Improved system for helper-dependent adenoviral vector production Donna Palmer, Philip Ng  Molecular Therapy  Volume 8, Issue 5, Pages 846-852 (November 2003) DOI: 10.1016/j.ymthe.2003.08.014 Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 1 (A) The HV AdLC8cluc contains loxP sites flanking ψ and a DNA stuffer replacing the E3 region. (B) The HV AdNG163R-2 contains a reverse orientation ψ flanked by loxP sites and a DNA stuffer inserted into the wild-type E3 region. (C) The HDAd HDΔ28E4LacZ contains a MCMV–LacZ expression cassette. (A to C) The sizes of the relevant restriction enzyme fragments and the positions of probe ψ, HV-specific probe SB, and HDAd-specific probe H used for Southern analysis are shown and described in detail elsewhere [11,13]. The orientation of ψ is indicated, small triangles represent the orientation and position of the loxP sites, and the small horizontal arrows represent the viral ITRs. (D) The plasmid pNG159 was used to generate the 116 producer cell line. All restriction fragment sizes are shown in kb. Molecular Therapy 2003 8, 846-852DOI: (10.1016/j.ymthe.2003.08.014) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 2 (A) Southern analysis for efficiency of ψ excision. The indicated cell lines were infected with AdLC8cluc and total intracellular DNA was extracted 48 h postinfection, digested with BglI, and analyzed by Southern hybridization with probe SB. (B) Western analysis for Cre. Total protein was extracted from the indicated cell lines and 10 μg was analyzed for the 39-kDa Cre protein. Molecular Therapy 2003 8, 846-852DOI: (10.1016/j.ymthe.2003.08.014) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 3 (A) Amplification of HDΔ28E4LacZ with 116 cells and AdNG163R-2 (circles) or with 293Cre4 cells and AdLC8cluc (squares). The total yield of HDΔ28E4LacZ per 60-mm dish in blue-forming units (BFU) was determined at each serial passage. (B) Southern analysis of HDΔ28E4LacZ amplification with 293Cre4 cells and AdLC8cluc. Total intracellular DNA was extracted from coinfected cells from each serial passage shown in (A), digested with BglI, and hybridized simultaneously with probes SB and H. Lane a contains DNA from 293 cells infected with AdLC8cluc. Lane b contains DNA from 293Cre4 cells infected with AdLC8cluc. Lane c contains the HDAd plasmid pΔ28E4LacZ digested with BglI and PmeI. Lane d contains the plasmid pCA36 [17] digested with AvaII, resulting in a 2.3-kb fragment encompassing probe SB and a 3.1-kb fragment encompassing probe H. (C) Southern analysis of HDΔ28E4LacZ amplification with 116 cells and AdNG163R-2. Total intracellular DNA was extracted from coinfected cells from each serial passage shown in (A), digested with BglI, and hybridized simultaneously with probes SB and H. Lane a contains DNA from 293 cells infected with AdNG163R-2. Lane b contains DNA from 116 cells infected with AdNG163R-2. Lanes c and d are as described for (B). Molecular Therapy 2003 8, 846-852DOI: (10.1016/j.ymthe.2003.08.014) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Overview of the rescue (P0), amplification (P1 to P3), and large-scale production (P4) of HDAd (see Materials and Methods for details). The entire process beginning with vector rescue (P0) to the final purified HDAd takes less than 2 weeks. Molecular Therapy 2003 8, 846-852DOI: (10.1016/j.ymthe.2003.08.014) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 5 Detailed analysis of a representative example of HDΔ28E4LacZ produced from 3 liters of 116 cells using AdNG163R-2 following the protocol shown in Fig. 4. (A) HDΔ28E4LacZ was purified by one step and two continuous CsCl gradients (cont. 1 and cont. 2). Total viral particle (vp) yield, specific vp yield per cell (vp/cell), and infectivity (particle-to-infectious unit ratio expressed as vp:BFU) are shown. The percentage HV contamination from virion DNA extracted from the step and continuous CsCl gradients was determined by TaqMan quantitative PCR. The lowest band in the CsCl step gradient contains the virions. (B) Analysis of HDΔ28E4LacZ DNA. DNA was extracted from the virions obtained from the step and the two continuous CsCl gradients and digested with ApaLI. The structure of HDΔ28E4LacZ DNA (labeled HDAd) is indistinguishable from that of the parental pΔ28E4LacZ plasmid (labeled pHDAd), except for the expected absence of the 2.5-kb ApaLI–PmeI fragment bearing the bacterial plasmid sequences present in pΔ28E4LacZ. Southern analysis with probe ψ revealed the expected 6.7-kb HDΔ28E4LacZ-specific band but no detectable AdNG163R-2-specific 1.5-kb band in all three CsCl gradient fractions. The HV lane contains AdNG163R-2 DNA. Control lanes (labeled pHDAd + pHV) consisted of ApaLI- and PmeI-digested pΔ28E4LacZ mixed with 10-fold serial dilutions of ApaLI- and PacI-digested HV plasmid pNG163R-2 (pHV). Extraneous bands in the HDAd lanes are not rearrangement products because they are also present in the pHDAd lane and thus represent nonspecific hybridization of the probe to the other vector bands. Note that at 5% contamination, the HV-specific bands are visible by ethidium bromide staining. Molecular Therapy 2003 8, 846-852DOI: (10.1016/j.ymthe.2003.08.014) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 6 (A) Three additional examples of HDΔ28E4LacZ obtained from 3 liters of 116 cell using AdNG163R-2. The first continuous CsCl gradients are shown. Total vector yield per 3-liter culture was consistently >1 × 1013 vp, with specific yields of >10,000 vp/cell. Vector infectivities (vp:BFU) are no more than 15:1. Percentage HV contamination in the step and two continuous gradients was determined by TaqMan PCR. (B and C) Genomic structure of HDΔ28E4LacZ. DNA was extracted from the four HDΔ28E4LacZ preparations presented in (A) and in Fig. 5, digested with (B) ApaLI or (C) HpaI, and analyzed by Southern blotting using pΔ28E4LacZ as the probe. The genomic structures of all four HDΔ28E4LacZ preparations (labeled HDAd) are indistinguishable from that of pΔ28E4LacZ (labeled pHDAd) except for the expected absence of the 3.0-kb PmeI fragment bearing the bacterial plasmid sequences in pΔ28E4LacZ. The bands visible in the AdNG163R-2 lane (labeled HV) represent the terminal fragments that hybridize to the homologous ψ and ITR sequences present in the pΔ28E4LacZ probe. 1KB PLUS (Life Technologies) standard shown. Molecular Therapy 2003 8, 846-852DOI: (10.1016/j.ymthe.2003.08.014) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions