Safe and efficient local gene delivery into skeletal muscle via a combination of Pluronic L64 and modified electrotransfer

Abstract

Efficient DNA electrotransfer into muscles can be achieved by combining two types of electronic pulses sequentially: short high-voltage (HV) pulse for the cell electropermeabilization and long low-voltage (LV) pulse for the DNA electrophoresis into cells. However, the voltages currently applied can still induce histological and functional damages to tissues. Pluronic L64 has been considered as a molecule possessing cell membrane-disturbing ability. For these reasons, we hope that L64 can be used as a substitute for the HV pulse in cell membrane permeabilization, and a safe LV pulse may still keep the ability to drive plasmid DNA across the permeabilized membrane. In this work, we optimized the electrotransfer parameters to establish a safe and efficient procedure using a clinically applied instrument, and found out that the critical condition for a successful combination of electrotransfer with L64 was that the injection of plasmid/L64 mixture should be applied 1 h before the electrotransfer. In addition, we revealed that the combined procedure could not efficiently transfer plasmid into solid tumor because the uncompressed plasmid may rapidly permeate the leaky tumor vessels and flow away. Altogether, the results demonstrate that the combined procedure has the potential for plasmid-based gene therapy through safe and efficient local gene delivery into skeletal muscles.

Intramuscular Expression of Plasmid-Encoded FVII-Fc Immunoconjugate for Tumor Immunotherapy by Targeting Tumoral Blood Vessels and Cells

“…We further established a more effective method by combining L64 and low-voltage electropulse. In this method, pre-injection of L64 may increase the permeability of in situ muscle cells and the subsequent electric field power may drive the negatively charged plasmids to translocate into the cells (23).…”

The proper strategy to compress and protect plasmid DNA in the Pluronic L64-electropulse system for enhanced intramuscular gene delivery

“…The plasmids encoding β- d -galactosidase (pCMV-LacZ), luciferase (pCMV-Luc), far-red fluorescent protein (pCMV-E2) and growth-hormone-releasing hormone (pCMV-GHRH) were previously constructed in our laboratory [18, 23, 25]. The plasmids were amplified in E. coli DH5α and extracted using the Endo-Free Plasmid Kit (Invitrogen, Carlsbad, CA, USA).…”

“…Ten micrograms of reporter plasmids (pCMV-LacZ, pCMV-Luc and pCMV-E2) or 25 µg of pCMV-GHRH in 40 µl solution were used for each intramuscular injection. PEI/pDNAs or EGCG/pDNAs were mixed with 0.4% (w/v) of Pluronic L64 diluted in saline to get the final concentration of 0.1% L64, kept for 5 min at room temperature and injected into each side of the tibialis anterior (TA) muscle using a 29-gauge BD ultra-fine insulin syringe (BD, Franklin Lakes, NJ, USA) for 2–5 s. The transcutaneous electrotransfer was subsequently administrated using previous protocol [18]. The different systems were constructed and named as L/E, L/E/P and L/E/G respectively.…”

“…In a previous study, we created an efficient and safe intramuscular gene delivery method by combining Pluronic L64 and low-voltage electropulse [18]. In this procedure, Pluronic L64 could interact with the cell membrane, disturb its integrity and increase its permeability, which consequently facilitated the migration of pDNAs driven by the low-voltage electropulse across the permeabilized membrane.…”

An efficient method for in vitro gene delivery via regulation of cellular endocytosis pathway

“…23 Cells were treated with PEI/ TOTO ® -3-pDNA polyplexes using the standard and modified methods for 4 hours and 1.5 hours, respectively. To observe the intracellular traffic of labeled pDNA, cells were washed with PBS twice and analyzed using confocal laser scanning microscopy (Leica TCP SP5) with the excitation wavelength at 642 nm and emission wavelength at 660 nm.…”