Volume 4, Issue 6, Pages 534-542 (December 2001) Ablating Adenovirus Type 5 Fiber–CAR Binding and HI Loop Insertion of the SIGYPLP Peptide Generate an Endothelial Cell-Selective Adenovirus  Stuart A. Nicklin, Dan J. Von Seggern, Lorraine M. Work, Don C.K. Pek, Anna F. Dominiczak, Glen R. Nemerow, Andrew H. Baker  Molecular Therapy  Volume 4, Issue 6, Pages 534-542 (December 2001) DOI: 10.1006/mthe.2001.0489 Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 1 Analysis of CAR-mediated transduction of primary vascular cells. (A) Primary HUVEC, HSVEC, and VSMC were infected with increasing particles/cell of AdCTL or CAR-ablated virus AdKO2G and analyzed by immunofluorescence (at 5000 viral particles/cell). Reporter gene expression was quantified by fluorimetry for HUVEC (B), HSVEC (C), or VSMC (D). *P < 0.05 versus AdKO2G. Results are representative of three independent experiments. Molecular Therapy 2001 4, 534-542DOI: (10.1006/mthe.2001.0489) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 2 Generation of peptide-modified fibers. (A) Side and top view demonstrating structure of the Ad5 fiber (adapted from [37]), showing the exposed surface HI loop in the fiber knob (site of peptide insertion). (B) Immunoblot analysis of recombinant fibers demonstrating efficient trimerization with either the SIGYPLP or LSNFHSS peptides incorporated into the HI loop. Molecular Therapy 2001 4, 534-542DOI: (10.1006/mthe.2001.0489) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 3 Production and characterization of peptide-modified fibers. (A) The 293T cells are transfected with a plasmid encoding the desired fiber, and then infected 24 h later with an eGFP-expressing, fiber-gene-deleted Ad vector, Ad5.GFP.δF. Progeny virus incorporates only the fiber encoded by the plasmid. (B) Immunoblot analysis of Ad particles with the peptide-modified fibers. Blots were probed with an anti-Ad2 fiber antibody (which cross-reacts with the Ad5 fiber (top)) and then reprobed with anti-penton base protein antibody as a loading control (bottom). Molecular Therapy 2001 4, 534-542DOI: (10.1006/mthe.2001.0489) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 4 Enhanced EC selectivity by SIGYPLP but not LSNFHSS fiber-modified Ad vectors. EGFP expression was quantified following infection with AdCTL, AdSIG, or AdLSN. (A) Infection ratio of HUVEC relative to HepG2 hepatocytes. (B) Infection ratio of HSVEC relative to HepG2 heptocytes. (C) Flow cytometric assessment of HUVEC transduction relative to HepG2 hepatocytes. (D) Infection ratio of VSMC relative to HepG2 hepatocytes. **P < 0.01 versus AdCTL. *P < 0.05 versus AdCTL. AdLSN versus AdCTL, P = 0.065 in VSMC; AdSIG versus AdCTL, P = 0.17 in VSMC. Results are representative of three independent experiments. NS, not significant. Molecular Therapy 2001 4, 534-542DOI: (10.1006/mthe.2001.0489) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 5 Simultaneous detargeting and retargeting of adenoviral vectors. (A) Immunoblot analysis of Ad particles containing fibers with CAR-blocking mutations and SIGYPLP peptide insertion (top panel is fiber; bottom panel is penton). (B–E) Quantification of reporter gene expression using fluorimetric analysis of lysates from HUVEC (B) and HSVEC (C). (D) Western blot for eGFP in HUVEC lysates. (E) FACScan analysis for eGFP expressing HUVEC. (F) Competitive inhibition of Ad infection with soluble Ad5 fiber knob. (G) Quantification of reporter gene expression by fluorimetry in HAEC. *P < 0.05 versus AdCTL. Results are representative of three independent experiments. Molecular Therapy 2001 4, 534-542DOI: (10.1006/mthe.2001.0489) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 6 Analysis of EC selectivity mediated by AdKOISIG. VSMC (A) and HepG2 (B) lysates were quantified for eGFP expression by fluorimetry following infection with AdCTL, AdKO1, or AdKO1SIG. (C) Ratio of reporter gene expression in AdCTL, AdKO1SIG, or AdSIG transduced EC versus HepG2 hepatocytes. *P < 0.05 versus AdCTL. Representative of three independent experiments. NS, not significant. Molecular Therapy 2001 4, 534-542DOI: (10.1006/mthe.2001.0489) Copyright © 2001 American Society for Gene Therapy Terms and Conditions