A novel system for in vivo neprilysin gene delivery using a syringe electrode.
Existing methods for in vivo gene transfer are generally inefficient and have several technical problems. In the present study we aimed to develop a safe, simple and efficient gene-delivery system for in vivo therapeutic applications. We focused on delivery of a plasmid encoding human neprilysin (hNEP), an enzyme implicated in the degradation of toxic amyloid-beta (Abeta) peptide, with potential application in Alzheimer's disease therapy. We report the development of a syringe electrode device in which DNA is injected via one of the two syringes and DNA uptake is stimulated by application of a brief pulsed square-wave electrical field between the two syringe needles. Using this device, purified plasmid DNA encoding hNEP was injected into hindlimb skeletal muscle of 6-week-old KunMing mice and electrostimulation (50V/cm, 6 pulses, 20ms per pulse) was applied to the syringe needles. hNEP protein was detected in muscle, serum and brain of treated mice by western blotting and ELISA at 7, 14 and 30 days post-transfer. Importantly, hNEP levels following DNA injection alone, but without electrostimulation, were barely above background. Only low levels of muscle damage were detected following DNA injection and electrotransfer. These results demonstrate that DNA delivery by the syringe electrode technique can give rise to efficient long-term expression of the encoded polypeptide, and that the electrotransfer protocol is essential for effective plasmid DNA uptake and expression. This technique provides a safe and efficient non-viral method for in vivo gene delivery with potential applications in both basic research and in gene therapy of neuronal disease.
Li
Yuanli
Y
The Key Laboratory of Biomedical Information Engineering of Ministry of Education, Institute of Biomedical Engineering, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an, PR China.
Wang
Jue
J
Liu
Jiankang
J
Liu
Feng
F
D000818:Animals; D001921:Brain / Q000378:metabolism / Q000473:pathology; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004195:Disease Models, Animal; D004558:Electric Stimulation / Q000379:methods; D004566:Electrodes; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005070:Evans Blue; D018014:Gene Transfer Techniques / Q000295:instrumentation; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D051379:Mice; D008815:Mice, Inbred Strains; D018482:Muscle, Skeletal / Q000378:metabolism / Q000473:pathology; D009135:Muscular Diseases / Q000473:pathology / Q000628:therapy*; D015260:Neprilysin / Q000235:genetics* / Q000378:metabolism; D013594:Syringes; D013997:Time Factors
D005070:Evans Blue; D004247:DNA; D015260:Neprilysin