Improved system for producing adenoviral vectors

第一代非复制型腺病毒载体显示出作为抗癌剂和作为抗肿瘤药物的前景。 预防性疫苗。目前用于制造这些载体的方法使用293细胞 细胞系或PER.这些生产细胞提供互补的E1 a和E1 b病毒 在细胞中对失能的腺病毒载体起作用。293细胞系非常有用， 广泛用于制备用于早期临床试验的相对小批量的载体。然而 它产生可复制腺病毒的频率，从10到20， 50%的批次取决于载体，这意味着，随着制造工艺的规模化， 到了后期的临床试验，或者是市场化的时候，就越来越不被人接受了。随着水垢 增加，越来越多的批次超过FDA规定的允许污染 水平的1 RCA/3x 10 e10病毒颗粒，这样的生产工艺不能声称是 ?控制？如果故障率大于~ 10%，则按照FDA标准。PER.C6细胞使用 特别设计的匹配载体，以消除生产系和 vector.该系统与大多数当前临床载体不兼容， 学术研究人员或小公司。RCA频率降低10倍， 在293个细胞中观察到的，如果载体生产率是 相当于我们以人肿瘤细胞系A549为基础，构建了一个名为c24的细胞系， 其生产能力与293个细胞一样，是稳定的，不含支原体，并携带1-2个拷贝的 E1表达质粒与常规腺病毒载体重叠非常有限。我们提出 为了严格测试c24与293细胞相比的RCA频率，并使其适用于无血清 增长如果RCA生成可以减少>10倍，并且在无血清环境中，则 C24可以为具有进一步改进的安全性的可靠的载体生产系统提供基础 可以广泛用于生产腺病毒载体产品的特征，包括 是吗？自己的后期管道。

First generation non-replicative adenoviral vectors show promise as anti-cancer agents and as prophylactic vaccines. Current methods for manufacturing these vectors use either the 293 cell line or the PER.C6 cell line. These producer cells provide complementary E1a and E1b viral function in the cell for disabled adenoviral vectors. The 293 cell line has been very useful and widely used for making relatively small batches of vector for early stage clinical trials. However the frequency with which it generates replication competent adenovirus, which disqualifies from 10 to 50% of batches depending on the vector, means that, as the manufacturing processes are scaled up in later stage clinical trials, or for marketing, it becomes less and less acceptable. As scale increases, increasing numbers of batches exceed the FDA mandated allowable contamination level of 1RCA/3x10e10 viral particles, and such a manufacturing process cannot be claimed to be ?controlled? by FDA standards if the failure rate is greater than ~ 10%. PER.C6 cells use a specifically designed matched vector to eliminate sequence overlap between producer line and vector. The system is not compatible with most current clinical vectors and is not available to academic researchers or small companies. A modest 10 fold reduction in RCA frequency over that seen in 293 cells would provide a robust production system if the vector productivity were equivalent. We have constructed a cell line named c24 based on the human tumor line A549, which is as productive as 293 cells, is stable, is free of mycoplasma and carries 1-2 copies of an E1 expression plasmid with very limited overlap with conventional adenoviral vectors. We propose to rigorously test the RCA frequency in c24 compared to 293 cells, and to adapt it to serum free growth. If the RCA generation can be reduced by >10-fold, and in a serum free environment, then c24 can provide the basis for a reliable vector production system with a further improved safety profile that can be widely used to manufacture adenoviral vector products, including Advantagene?s own late stage pipeline.