Recombinant Adeno Associated Virus

非致病性人依赖病毒，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，因此整个AAV基因组可以用感兴趣的基因替换。然后可以在细胞培养物中瞬时产生所得重组AAV（rAAV），并基于病毒体的物理性质浓缩和纯化rAAV颗粒。已建立的rAAV生产方法依赖于贴壁细胞的瞬时转染，贴壁细胞以约100，000个细胞/cm生长（2）。如果每个转染细胞产生10，000个rAAV颗粒，则每cm（2）的产量为每cm（2）10 e9个颗粒或每15 cm直径板约2x 10 e11个颗粒。尽管rAAV的每细胞产量相对较高，但随着生产载体所需细胞的指数增加，迅速扩大生产的能力变得难以维持。因此，生产10 e15颗粒需要5，000至10，000个组织培养板。为了利用悬浮生长的细胞，我们开发了利用重组苜蓿银纹夜蛾核型多角体病毒（AcNPV或杆状病毒）将AAV rep和cap基因以及rAAV载体DNA递送到昆虫细胞系草地贪夜蛾（Spodoptera frugiperda，Sf 9）细胞中的rAAV生产系统。这些细胞以每毫升10 e6至> 10 e7个细胞的密度生长。我们证明了在AAV Rep 78的存在下，rAAV DNA从质粒或杆状病毒中被拯救和复制，并且rAAV DNA复制的水平超过了在HEK 293细胞中观察到的水平。类似地，Sf 9细胞提取物以比HEK 293细胞更大的量表达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，我们能够获得出现在成熟病毒体中的VP表达量。两种AAV编码的复制蛋白Rep 78和Rep 52是AAV DNA复制所必需的。通过利用强和弱昆虫病毒启动子，我们能够实现相对高水平的Rep 52和低水平的Rep 78。用三种重组杆状病毒：Bac-Rep、Bac-VP和Bac-ITR感染Sf 9细胞，每个Sf 9细胞产生大于10 e4个rAAV 2的DNA酶抗性颗粒。以2xl 0 e6个细胞/ml感染Sf 9细胞导致> 10 e10个颗粒/ml。我们分析了颗粒，并基于以下测定确定Sf 9产生的rAAV与HEK 293产生的rAAV不可区分：电子显微镜2.蛋白质组成3.浮力密度4.用抗AAV 2衣壳mAb中和5. 293细胞的转导6.肝素7对转导的抑制。鼠视网膜和骨骼肌的体内转导我们已经将该过程扩展到包括AAV血清型1、4和5。然而，使用为rAAV 2生产开发的方法，没有先验原因会阻止在Sf 9细胞中产生任何AAV血清型。

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.