Core--Vector

核心--向量

• 批准号: 6853361
• 负责人: Richard O Snyder
• 金额: $ 13.01万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6853361
• 关键词: adeno associated virus group; affinity chromatography; biomedical facility; biotechnology; chloride channels; cystic fibrosis; gene therapy; heparan sulfate; technology /technique development; 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 centrifugation 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 routine and large-scale production and purification of other AAV serotypes, including AAV1, AAV5, AAV7, and AAV8 vectors, and capsid mutants of AAV serotypes 2, 1, and 5. Assays and specific reagents will be developed to accurately determine the titers of the different serotype vectors and the capsid mutants. The third objective of this proposal is to develop viral vectors targeted to bronchial epithelial cells in order to provide a highly specific delivery of the cystic fibrosis (CF) gene, as well as to diminish the transduction of collateral tissues thereby reducing the required dose of vector to the patients.

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 载体的小规模制备物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常规和大规模生产和纯化其他 AAV 血清型，包括 AAV1、AAV5、AAV7 和 AAV8 载体，以及 AAV 血清型 2、1 和 5 的衣壳突变体。 测定和特定试剂将开发用于准确测定不同血清型载体和衣壳突变体的滴度。该提案的第三个目标是开发针对支气管上皮细胞的病毒载体，以提供囊性纤维化（CF）基因的高度特异性递送，并减少侧支组织的转导，从而减少所需的载体剂量给患者。