Cationic microbubbles and antibiotic-free miniplasmid for sustained ultrasound–mediated transgene expression in liver

Abstract

Despite the increasing number of clinical trials in gene therapy, no ideal methods still allow non-viral gene transfer in deep tissues such as the liver. We were interested in ultrasound (US)-mediated gene delivery to provide long term liver expression. For this purpose, new positively charged microbubbles were designed and complexed with pFAR4, a highly efficient small length miniplasmid DNA devoid of antibiotic resistance sequence. Sonoporation parameters, such as insonation time, acoustic pressure and duration of plasmid injection were controlled under ultrasound imaging guidance. The optimization of these various parameters was performed by bioluminescence optical imaging of luciferase reporter gene expression in the liver. Mice were injected with 50μg pFAR4-LUC either alone, or complexed with positively charged microbubbles, or co-injected with neutral MicroMarker™ microbubbles, followed by low ultrasound energy application to the liver. Injection of the pFAR4 encoding luciferase alone led to a transient transgene expression that lasted only for two days. The significant luciferase signal obtained with neutral microbubbles decreased over 2days and reached a plateau with a level around 1 log above the signal obtained with pFAR4 alone. With the newly designed positively charged microbubbles, we obtained a much stronger bioluminescence signal which increased over 2days. The 12-fold difference (p<0.05) between MicroMarker™ and our positively charged microbubbles was maintained over a period of 6months. Noteworthy, the positively charged microbubbles led to an improvement of 180-fold (p<0.001) as regard to free pDNA using unfocused ultrasound performed at clinically tolerated ultrasound amplitude. Transient liver damage was observed when using the cationic microbubble-pFAR4 complexes and the optimized sonoporation parameters. Immunohistochemistry analyses were performed to determine the nature of cells transfected. The pFAR4 miniplasmid complexed with cationic microbubbles allowed to transfect mostly hepatocytes compared to its co-injection with MicroMarker™ which transfected more preferentially endothelial cells.

Photoacoustic transfection of DNA encoding GFP

“…The low P max− and the high frequency components of the PA waves used in this work place their mechanical index below the onset of cavitation, MI = P max− /√ f < 0.7, where the pressure is in MPa and the frequency in MHz 36 . This contrasts with sonophoresis ( f < 1 MHz) where the cavitation of microbubbles is responsible for transfection 37 or dermal delivery 38 . On the other hand, the high frequencies and high P max+ of PA waves lead to high stress gradients and high impulses that enable less common mechanisms of interaction between US and cells.…”

Sonoporation by microbubbles as gene therapy approach against liver cancer

“…This study confirmed how the optimization of ultrasound parameters and of the chemical and electrostatic structure of the microbubbles could play a pivotal role in increasing the efficacy of gene delivery. Moreover, the sonoporation method by Sonovue has already provided encouraging results in a recent human trial, whereas the gemcitabine treatment in inoperable pancreas cancer has been improved, extending the survival of patients without additional toxicity [ 32 ].…”

Development of Theranostic Cationic Liposomes Designed for Image-Guided Delivery of Nucleic Acid

“…Finally, no effect of in vivo transfection could be obtained with a low amount of DNA, which usually requires µg of plasmid. Here, we ought to use a small plasmid that already showed its ability to transfect the liver after a single injection of positively charged microbubbles in vivo [ 41 ].…”