Gene Therapy Vector Core

基因治疗载体核心

• 批准号: 8466892
• 负责人: JOSEPH E RABINOWITZ
• 金额: $ 14.58万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8466892
• 关键词: Adenoviruses; Affect; Aliquot; American; Animal Model; Antibiotics; Biological; Biological Testing; Cardiac; Cardiac Myocytes; Cell Line; Cells; Centrifugation; Cesium; Cloning; Core Facility; Cytomegalovirus; Dependovirus; Development; Dialysis procedure; Economics; Enhancers; Feedback; Gene Transduction Agent; Germany; Goals; Heart failure; Hela Cells; In Vitro; Individual; Instruction; Laboratories; Methods; MicroRNAs; Molecular; Myocardial Infarction; Neonatal; Participant; Plasmids; Polyethylene Glycols; Precipitation; Preparation; Procedures; Production; Psyche structure; Quality Control; Rattus; Reagent; Recommendation; Serotyping; Services; Silver Staining; Subfamily lentivirinae; Sucrose; Testing; Therapeutic; Time; Titrations; Transgenes; Viral; Viral Vector; Virion; Virus; Western Blotting; absorption; base; cell preparation; cost; design; design and construction; experience; fast protein liquid chromatography; heart function; improved; in vitro testing; in vivo; iodixanol; large scale production; member; novel therapeutics; particle; polyacrylamide gels; programs; promoter; recombinant virus; therapeutic gene; transduction efficiency; transgene expression; vector

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.

项目概要（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）设计和构建增强子/启动子和转基因，用于包装在病毒载体内； (2) 这些病毒载体类型的生产、纯化和测试。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，这些将在构建每个新的包装质粒之前进行检查。 B) 每个包装质粒的构建将通过限制性消化和测序来确认，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 已通过碘克沙醇梯度离心、快速蛋白液相色谱 (FPLC)、随后透析进行纯化。 /\AV9 已通过聚乙二醇沉淀纯化，然后铯逐步梯度，然后铯连续梯度，然后透析。对于每种纯化方法，我们都使用转基因引物和实时 PCR 来量化病毒的滴度。为了检查病毒的纯度，我们使用 SDS 聚丙烯酰胺凝胶进行银染色。为了测试生物污染物，我们将等份的纯化病毒添加到不含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​​准备将被丢弃。虽然 2 x 109 细胞制剂中的 5000 个病毒粒子/细胞将产生 1 x 1013 颗粒，但这些制剂在体外或体内表现不佳。 为了纯化腺病毒，我们使用两种方法。首先采用传统的铯阶梯梯度法，然后采用连续铯梯度法，然后进行透析。第二种方法使用 Vivapure AdenoPACK 试剂盒（德国哥廷根）的商业柱纯化方法。我们利用紫外吸收进行定量（Liebermann 和 Mental 1994）以及实时 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；圣地亚哥，加利福尼亚州）。 C) 对每种病毒的测试包括： i) 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 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测试限制性（心脏）启动子的体外转导效率。 iii) Western blot analysis will be used to establish expression of viral transgenes prior to delivery to project leaders. 该核心将生产和纯化 AAV 血清型 1-9、腺病毒 2 型和慢病毒。具体来说，我们将根据 PPG 参与者的需求开发和维护用于大规模生产（15 厘米板范围内 100-200 个）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RNA和微小RNA的亚克隆相关的分子生物学支持，以及用于病毒发育的增强子-启动子配置。 Core D的主要功能是； 1) 腺病毒和腺相关病毒的生产，2) 这些病毒的纯化，以及 3) 在转移给科学计划成员之前测试病毒。另一项功能是根据科学计划成员的需要生产 VSVG 假型慢病毒。