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Journal of Virology, February 2002, p. 1904-1913, Vol. 76, No. 4
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.4.1904-1913.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Feasibility of Generating Adeno-Associated Virus Packaging Cell Lines Containing Inducible Adenovirus Helper Genes

Chunping Qiao, Juan Li, Anna Skold, Xudong Zhang, and Xiao Xiao*

Department of Molecular Genetics and Biochemistry and Gene Therapy Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

Received 8 August 2001/ Accepted 25 October 2001

The adeno-associated virus (AAV) vector system is based on nonpathogenic and helper-virus-dependent parvoviruses. The vector system offers safe, efficient, and long-term in vivo gene transfer in numerous tissues. Clinical trials using AAV vectors have demonstrated vector safety as well as efficiency. The increasing interest in the use of AAV for clinical studies demands large quantities of vectors and hence a need for improvement in vector production. The commonly used transient-transfection method, although versatile and free of adenovirus (Ad), is not cost-effective for large-scale production. While the wild-type-Ad-dependent AAV producer cell lines seem to be cost-effective, this method faces the problem of wild-type Ad contamination. To overcome these shortcomings, we have explored the feasibility of creating inducible AAV packaging cell lines that require neither transfection nor helper virus infection. As a first step toward that goal, we have created a cell line containing highly inducible Ad E1A and E1B genes, which are essential for AAV production. Subsequently, the AAV Rep and Cap genes and an AAV vector containing a green fluorescent protein (GFP) reporter gene were stably introduced into the E1A-E1B cell line, generating inducible AAV-GFP packaging cell lines. Upon induction of E1A and E1B genes and infection with replication-defective Ad with E1A, E1B, and E3 deleted, the packaging cells yielded high-titer AAV-GFP vectors. Finally, the E2, E4, and VA genes of Ad, under the control of their endogenous promoters, were also introduced into these cells. A few producer cell lines were obtained, which could produce AAV-GFP vectors upon simple drug induction. Although future improvement is necessary to increase the stability and vector yield of the cells, our study has nonetheless demonstrated the feasibility of generating helper-virus-free inducible AAV producer cell lines.

* Corresponding author. Mailing address: Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Room W1244 BST, Pittsburgh, PA 15261. Phone: (412) 648-9487. Fax: (412) 648-9610. E-mail: xiaox{at}pitt.edu.

Journal of Virology, February 2002, p. 1904-1913, Vol. 76, No. 4
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.4.1904-1913.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Matsushita, T., Okada, T., Inaba, T., Mizukami, H., Ozawa, K., Colosi, P. (2004). The adenovirus E1A and E1B19K genes provide a helper function for transfection-based adeno-associated virus vector production. J. Gen. Virol. 85: 2209-2214 [Abstract] [Full Text]  
  • Qiao, C., Wang, B., Zhu, X., Li, J., Xiao, X. (2002). A Novel Gene Expression Control System and Its Use in Stable, High-Titer 293 Cell-Based Adeno-Associated Virus Packaging Cell Lines. J. Virol. 76: 13015-13027 [Abstract] [Full Text]  


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