356. Sonoporation Increases Non-Viral Gene Transfer to the Murine Lung

Abstract

Related to our programme of cystic fibrosis gene therapy, we assessed whether ultrasound (U/S) can increase non-viral lung transfection. U/S penetration in the air-filled lung is limited due to significant energy losses at the air/tissue interfaces. Thus, using an ex vivo rat lung model, we have shown that more than 99% of the U/S energy is lost. Here, we assessed, whether the remaining energy that penetrates the tissue is sufficient to increase non-viral gene transfer. Naked plasmid DNA (pDNA) and polyethylenimine (PEI)/pDNA complexes (100 |[mu]|g pDNA and 10 |[mu]|g pDNA, respectively) were administered to Balb/c mice via intranasal instillation, in conjunction with Optison (1:1 v:v), an U/S contrast agent shown to improve sonoporation (formation of transient pores). U/S (1 MHz, 20% duty cycle, 3 W/cm2) was then applied externally onto the chest and the back. For naked pDNA, a 2-min U/S exposure, led to a significant 6-fold increase in luciferase (Lux) expression (no U/S: 1.0|[plusmn]|0.2 RLU/mg protein, 2-min U/S: 6.3|[plusmn]|0.8 RLU/mg protein; p<0.001; n=8). This was further enhanced (4-fold) after a 20-min U/S exposure (28.0|[plusmn]|7.8 RLU/mg protein; p<0.05, compared to 2-min U/S; n=8). The positive U/S effect on naked pDNA is, however, dependent on the presence of Optison. Without it even a 20-min U/S exposure has no effect (naked pDNA: 4.1|[plusmn]|0.8 RLU/mg protein, pDNA + 20-min U/S: 6.1|[plusmn]|0.8 RLU/mg protein; n=6-10). U/S exposure did not increase PEI/pDNA transfection efficiency, suggesting that U/S has a vector-specific effect (no U/S: 3.1|[plusmn]|0.9 RLU/mg protein, 20-min U/S: 3.0|[plusmn]|0.5 RLU/mg protein; n=8). While the use of Optison was not detrimental to naked pDNA gene transfer in the absence of U/S (pDNA alone: 0.9|[plusmn]|0.3 RLU/mg protein, pDNA+Optison: 1.0|[plusmn]|0.2 RLU/mg protein; n=8), when added to PEI/pDNA complexes it resulted in an 8-fold decrease in gene expression (PEI/pDNA: 24.7|[plusmn]|10.4 RLU/mg protein, PEI/pDNA+Optison: 3.1|[plusmn]|0.9 RLU/mg protein; p<0.01; n=8), again emphasising vector-specific effects. To reduce U/S energy losses and thus attempt to enhance transfection efficiency further, we are also investigating the use of perfluorocarbon (PFC) liquid. Ex vivo measurements in rat lungs showed that PFC allows 20-30% of U/S energy to penetrate the lung tissue. Magnetic resonance imaging is currently being used to determine the distribution, and the attenuation time of PFC in the lung of spontaneously breathing mice, to indicate the optimal conditions for U/S application for future gene transfer studies.