1. Volume 7, Issue 4, Pages 526-534 (April 2003)
Multigene expression from a replicating adenovirus using native viral promoters 
Maxine Bauzon, Daniel Castro, Michael Karr, Lynda K Hawkins, Terry W Hermiston 
Molecular Therapy 
Volume 7, Issue 4, Pages (April 2003)
DOI: /S (03)
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Diagram of vector construction. The Ad5 E3 region transcription unit, adjacent regions, and viruses used in these studies are represented diagrammatically. The restriction sites that were used for cloning hMCP-3 and/or mTNF are shown above the line and the corresponding nucleotide number relative to the Ad5 genome is indicated. The boxes listed below the line indicate the genes for identified proteins in the E3 region. They include the 3′ end of the L4 message for pVIII, 12.5K, gp19K, ADP, and the genes in the E3B region (RIDα, RIDβ, and 14.7K). The viruses used in this study have transgenes in place of some of the E3 endogenous genes. ONYX-320 carries mTNF in place of ADP. ONYX-371 carries MCP-3 instead of the 6.7K and gp19K genes, and ONYX-372 carries both of these transgenes, MCP-3 in the 6.7K/gp19K region and mTNF in the ADP region.
Molecular Therapy 2003 7, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Transgene protein expression. Western blot analysis was performed on protein extracts from Ad5-infected cells and from conditioned cell culture medium from ONYX-371-, -320-, and -372-infected cells. The numbers above each blot indicate the time postinfection. The “M” and “A” indicate mock-infected and Ad5-infected cells, respectively, harvested at 24 h postinfection. For analysis of MCP-3 (A) and mTNF (B), expression represents the protein secreted into the medium 1 h prior to the time point indicated (see Materials and Methods for details). (A) Endogenous gp19K protein expression for Ad5 or transgene (MCP-3) protein expression from viruses ONYX-371 and ONYX-372. (B) Endogenous ADP expression for Ad5 and mTNF expression from viruses ONYX-320 and ONYX-372.
Molecular Therapy 2003 7, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Quantitation of MCP-3 and mTNF. An ELISA was performed to quantitate the amounts of MCP-3 and mTNF secreted into the culture medium during a 1-h period over the course of the viral infection. Values are expressed as nanograms of MCP-3 or mTNF synthesized per million cells per hour. This experiment was done in replicates of 4 and the standard deviation is shown by error bars. The numbers on the x axis represent the time postinfection that the samples were collected.
Molecular Therapy 2003 7, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
Western blot analysis of Ad5 E3 endogenous proteins. Western blot analysis was performed on protein extracts from Ad5-, ONYX-371-, ONYX-320-, and ONYX-372-infected cells, examining the expression of (A) gp19K, (B) ADP, (C) RID-β, (D) 14.7K, and (E) the L5 region protein fiber (see Fig. 5 for map and position of the E3 genes and fiber relative to transgene insertion sites in the E3 region). The numbers above each blot indicate the time postinfection. The “M” and “A” indicate mock-infected and Ad5-infected cells, respectively, harvested at 24 h postinfection. The fiber protein is synthesized from the downstream L5 region and is shown to look for any effects of transgene insertion on the region immediately downstream of the E3 region.
Molecular Therapy 2003 7, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
Time course of infected A549 cells. A549 cells were infected at 10 pfu/cell with the indicated virus. Photographs were taken at 24, 48, and 60 h postinfection.
Molecular Therapy 2003 7, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions