Core--Vector

核心--向量

• 批准号: 7311631
• 负责人: Richard O Snyder
• 金额: $ 21.34万
• 依托单位: UNIVERSITY OF FLORIDA
• 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/7311631
• 关键词: adeno associated virus group; biomedical facility; biotechnology; high throughput technology; transfection /expression vector

It is essential that investigators affiliated with this program be provided with consistently high titer, pure rAAV preparations. Therefore, the Vector Core Laboratory at the University of Florida has three objectives. The first objective of the Vector Core Laboratory is to make the highest quality preclinical vector for the investigators affiliated with this program conducting pre-clinical gene transfer experiments, as well as, safety and toxicology studies to support the AAV platform. Each virus preparation is now produced using a mini-Ad plasmid DNA system to eliminate Ad contamination. Small-scale preparations of rAAV 1, 2, and 5 vectors are purified by iodixanol gradient centrifiigation followed by heparin affinity or anion exchange chromatography. Large-scale preparations of rAAV2 are purified by a newly developed method that uses FPLC chromatography on heparin sulfate, phenyl Sepharose, and cation exchange columns. All virus stocks are subjected to stringent quality control assays to assess purity, particle titer, infectious titer, particle to infectivity ratio and potential contamination by rcAAV. The second objective of the vector core is the establishment of routine small-scale high-throughput production and purification of other AAV serotypes, including AAV6, AAV7, AAV8, AAV9, AAV 10, and hybrid B19 vectors, and capsid mutants. Assays and specific reagents will be developed to accurately determine the titers and purity characteristics of the different serotype vectors and the capsid mutants. The large-scale production and purification methods that have been developed thus far are for AAV serotype 2 vectors. The third objective of the vector core is to develop large-scale production and purification methods for other AAV serotype vectors to support these projects and eventual human trials.

必须为参与该计划的研究人员提供一致的高滴度、纯 rAAV 制剂。因此，佛罗里达大学的Vector Core实验室有三个目标。载体核心实验室的首要目标是为该项目附属的研究人员制作最高质量的临床前载体，以进行临床前基因转移实验以及安全性和毒理学研究，以支持 AAV 平台。现在每种病毒制剂均使用微型 Ad 质粒 DNA 系统生产，以消除 Ad 污染。 rAAV 1、2 和 5 载体的小规模制剂通过碘克沙醇梯度离心纯化，然后进行肝素亲和或阴离子交换层析。 rAAV2 的大规模制备物通过新开发的方法进行纯化，该方法使用硫酸肝素、苯基琼脂糖凝胶和阳离子交换柱上的 FPLC 色谱法。所有病毒储液均经过严格的质量控制测定，以评估纯度、颗粒滴度、传染性滴度、颗粒与传染性比率以及 rcAAV 的潜在污染。载体核心的第二个目标是建立常规小规模高通量生产和 其他 AAV 血清型的纯化，包括 AAV6、AAV7、AAV8、AAV9、AAV 10 和杂交 B19 载体以及衣壳突变体。将开发测定方法和特异性试剂，以准确测定不同血清型载体和衣壳突变体的滴度和纯度特征。迄今为止已开发的大规模生产和纯化方法是针对AAV血清型2载体。载体核心的第三个目标是开发其他 AAV 血清型载体的大规模生产和纯化方法，以支持这些项目和最终的人体试验。