695. Low-Frequency Ultrasound Increases Non- Viral Lung Gene Transfer

LUNG AND RESPIRATORY DISEASE heparan sulfate (HS) and chondroitin sulfate B in gene transfer mediated by a VSV-G pseudotyped LV vector in a respiratory epithelial cell line of tracheal origin (CFT1-C2). The HIV-1 derived LV vector PPT-GFP carries the Green Fluorescence Protein (GFP) reporter gene under the control of the CMV promoter (Follenzi, 2000). To evaluate the interaction between GAGs expressed on the apical surface of respiratory epithelial cells and lentiviral particles, the LV vector (3.2x10E8 TU, corresponding to MOI 2000) was pre-incubated with heparin or chondroitin sulfate B at different concentrations (10-250 micrograms/ml) for 60 minutes in binding buffer and then added to polarized CFT1-C2 cells (polarization was evaluated by the measure of transepithelial resistance and apical expression of ZO-1). The virus-containing solution was incubated with cells for 24 hours and GFP expression was analyzed by flow cytometry 72 hours after. Heparin and chondroitin sulfate B determined a dose-dependent inhibition of transduction efficiency up to 86% and 83% of untreated vector, respectively. HS expression in CFT1-C2 cells was analyzed by flow cytometry and confocal microscopy. HS was expressed by 85-88% of cells, the expression being much stronger on the basolateral side of polarized cells. Cells were incubated with 12 mM EGTA before LV administration to disrupt the epithelial tight junctions and allow the lentiviral particles to access the basolateral side of the epithelium. In the absence of EGTA, 60% of cells resulted GFP positive, whereas the pretreatment with EGTA increased the percentage of GFP-expressing cells up to 80.5%. Our data strongly indicate that the little amount of GAGs restricted to the apical surface might be sufficient to mediate transduction of respiratory epithelial cells by a VSV-G pseudotyped LV vector. However, we can not exclude that other receptor(s) could be involved. References 1. Guibinga GH et al., Mol Ther 5: 538-546, 2002 2. Follenzi A et al., Nat Genetics 25: 217-222, 2000. This work has been supported by a grant from the Italian Cystic Fibrosis Research Foundation and by the Associazione Lombarda Fibrosi Cistica – ONLUS.

695. Low-Frequency Ultrasound Increases NonViral Lung Gene Transfer Stefania Xenariou,1,4 Hai-Dong Liang,2 Uta Griesenbach,1,4 Jie Zhu,1 Raymond Farley,1,4 Lucinda Somerton,1,4 Charanjit Singh,1,4 Peter K. Jeffery,1 Ronald K. Scheule,3 Seng H. Cheng,3 Duncan M. Geddes,1,4 Martin Blomley,2 Eric W. F. W. Alton.1,4 1 Gene Therapy, National Heart and Lung Institute, Imperial College, London, United Kingdom; 2Ultrasound Group, Imaging Sciences Department, Imperial College, London, United Kingdom; 3Genzyme Corporation, Framingham; 4UK Cystic Fibrosis Gene Therapy Consortium. We have previously shown that high-frequency (1 MHz) ultrasound (U/S) increases naked plasmid DNA (pDNA) gene transfer in the mouse lung and that this effect requires microbubbles (Optison) to be present in the formulation. Here, we assessed lowfrequency (30-35 kHz) U/S, in an attempt to further increase nonviral gene delivery to the lung in vivo. The potential advantages of lowering U/S frequency include: a) increased cavitation, the mechanism responsible for the formation of transient pores on cell membranes and b) enhanced penetration of U/S energy into the lung, by reducing wave scattering and heat losses. Cationic lipid GL67 (GL67)/pDNA (80 µg/100 µl), polyethylenimine (PEI)/pDNA (20 µg/100 µl) and naked pDNA (100 µg/100 µl) were delivered to Balb/ c mice via intranasal instillation. Low-frequency U/S (30-35 kHz, 0.07 or 0.1 Mpa, 10 min) was then applied onto the chest and back. Sonoporation did not enhance GL67/pDNA and PEI/pDNA gene transfer. However, U/S exposure at 0.1 Mpa, but not at 0.07 Mpa, increased naked pDNA transfection approximately 4-fold (pDNA: Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy

