Core--Vector

核心--向量

• 批准号: 6754329
• 负责人: NICHOLAS MUZYCZKA
• 金额: $ 31.21万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-12-01 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6754329
• 关键词: adeno associated virus group; biomedical facility; biotechnology; cell line; helper virus; 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 are purified by a novel method utilizing iodixanol gradient centrifugation followed by heparin affinity chromatography. Large-scale preparations are purified by a newly developed method that uses FPLC chromatography on heparin sulfate and phenyl Sepharose 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 core is also capable of providing support to the projects requiring lentiviral and adenoviral vectors. The second objective of the Vector Core is to implement an improved method for scale up of rAAV production that relies on the infection of AAV producer cell lines carrying a rAAV provirus, with a defective helpervirus. In the first scenario, a defective herpesvirus carrying the AAV rep and cap genes is used to infect the proviral cell lines. In a second approach, adenovirus is used to infect cell lines harboring the AAV provirus and the AAV rep and cap genes. These approaches provide increased vector yield on a per cell basis compared to currently used plasmid transfection methods, and allows scale-up of production. Because these approaches require the isolation of producer cells lines, it has the disadvantage of requiring increased set up time. These methods are currently being adapted to high capacity bioreactor production. As this method becomes available, it will be used for preclinieal virus preparations that require large amounts of a single vector type (in excess of 10[14] infectious units equal to 10[15] to 10[16] vector particles). The production and purification methods that have been developed thus far are for AAV serotype 2 vectors. The third objective of the vector core is the routine and large-scale production and purification of other AAV serotypes, including AAV1 and AAV5 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.

必须为该项目的研究人员提供始终如一的高滴度、纯rAAV制剂。因此，佛罗里达大学的载体核心实验室有三个目标。载体核心实验室的首要目标是为该项目下属的研究人员提供最高质量的临床前载体，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