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J Virol, March 1998, p. 2224-2232, Vol. 72, No. 3
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Production of High-Titer Recombinant Adeno-Associated Virus Vectors in the Absence of Helper Adenovirus

Xiao Xiao,1,2,* Juan Li,1 and Richard Jude Samulski1,3

Gene Therapy Center,1 Division of Pharmaceutics,2 and Department of Pharmacology,3 University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

Received 22 September 1997/Accepted 24 November 1997

Recently, efficient and long-term in vivo gene transfer by recombinant adeno-associated virus type 2 (rAAV) vectors has been demonstrated in a variety of tissues. Further improvement in vector titer and purity will expedite this in vivo exploration and provide preclinical information required for use in human gene therapy. In an effort to obtain higher titers, we constructed a novel AAV helper plasmid which utilizes translational control of AAV Rep genes (J. Li et al., J. Virol. 71:5236-5243, 1997). To address the issue of purity, in this study we report the first rAAV production method which is completely free of adenovirus (Ad) helper virus. The new production system uses a plasmid construct which contains a mini-Ad genome capable of propagating rAAV in the presence of AAV Rep and Cap genes. This construct is missing some of the early and most of the late Ad genes and is incapable of producing infectious Ad. Transfection of 293 cells with the new mini-Ad helper and AAV packaging plasmids results in high-titer rAAV vectors with yields greater than 1,000 transducing units, or 105 viral particles per cell. When rAAV vectors were produced by using this production scheme and compared to traditional heat-inactivated rAAV preparations in vitro and in vivo, we observed transduction equivalent to or better than normal levels. The complete removal of infectious Ad from AAV production should facilitate a better understanding of immune response to AAV vectors in vivo, eliminate the need for developing replication-competent Ad assays, and provide a more defined reagent for clinical use.

* Corresponding author. Mailing address: Gene Therapy Center, G44 Wilson Hall, CB 7352, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599. Phone: (919) 962-1221 or (919) 962-1350. Fax: (919) 962-1313. E-mail: xxi{at}med.unc.edu.




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