Abstract
A human embryonic kidney cell line 293 is widely used for adenovirus production and propagation. With this cell line, however, replication-competent virus (RCV) is frequently generated, especially during large-scale production and successive propagation because 293 cells contain not only E1 gene but also non-E1 adenovirus gene. Homologous recombination between non-E1 region of 293 genomic DNA and its homologous region in the recombinant adenoviral vector generate RCV. To overcome this problem, we developed a new packaging cell line, Hela-E1, which contains minimum E1 region and from which non-E1 adenoviral region that is homologous with recombinant adenovirus vector was excluded. No RCV was detected during adenovirus propagation in Hela-E1 compared to in 293. In addition, adenovirus-p53 produced in HeLa-E1 was able to overexpress p53 protein when introduced into an ovarian cancer cell line, SKOV3. These results may have a significant impact on the development of packaging cell lines for replication-deficient adenovirus production.
Highlights
A recombinant adenovirus vector has been extensively utilized as a delivery vehicle for gene transfer, vaccination, and gene therapy (Zhang, 1999)
Adenovirus-p53 (Ad-p53) produced in HeLa cell that expresses adenovirus E1 gene (HeLa-E1) induced an overexpression of p53 protein when transfected into an ovarian cancer cell line, SKOV3. These results indicate that HeLa-E1 can act as a packaging cell line that excludes replication competent virus (RCV) generation and the strategy used here can be useful for making other new packaging cell lines for recombinant adenovirus production
The most widely used adenovirus packaging cell line is 293, which was derived from human embryonic kidney (HEK) cells by transformation with Adenovirus type5 (Ad5) fragments. 293 cells contain the left 11% of Ad5 genome
Summary
A recombinant adenovirus vector has been extensively utilized as a delivery vehicle for gene transfer, vaccination, and gene therapy (Zhang, 1999). The widely used recombinant adenovirus vector was derived from subgroup C and is highly infectious and capable of delivering therapeutic genes to many different cell types. RCV production was often detected during a large-scale adenovirus propagation or successive production in universal packaging cell line, 293 (Lochmuller et al, 1994). This is due to a fact that 293 cells contain and express the left 11% of the Adenovirus type (Ad5) genome and this region contains a part of adenovirus E1 region (1.6-9.7 m.u.) as well as non-E1 region (0-1.6 m.u., 9.7-11 m.u.). RCV is presumably generated by the homologous recombination between non-E1 region of 293 genomic DNA and homologous region of a recombinant adenovirus vector (Figure 1)
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