495. Efficient In Vivo Transduction of Mouse Airway Epithelial Cells by the Simian Immunodeficiency Virus Vector Pseudotyped with Sendai Virus F and HN Proteins
Authors: Gerry McLachlan, Uta Griesenbach, Toshi Owaki, Liz Hillery, John Williams, Zhu Yafeng, Chris Boyd, Deborah Gill, Mamoru Hasegawa, Eric W. F. W. Alton, David Collie
Journal: Molecular Therapy (2004)

We have described gene delivery in sheep as a large animal model for airway gene transfer. This model allows comparison of different GTAs in terms of efficacy and safety using doses, delivery methods and assays, relevant to human studies. Proof-of-principle for non-viral gene transfer has been established however, levels of transgene expression are too low on a per cell basis to characterise which cells are targeted. Our preferred method for delivery is by aerosol however, not all GTAs are stable following aerosolisation in conventional jet or ultrasonic nebulisers and the cost of producing GTAs in sufficient quantities for whole lung aerosol studies can be prohibitive. These issues were behind our initial studies with delivery by instillation to individual segments. Unfortunately this approach is associated with increased toxicity as a result of pooling of material in the terminal bronchiole/alveolar duct regions. The Trudell AeroProbe* catheter develops an aerosol at the tip and can be passed through a bronchoscope. This allows localised delivery of GTAs, reducing the amount of material required. A further advantage of the AeroProbe* is that it is a single-pass aerosol device and subjects the GTA to less shear stress. An important question to be addressed when comparing delivery with different devices, is how their respective distribution patterns compare. Here we used a recombinant Sendai virus (DF/SeV-LacZ) vector, shown to be highly efficient at transducing airway epithelial cells in a variety of species, to investigate distribution patterns of delivery methods. SeV was delivered either by instillation to single lung segments via a polyethylene catheter (PEC), by AeroProbe* directed at segments or by AeroProbe* directed at the whole lung. In terms of stability, 100% and 49 +/- 3% of infectious virus particles survived the PEC and AeroProbe* respectively (n=3/group). In vivo studies show the variation in distribution of the different delivery methods. Instillation of 5ml SeV (3e9 CIU) to the same segment of different animals (n=3) results in varied distribution. Data from these animals confirm the absence of spillover as determined by histological assessment of adjacent segments. Segmentally-directed delivery of 5ml SeV (1e10 CIU) via AeroProbe* gives a different distribution. We observed areas of expression at airway bifurcations from impaction of aerosol particles at these sites and expression around sub-mucosal gland openings and within the glands themselves. Spillover to adjacent segments was observed in this case. Delivery of 24ml SeV (4e10 CIU) to the whole lung via the AeroProbe* gave a different type of expression pattern again. SeV was distributed throughout the lung. An even pattern of distribution all the way to the peripheral alveolar regions can be observed in several segments although the extent varies. These distribution patterns will have implications for sampling. It is clear from these studies that the properties of SeV make it an ideal vector to study the distribution pattern of different devices in our ovine model.