Gene Therapy Vector Core

基因治疗载体核心

• 批准号: 8650323
• 负责人: JOSEPH E RABINOWITZ
• 金额: $ 15万
• 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/8650323
• 关键词: Adenoviruses; Affect; Aliquot; American; Animal Model; Antibiotics; Biological; Biological Testing; Cardiac; Cardiac Myocytes; Cell Line; Cells; Centrifugation; Cesium; Cloning; Cytomegalovirus; Dependovirus; Development; Dialysis procedure; Enhancers; Gene Transduction Agent; Germany; Goals; Heart failure; Hela Cells; In Vitro; Individual; Instruction; Methods; MicroRNAs; Molecular; Myocardial Infarction; Neonatal; Participant; Plasmids; Polyethylene Glycols; Precipitation; Production; Psyche structure; Rattus; Recommendation; Serotyping; Services; Silver Staining; Subfamily lentivirinae; Sucrose; Testing; Therapeutic; Time; Titrations; Transgenes; Viral; Viral Vector; Virion; Virus; Western Blotting; absorption; base; cell preparation; 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.

项目摘要（请参阅说明）：此病毒矢量核心功能是为三个项目提供两个主要服务：（1）增强子/启动子和转基因的设计和构建，用于在病毒矢量中包装； （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）每个病毒矢量具有特定的设计要求，包括包装能力，血清型和伪型建议，将在构建每个新包装质粒之前对其进行检查。 b）每个包装质粒的构建将通过限制消化和测序确认，然后对转基因表达进行适当测试。生产，纯化和测试包括：a）生产： - /\ AV血清型1-9-腺病毒2 -VSVG假病毒假病变型慢病毒b） /\ av的纯化将取决于血清型。 AAV血清型1-8已通过碘级梯度离心纯化，然后是快速蛋白质液相色谱（FPLC），然后进行透析。 /\ av9已通过聚乙烯甘油沉淀纯化，然后是剖腹梯度，然后是剖腹连续梯度，然后是透析。对于每种纯化方法，我们都使用了转基因引物和实时PCR来量化病毒的滴度。为了检查病毒的纯度，我们使用SDS聚丙烯酰胺凝胶的银色染色。为了测试生物污染物，我们在没有抗生素的培养细胞中添加了纯化病毒的等分试样。经验证明，如果从每个单元中获得的病毒颗粒的数量小于5000，则将丢弃准备。尽管2 x 109细胞制备中的5000个病毒粒子/细胞会产生1 x 1013颗粒，但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，传统的铯阶梯梯度方法，然后是连续的铯梯度，然后是透析。第二种方法使用Vivapure Adenopack套件（德国Goettingen）的商业柱纯化方法。我们已经利用了紫外线吸收来定量（Liebermann和Chenter 1994）以及实时PCR。慢病毒的纯化利用了蔗糖垫梯度。另外，还有许多可商购的慢病毒浓度和纯化试剂盒（Cell Biolabs； CA）。 c）每个病毒的测试包括： i）在向项目领导者交付之前透析后对病毒的滴定。 ii）测试CMV或其他强启动子或原发性新生大鼠心肌细胞（心脏）启动子的HeLa细胞中病毒的体外转导效率。 iii）将使用Western印迹分析来建立病毒转基因的表达，然后再向项目领导者传递。 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，如PPG参与者的需求所示，我们将开发和维护特定载体和转基因的大规模生产（100-200 cm板范围）的细胞系。我们将提供与新的治疗基因，抑制性和微RNA以及根据项目负责人所需的病毒发育的增强子促进型构型相关的分子生物学支持。核心D的主要功能是； 1）产生腺相关病毒和腺相关病毒，2）这些病毒的纯化，以及3）在转移到科学计划成员之前测试病毒。另一个功能将是科学计划成员需要的VSVG假病毒的生产。