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.
Liang
K W
KW
Center for Pharmacogenetics, School of Pharmacy, University of Pittsburgh, Pennsylvania 15261, USA.
Hoffman
E P
EP
Huang
L
L
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