5.9±1.2 RLU/mg protein, pDNA + 0.1 Mpa: 22.6±3.8 RLU/mg protein, p<0.05, n=7-8). Importantly, low-frequency U/S increased lung gene transfer in the absence of microbubbles. Addition of Optison did not further enhance this effect, due to its inhibitory effects on non-viral lung gene transfer (pDNA + Optison: 0.4±0.1 RLU/mg protein, pDNA + Optison + 0.1 Mpa: 2.5±0.7 RLU/mg protein, n=7-8). Haemorrhage, a common U/S-induced side-effect, was also assessed and increased with pressure in a dose-dependent manner. Interestingly, haemorrhage occurred throughout the lung, indicating that U/S may penetrate deep into the tissue. We have, thus, established for the first time that low-frequency sonoporation can enhance non-viral gene delivery to the mouse lung in vivo.

696. Development of Lipid/Peptide (Lip/Tide) Vectors for Respiratory Gene Transfer Stephen L. Hart,1 Aris Tagalakis,1 Michele J. Writer,1 James Devaney,1 Helen C. Hailes,2 Alethea B. Tabor,2 John B. Wong,2 Steve Bottoms,3 Robin J. McAnulty,3 Ron Scheule,4 Seng Cheng,4 Adam Jaffe.1 1 Molecular Immunology Unit, UCL Institute of Child Health, London, United Kingdom; 2Department of Chemistry, University College London, London, United Kingdom; 3Centre for Respiratory Research, University College London, London, United Kingdom; 4Genetic Diseases Science, Genzyme Corporation, Framingham, MA. We are developing synthetic,targeted vector systems for the respiratory system to treat diseases such as cystic fibrosis (CF). Lip/Tide-I vectors comprised an integrin-targeting peptide and DOTMA/DOPE which displayed transfected human epithelial cells with low receptor specificity due to a lack of integrin receptors. The integrin-targeting motif was replaced with an epithelial targeting peptide selected by phage display panning on human airway epithelial cells. The cationic lipid was optimised by substituting DOTMA, which has an eighteen-carbon alkyl tail (C18), with a C16 cationic lipid. The epithelial-optimised Lip/Tide vector (Lip/Tide-E) was compared with Lip/Tide-I in transfections of cultured human airway epithelial cells and in vivo by tracheal instillation into murine lungs of fifty microlitres of vector containing 8-16 micrograms of DNA. Lip/Tide-E transfection in human epithelial cells was eight-fold higher than Lip/Tide-I with high specificity of receptor-mediated transfection but did not enhance transfection in vivo owing to species specificity. The lipid enhanced luciferase transfection in human epithelial cells about ten-fold and in murine lung in vivo by twofold. Luciferase expression in mouse whole lung extracts was compared after one and three doses of Lip/Tide-E vector. Single dose expression levels of Lip/Tide (2,400 RLU/mg) were similar to levels with GL67 (Genzyme), a vector used in CF gene therapy trials, and about twice the level achieved with polyethylenimine (PEI 25 kDa). After three weekly doses of Lip/Tide vector, luciferase expression was equivalent to single dose levels. Immunohistochemical staining of lung sections revealed that the Lip/Tide-transfected luciferase activity was located mainly in airway epithelium, with some further transfection of macrophages, while GL67 activity was largely in macrophages. A moderate Lip/Tide-mediated inflammatory cell infiltration was apparent and levels of inflammatory cytokines from bronchoalveolar lavage fluids, particularly IL-6 and IL-12, were elevated 24 h after transfection. The vector retained its transfection efficiency after nebulisation as evaluated in luciferase assays after in vitro and in vivo administration. In summary, the Lip/Tide-E vector transfection efficiency in human epithelial cells in vitro and in the airway epithelium of mouse lung is superior to Lip/Tide-I and compares well with established vectors such as GL67 and PEI. The modest inflammatory response and the S269