Evaluation of polyethylene glycol modification of first-generation and helper-dependent adenoviral vectors to reduce innate immune responses  Hoyin Mok, Donna J. Palmer, Philip Ng, Michael A. Barry  Molecular Therapy  Volume 11, Issue 1, Pages 66-79 (January 2005) DOI: 10.1016/j.ymthe.2004.09.015 Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 1 Degree of capsid modification with various amounts of PEG and its impact on transduction ex vivo. (A) 1012 AdGFPluc were modified with 0.1, 0.25, 0.5, 1.0, 2.0, or 10.0 mg/ml SPA-PEG. The amount of conjugation was quantified by fluorescamine assay. The degree of modification was compared to unmodified vectors and is expressed as a percentage of the amino groups remaining. (B) PEG-modified vectors (⋄) were used to transduce HeLa cells at 10 particles per cell. Levels of luciferase expression were measured 24 h posttransduction and are expressed as RLU. Unreacted mPEG (□) was mixed with AdGFPluc as a control. Molecular Therapy 2005 11, 66-79DOI: (10.1016/j.ymthe.2004.09.015) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 2 Biodistribution of PEG-modified vectors in vivo through intravenous administration. Each mouse received an intravenous dose of 1 × 1011 viral particles (n = 5). (A) Organ transduction. Mice were injected with unmodified FG-Ad or vectors modified with PEG at 10 mg/ml concentration. Organs were harvested after 24 h. Levels of luciferase expression were measured, normalized by protein content, and expressed as RLU/mg protein. (B) Viral genomes were measured in the organs by quantitative real-time PCR and expressed as copies/μg DNA. (C) Liver transduction was then compared using vectors with various degrees of capsid modifications as in (A). Each column represents five mice in the experiment. (D) LacZ staining of frozen liver section from mice injected with unmodified or PEGylated HDΔ28E4LacZ, expressing β-galactosidase [18]. Molecular Therapy 2005 11, 66-79DOI: (10.1016/j.ymthe.2004.09.015) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 3 Effects of physical force and receptor interactions by PEGylated vectors in vitro. (A) 109 viral particles of FG-Ad and FG-Ad-PEG were incubated with CHO cells for 2 h with or without centrifugation. Transduction was determined by flow cytometry after 48 h. (B) CHO cells treated with heparinase I were incubated with 109 particles of FG-Ad or PEG-modified FG-Ad for 48 h. The amount of transduction was measured after 48 h and compared to untreated CHO controls. *P < 0.05. (C) 109 particles of FG-Ad or FG-Ad-PEG-biotin encoding GFP were incubated with streptavidin magnetic beads and placed on CHO cells in the absence or presence of magnet. The amounts of fluorescence were noted after 48 h of incubation. (D) Western blot detecting KKTK motif on fiber shaft of boiled (B) and unboiled (U) samples of FG-Ad and PEG-modified FG-Ad. (E) Transduction of 109 viral particles of FG-Ad or FG-Ad-PEG-biotin with streptavidin magnetic beads was compared on CHO incubated with RGD4C peptides and untreated control. **P < 0.01. Molecular Therapy 2005 11, 66-79DOI: (10.1016/j.ymthe.2004.09.015) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 4 Serum liver enzymes (ALT and AST) levels 5 days postadministration. Serum was obtained 5 days after vector administration through retro-orbital bleeds. The amounts of ALT and AST are expressed as units/L. The levels of ALT and AST from animals injected with buffer were taken as baseline controls. Note that FG-Ad and HD-Ad experiments were performed separately under conditions under which ALT and AST levels from control mice were identical. Note also that the FG-Ad vector expressed GFP-LUC, whereas the HD-Ad vector HDΔ28E4LacZ expresses β-galactosidase [18] and that both vectors were prepared by different methods. While these gene and preparation variables exist, the relative liver damage observed between FG-Ad and HD-Ad vectors is consistent with previous comparisons [8,9]. Molecular Therapy 2005 11, 66-79DOI: (10.1016/j.ymthe.2004.09.015) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Serum interleukin-6 levels and kinetics post-vector administration. Mice were injected with 3 × 1011 vp (1 × 1013 vp/kg) of modified or unmodified vector intravenously. Serum IL-6 levels were measured at 6, 12, 24, and 48 h after injection using a murine IL-6 ELISA kit. (A) Serum IL-6 levels 6 h after injection of increasing dose of unmodified viral particles into ICR-1 mice. (B) IL-6 levels between unmodified (light gray) and PEG-modified (black) vectors. Each column represents an experimental group of five to nine mice. **P < 0.01 compared to unmodified vectors. (C) The kinetics of IL-6 levels is shown with unmodified FG-Ad and PEG-modified FG-Ad viruses, compared to buffer-injected controls. *P < 0.05 comparing Ad-PEG with unmodified Ad. Each data point represents an experimental group of five mice. Note that FG-Ad and HD-Ad experiments were performed separately under conditions under which IL-6 levels from control mice were identical. Note also that the FG-Ad vector expressed GFP-LUC, whereas the HD-Ad vector HDΔ28E4LacZ expresses β-galactosidase [18] and that both vectors were prepared by different methods. While these gene and preparation variables exist, the relative IL-6 responses observed between FG-Ad and HD-Ad vectors are consistent with previous results. Molecular Therapy 2005 11, 66-79DOI: (10.1016/j.ymthe.2004.09.015) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 6 Viral uptake and macrophage IL-6 expression in vitro by adenoviral transduction. 1 × 1010 either unmodified or PEG-modified adenoviruses were used to stimulate RAW264.7 cells for 6 or 36 h. (A) Supernatants were harvested and tested for IL-6 concentrations. (B) Real-time PCR analysis of Ad- and Ad-PEG-infected RAW cells. Whole cell DNA was isolated from infected RAW cells and used as a template for real-time PCR. Amplified hexon DNA products were normalized to GAPDH cellular controls. ***P < 0.001 compared to unmodified vectors. Experimental group n = 3. Molecular Therapy 2005 11, 66-79DOI: (10.1016/j.ymthe.2004.09.015) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 7 Distribution of AlexaFluor555-labeled unmodified or PEGylated viruses in the liver 30 min post-intravenous injection. AlexaFluor555 (red-orange)-labeled virions were injected (1 × 1011 viral particles/mouse) through the tail vein and livers were harvested 30 min later. Unmodified virions (D, E, and F) and PEGylated virions (G, H, and I) are compared with PBS control (A, B, and C). Virions are seen in orange (D and G). To visualize Kupffer cells, liver sections were stained with the F4/80 antibody and detected with AlexaFluor488 anti-mouse antibody (detected in green) (B, E, and H). Overlay images (C, F, and I) from fluorescent virions and F4/80 staining are included. Large white arrow shows an example of colocalization of virions with F4/80-positive Kupffer cells. Small white arrows show an example of localization of virions separate from F4/80-positive Kupffer cells. All cryosections were evaluated by fluorescence microscopy. Original magnification 400×. Molecular Therapy 2005 11, 66-79DOI: (10.1016/j.ymthe.2004.09.015) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 8 Schematic representation of receptor interaction of unmodified Ad and PEG-modified Ad vectors. Unmodified Ad5 binds to CAR, αv integrins, and heparin sulfate proteoglycans (HSP) for cell-mediated endocytosis. Transduction of PEG-modified vectors in vitro appears to fail because PEG inhibits interactions between these ligands and receptors on the cell surface due to steric hindrance. Receptor binding can be partially rescued in vitro by applying force to displace the flexible PEG chains when integrin and HSP binding domains are exposed for cell interaction. In vivo, pharmacodynamic forces due to “forced sieving” and “endothelial massage” [21] may enable these interactions on hepatocytes, since they bear integrins and HSPs. In contrast, these same forces may not act on macrophages or Kupffer cells to the same degree, since these cells lack most receptors for Ad. Molecular Therapy 2005 11, 66-79DOI: (10.1016/j.ymthe.2004.09.015) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions