺尤为关键,因为该类杂质的性质与真正的rAAV载体极其相似,一旦存在便难以去除,且会引起严重不良反应。以下总结了该类杂质形成的过程及有别于rAAV载体的特点,并对可以防止其生成或将其与rAAV载体有效分离的技术手段进行了评价。     

A novel and highly efficient production system for recombinant adeno-associated virus vector

Recombinant adeno-associated virus(rAAV) has proven to be a promising gene delivery vector for human gene therapy. However, its application has been limited by difficulty in obtaining enough quantities of high-titer vector stocks. In this paper, a novel and highly efficient production system for rAAV is described. A recombinant herpes simplex virus type 1(rHSV-1) designated HSV1-rc/△UL2, which expressed adeno-associated virus type2(AAV-2) Rep and Cap proteins, was constructed previously. The data confirmed that its functions were to support rAAV replication and packaging, and the generated rAAV was infectious. Meanwhile, an rAAV proviral cell line designated BHK/SG2, which carried the green fluorescent protein(GFP) gene expression cassette, was established by transfecting BHK-21 cells with rAAV vector plasmid pSNAV-2-GFP. Infecting BHK/SG2 with HSV1-rc/△UL2 at an MOI of 0.1 resulted in the optimal yields of rAAV, reaching 250 transducing unit(TU) or 4.28×104 particles per cell. Therefore, compared with the conventional transfection method, the yield of rAAV using this "one proviral cell line, one helper virus" strategy was increased by two orders of magnitude. Large-scale production of rAAV can be easily achieved using this strategy and might meet the demands for clinical trials of rAAV-mediated gene therapy.     

A novel and highly efficient production system for recombinant adeno-associated virus vector

Recombinant adeno-associated virus (rAAV) has proven to be a promising gene delivery vector for human gene therapy. However, its application has been limited by difficulty in obtaining enough quantities of high-titer vector stocks. In this paper, a novel and highly efficient production system for rAAV is described. A recombinant herpes simplex virus type 1 (rHSV-1) designated HSV1-rc/AUL2, which expressed adeno-associated virus type2 (AAV-2) Rep and Cap proteins, was constructed previously. The data confirmed that its functions were to support rAAV replication and packaging, and the generated rAAV was infectious. Meanwhile, an rAAV proviral cell line designated BHK/SG2, which carried the green fluorescent protein (GFP) gene expression cassette, was established by transfecting BHK-21 cells with rAAV vector plasmid pSNAV-2-GFP. Infecting BHK/SG2 with HSV1-rc/AUL2 at an MOI of 0.1 resulted in the optimal yields of rAAV, reaching 250 transducing unit (TU) or 4.28×104 particles per cell. Therefore, compared     

测定重组腺相关病毒基因组滴度的qPCR新方法

腺相关病毒(Adeno-associated virus,AAV)在基因治疗应用中具有很多优势,但是其生物学滴度的测定仍很繁琐,不同实验室使用各自的方法和参照,这些都影响了重组腺相关病毒(rAAV)载体在临床前和临床上的应用.反向末端重复序列(Inverted terminal repeats,ITR)是重组腺相关病毒载体中不可或缺的顺式作用元件,针对ITR2以及ITR2-CMV设计的qPCR检测方法可以快速、准确地得到rAAV2的基因组滴度,由于该方法可以广泛适用,因此对推动AAV滴度检测的标准化有重要意义.     

Gene therapy for hemophilia B mediated by recombinant adeno-associated viral vector with hFIXR338A, a high catalytic activity mutation of human coagulation factor IX

A mutant human factor IX with arginine at 338 residual changed to alanine (hFIXR338A) by site-directed mutagenesis was introduced into AAV vectors, and a recombinant adeno-associ- ated viral vector containing hFIXR338A, prepared by rHSV/AAV hybrid helper virus system, was directly introduced to the hind leg muscle of factor IX knock out mice. The expression and the biological activity of human factor IX mutant, hFIXR338A, and the immune response against it in the treated mice were assayed and detected. The results showed that (i) the high-level expression of human factor IX mutant protein, hFIXR338A, has been detected in rAAV-hFIXR338A treated hemophilia B mice and lasted more than 15 weeks; (ii) the clotting activity of hFIXR338A in plasma is 34.2%± 5.23%, which is remarkably higher than that of (14.27% ± 3.4%) of wild type hFIX treated mice in the activated partial thromboplastin assay; (iii) immune response against factor IX R338A was absent, with no factor IX mutant protein (hFIXR338A) inhibitors development in the treated mice; and (iv) no local or systemic side-effects and toxicity associated with the gene transfer were found. It demonstrated the potential use of treating hemophilia B by recombinant adeno-associated viral vectors with mutant hFIXR338A gene, an alternative strategy for hemophilia B gene therapy to wild-type human factor IX.     

重组腺相关病毒载体基因表达效率与辐射的关系

重组腺相关病毒(rAAV)载体被认为是最有希望的临床应用基因治疗载体之一。rAAV载体基因传递的有效性与病毒衣壳蛋白的免疫原性之间的矛盾是制约该类基因药物开发的关键难题,提高其表达效率是rAAV载体研发的主攻方向之一。研究发现,紫外线、γ射线等辐射可以提高rAAV载体的基因表达效率,其作用效果与辐射种类和剂量、细胞种类和状态、载体感染指数和感染时间等因素有关。其机制可能与DNA损伤反应,特别是DNA双链断裂修复有关。对辐射与rAAV载体的表达效率之间关系的深入了解,为两者的联合应用打开方便之门。本文将对UV、γ射线等辐射提高rAAV载体表达效率的机制、影响因素及其在基因治疗领域的应用进展进行综述。     

Gene therapy for hemophilia B mediated by recombinant adeno-associated viral vector with hFIXR338A, a high catalytic activity mutation of human coagulation factor IX

A mutant human factor IX with arginine at 338 residual changed to alanine (hFIXR338A) by site-directed mutagenesis was introduced into AAV vectors, and a recombinant adeno-associ-ated viral vector containing hFIXR338A, prepared by rHSV/AAV hybrid helper virus system, was directly introduced to the hind leg muscle of factor IX knock out mice. The expression and the biological activity of human factor IX mutant, hFIXR338A, and the immune response against it in the treated mice were assayed and detected. The results showed that (i) the high-level expression of human factor IX mutant protein, hFIXR338A, has been detected in rAAV-hFIXR338A treated hemophilia B mice and lasted more than 15 weeks; (ii) the clotting activity of hFIXR338A in plasma is 34.2%± 5.23%, which is remarkably higher than that of (14.27%±3.4%) of wild type hFIX treated mice in the activated partial thromboplastin assay; (iii) immune response against factor IX R338A was absent, with no factor IX mutant protein (hFIXR338A) inhibitors deve