Targeted Delivery of Plasmid DNA to Myogenic Cells via Transferrin-Conjugated Peptide Nucleic Acid

Abstract

We describe a novel approach to conjugate a targeting ligand to plasmid DNA without affecting either its supercoiled conformation or its ability to be efficiently transcribed. A 14-mer peptide nucleic acid (PNA) containing lysine and cysteine on each end was designed to target to a unique sequence located at the antibiotic resistance gene of the plasmid. The binding of PNA to the plasmid was found to be dose-dependent and sequence-specific and not to change the conformation of the plasmid. Transferrin (Tf) was conjugated with PNA via a reversible disulfide bond using N-succinimidyl-3-(2-pyridyldithio)propionate. Tf-PNA retained the ability to the plasmid in a sequence-specific manner. The efficiency of this bioconjugate for delivering plasmid was examined in cultured myoblasts and myotubes. Naked DNA and Tf-PNA/DNA showed no transfection activity in either myoblasts or myotubes. Polyethyleneimine (PEI) is required for significant increase of the transfection efficiency. At N:P ratio of 5, Tf-PNA enhanced gene transfection about fourfold over that of the DNA/PEI complex in both myoblasts and myotubes. This enhancement could be inhibited by excess free Tf, indicating that the enhancement of transfection was through Tf-mediated endocytosis. These findings suggest that this targeting system may have the potential for gene transfer to myogenic cells in vivo. © 2000 American Society for Gene Therapy.