A novel system for the production of fully deleted adenovirus vectors that does not require helper adenovirus
A novel system for the production of fully deleted adenovirus vectors that does not require helper adenovirus
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DOI：
10.1038/sj.gt.3301459
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发表时间：
2001-06-01
期刊：
影响因子：
5.1
通讯作者：
Krougliak, VA
中科院分区：
文献类型：
--
作者：
Cheshenko, N;Krougliak, N;Krougliak, VA
全删除腺病毒载体(FD-ADV)有望成为基因治疗的新工具。由于这些载体删除了所有的腺病毒基因，它们需要辅助腺病毒来繁殖。辅助者对载体制剂的污染限制了目前现有的FD-ADV在基因治疗中的应用。我们已经开发了一种替代的FD-ADV增殖系统，在该系统中，复制和包装载体所必需的腺病毒基因通过杆状病毒-腺病毒杂交体输送到生产细胞中。构建了携带包装缺陷型腺病毒基因组Cre重组酶可切除拷贝的混合杆状病毒Bac-B4。虽然Bac-B4中的DNA插入片段总大小为38kb，但该重组杆状病毒的遗传结构稳定。BAC-B4在Sf9昆虫细胞中的产量很高，浓缩前的滴度为5×10(8)p.f.fu/ml。用表达LacZ的Fd-AdV质粒DNA转染293-Cre细胞，然后以2000p.f.u/ml的MOI感染Bac-B4，可挽救无辅助子的载体。随后使用Bac-B4作为辅助传代获得的Fd-Adv导致每一代的载体滴度类似于100倍的增加。得到的载体完全没有辅助病毒，能够转导培养的293细胞。然而，复制能力腺病毒(RCA)的最终出现阻止了FD-ADV生产的放大。目前正在进行实验，以优化该系统以大规模生产无辅助病毒的FD-ADV，并将载体生产过程中产生复制能力强的腺病毒(RCA)的可能性降至最低。这种以杆状病毒为基础的系统将是生产FD-ADV的一种非常有用的替代方法。
Fully deleted adenovirus vectors (FD-AdVs) would appear to be promising tools for gene therapy. Since these vectors are deleted of all adenoviral genes, they require a helper adenovirus for their propagation. The contamination of the Vector preparation by the helper limits the utility of currently existing FD-AdVs in gene therapy applications. We have developed an alternative system for the propagation of FD-AdVs, in which the adenoviral genes essential for replication and packaging of the vector are delivered into producer cells by a baculovirus-adenovirus hybrid. A hybrid baculovirus Bac-B4 was constructed to carry a Cre recombinase-excisable copy of the packaging-deficient adenovirus genome. Although the total size of the DNA insert in Bac-B4 was 38 kb, the genetic structure of this recombinant baculovirus was stable. Bac-B4 gave high yields in Sf9 insect cells, with liters of 5 x 10(8) p.f.u./ml before concentration. Transfection of 293-Cre cells with lacZ-expressing FD-AdV plasmid DNA followed by infection by Bac-B4 at a MOI of 2000 p.f.u./ml resulted in rescue of the helper-free vector. Subsequent passaging of the obtained FD-AdV using Bac-B4 as a helper resulted in similar to 100-fold increases of the vector titer at each passage. This resulting Vector was completely free of helper virus and was able to transduce cultured 293 cells. However, scaling-up of FD-AdV production was prevented by the eventual emergence of replication-competent adenovirus (RCA). Experiments are underway to optimize this system for the large-scale production of helper virus-free FD-AdVs and to minimize the possibility of generation of replication-competent adenovirus (RCA) during vector production. This baculovirus-based system will be a very useful alternative to current methods for the production of FD-AdVs.