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Recombinant adenovirus expressing adeno-associated virus cap and rep proteins supports production of high-titer recombinant adeno-associated virus

Gene Therapy volume 8, pages 704–712 (2001)Cite this article

Abstract

It has been difficult to produce a chimeric vector containing both Ad and AAV rep and cap, and to grow such chimeric vectors in 293 cells. By recombination in vitro in a bacterial host, we were able to produce recombinant plasmid AdAAV (pAdAAVrep-cap), which could be used to generate recombinant AdAAV (rAdAAVrep-cap) after transfection into 293 cells. A recombinant adenovirus, rAdAAVGFP, in which the green fluorescent protein (GFP) gene is flanked by the AAV terminal repeats cloned into the E1-deleted site of Ad was also generated. Co-infection of rAdAAVrep-cap together with rAdAAVGFP into 293 cells resulted in production of high titers of rAAV expressing GFP. It was noted that the titer of rAdAAVrep-cap was lower than the titer of control AdCMVLacZ. The lower titer of rAdAAvrep-cap was associated with expression of Rep protein. Non-homologous recombination occurs after high passage and results in deletions within the AAV rep genes. These results indicate that (1) rAdAAVrep-cap can be produced; (2) rAdAAVrep-cap + rAdAAVGFP is a convenient and efficient way to transfect 293 cells to grow high titer rAAV; and (3) frozen stock is required to avoid propagation of rep-deleted pAdAAVrep-cap.

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Acknowledgements

We thank Dr James Wilson for providing B50 cell line, Dr J Samulski for plasmid psub201, Ad309 and Dr Bert Vogelstein for plasmid DNAs pshuttle, pAdeasy1. We also thank Mr M Spell and Dr T Rogers at the FACS Core Facility at UAB for helping to do the FACS analyses. We also thank M Linda Flurry for excellent secretarial work and Dr Fiona Hunter for editorial assistance. This work is supported by NIH grants R01 AG 11653, RO1-AI-42900, N01 AR 6–2224, and CA20468, and a Birmingham VAMC Merit Review Grant. Huang-Ge Zhang is a recipient of an Investigation award from American Arthritis Foundation, and Hui-Chen Hsu is a recipient of a postdoctoral fellowship from Arthritis Foundation.

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Authors and Affiliations

  1. Department of Medicine, Division of Clinical Immunology and Rheumatology, The University of Alabama at Birmingham, Birmingham, AL, USA

    H-G Zhang, Y M Wang, J F Xie, X Liang, H-C Hsu & X Zhang

  2. Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL, USA

    J Douglas, D T Curiel & J D Mountz

  3. Veterans Administration Medical Center, Birmingham, AL, USA

    J D Mountz

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Zhang, HG., Wang, Y., Xie, J. et al. Recombinant adenovirus expressing adeno-associated virus cap and rep proteins supports production of high-titer recombinant adeno-associated virus. Gene Ther 8, 704–712 (2001). https://doi.org/10.1038/sj.gt.3301454

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