Gene Therapy Vector Core

项目概述（见说明书）：该病毒载体的核心功能是为三个项目中的每一个提供两项主要服务：（1）设计和构建用于包装在病毒载体内的增强子/启动子和转基因;（2）生产、纯化和测试这些病毒载体类型。包装质粒的设计和构建包括：A）每个病毒载体具有特定的设计要求，包括包装能力、血清型和假型建议，这些将在构建每个新的包装质粒之前进行检查。B）通过限制性内切酶和测序确认每个包装质粒的构建，然后适当地测试转基因表达。生产、纯化和测试包括：A）生产：-AV血清型1-9 -腺病毒2型- VSVG假型慢病毒B）AV的纯化将取决于血清型。AAV血清型1-8已经通过碘克沙醇梯度离心，然后快速蛋白质液相色谱（FPLC），接着透析来纯化。已经通过聚乙二醇沉淀，然后铯分步梯度，然后铯连续梯度，接着透析来纯化AV 9。对于这些纯化方法中的每一种，我们使用转基因引物和实时PCR来定量病毒的滴度。为了检查病毒的纯度，我们使用SDS聚丙烯酰胺凝胶的银染色。为了检测生物污染物，我们将纯化的病毒加入到不含抗生素的培养细胞中。经验表明，如果从每个细胞中获得的病毒颗粒数量小于5000个，则将丢弃制备物。虽然在2 X IO 9个细胞制备物中5000个病毒粒子/细胞将产生I X IO 13个颗粒，但这些制备物在体外或体内表现不佳。 对于腺病毒的纯化，我们使用两种方法。传统的方法先用铯分步梯度法再用铯连续梯度法，然后用透析法。第二种方法使用来自Vivapure AdenoPACK试剂盒（Goettingen，德国）的商业柱纯化方法。我们已经利用UV吸收进行定量（Liebermann和Mental 1994），以及真实的时间PCR。慢病毒的纯化利用蔗糖缓冲梯度。此外，还有许多市售的慢病毒浓缩和纯化试剂盒（Cell Biolabs; San Diego，CA）。C）测试每种病毒，包括： i）在透析后，在交付给项目负责人之前，对病毒进行滴定。 ii）测试病毒在HeLa细胞中对CMV或其他强启动子的体外转导效率，或在原代新生大鼠心肌细胞中对限制性（心脏）启动子的体外转导效率。 iii）在交付给项目负责人之前，将使用蛋白质印迹分析来确定病毒转基因的表达。 该核心将生产和纯化AAV血清型1-9、腺病毒2型和慢病毒。具体而言，我们将根据PPG参与者的需求开发和维护用于大规模生产（100-200个15 cm平板范围）特定载体和转基因的细胞系。我们将根据项目负责人的需要，提供与新型治疗基因、抑制性和微小RNA的亚克隆以及病毒开发的增强子-启动子配置相关的分子生物学支持。核心D的主要功能是：1）生产腺相关病毒和腺相关病毒，2）纯化这些病毒，3）在转移给科学计划成员之前检测病毒。一个额外的功能将是生产VSVG假型慢病毒，如科学计划成员所需。

PROJECT SUMMARY (See instructions): This viral vector core function is to provide each of the three projects two major services: (1) Design and construction of enhancer/promoters and transgenes for packaging within viral vectors; (2) Production, purification and testing of those viral vector types. Design and construction of packaging plasmids include: A) Each viral vector has specific design requirements including packaging capacity, serotype and pseudotype recommendations, these will be examined prior to construction of each new packaging plasmid. B) The construction of each packaging plasmid will be confirmed by restriction digests and sequencing, and then appropriately tested for transgene expression. Production, purification and testing include: A) Production of: - /\AV serotypes 1-9 - Adenovirus type 2 - VSVG pseudotyped Lentivirus B) Purification of /\AV will depend on serotype. AAV serotypes 1-8 have been purified by iodixanol gradient centrifugation then fast protein liquid chromatography (FPLC), followed by dialysis. /\AV9 has been purified by polyethylene glycol precipitation, then a cesium step gradient then a cesium continuous gradient, followed by dialysis. For each of these purification methods we have used transgene primers and real-time PCR to quantify the titer of the virus. To examine the purity of the virus we use silver staining of SDS polyacrylamide gels. To test for biological contaminants we add aliquots ofthe purified virus to cells in culture without antibiotics. Experience has demonstrated that if the number of viral particles obtained from each cell is less than 5000 for /\AV then the prep will be discarded. While 5000 virions/cell in a 2 x 109 cell preparation will produce 1 x 1013 particles these preps do not perform well in vitro or in vivo. For the purification of Adenovirus we use two methods. First the traditional method of cesium step gradient then by continuous cesium gradient, followed by dialysis. The second approach uses commercial column purification methods from Vivapure AdenoPACK kits (Goettingen, Germany). We have utilized the UV absorption for quantification (Liebermann and Mental 1994), as well as real time PCR. The purification of lentivirus utilizes a sucrose cushion gradient. In addition there are a number of commercially available lentivirus concentration and purification kits (Cell Biolabs; San Diego, CA). C) Testing of each virus that will include: i) Titration of the viruses after dialysis prior to delivery to project leaders. ii) Testing of the in vitro transduction efficiency of viruses in HeLa cells for CMV or other strong promoters, or primary neonatal rat cardiomyocytes for restricted (cardiac) promoters. iii) Western blot analysis will be used to establish expression of viral transgenes prior to delivery to project leaders. This core will manufacture and purify AAV serotypes 1-9, Adenovirus type 2 and Lentivirus. Specifically, we will develop and maintain cell lines for large scale production (100-200 of 15-cm plate range) of specific vectors and transgenes as dictated by PPG participant needs. We will provide the molecular biological support related to sub-cloning of novel therapeutic genes, inhibitory and micro RNAs, as well as enhancer-promoter configurations for viral development as needed by the Project Leaders. The main functions of Core D are; 1) Production of Adeno- and Adeno-associated viruses, 2) Purification of these viruses, and 3) Testing viruses prior to transferring to members of the Scientific Program. An additional function will be the production of VSVG pseudotyped lentivirus, as needed by members ofthe Scientific Program.