Background and aims: Transient ultrasound-induced increase in permeability of cell membrane and in some cases may enhance gene transfer. The objective of this work is to evaluate transfection efficiency and safety for intrahepatic gene delivery by sonoporation.
Methods: Polyplexes of expression vector DNAs complexed to galactose-bearing polyethylenimine have been used. Expression vectors are two plasmid containing expression cassette for full-size human preproinsulin gene controlled with CMV promoter and flanked by inverted terminal repeats of AAV (pTRhins) and the same cassette for marker gfp gene(pTRegfp). Animals were injected under US guidance with polyplexes in a dose of 40 Âµg pTRegfp /0.7 ml (rat) and of 15 Âµg pTRhins /0.15 ml (diabetic mouse) into the liver parenchyma of subdiaphragmal segments using 31 G needles. Afterwards injection locus in depth of 1 cm during 180 sec was insonated by 130 Db ultrasound using multifrequency 3-8 MHz probe.
Results: Sonoporation is able to enhance polyplex intrahepatic gene delivery to transfecting liver cells in vivo. Flow cytometry analysis of primary hepatocytes isolated from the liver of experimental rats showed that ultrasound -enhanced polyplex gene transfer was highly localized, and was superior to all controls. At least 42% of the liver cells in vivo can be transfected in this way with ultrasound exposure versus 1.2% without it. Hypoglycemic effect of the insulin gene delivery followed by 3 min US exposure was observed on the third day: glucose level of diabetic mice (hyperglycemic 6- week) decreased on average by 30%. A week after the procedure serial sections from rat liver injected with polyplexes containing 40 Âµg marker plasmid or saline solution alone exposed to US and without it were analysed for the presence of inflammation. Pathomorphological and histological analysis of experimental livers revealed no inflammatory processes in tissues, and any detectable side effects of US-enhanced gene delivery were seen. Experimental mice and rat liver DNAs were positive in transgene PCR for 1 month after gene delivery.
Conclusions: Our results demonstrate that polyplex gene transfer by US exposure is effective, robust and feasible and can become the new relevant technology for gene therapy.
Keywords: intraheptic intervention, gene delivery, sonoporation, animal model