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

Abstract

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 HI, 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.

Overview summary

Nonviral gene therapy strategies are limited by the inability to specifically deliver DNA to target cells, and induce meaningful cellular responses, such as cytotoxicity. Nonspecific delivery can be overcome by employing the natural process of ligand-mediated endocytosis. Efficacy can be increased through the use of highly potent cytotoxic molecules. In this study, the ligand basic fibroblast growth factor (FGF2) was used to transport therapeutic genes to cells bearing the high-affinity FGF receptors. Two mechanistically distinct proteins were investigated: saporin, a ribosomal inactivating protein, and herpes simplex virus thymidine kinase, a conditionally cytotoxic protein. These studies demonstrate that a ligand, together with its high-affinity cognate receptor, can serve to selectively deliver DNA encoding therapeutic proteins to cells.