Cheshenko N, Krougliak N, Eisensmith R C, Krougliak V A
Institute for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA.
Gene Ther. 2001 Jun;8(11):846-54. doi: 10.1038/sj.gt.3301459.
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 titers 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 approximately 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.
完全缺失腺病毒载体（FD-AdVs）似乎是基因治疗的有前景的工具。由于这些载体缺失了所有腺病毒基因，它们的繁殖需要辅助腺病毒。辅助病毒对载体制剂的污染限制了现有FD-AdVs在基因治疗应用中的效用。我们开发了一种用于FD-AdVs繁殖的替代系统，其中载体复制和包装所必需的腺病毒基因由杆状病毒-腺病毒杂交体递送至生产细胞。构建了一种杂交杆状病毒Bac-B4，以携带包装缺陷型腺病毒基因组的可被Cre重组酶切除的拷贝。尽管Bac-B4中插入的DNA总大小为38 kb，但这种重组杆状病毒的遗传结构是稳定的。Bac-B4在Sf9昆虫细胞中产生高产量，浓缩前滴度为5×10⁸ p.f.u./ml。用表达lacZ的FD-AdV质粒DNA转染293-Cre细胞，然后以2000 p.f.u./ml的感染复数用Bac-B4感染，导致无辅助病毒载体的拯救。随后使用Bac-B4作为辅助病毒对获得的FD-AdV进行传代，每次传代载体滴度增加约100倍。所得载体完全不含辅助病毒，并且能够转导培养的293细胞。然而，有复制能力的腺病毒（RCA）的最终出现阻碍了FD-AdV生产的扩大规模。正在进行实验以优化该系统用于大规模生产无辅助病毒的FD-AdVs，并尽量减少载体生产过程中产生有复制能力的腺病毒（RCA）的可能性。这种基于杆状病毒的系统将是目前生产FD-AdVs方法的非常有用的替代方法。
