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Adenovirus vector production using low-multiplicity infection of 293 cells.

ABSTRACT: The use of low-multiplicity infection of 293 cells in static culture with regular medium replacement was investigated for efficient large-scale production of adenovirus vectors for gene therapy applications. An adenovirus vector carrying the enhanced green fluorescent protein gene (Ad EGFP) was used to infect 293-F cells at a low multiplicity of infection (MOI) of 0.00001-0.1 transductional unit (TU) cell(-1). The cells, which have the ability to grow in suspension, were incubated in T-flasks and the serum-free culture medium was replaced with fresh medium via centrifugation every 2 days. Because only a small proportion of cells were initially infected at low MOIs (<1 TU cell(-1)), uninfected cells continued to grow until they were infected by progeny adenoviruses released from previously infected cells. When 293-F cells at a relatively low density of 1 x 10(5) cells cm(-3) were infected with Ad EGFP at a low MOI of 0.001 TU cell(-1), the vector yield was 2.7-fold higher than the maximum yield obtained with high-multiplicity infection (MOI = 10 TU cell(-1)) in batch culture. These results indicate that efficient adenovirus vector production using low MOIs is achieved by minimization of either nutrient depletion and/or accumulation of inhibitory metabolites in the culture medium.

SUBMITTER: Yamada K

PROVIDER: S-EPMC2774567 | biostudies-other | 2009 Apr

REPOSITORIES: biostudies-other

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Project description:Recombinant adenovirus (Ad) gene transfer vectors are effective at transferring exogenous genes to a variety of cells and tissue types both in vitro and in vivo. However, in the process of gene transfer, the Ad vectors induce the expression of target cell genes, some of which may modify the function of the target cell and/or alter the local milieu. To develop a broader understanding of Ad vector-mediated induction of endogenous gene expression, genes induced by first-generation E1(-) E4(+) Ad vectors in primary human umbilical vein endothelial cells were identified by cDNA subtraction cloning. The identified cDNAs included signaling molecules (lymphoid blast crisis [LBC], guanine nucleotide binding protein alpha type S [Galpha-S], and mitogen kinase [MEK5]), calcium-regulated/cytoskeletal proteins (calpactin p11 and p36 subunits, vinculin, and spinocerebellar ataxia [SCA1]), growth factors (insulin-like growth factor binding protein 4 and transforming growth factor beta2), glyceraldehyde-6-phosphate dehydrogenase, an expressed sequence tag, and a novel cDNA showing homology to a LIM domain sequence. Two- to sevenfold induction of the endogenous gene expression was observed at 24 h postinfection, and induction continued up to 72 h, although the timing of gene expression varied among the identified genes. In contrast to that observed in endothelial cells, the Ad vector-mediated induction of gene expression was not found following Ad vector infection of primary human dermal fibroblasts or human alveolar macrophages. Empty Ad capsids did not induce endogenous gene expression in endothelial cells. Interestingly, additional deletion of the E4 gene obviated the upregulation of genes in endothelial cells by the E1(-) E3(-) Ad vector, suggesting that genes carried by the E4 region play a central role in modifying target cell gene expression. These findings are consistent with the notion that efficient transfer of exogenous genes to endothelial cells by first-generation Ad vectors comes with the price that these vectors also induce the expression of a variety of cellular genes.

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Adenovirus vector production using low-multiplicity infection of 293 cells.

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