Volume 2, Issue 4, Pages 394-403 (October 2000) Selective Rep-Cap Gene Amplification as a Mechanism for High-Titer Recombinant AAV Production from Stable Cell Lines  Xingluo Liu, Frosso Voulgaropoulou, Ruju Chen, Philip R. Johnson, K. Reed Clark  Molecular Therapy  Volume 2, Issue 4, Pages 394-403 (October 2000) DOI: 10.1006/mthe.2000.0132 Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 1 Comparison of infectious rAAV/β-gal yield produced from D6 and cotransfected HeLa cells. The yield of infectious rAAV/β-gal from 3 × 105 D6 (gray bars) or plasmid cotransfected HeLa cells (black bars) 48 h postadenovirus infection is shown with respect to the adenovirus m.o.i. Cotransfected HeLa cells received 1 μg each of plasmids pAAV/CMV/β-gal and pBS/rep-cap 3 h prior to adenovirus infection. The viral yield is reported as the total number of blue forming units (Bfu) produced per treatment. Values represent averages of three independent experiments (error bars represen Molecular Therapy 2000 2, 394-403DOI: (10.1006/mthe.2000.0132) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 2 Western blot analysis of Rep and Cap protein expression in D6 and cotransfected HeLa cells. Total cellular protein (200 ng) was prepared from D6 or cotransfected HeLa cells 48 h after adenovirus infection and analyzed by Western blot. As a positive control, 20 ng of total protein from wt AAV coinfected Hela cells (wt) was used to identify Rep and Cap proteins (lane 1). (A) Rep protein expression as a function of adenovirus m.o.i. D6 cells or plasmid cotransfected HeLa cells (Tx) were either mock infected or adenovirus infected at the indicated m.o.i. HeLa cells infected with adenovirus alone (m.o.i. = 10) served as the negative control (lane 2). (B) Cap protein expression as a function of adenovirus m.o.i. Duplicate filter as seen in A, except an anti-AAV-2 cap primary antibody was used for detection. Upon extended exposure, all adenovirus infected samples possessed detectable VP3 protein. Molecular Therapy 2000 2, 394-403DOI: (10.1006/mthe.2000.0132) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 3 Cell line-specific rep-cap gene amplification. (A) Rep-cap gene amplification in C12 and D6 cells. Total cellular DNA (10 μg) was isolated from C12 (lanes 2, 3, and 4) or D6 cells (lanes 5, 6, and 7) 48 h after adenovirus infection at the indicated m.o.i., and subsequently digested with SpeI (cuts once in the plasmid) and hybridized with the 4.3-kb rep-cap DNA fragment. HeLa cells infected with adenovirus served as a negative hybridization control (lane 1). (B) Failure of transfected rep-cap plasmid sequences to amplify in response to adenovirus infection. Total cellular DNA (10 μg) was isolated from the indicated adenovirus infected cells (m.o.i. = 100), digested with SpeI, and either run directly on the gel (–) or restriction digested with the methylation sensitive enzyme DpnI, as indicated (+). GE7 cells are another rAAV producer cell line, while “Co-tx” represents HeLa cells cotransfected with plasmids pAAV/CMV/β-gal and pBS/rep-cap. “Tx” denotes HeLa cells transfected with the tripartite producer plasmid that was used to generate the D6 cell line. Hybridization with the 4.3-kb rep-cap DNA fragment revealed complete DpnI digestion of input rep-cap containing plasmids (lanes 6 and 8). (C) Rep gene amplification in D6 cells over time. Total cellular DNA (100 ng) from adenovirus infected D6 cells was subjected to quantitative PCR and rep copies per cell calculated by assuming 6 pg of DNA/cell. Data are averages of samples run i Molecular Therapy 2000 2, 394-403DOI: (10.1006/mthe.2000.0132) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 4 Episomal rep-cap gene amplification in adenovirus infected D6 cells. (A) Total cellular (C; lanes 1 and 3) or Hirt (H; lanes 2 and 4) DNA was prepared from uninfected (lanes 1 and 2) or adenovirus infected (lanes 3 and 4) D6 cells. DNA was restriction digested with SpeI and hybridized with the rep-cap DNA fragment. We note that two times more cell equivalents worth of Hirt DNA was loaded (lanes 2 and 4) relative to the cellular DNA samples. Autoradiographic exposure was 24 h. (B) PCR quantitation of rep gene copies per cell in total cellular DNA (100 ng) or Hirt DNA (equivalent yield from 16,60 Molecular Therapy 2000 2, 394-403DOI: (10.1006/mthe.2000.0132) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 5 Adenovirus induced gene amplification of flanking cellular DNA. Total cellular (C; lanes 1 and 3) or Hirt (H; lanes 2 and 4) DNA was prepared from uninfected (lanes 1 and 2) or adenovirus infected (lanes 3 and 4) D6 cells and restriction digested with PmeI (no sites within the plasmid). The hybridization probe was a 274-bp PCR amplified portion of the AAVS1 locus (1310–1584 bp) corresponding to the previously published right flank probe (37). Autoradiographic exposure was 4 Molecular Therapy 2000 2, 394-403DOI: (10.1006/mthe.2000.0132) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 6 Lack of detectable wild-type AAV replication in D6 derived rAAV/β-gal. A passage assay was employed to detect the presence of infectious wild-type like AAV in a D6 derived rAAV/β-gal stock. HeLa cells were initially infected with adenovirus (m.o.i. = 10) and 1011 DRP of rAAV/β-gal. To determine the detection sensitivity of the assay, HeLa cells were also coinfected with adenovirus and the indicated amount of infectious wild-type AAV-2 (0.01–100 IU). Clarified cell lysates were generated 48 h postinfection (heat treated at 56°C) and used to coinfect naive HeLa cells in the presence of adenovirus (m.o.i. = 10). Hirt DNA was isolated from these cells (P2) 48 h after infection and used for Southern blot hybridization with a 300-bp fragment of the AAV cap coding sequence. Indicated are the replicating monomeric (MF) and dimeric (DF) wild-type AAV-2 forms in the control samples. Autoradiographic exposure was 9 Molecular Therapy 2000 2, 394-403DOI: (10.1006/mthe.2000.0132) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 7 In situ hybridization analysis of D6 cells. D6 and HeLa cells were mock or adenovirus infected (m.o.i. = 10), 48 h later 105 cells were collected and loaded onto a microscope slide using a cytospin centrifuge device. Cells were hybridized with sense or antisense β-gal riboprobes as described under Materials an Molecular Therapy 2000 2, 394-403DOI: (10.1006/mthe.2000.0132) Copyright © 2000 American Society for Gene Therapy Terms and Conditions