Wenbin Ying

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

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, αV 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.—Doukas, J., Hoganson, D. K., Ong, M., Ying, W., Lacey, D. L., Baird, A., Pierce, G. F., Sosnowski, B. A. Retargeted deliveryof adenoviral vectors through fibroblast growth factor receptors involves unique cellular pathways. FASEB J. 13, 1459–1466 (1999)

Targeted Delivery of DNA Encoding Cytotoxic Proteins through High-Affinity Fibroblast Growth Factor Receptors

Nonviral DNA delivery strategies for gene therapy have generally been limited by a lack of specificity and efficacy. However, ligand-mediated endocytosis can specifically deliver DNA in vitro to cells bearing the appropriate cognate receptors. Similarly, in order to circumvent problems related to efficacy, DNA must encode proteins with high intrinsic activities. We show here that the ligand basic fibroblast growth factor (FGF2) can target FGF receptor-bearing cells with DNA encoding therapeutic proteins. Delivery of genes encoding saporin, a highly potent ribosomal inactivating protein, or the conditionally cytotoxic herpes simplex virus thymidine kinase, a protein that can kill cells by activating the prodrug ganciclovir, is demonstrated. The saporin gene was codon optimized for mammalian expression and demonstrated to express functional protein in a cell-free assay. FGF2-mediated delivery of saporin DNA or thymidine kinase DNA followed by ganciclovir treatment resulted in a 60 and 75% decrease in cell number, respectively. Specificity of gene delivery was demonstrated in competition assays with free FGF2 or with recombinant soluble FGF receptor. Alternatively, when histone H1, a ligand that binds to cell surface heparan sulfate proteoglycans (low-affinity FGF receptors), was used to deliver DNA encoding thymidine kinase, no ganciclovir sensitivity was observed. These findings establish the feasibility of using ligands such as FGF2 to specifically deliver genes encoding molecular chemotherapeutic agents to cells.

CTb targeted non-viral cDNA delivery enhances transgene expression in neurons

Efficient neuronal gene therapy is a goal for the long‐term repair and regeneration of the injured central nervous system (CNS). We investigated whether targeting cDNA to neurons with cholera toxin b chain conjugated non‐viral polyplexes led to increased efficiency of non‐viral gene transfer in the CNS. Here, we illustrate the potential for this strategy by demonstrating enhanced transfection of a differentiated neuronal cell type, PC12.

Anti-B16-F10 melanoma activity of a basic fibroblast growth factor-saporin mitotoxin.

Background. The authors attached basic fibroblast growth factor (FGF‐2), a growth factor for numerous tumors and normal cell types, to saporin (SAP), a ribosomeinactivating protein isolated from the plant Saponaria officinalis. The conjugate (FGF‐SAP) then was tested for antitumor activity using B16‐F10 melanoma cells. This rapidly growing murine melanoma cell line has been used classically as a model to screen antitumor agents.

Saporin toxins directed to basic fibroblast growth factor receptors effectively target human ovarian teratocarcinoma in an animal model

Background. The antitumor activity of the chemical conjugate and recombinant forms of the mitotoxin basic fibroblast growth factor (bFGF) saporin (SAP) and the bFGF receptor‐directed immunotoxin 11A8‐SAP against human ovarian teratocarcinoma PA‐1 was examined in athymic nude mice. Alternative administration schedules to prolong therapeutic efficacy were explored.