Core--Vector

重要的是，研究人员隶属于该计划提供一贯的高滴度，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与该计划有关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，以及支持AAV的安全性和毒理学研究。 平台现在使用mini-Ad质粒DNA系统生产每种病毒制剂以消除Ad污染。小规模制备的rAAV纯化的一种新的方法，利用碘克沙醇梯度离心，然后肝素亲和层析。大规模的制剂是通过一种新开发的方法，使用FPLC纯化 在硫酸肝素和苯基琼脂糖柱上进行柱层析。对所有病毒原液进行严格的质量控制测定，以评估纯度、颗粒滴度、感染滴度、颗粒与感染性比率和rcAAV的潜在污染。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。Vector Core的第二个目的是实施一种用于rAAV生产规模扩大的改进方法， 依赖于用缺陷型辅助病毒感染携带rAAV原病毒的AAV生产细胞系。在第一种情况下，使用携带AAV rep和cap基因的缺陷型疱疹病毒感染前病毒细胞系。在第二种方法中，使用腺病毒感染携带AAV前病毒和AAV rep和cap基因的细胞系。与目前使用的质粒转染方法相比，这些方法在每个细胞的基础上提供了增加的载体产率，并允许扩大生产。因为这些方法需要分离生产细胞系，所以其具有需要增加的建立时间的缺点。这些方法目前正在适应高容量生物反应器生产。随着该方法的可用，它将用于需要大量单一载体类型（超过10[14]个感染单位等于10[15]至10[16]个载体颗粒）的临床前病毒制备。 迄今为止已经开发的生产和纯化方法用于AAV血清型2载体。载体核心的第三个目的是常规和大规模生产和纯化其他AAV血清型，包括AAV 1和AAV 5载体，以及AAV血清型2、1和5的衣壳突变体。将开发测定和特异性试剂，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.