1. Receptor-Targeted Gene Delivery Using Multivalent Phagemid Particles
David Larocca, Kristen Jensen-Pergakes, Michael A. Burg, Andrew Baird 
Molecular Therapy 
Volume 3, Issue 4, Pages (April 2001)
DOI: /mthe
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

2. FIG. 1
EGF display phagemid vectors for mammalian cell gene delivery and ligand display strategy. A CMV-GFP expression cassette with SV40 origin (SV-CMV-GFP) and an EGF N-terminal (NT) pIII fusion gene from the MG4 phage vector were inserted into a pUC119 phagemid to make pUCMG4-EGF. The pUC119 phagemid contains both a plasmid origin of replication (pUC ori) and a phage origin of replication (M13 ori) and an ampicillin-resistance gene (Ampr). The EGF-pIII gene is regulated by the lac operon in pUC119. The pUCMG4CT-EGF phagemid was created by fusing the EGF gene to the C-terminal domain-encoding sequence of the pIII gene (CT) (A) The phagemid particles produced when either vector is rescued with wild-type R408 helper phage are predicted to display one or fewer EGF molecules per particle (monovalent display). (B) The phagemid particles produced by vector rescued with R408d3 are predicted to display three to five EGF molecules per particle (multivalent display).
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

3. FIG. 2
Western blot analysis of EGF-pIII fusion proteins displayed on phage and phagemid particles. Protein extracts from equivalent titers of CsCl-banded phage particle preparations were separated on PAGE gels and electrophoretically blotted onto nitrocellulose filters. The filters were blocked and probed with antibody and reactive proteins visualized using chemiluminescence. Duplicate blots were probed with (A) anti-pIII antibody and (B) anti-EGF antibody. Lanes A, R408 helper phage; lanes B, MG4-EGF phage; lanes C, pUCMG4-EGF (R408); lanes D, pUCMG4-EGF (R408d3); lanes E, pUCMG4CT-EGF (R408); lanes F, pUCMG4CT-EGF (R408d3). Molecular weights (kDa) of prestained protein size markers are indicated on the left side of each blot.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

4. FIG. 3
Increased internalization of multivalent phagemid particles in PC-3 cells. Cells were incubated with phage/phagemid particles (1 × 1010 cfu/ml) for 2 h and stained for internalized phage as described in the text. (A) pUCMG4CT-EGF (R408 rescue), (B) pUCMG4CT-EGF (R408d3 rescue), (C) pUCMG4-EGF (R408 rescue), (D) pUCMG4-EGF (R408d3 rescue), (E) MG4-EGF phage, (F) pUCMG4CT (R408d3 rescue), (G) pUCMG4 (R408d3 rescue), (H) MG4 phage.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

5. FIG. 4
Increased transduction efficiency of multivalent phagemid particles in PC-3 cells. PC-3 cells were transfected with pUCMG4-EGF (squares) and pUC MG4CT-EGF (circles) phagemid particles that were rescued with either R408 helper phage (filled symbols) or R408d3 helper phage (open symbols). PC-3 cells were also transfected with MG4-phage (solid triangles) for comparison to phagemid transfection. Cells were incubated with phage at the concentrations indicated, harvested at 72 h after phage addition, and analyzed by FACS for GFP expression. Transduction efficiency was measured as % GFP-positive cells.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

6. FIG. 5
Specificity of multivalent phagemid particle targeting. PUCMG4CT-EGF or pUCMG4CT control particles (1010 cfu/ml) were incubated with PC-3 cells in the presence or absence of added EGF ligand. Cells were harvested at 96 h after phage addition and analyzed by FACS for GFP expression. Specific transduction of PC-3 cells is inhibited in the presence of excess free EGF (2 ug/ml). Transduction efficiency was measured as % GFP-positive cells.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions