Volume 8, Issue 6, Pages 867-872 (December 2003) A new peptide ligand that targets particles and heterologous proteins to hepatocytes in vivo  Alex V Sokoloff, So C Wong, James J Ludtke, Magdolna G Sebestyén, Vladimir M Subbotin, Guofeng Zhang, Tatyana Budker, Marcus Bachhuber, Yoshihiro Sumita, Jon A Wolff  Molecular Therapy  Volume 8, Issue 6, Pages 867-872 (December 2003) DOI: 10.1016/j.ymthe.2003.09.009 Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Structural features of T7 phage. (A) A cartoon of T7 phage. The tail fiber site incorporating mutations that disrupt phage targeting to hepatocytes and putatively containing a hepatocyte-targeting determinant is circled. (B) A cross-sectional view of the p17 triple-stranded coiled-coil domain (rod domain) containing a putative hepatocyte-targeting determinant. The hydrophobic interactions between amino acid residues in the a and d positions and electrostatic interactions between positive and negative residues in the e and g positions are shown as dashed lines. (C) The three major domains of p17 are shown. The putative coiled-coil rod domain includes residues 151–267. The location of K211 is indicated. Molecular Therapy 2003 8, 867-872DOI: (10.1016/j.ymthe.2003.09.009) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Immunohistochemical localization of T7 phage and phage p17 derivatives in mouse and rhesus monkey liver. T7 phage (1011 pfu in 400 μl of PBS), cloned p17 (5 μg in 250 μl of PBS), p17 fragments (75 μg in 250 μl of PBS), p17/EYFP fusion proteins (75 μg in 250 μl of PBS), and p17/IFN fusion proteins (50 μg in 250 μl of PBS) were slowly injected into mice through the tail vein and the livers were removed at a certain time point. The injection of p17 (500 μg in 30 ml μl of PBS) into adult rhesus monkeys was performed through the subcutaneous hand vein. Approximately 5-mm blocks of liver tissue from the left lateral lobe were snap-frozen in OCT compound and sectioned (5–7 μm thick) using a Microm HM 505 N cryostat (Carl Zeiss). Sections, mounted on Superfrost Plus Fisher slides and air-dried overnight, were fixed in 4% formaldehyde for 20 min, washed with PBS, and blocked with 10% donkey serum in PBS for 30 min. All following incubation steps were done at room temperature for 60 min, using antibody solutions in PBS containing 2 mg/ml BSA. Blocked liver sections were incubated with rabbit anti-T7 antibody (IgG fraction) raised against the Novagen T7Select415-1b vector phage or, in the case of IFN/p17 proteins, with rabbit anti-human IFN-α (1:250; Accurate), washed three times with PBS, and incubated with 3.5 μg/ml Cy3-conjugated donkey anti-rabbit antibody (The Jackson Laboratory). Sections were washed three times with PBS and counterstained for 20 min with 13 nM To-Pro-3 and 16.5 nM Alexa 488–phalloidin (Molecular Probes) to visualize nuclei and actin filaments [13], rinsed three times with PBS, sealed, and analyzed under a confocal Zeiss LSM 510 microscope (Zeiss) using 560–615 and 505–550 nm BP filters and a 650-nm LP filter. EYFP was detected without immunostaining using a 505–550 nm BP filter. Images were reconstructed as a flattened projection of a stack of eight 0.4-μm optical sections. Reconstructed confocal fluorescence images acquired with a 63× objective are shown. The image area is 75 × 75 μm unless indicated otherwise. Phage and p17 proteins, with the exception of EYFP fusion products, are shown in red, nuclei in blue, and F-actin in gray near basolateral membranes and in white near bile capillary membranes. (A) A1, 20-6 phage (p17-E211) at 30 min after injection; A2, 20-6F phage (p17-K211) at 5 min after injection; A3, 20-6F phage at 30 min after injection. (B) B1, A single optical section (30 × 30 μm area) for phage 20-6F at 30 min after injection; B2, p17-K211 at 15 min after injection; B3, p17-E211 at 15 min after injection. (C) C1, EYFP/p17-K211 at 15 min after injection; C2, EYFP/p17-E211 at 15 min after injection; C3, IFN/p17-K211 stained with anti-T7 antibody at 15 min after injection. (D) D1, IFN/p17-E211 stained with anti-T7 antibody at 15 min after injection; D2, IFN/p17-K211 stained with anti-IFN antibody at 15 min after injection; D3, IFN/p17-E211 stained with anti-IFN antibody at 15 min after injection. (E) E1, background staining with anti-IFN antibody in the absence of injected protein; E2, p17-K211 at 60 min after injection into a rhesus monkey; E3, p17-E211 at 60 min after injection into a rhesus monkey. Molecular Therapy 2003 8, 867-872DOI: (10.1016/j.ymthe.2003.09.009) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions