Vector Core

本应用项目中提出的实验要求从单个实验室设计的质粒生产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犬的临床前研究。该中心将根据个别项目的要求，确保重组AAV用于表达治疗性转基因或报告基因的可用性。AAV载体是在无辅助病毒的系统中产生的，该系统基于大规模转染HEK-293细胞，使用两个提供AAV rep/cap和腺病毒辅助功能的辅助质粒和第三个编码重组载体的质粒。这将允许生产各种不同的载体（包括不同的转基因、表达）

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.