Recombinant Adeno Associated Virus

重组腺相关病毒

• 批准号: 6966931
• 负责人: Robert Kotin
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 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/6966931
• 关键词: DNA replication origin; adeno associated virus group; capsid; human tissue; microorganism culture; plasmids; recombinant virus; transfection /expression vector; virus genetics; virus replication

The non-pathogenic human dependoviruses, adeno-associated viruses (AAV), infect either proliferating or post-mitotic cells. Since all the AAV and helper virus genes can be provided in trans, the entire AAV genome may be replaced with the gene of interest. The resulting recombinant AAV (rAAV) may then be produced transiently in cell culture and the rAAV particles concentrated and purified based on physical properties of the virion. The established rAAV production methods rely on transient transfection of adherent cells which grow at approximately 100,000 cells per cm(2). If each transfected cell produces 10,000 particles of rAAV, then the yield per cm(2) is 10e9 particles per cm(2) or approximately 2x10e11 particles per 15 cm diameter plate. Although the per cell yield of rAAV is relatively high, the ability to scale-up production rapidly becomes untenable with exponential increase of cells required to produce vector. Thus, production of 10e15 particles requires 5,000 to 10,000 tissue culture plates. In order to take advantage of cells growing in suspension, we developed an rAAV production system that utilizes recombinant Autographa californica nuclear polyhedrosis virus, (AcNPV or baculovirus) to deliver the AAV rep and cap genes as well as the rAAV vector DNA into the insect cell line, Spodoptera frugiperda (Sf9) cells. These cells grow at densities of 10e6 to >10e7 cells per ml. We demonstrated that rAAV DNA is rescued and replicated from either plasmid or baculovirus in the presence of AAV Rep 78 and that the level of rAAV DNA replication exceeds the level observed in HEK 293 cells. Similarly, Sf9 cell extracts express the AAV structural proteins, VP-1, VP-2, and VP-3 in greater amounts than HEK 293 cells. By manipulating the initiation codon and contexts, we were able to obtain VP expression in amounts that appear in the mature virion. Two AAV encoded replication proteins, Rep 78 and Rep 52, are necessary for AAV DNA replication. By utilization of a strong and weak insect virus promoters, we were able to achieve relatively high levels of Rep 52 and low levels of Rep 78. Infecting Sf9 cells with three recombinant baculoviruses: Bac-Rep, Bac-VP, and Bac-ITR, produced greater than 10e4 DNAse resistant particles of rAAV2 per Sf9 cells. Infecting the Sf9 cells at 2x10e6 cells per ml resulted in >10e10 particles per ml. We analyzed the particles and have determined that the Sf9 produced rAAV was indistinguishable from HEK 293 produced rAAV based on the following assays: 1. Electron microscopy 2. Protein composition 3. Buoyant density 4. Neutralization with an anti-AAV2 capsid mAb 5. Transduction of 293 cells 6. Inhibition of transduction with heparin 7. In vivo transduction of murine retina and skeletal muscle We have extended the process to include AAV serotypes 1, 4, and 5. However, using the processes developed for rAAV2 production, there are no a priori reasons that would prevent the production of any AAV serotype in Sf9 cells.

非致病性人类依赖病毒，腺相关病毒(AAV)，感染增殖或有丝分裂后的细胞。由于所有的AAV和辅助病毒基因都可以反式提供，AAV的整个基因组可能会被目标基因取代。得到的重组AAV(RAAV)然后可以在细胞培养中瞬时产生，并根据病毒粒子的物理性质浓缩和纯化rAAV颗粒。已建立的rAAV生产方法依赖于瞬时转染贴壁细胞，贴壁细胞的生长速度约为每厘米100,000个细胞(2)。如果每个转基因细胞产生10,000个rAAV颗粒，那么每厘米(2)的产量是每厘米(2)10e9个颗粒，或大约每15厘米直径平板2x10e11个颗粒。虽然rAAV的单位细胞产量相对较高，但随着生产载体所需细胞的指数增长，迅速扩大生产的能力变得难以维持。因此，生产10E15颗粒需要5,000到10,000块组织培养板。为了更好地利用悬浮生长的细胞，我们建立了一种利用重组链球菌核型多角体病毒(AcNPV或杆状病毒)将AAV rep和capp基因以及rAAV载体DNA导入昆虫细胞系Spotoptera rugiperda(Sf9)的rAAV生产系统。这些细胞以每毫升10e6到10e7个细胞的密度生长。我们证明了在AAV Rep 78存在的情况下，rAAVDNA可以从质粒或杆状病毒中拯救和复制，并且rAAVDNA复制水平超过了在HEK 293细胞中观察到的水平。同样，Sf9细胞提取物比HEK 293细胞更多地表达AAV结构蛋白VP-1、VP-2和VP-3。通过操纵起始密码子和上下文，我们能够获得成熟病毒粒子中出现的数量的VP表达。两个AAV编码的复制蛋白Rep 78和Rep 52是AAV DNA复制所必需的。通过利用强弱两种昆虫病毒启动子，我们能够获得相对较高水平的Rep 52和较低水平的Rep 78。用Bac-Rep、Bac-VP和Bac-ITR三种重组杆状病毒感染Sf9细胞，每Sf9细胞产生大于10e4DNase抗性的rAAV2颗粒。以每毫升2×10e6个细胞感染Sf9细胞产生&GT；10e10颗粒/毫升。我们分析了这些颗粒，并根据以下检测确定Sf9产生的rAAV与HEK 293产生的rAAV没有区别：1.电子显微镜2.蛋白质组成3.浮力密度4.用抗AAV2衣壳单抗中和5.293细胞的转导6.肝素抑制转导7.小鼠视网膜和骨骼肌的体内转导我们已经扩展了这个过程到包括AAV血清型1、4和5。然而，使用为生产rAAV2而开发的方法，没有先验的原因可以阻止任何AAV血清型在Sf9细胞的产生。