Gene Therapy Vector Core

项目概述（见说明）：该病毒载体的核心功能是为三个项目提供两项主要服务：(1)设计和构建增强子/启动子以及包装在病毒载体内的转基因；(2)病毒载体类型的制备、纯化和检测。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、血清型和伪型建议，这些将在构建每个新的包装质粒之前进行检查。B)每个包装质粒的结构将通过酶切和测序确认，然后进行适当的转基因表达测试。生产、纯化和检测包括：A)生产：- /\AV血清型1-9 -腺病毒2 - VSVG伪型慢病毒B) /\AV的纯化取决于血清型。采用碘二醇梯度离心、快速蛋白液相色谱（FPLC）、透析纯化AAV血清型1-8。/\AV9经聚乙二醇沉淀法纯化，然后是铯阶梯梯度，然后是铯连续梯度，最后是透析。对于每一种纯化方法，我们都使用转基因引物和实时PCR来量化病毒的滴度。为了检验病毒的纯度，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，我们将等量的纯化病毒添加到没有抗生素的培养细胞中。经验表明，如果从每个细胞获得的病毒颗粒数量少于5000个/\AV，则该制剂将被丢弃。虽然5000个病毒粒子/细胞在2 × 109细胞制剂中会产生1 × 1013个颗粒，但这些制剂在体外或体内都表现不佳。

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.