Stefania Xenariou BS, Abstract Co-Author: Nothing to Disclose

Haidong Liang PhD, Presenter: Nothing to Disclose

Uta Griesenbach PhD, Abstract Co-Author: Nothing to Disclose

Duncan Geddes MD, Abstract Co-Author: Nothing to Disclose

Martin John Blomley MD, Abstract Co-Author: Nothing to Disclose

Eric Alton MD, Abstract Co-Author: Nothing to Disclose

We are investigating physical delivery systems for cystic fibrosis gene therapy. We hypothesised ultrasound (US) could increase non-viral lung transfection, despite the known problem of limited US penetration in the air-filled lung using relatively low frequency ultrasound (1MHz).

Naked plasmid DNA (pDNA) and polyethylenimine (PEI)/pDNA complexes (100 µg pDNA and 10 µg 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 by us and others to improve ultrasound mediated permeabilisation (formation of transient pores). U/S (1 MHz, 20% duty cycle, 3 W/cm2) was applied externally onto the chest and the back. We used luciferase as the reporter gene.

: 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±0.2 RLU/mg protein, 2-min US: 6.3±0.8 RLU/mg protein; p<0.001; n=8). This was further enhanced (4-fold) after a 20-min US exposure (28.0±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±0.8 RLU/mg protein, pDNA + 20-min U/S: 6.1±0.8 RLU/mg protein; n=6-10). US exposure did not increase PEI/pDNA transfection efficiency, suggesting that US has a vector-specific effect (no U/S: 3.1±0.9 RLU/mg protein, 20-min U/S: 3.0±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±0.3 RLU/mg protein, pDNA+Optison: 1.0±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±10.4 RLU/mg protein, PEI/pDNA+Optison: 3.1±0.9 RLU/mg protein; p<0.01; n=8), again emphasising the vector-specific effects

Microbubble ultrasound shows encouraging promise as a physical delivery system to improve gene transfer efficiency to the lung, encouraging further optimisation studies.

Xenariou, S, Liang, H, Griesenbach, U, Geddes, D, Blomley, M, Alton, E, Microbubble Ultrasound Increases Non-viral Gene Transfer to the Murine Lung : Work in Progress.  Radiological Society of North America 2005 Scientific Assembly and Annual Meeting, November 27 - December 2, 2005 ,Chicago IL. http://archive.rsna.org/2005/4414539.html