title: 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 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.
authors.affiliation: Selective Genetics, Inc., San Diego, CA 92121, USA.
authors.forename: D K
authors.initials: DK
authors.lastname: Hoganson
authors.forename: L A
authors.initials: LA
authors.lastname: Chandler
authors.forename: G A
authors.initials: GA
authors.lastname: Fleurbaaij
authors.forename: W
authors.initials: W
authors.lastname: Ying
authors.forename: M E
authors.initials: ME
authors.lastname: Black
authors.forename: J
authors.initials: J
authors.lastname: Doukas
authors.forename: G F
authors.initials: GF
authors.lastname: Pierce
authors.forename: A
authors.initials: A
authors.lastname: Baird
authors.forename: B A
authors.initials: BA
authors.lastname: Sosnowski
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002470:Cell Survival / Q000235:genetics; D002474:Cell-Free System; D006224:Cricetinae; D004247:DNA / Q000008:administration & dosage*; D018076:DNA, Complementary; D000922:Immunotoxins; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases; D010940:Plant Proteins / Q000008:administration & dosage / Q000235:genetics*; D017468:Receptors, Fibroblast Growth Factor / Q000235:genetics*; D054789:Ribosome Inactivating Proteins, Type 1; D012270:Ribosomes; D000076985:Saporins; D018139:Simplexvirus / Q000201:enzymology; D013937:Thymidine Kinase / Q000008:administration & dosage / Q000235:genetics*; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D000922:Immunotoxins; D010940:Plant Proteins; D017468:Receptors, Fibroblast Growth Factor; D054789:Ribosome Inactivating Proteins, Type 1; D004247:DNA; D013937:Thymidine Kinase; D009699:N-Glycosyl Hydrolases; D000076985:Saporins