Gene Therapy Vector Core

项目摘要(见说明)：这一病毒载体的核心功能是为三个项目中的每一个项目提供两项主要服务：(1)设计和构建增强子/启动子和转基因包装在病毒载体中；(2)生产、纯化和检测这些病毒载体类型。包装质粒的设计和构建包括：a)每个病毒载体都有特定的设计要求，包括包装容量、血清型和假型建议，在构建每个新的包装质粒之前将对这些要求进行检查。B)每个包装质粒的构建将通过限制性内切酶和测序来确认，然后适当地检测转基因表达。生产、纯化和检测包括：A)生产-/\AV血清型1-9-腺病毒2型-VSVG假型慢病毒B)/\AV的纯化取决于血清型。AAV血清型1-8型用碘二醇梯度离心法纯化，然后用快速蛋白质液相色谱(FPLC)分离，然后透析。/\AV9经聚乙二醇沉淀、铯分级梯度、铯连续梯度、透析法纯化。对于每一种纯化方法，我们都使用了转基因引物和实时荧光定量聚合酶链式反应来定量病毒的滴度。为了检测病毒的纯度，我们使用了银染的十二烷基硫酸钠-聚丙烯酰胺凝胶。为了检测生物污染物，我们在不含抗生素的培养细胞中加入等量的纯化病毒。经验表明，如果从每个细胞中获得的病毒颗粒数量少于5000个/\AV，则PREP将被丢弃。虽然在2x109细胞中每个细胞5000个病毒粒子会产生1x1013个颗粒，但这些制备在体外或体内都表现不佳。 对腺病毒的提纯，我们采用了两种方法。先用传统的铯梯度法，再用连续铯梯度法，再进行透析。第二种方法使用ViVapure AdenoPACK试剂盒(德国哥廷根)的商用柱纯化方法。我们利用紫外线吸收进行定量(Liebermann和Mind1994)，以及实时聚合酶链式反应(Real Time PCR)。慢病毒的提纯采用蔗糖垫梯度。此外，还有一些商业上可用的慢病毒浓缩和纯化试剂盒(Cell Biolabs，加利福尼亚州圣地亚哥)。C)每种病毒的测试将包括： I)在交付给项目负责人之前，对透析后的病毒进行滴定。 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)启动子的转导效率。 Iii)在交付给项目负责人之前，将使用Western印迹分析来确定病毒转基因的表达。 该核心将制造和提纯AAV血清型1-9、腺病毒2型和慢病毒。具体地说，我们将根据PPG参与者的需求开发和维护大规模生产特定载体和转基因的细胞系(100-200个15厘米平板范围)。我们将根据项目负责人的需要，提供与亚克隆新的治疗基因、抑制基因和微RNA相关的分子生物学支持，以及用于病毒开发的增强子-启动子配置。核心D的主要功能是：1)生产腺病毒和腺相关病毒，2)提纯这些病毒，以及3)在将病毒转移给科学计划成员之前对病毒进行测试。根据科学计划成员的需要，另一项功能将是生产VSVG假型慢病毒。

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.