Retargeted delivery of adenoviral vectors through fibroblast growth factor receptors involves unique cellular pathways

Abstract

A major goal of gene therapy is to improve target specificity by delivering vectors through alternative cellular receptors. We previously reported that adenoviral vector delivery through basic fibroblast growth factor (FGF2) receptors enhances both cellular transduction and in vivo efficacy. We now present studies addressing the cellular pathways and mechanisms underlying these events. Cellular receptors for adenoviruses are not required for transduction by FGF2-retargeted vectors. Moreover, alpha(V) integrins can antagonize FGF2 retargeting, in contrast to their obligatory role in non-retargeted vector delivery. By contrast, high-affinity FGF receptors, which are overexpressed on potential tumor targets, are required for FGF2-retargeted transduction. Low-affinity heparan sulfate proteoglycan interactions, however, are not a prerequisite, in marked contrast to their obligatory role in FGF2 mitogenic signaling. By comparing receptor expression and ligand binding with transgene expression, we also demonstrate that FGF2 retargeting enhances transduction by mechanisms other than increasing the number of targeted cells. Rather, the use of alternative targeting ligands supports the conclusion that specific receptor interactions and intracellular events serve to enhance transgene expression. Together, these studies highlight the unique delivery and transduction pathways used by FGF2-retargeted adenoviruses, and help define the basis for their enhanced in vivo efficacy.

Targeting of suicide gene delivery in pancreatic cancer cells via FGF receptors

“…9 -16,32 This anticancer suicide gene therapy has been successfully employed in a number of in vitro and in vivo studies and has found its way into clinical trials. Its potential usefulness in pancreatic cancer cells in vitro, 13,16 as well as in animal tumor models for pancreatic cancer, 20,33 -35 has been previously demonstrated. However, the specificity of this approach in relation to the expression profile of FGFRs in PDAC has not been yet been clearly documented.…”

“…Recombinant E1A -deleted and CMV-driven adenovirus expressing firefly luciferase (AdLuc ) or green fluorescent protein ( AdGFP ), an E1 -deleted Ad5 vector expressing the CMV-driven HSV-TK gene (AdTK ), and the FGF2 -Fab 0 conjugate were constructed as previously reported 16,17 and provided by one of the authors on this study ( Dr B Sosnowski ). The FGF2 -Fab 0 conjugate consists of a modified recombinant FGF2 linked with the Fab 0 fragment from a blocking monoclonal antibody generated against the adenovirus 5 knob region.…”

“…The FGF2 -Fab 0 conjugate consists of a modified recombinant FGF2 linked with the Fab 0 fragment from a blocking monoclonal antibody generated against the adenovirus 5 knob region. 16,17 The specificity of the interactions of FGF2 -Fab 0 with FGFRs was previously demonstrated by neutralizing its activities with an anti -FGFR -1 antibody. 16 Cell culture and infection Cells were grown in DME medium (COLO -357, MIAPaCa -2, PANC -1, and L6 cells) or RPMI medium ( ASPC -1, CAPAN -1, and T3M4 ) at 378C in humidified air with 5% CO 2 .…”

“…16,17 The specificity of the interactions of FGF2 -Fab 0 with FGFRs was previously demonstrated by neutralizing its activities with an anti -FGFR -1 antibody. 16 Cell culture and infection Cells were grown in DME medium (COLO -357, MIAPaCa -2, PANC -1, and L6 cells) or RPMI medium ( ASPC -1, CAPAN -1, and T3M4 ) at 378C in humidified air with 5% CO 2 . Media were supplemented with penicillin G (100 U /mL ), streptomycin ( 100 g /mL ), and 8% FBS.…”

“…17 Pancreatic cancer cells are susceptible to transduction of adenoviral -mediated delivery of the HSV-TK gene. 10,13,16,20 In addition, PDACs overexpress the two Ig -like variants of the FGFR -1 and FGFR -2 21,22 and multiple FGFs. 23 We therefore sought to determine whether an adenoviral -based gene delivery system targeting FGF receptors may be of potential benefit in PDAC, by studying six human pancreatic cancer cell lines and by characterizing FGFR expression in PDAC samples.…”