Targeted delivery of plasmid DNA to myogenic cells via transferrin-conjugated peptide nucleic acid.
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.
Center for Pharmacogenetics, School of Pharmacy, University of Pittsburgh, Pennsylvania 15261, USA.
K W
KW
Liang
E P
EP
Hoffman
L
L
Huang
D002460:Cell Line; D004220:Disulfides; D004705:Endocytosis; D017930:Genes, Reporter; D008024:Ligands; D008156:Luciferases / Q000235:genetics; D018482:Muscle, Skeletal / Q000166:cytology* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D010957:Plasmids; D011990:Receptors, Transferrin / Q000235:genetics / Q000502:physiology; D014162:Transfection / Q000379:methods*; D014168:Transferrin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D004220:Disulfides; D008024:Ligands; D020135:Peptide Nucleic Acids; D011990:Receptors, Transferrin; D014168:Transferrin; D008156:Luciferases