Vector Core

在本申请的项目中提出的实验需要从各个实验室设计的质粒中生产AAV载体。载体核心设施的目标是生产研究级AAV载体，用于止血正常和血友病小鼠、非人类灵长类动物和血友病狗的临床前研究。该核心将确保根据个别项目的要求，提供用于表达治疗性转基因或报告基因的重组AAV。AAV载体是在大规模转染HEK-293细胞的基础上，利用提供AAV rep/帽和腺病毒辅助功能的两个辅助质粒和编码重组载体的第三个质粒，在无辅助病毒系统中产生的。这将允许产生各种不同的载体(包括不同的转基因、表达 卡带和衣壳/血清型)供调查人员使用。核心设备的使用将提供可重复的媒介产量和纯度，并且将比单独实验室的媒介生产更具成本效益，特别是对于涉及大型动物模型的实验。Core的服务将包括大规模制备辅助和载体载体，使用磷酸钙沉淀法大规模转染HEK-293细胞，通过细胞裂解、差速沉淀和梯度离心法回收和纯化重组AAV，透析、无菌过滤和储存纯化的载体，以及定量狭缝杂交。该中心将对载体制剂进行质量控制，并协助不同的项目进行功能分析。标准载体制剂预计可产生约10[14]个载体 每10个滚筒瓶或同等的组织培养器皿的基因组。使用滚瓶法的媒介生产规模已经得到优化(BioTechniques 34：1,2003)，并将应用于大型动物研究媒介的生产。核心将产生不同血清型的AAV载体，包括AAV-2、AAV-8和混合载体。

Experiments proposed in the projects of this application require production of AAV vectors from plasmids designed by the individual laboratories. The goal of the Vector Core Facility is the production of research grade AAV vector for pre-clinical studies in hemostatically normal and hemophilic mice, non-human primates, and in hemophilic dogs. The Core will ensure the availability of recombinant AAV for expression of therapeutic transgenes or reporter genes as required by the individual projects. AAV vector is produced in a helper virus-free system based on large-scale transfection of HEK-293 cells using two helper plasmids that supply AAV rep/cap and adenoviral helper functions and a third plasmid encoding the recombinant vector. This will allow production of a variety of different vectors (including different transgenes, expression cassettes and capsids/serotypes) for the investigators. The use of a Core Facility will provide reproducible yield and purity of vector, and will be more cost-effective than vector production in individual laboratories, in particular for experiments that involve large animal models. The service of the Core will include large-scale preparation of helper and vector plasmids, large-scale transfection of HEK-293 cells using calcium phosphate precipitation, recovery and purification of recombinant AAV by cell lysis followed by differential precipitation and gradient centrifugation, dialysis, sterile filtration, and storage of purified vector, and quantitative slot blot hybridization. The Core will carry out quality control of vector preparations and assist the different projects with functional assays. Standard vector preparations are expected to yield about 10[14] vector genomes per 10 roller bottles or equivalent tissue culture vessels. Scale-up of vector production using a roller bottle method has been optimized (Biotechniques 34:1, 2003) and will be applied to production of vector for large animal studies. The Core will produce AAV vector of different serotypes including AAV-2, and -8, and hybrid vectors.