Recombinant Adeno Associated Virus

生物技术-开发重组腺相关病毒的规模化生产工艺。 为了将我们的治疗性基因转移的努力扩展到体外研究和小动物模型之外，必须克服使用贴壁细胞生产rAAV的障碍。在无脊椎动物细胞中产生生物活性的重组依赖性病毒起源于该实验室，其关键和意外的实验结果表明AAV Rep 78支持Sf 9细胞中的AAV DNA复制。尽管已知哺乳动物病毒结构蛋白在昆虫细胞中组装成病毒样颗粒（VLP），例如乳头瘤病毒科，但从未在无脊椎动物细胞中检查过病毒或载体DNA复制和包装。生产其他缺乏病毒基因的重组病毒载体似乎是可能的，特别是对于无包膜的DNA病毒。 与哺乳动物细胞中的rAAV生产类似，昆虫细胞需要来自AAV的顺式和反式因子用于复制、包装和颗粒形成。AAV末端回文序列，被称为反向末端重复序列（ITR），用作DNA合成的引物，并且被认为对于腺苷酸化是重要的。复制和包装需要四种AAV非结构蛋白中的两种：一种p5蛋白，Rep 78或Rep 68，以及一种p19蛋白，Rep 52或Rep 40。 目前，我们能够在昆虫细胞中使用BEV产生AAV血清型1、2、4、5、6和8载体。利用在线介电常数测量、感染多重性分析、感染时的细胞密度和杆状病毒稳定性研究对生产工艺进行的新开发的改进增加了我们对生产工艺以及rAAV产量的理解。我们现在获得3x 1013个颗粒/l的rAAV，因此350 l生产运行可产生1016个rAAV颗粒，足以用于毒理学研究和临床前，大型动物模型中的剂量递增实验。 重要的是，我们已经在神经退行性疾病（如脊髓小脑共济失调和亨廷顿病）和癌症的小动物模型中验证了用昆虫细胞系统产生的治疗性rAAV载体。大规模AAV生产的这些进展将使我们能够解决特定的灾难性，难治性人类疾病（如杜氏肌营养不良症），其中大量的重组载体是成功的结果必不可少的。

Biotechnology-developing scalable production processes for recombinant adeno-associated virus. To extend our efforts of therapeutic gene transfer beyond in vitro studies and small animal models, the impediment of using adherent cells for rAAV production had to be overcome. The production of biologically active, recombinant dependovirus in invertebrate cells originated in this laboratory following a critical and unexpected experimental result demonstrating that AAV Rep 78 supported AAV DNA replication in Sf9 cells. Although it was known that mammalian virus structural proteins assemble into virus-like particles (VLPs) in insect cells, e.g. Papillomaviridae, virus or vector DNA replication and packaging was never examined in invertebrate cells. Producing other recombinant virus vectors devoid of virus genes appears possible especially for non-enveloped, DNA viruses. Similar to rAAV production in mammalian cells, insect cells require both cis and trans factors from AAV for replication, packaging, and particle formation. The AAV terminal palindromes, referred to as the inverted terminal repeats (ITRs), function as primers for DNA synthesis and are considered important for encapsidation. Replication and packaging require two of the four AAV non-structural proteins: one of the p5 proteins, either Rep 78 or Rep 68, and one of the p19 proteins, either Rep 52 or Rep 40. Currently, we are able to produce AAV serotypes 1, 2, 4, 5, 6, and 8 vectors using BEVs in insect cells. Newly developed improvements to production processes utilizing on-line permittivity measurements, multiplicity of infection analysis, cell density at time of infection, and baculovirus stability studies, have increased both our understanding of the production process as well as the yields of rAAV. We now obtain ≥ 3x1013 particles /l of rAAV, thus a 350 l production run may yield ≈ 1016 rAAV particles, sufficient for toxicological studies, and preclinical, dose escalation experiments in large animal models. Importantly, we have validated therapeutic rAAV vectors produced with the insect cell system in small animal models of neurodegenerative diseases (such as spinocerebellar ataxia and Huntingtons disease) and cancer. These advances in large-scale AAV production will allow us to address specific catastrophic, intractable human diseases (such as Duchenne muscular dystrophy) for which large amounts of recombinant vector are essential for successful outcome.