Recombinant Adeno Associated Virus

非致病性人类依赖病毒，腺相关病毒（AAV），感染增殖细胞或有丝分裂后细胞。由于所有AAV和辅助病毒基因都可以反式提供，因此整个AAV基因组可能被感兴趣的基因所取代。然后可以在细胞培养中瞬时产生重组AAV (rAAV)，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生产方法依赖于贴壁细胞的瞬时转染，其生长速度约为每厘米100,000个细胞(2)。如果每个转染细胞产生10,000个rAAV颗粒，则每厘米(2)的产量为每厘米(2)10e9个颗粒或每15厘米直径板约2 × 10e11个颗粒。虽然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5000到10000个组织培养板。为了利用细胞悬浮生长的优势，我们开发了一种rAAV生产系统，该系统利用重组加州自签名核多角体病毒（AcNPV或杆状病毒）将AAV rep和cap基因以及rAAV载体DNA传递到昆虫细胞系Spodoptera frugiperda （Sf9）细胞中。这些细胞以每ml 10e6到10e7个细胞的密度生长。我们证明，在AAV Rep 78存在的情况下，rAAV DNA从质粒或杆状病毒中获救并复制，并且rAAV DNA复制的水平超过了在HEK 293细胞中观察到的水平。同样，Sf9细胞提取物比HEK 293细胞表达更多的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VP表达量。两个AAV编码的复制蛋白，Rep 78和Rep 52，是AAV DNA复制所必需的。利用强弱两种昆虫病毒启动子，可获得较高水平的Rep 52和较低水平的Rep 78。用三种重组杆状病毒：Bac-Rep、Bac-VP和Bac-ITR感染Sf9细胞，每个Sf9细胞产生超过10e4个耐dna酶的rAAV2颗粒。以每ml 2 × 10e6个细胞的速度感染Sf9细胞，结果每ml产生bbb10e10个颗粒。我们对颗粒进行了分析，并根据以下实验确定Sf9产生的rAAV与HEK 293产生的rAAV无法区分。电子显微镜；蛋白质组成浮力密度4。用抗aav2衣壳mAb 5中和。293细胞的转导肝素对转导的抑制作用小鼠视网膜和骨骼肌的体内转导我们已经扩展了这一过程，包括AAV血清型1、4和5。然而，使用为生产rAAV2开发的工艺，没有先验的原因可以阻止Sf9细胞中任何AAV血清型的生产。

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.