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. 1998 Mar;72(3):2224-32.
doi: 10.1128/JVI.72.3.2224-2232.1998.

Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus

X Xiao  1 , J Li, R J Samulski
Affiliations

Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus

X Xiao et al. J Virol. 1998 Mar.

Abstract

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 10(5) 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.

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Figures

FIG. 1
FIG. 1
Construction of AAV packaging and Ad helper plasmids. (A) AAV packaging plasmids AAV/Ad, ACG-2, XX2, and XX2-LacZ all contain the endogenous promoter p5 (open box) and Rep and Cap genes (shaded box). Plasmids ACG-2, XX2, and XX2-LacZ utilize an artificial ACG start codon for Rep protein synthesis (27). In addition, plasmids XX2 and XX2-LacZ contain an extra copy of the p5 promoter element downstream of the Cap gene. Also included in pXX2-LacZ is an AAV-LacZ vector containing a cytomegalovirus (CMV)-LacZ gene cassette (hatched box) flanked by the AAV ITRs (solid arrow). (B) Construction of Ad helper plasmids. The thick solid lines represent Ad DNA sequences. The dotted lines are the Ad DNA sequences deleted in constructs pBHG10, pXX5, and pXX6. The arrowhead lines are the Ad early and late genes or RNA transcripts. L1 to L5 are the Ad late gene transcripts made by the Ad major late promoter (MLP). Ad E1A, E1B, E2A, E4, and VA RNA genes (large hatched arrows) are the essential genes supplying the helper functions for AAV production. DBP, single-stranded DNA binding protein.
FIG. 2
FIG. 2
Western analysis of the Ad5 fiber protein with an antibody against the C-terminal knob domain (19). Lanes: 1, CsCl gradient purified Ad particles as the positive control; 2, lysate of Ad5-infected 293 cells; 3 to 5, lysates of 293 cells transfected with plasmids pBHG10, pXX5, and pXX6 respectively; 6, lysate of mock-transfected 293 cells.
FIG. 3
FIG. 3
Comparison of transfection efficiencies with different vector/carrier DNA ratios. Samples of 293 cells were stained with X-Gal at 24 h after transfection with 25 μg of vector plasmid pdx31-LacZ (1:0) (A), 4 μg of pdx-31-LacZ and 21 μg of carrier plasmid pBluescript KS (1:5.25) (B), or 2.5 μg of pdx31-LacZ and 22.5 μg of pBluescript KS (1:9 ratio) (C). No obvious difference in percentage and intensity of the blue cells can be noticed at different vector/carrier DNA ratios.
FIG. 4
FIG. 4
Comparison of different vector/packaging/Ad helper plasmid ratios on the yields of rAAV vectors.
FIG. 5
FIG. 5
Time course of rAAV yields, using Ad plasmid pXX6 as the helper.
FIG. 6
FIG. 6
In vivo transduction of muscle tissues by an rAAV-LacZ vector generated with the Ad-free method. The muscle tissues were cryosectioned and X-Gal stained at 3 weeks (A) and 4 months (B) following intramuscular delivery of 107 infectious units of rAAV vector, which was purified by double CsCl gradient centrifugation.
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