Volume 20, Issue 12, Pages 2268-2281 (December 2012) Ad5:Ad48 Hexon Hypervariable Region Substitutions Lead to Toxicity and Increased Inflammatory Responses Following Intravenous Delivery  Lynda Coughlan, Angela C Bradshaw, Alan L Parker, Hollie Robinson, Katie White, Jerome Custers, Jaap Goudsmit, Nico Van Roijen, Dan H Barouch, Stuart A Nicklin, Andrew H Baker  Molecular Therapy  Volume 20, Issue 12, Pages 2268-2281 (December 2012) DOI: 10.1038/mt.2012.162 Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Pre-existing neutralizing antibodies in Scottish cohort of patients. A dilution of 2.5% patient sera (n = 63) was used to inhibit adenovirus (Ad)-mediated luciferase transduction in HepG2 cells. Sera which inhibited >90% transduction compared to no serum control, was considered to be neutralizing, as described previously.15,50 Molecular Therapy 2012 20, 2268-2281DOI: (10.1038/mt.2012.162) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Detection of virus in the liver by quantitative PCR (qPCR) and immunofluorescence. (a) MF1 mice were injected intravascularly (i.v.) with 3 × 1010 virus particles (vp) Ad5, Ad5HVR48(1-7), or Ad48 and livers harvested for analysis (1 hour and 48 hours). Animals were pretreated with 200 µl phosphate-buffered saline (PBS) (–CD) or clodronate liposomes (+CD) 48 hours prior. Genomes were detected by real-time qPCR. (b) Quantification of viral genomes in the liver following i.v. injection of 1 × 1011 vp. Data represent the mean ± SEM (n = 5–8 per group), ***P < 0.001, **P < 0.01, *P < 0.05. Significance indicators directly above histogram bars indicate comparison to Ad5, within same +CD treatment group. (c) Immunofluorescence detection of Alexa-488-labeled virus in the liver 1 hour postinjection of 1 × 1011 vp (+CD). Kupffer cells (KCs) were identified by F4/80+ staining (red) and Ad5-488, Ad5HVR48(1-7)-488 (abbreviated to HVR48-488 in figure) or Ad48-488 are shown in green. Nuclei were counterstained with DAPI (blue). Right hand columns of each treatment group represent magnifications of the boxed areas (outlined in white). Images are representative of multiple fields of view from different animals. White arrows indicate virus within KCs (–CD) or on the surface of hepatocytes (+CD). (d) Quantification of Alexa-488 in liver images. A total of 6–15 separate images from different animals (n = 3 animals/group; mean ± SD) were thresholded, analyzed and regions corresponding to Alexa-488-labeled virus quantified using ImageJ analysis software. (e) Quantification of Alexa-488 within KCs in liver images. KCs (F4/80+) were manually segmented and the amount of Alexa-488 within summed using ImageJ software (average number of KCs = 14 per image). Graphical representation of whole liver in e is the same as –CD in d, for ease of comparison (n = 3 animals/group; mean ± SD). HVR, hypervariable region. Molecular Therapy 2012 20, 2268-2281DOI: (10.1038/mt.2012.162) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Detection of virus in the spleen by quantitative PCR (qPCR) and immunofluorescence. (a) MF1 mice were injected intravascularly (i.v.) with 3 × 1010 virus particles (vp) Ad5, Ad5HVR48(1-7), or Ad48 and spleens harvested for analysis (1 hour and 48 hours). Animals were pretreated with 200 µl phosphate-buffered saline (PBS) (–CD) or clodronate liposomes (+CD) 48 hours before virus delivery and genomes detected by qPCR. (b) Quantification of viral genomes in the spleen following i.v. injection of 1 × 1011 vp. Data represent the mean ± SEM (n = 5–8 per group), ***P < 0.001, **P < 0.01, *P < 0.05. *Significance indicators directly above histogram bars indicate comparison to Ad5, within same CD treatment group. (c) Immunofluorescence detection of Alexa-488-labeled virus (1 × 1011 vp) in the spleen (1 hour). Note: Alexa-488 labeled Ad5HVR48(1-7) is abbreviated to HVR48-488 in figure. Spleen sections were stained for F4/80, MARCO or CD169 (red) and images captured under a ×10 objective (–CD). (d) Markers to cell types which are unaffected by macrophage-depletion (B220, ER-TR7, MAdCAM-1) were used for colocalization. Images shown were captured using a ×40 objective and are representative of multiple fields of view (n = 3 animals/group; mean ± SD). Areas of colocalization are indicated by white arrows. (e) Quantification of Alexa-488 in spleen sections. A total of 6–10 separate images from different animals were analyzed (×10 magnification) and regions corresponding to Alexa-488-labeled virus quantified using ImageJ analysis (n = 3 animals/group; mean ± SD). (f) Quantification of viral genomes from blood 10 minutes and 1 hour postinjection of 1 × 1011 vp by qPCR, using 10 ng total DNA for analysis (n = 5–8 animals/group; mean ± SEM), *P < 0.05. Significance indicators directly above histogram bars indicate comparison to Ad5, within same CD treatment group. HVR, hypervariable region. Molecular Therapy 2012 20, 2268-2281DOI: (10.1038/mt.2012.162) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Quantification of serum cytokines/chemokines (6 hours). (a–j) Various cytokines/chemokines analyzed in the serum. MF1 mice were injected intravascularly (i.v.) with 3 × 1010 virus particles (vp) or 1 × 1011 vp Ad5, Ad5HVR48(1-7), or Ad48. Separate groups were pretreated with 200 µl phosphate-buffered saline (PBS) (–CD) or clodronate liposomes (+CD) 48 hours prior to virus delivery. Cytokine/chemokine levels were quantified from serum (6 hours) using a multiplex luminex kit. Data represent the mean ± SEM (n = 5–8 per group), **P < 0.01, *P < 0.05, NS, not significant. Significance indicators with a straight bar above histogram indicates comparison with corresponding Ad5-injected groups. HVR, hypervariable region. Molecular Therapy 2012 20, 2268-2281DOI: (10.1038/mt.2012.162) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Quantification of liver gene transfer and hepatotoxicity (48 hours). (a) Luciferase expression in frozen liver sections (4 µm) was detected by immunofluorescence. (b) Serum transaminases, aspartate aminotransferase (AST) and (c) alanine aminotransferase (ALT) were quantified 48 hours postinjection of 1 × 1011 virus particles (vp). (d) Paraffin-embedded liver sections (5 µm) were stained with hematoxylin and eosin (H&E) and assessed by a pathologist. Images were captured using a ×10 and a ×40 objective. Data represent the mean ± SEM (n = 5–8 per group), **P < 0.01, *P < 0.05. Significance indicators directly above histogram bars indicate comparison to phosphate-buffered saline (PBS) control. Molecular Therapy 2012 20, 2268-2281DOI: (10.1038/mt.2012.162) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Phenotypic identification of accumulating cells in periportal areas. (a) Frozen liver sections were stained by immunofluorescence using antibodies to broad myeloid marker CD11b or (b) granulocyte marker Gr-1. Images were captured using a ×10 objective. (c) The amounts of CD11b+ or (d) Gr-1+ cells were quantified in multiple images from several different animals using ImageJ software. All thresholding and adjustments were applied equally across compared images. Data represent the mean ± SEM (n = 3–5 animals/group), ***P < 0.001, **P < 0.01. Significance indicators directly above histogram bars indicate comparison to Ad5, within same CD treatment group. Molecular Therapy 2012 20, 2268-2281DOI: (10.1038/mt.2012.162) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions