1. Volume 8, Issue 1, Pages 99-107 (July 2003)
Targeting adenoviral vectors using heterofunctional polyethylene glycol FGF2 conjugates 
Julia Lanciotti, Antonius Song, John Doukas, Barbara Sosnowski, Glenn Pierce, Richard Gregory, Samuel Wadsworth, Catherine O'Riordan 
Molecular Therapy 
Volume 8, Issue 1, Pages (July 2003)
DOI: /S (03)
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Schematic diagram of tresyl-PEG-maleimide (TMPEG-mal). The bifunctional PEG molecule TMPEG-mal is shown. This molecule has two reactive groups; the tresyl group is reactive toward ∈ amino groups on the capsid of adenovirus while the maleimide group is reactive toward available cysteines on the surface of the FGF2 molecule.
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Chromatographic profiles. (A) Separation of free TMPEG-mal from Ad/PEG-mal. The elution profile of Ad/PEG-mal following gel-filtration chromatography on a BioSec resin is shown. Ad/PEG-mal is recovered in the void volume while uncoupled TMPEG-mal is recovered in the included volume. (B) Purification of Ad/PEG/FGF2 by ion-exchange chromatography. Uncoupled FGF2 is recovered in the flowthrough of the resin, while Ad/PEG/FGF2 binds and is eluted from the resin following a salt gradient elution.
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Endothelial cell proliferation assay. Adult bovine endothelial cells were cultured in the presence of FGF2 protein, Ad/PEG/FGF2 conjugate, or Ad/PEG plus FGF2 protein. For FGF2 protein and FGF2 protein plus Ad/PEG groups, inputs were adjusted so as to achieve concentrations equivalent to those of the corresponding Ad/PEG/FGF2 groups. Cellular proliferation was determined by measuring cellular acid phosphatase content (based on enzymatic conversion of pNPP substrate) and is expressed as OD405.
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
Ad/PEG/FGF2 enhances gene expression in cells expressing the FGF receptor. SKOV3.ip1 cells were infected with equivalent particles (500 particles) of one of the following: unmodified Ad, Ad/PEG, or Ad/PEG/FGF2. Transgene expression (β-galactosidase) was measured 48 h later and is expressed as RLU (Relative Light Units).
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
Ad/PEG/FGF2 has reduced transduction efficiency for 293 cells. (A) 293 cells or (B) SKOV3.ip1 cells were infected with increasing particles of unmodified Ad or Ad/PEG/FGF2. Transgene expression (β-galactosidase) is expressed as RLU.
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

7. FIG. 6
Transduction of SKOV3.ip1 cells by Ad/PEG/FGF2 is independent of fiber knob but sensitive to FGF2 protein. SKOV3.ip1 cells were infected with equivalent particles of unmodified Ad or Ad/PEG/FGF2 (A) in the presence of increasing concentrations of purified Ad2 fiber knob protein (0–1 μg/ml) or (B) in the presence of 0.5 μg/ml excess FGF2. Transgene expression (β-galactosidase) is expressed as the percentage of native expression, i.e., expression measured in the absence of fiber knob protein (A). *P < 0.05 (B).
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

8. FIG. 7
Transduction of SKOV3.ip1 tumors in vivo. Tumor-bearing CB-17 SCID mice were injected intraperitoneally with equivalent particles (5 × 1010) of unmodified Ad, Ad/PEG, or Ad/PEG/FGF2. Two days later tumors were recovered and assayed for β-galactosidase levels. Data are expressed as RLU/mg protein. Points represent data from six individual animals, bars represent averages of values.
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

9. FIG. 8
Biodistribution of modified adenovirus following intraperitoneal administration. TaqMan (hexon) analysis of (A) liver and (B) spleen 48 h following intraperitoneal administration of Ad or Ad/PEG/FGF2 (5 × 1010 particles). Points represent data from six individual animals, bars represent averages of values. *P < and **P < 0.05 compared to unmodified Ad.
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

10. FIG. 9
Transduction of SKOV3.ip1 cells in vitro in the presence of neutralizing serum. SKOV3.ip1 cells were infected with Ad or Ad/PEG/FGF2 in the presence or absence of human anti-Ad neutralizing serum. Transgene expression (β-galactosidase) is expressed as RLU.
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

11. FIG. 10
Cytokine secretion profiles. Unmodified Ad or Ad/PEG (2.5 × 1010 particles) was administered systemically to Balb/C mice. Splenocytes were harvested 11 days after administration and were cultured in the presence or absence of inactivated unmodified Ad for 48 h. Cell free supernatants were analyzed for (A) IFN-γ and (B) IL-10 by ELISA. Values are derived from the combined spleens of six animals.
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions