Core--Vector

至关重要的是，必须为参与该计划的研究人员提供一贯的高滴度、纯rAAV制剂。因此，佛罗里达大学的矢量核心实验室有三个目标。载体核心实验室的第一个目标是为参与该计划的研究人员制造最高质量的临床前载体，进行临床前基因转移实验，以及支持AAV的安全性和毒理学研究 站台。每种病毒制剂现在都使用迷你Ad质粒DNA系统来生产，以消除Ad污染。采用碘二醇梯度离心和肝素亲和层析的新方法纯化小规模rAAV制剂。大规模制剂是通过一种新开发的使用FPLC的方法进行纯化的 硫酸肝素和苯基琼脂糖柱层析。所有储存的病毒都经过严格的质量控制分析，以评估纯度、颗粒滴度、感染性滴度、颗粒与传染性的比率以及可能受到rcAAV的污染。该核心还能够为需要慢病毒和腺病毒载体的项目提供支持。向量核心的第二个目标是实施一种改进的方法来扩大rAAV生产，该方法 依赖于携带rAAV前病毒的AAV产生细胞系感染缺陷的HelperVirus。在第一种情况下，携带AAV代表和帽基因的有缺陷的疱疹病毒被用来感染前病毒细胞系。在第二种方法中，腺病毒被用来感染含有AAV前病毒和AAV rep和capp基因的细胞系。与目前使用的质粒转染法相比，这些方法在每个细胞的基础上提供了更高的载体产量，并允许放大生产。因为这些方法需要隔离生产者细胞系，所以它具有需要增加建立时间的缺点。这些方法目前正被用于高容量生物反应器的生产。随着这种方法的出现，它将用于需要大量单一媒介类型(超过10[14]个感染单位，相当于10[15]到10[16]个媒介颗粒)的临床前病毒制剂。 到目前为止，已经开发的生产和纯化方法是针对AAV血清2型载体。载体核心的第三个目标是常规和大规模地生产和纯化其他AAV血清型，包括AAV1和AAV5载体，以及AAV2、1和5型AAV2、1和5型的衣壳突变体。将开发各种检测方法和特异性试剂，以准确测定不同血清型载体和衣壳突变体的滴度。

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.