Generation of Recombinant Adeno-associated Virus Vectors by a Complete Adenovirus- Mediated Approach  Xiuwu Zhang, Chuan-Yuan Li  Molecular Therapy  Volume 3, Issue 5, Pages 787-792 (May 2001) DOI: 10.1006/mthe.2001.0306 Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 1 A strategy for the production of rAAV vectors based on the exclusive use of adenovirus vectors. (A) A schematic drawing of adenovirus-based production of recombinant rAAV. ITR, inverted terminal repeat sequence of AAV. g.o.i, gene of interest. IP, inducible promoter. CMV, cytomegalovirus promoter. (B) The three adenovirus vectors to be used for the production of rAAV-CMVGFP by use of a tetracycline-inducible system for rep production. GFP, green fluorescence protein. The arrows on top of the bars, which represent coding sequences of genes, indicate promoters. AAVpA, AAV polyadenylation signal. SV40pA, SV40 polyadenylation signal. X represents mutations that disrupt the p40 promoter. Molecular Therapy 2001 3, 787-792DOI: (10.1006/mthe.2001.0306) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 2 Characterization of the AdTRErepCMVcap. (A) Western blot analysis of adenovirus-directed expression of cap and rep genes. Left shows constitutive cap gene expression in 293 cells with increasing doxycycline. Right shows inducible rep78/68 gene expression with increasing dox concentration. In the control, a low but significant background is present. This is consistent with earlier reports about the tet-ON system (31, 32). Please also notice the constitutive level of rep52/40. (B) H/ndMI digestion of AdTRErepCMVcap DNA from serially passaged virus preparations. DNA in each lane was derived by proteinase K digestion of 1012 virus particles and phenol-chloroform extraction. A 1-kb DNA marker (Life Technologies, Bethesda, MD) was used for size determination. The number on top of each lane represents passage number. Molecular Therapy 2001 3, 787-792DOI: (10.1006/mthe.2001.0306) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 3 Production and analysis of rAAVCMVGFP by use of the tetracycline-inducible three-vector adenovirus system. (A) The titers of rAAVCMVGFP produced with different concentrations of doxycycline (determined by GFP fluorescence microscopy, as described under Experimental Protocols). (B) A photograph showing a clear rAAVCMVGFP band (prepared from five 150-mm petri dishes of 293 cells) that has been centrifuged and stained with ethidium bromide (33). (C) A Southern blot analysis of extrachromosomal DNA (isolated by the Hirt (23) method) to detect monomeric and dimeric forms of replicating AAV genomes. Two Hirt DNA samples were obtained. The first sample was derived from infecting 293 cells with the recombinant rAAVCMVGFP particles isolated from our tri-adenovirus approach together with a wild-type adenovirus. The second sample was derived from infecting 293 cells with wild-type AAV particles and wild-type adenovirus. The Hirt DNA samples were isolated 48 h postinfection. Duplicate Southern blots were carried out to obtain two membranes with the same two samples. The two membranes were then probed with two separate probes. The first is an EGFP fragment from pEGFP-N1 (Clontech). The second is a 1.2-kb rep fragment derived by restriction digestion (NcoI and HindIII). In both blots, the monomeric (MMF) and the dimeric (DMF) forms of the replicating AAV genomes are clearly visible. Left: Results from GFP probe. Right: Results from the rep probe. (D) Replication center assay of the rAAVCMVGFP. Top filter, 32P-labeled EGFP was used to hybridize with rAAVCMVGFP- and AdTRErepCMVcap-infected 293 cells. About 104 total virus particles of rAAVCMVGFP was used to infect the cells. Lower filter, 32P-labeled rep fragment was used to hybridize with rAAV-GFP- and wild-type Ad5 virus-infected 293 cells. About 1010 total rAAVCMVGFP particles were used to infect 293 cells. Each of the black dots in the top filter represents an active rAAV particle with the EGFP gene. The absence of any visible signal in the lower filter indicates a lack of active virus particles with the rep gene (which should be carried by the wild-type AAV virus). Molecular Therapy 2001 3, 787-792DOI: (10.1006/mthe.2001.0306) Copyright © 2001 American Society for Gene Therapy Terms and Conditions