ORIGINAL ARTICLE Validation of recombinant Sendai virus in a non-natural host model U Griesenbach 1,4,5 , G McLachlan 2,4,5 , T Owaki 3 , L Somerton 1,4 , T Shu 3 , A Baker 2,4 , P Tennant 2,4 , C Gordon 2,4 , C Vrettou 2,4 , E Baker 2,4 , DDS Collie 2,4 , M Hasegawa 3 and EWFW Alton 1,4 We have previously shown that recombinant Sendai virus (SeV) vector, derived from murine parainfluenza virus, is one of the most efficient vectors for airway gene transfer. We have also shown that SeV-mediated transfection on second administration, although reduced by 60% when compared with levels achieved after a single dose, is still high because of the efficient transfection achieved by SeV vector in murine airways. Here, we show that these levels further decrease on subsequent doses. In addition, we validated SeV vector repeat administration in a non-natural host model, the sheep. As part of these studies we first assessed viral stability in a Pari LC Plus nebuliser, a polyethylene catheter (PEC) and the Trudell AeroProbe. We also compared the distribution of gene expression after PEC and Trudell AeroProbe administration and quantified virus shedding after sheep transduction. In addition, we show that bronchial brushings and biopsies, collected in anaesthetized sheep, can be used to assess SeV-mediated gene expression over time. Similar to mice, gene expression in sheep was transient and had returned to baseline values by day 14. In conclusion, the SeV vector should be strongly considered for lung-related applications requiring a single administration of the vector even though it might not be suitable for diseases requiring repeat administration. Gene Therapy (2011) 18, 182–188; doi:10.1038/gt.2010.131; published online 21 October 2010 Keywords: Sendai virus; cystic fibrosis; lung; gene transfer; sheep INTRODUCTION Sendai virus (SeV), a murine paramyxovirus, is one of the most efficient viral vectors for airway gene transfer. 1,2 SeV carrying the cystic fibrosis (CF) transmembrane conductance regulator cDNA is able partially to correct the characteristic CF transmembrane con- ductance regulator-dependent chloride transport defect in the nasal epithelium of CF knockout mice. 3 Several aspects of SeV biology may explain the high gene transfer efficiency into airway epithelial cells. Moreover, SeV uses cholesterol and sialic acid as receptors and both are present on the surface of most cell types. Further, SeV requires short contact time with the target cell for internalization, and replicates in the cytoplasm of transduced cells, circumventing the nuclear membrane barrier. In mice gene expression is transient, with peak expression approximately 48 h after transfection, generally returning to baseline values within 2 weeks of transfection. 1 A transmission-incompetent SeV vector has been developed by deleting the F-protein, which is essential for cellular entry of the viral genome (DF/SeV). 4 Moreover, this modification does not reduce transfection efficiency of the virus. 1,4 In general the level of transgene expression achieved from repeat delivery of a viral vector is greatly reduced when compared with that from a single dose due to induction of effective immune responses in the recipient. 5,6 We have previously shown that SeV-mediated gene expression is reduced by 60% on second dose, and that tolerization of mice against immune-dominant SeV epitopes does not improve efficacy. 7 However, given the extremely high transfection efficiency of SeV, the levels of gene expression achieved after repeat administra- tion may still be of sufficient therapeutic value, if retained on subsequent administrations. Here, we assessed SeV-mediated trans- fection efficiency after three doses of the virus and compared residual levels of gene expression with that achieved using plasmid DNA complexed to the cationic lipid GL67A, currently being used in a clinical trial by the UK CF Gene Therapy Consortium (http://www.cfgenetherapy.org.uk/). The vast majority of repeat administration studies have been performed in mouse inbred strains, such as Balb/C and C57BL/6. Given that SeV has a natural tropism for the murine lung leading to pneumonia, we were concerned that assessment of repeat adminis- tration in mice may not be representative of non-natural host models, including man. The greater degree of similarity in size, structure and physiology between the sheep and human lung led us to develop sheep as an intermediate model for airway gene transfer, 8 to bridge the gap between studies in rodents and the clinic. Here, we have also assessed the transduction efficiency of both single and repeat SeV vector administration in the ovine lung. RESULTS Levels of gene expression after repeat administration of DF/SeV with short-term intervals to mouse lung are similar to non-viral gene transfer We have previously shown that SeV-mediated transfection on second administration, although reduced by 60% when compared with levels Received 19 May 2010; revised 27 July 2010; accepted 9 August 2010; published online 21 October 2010 1 Department of Gene Therapy, Imperial College London, National Heart and Lung Institute, London, UK; 2 The Wellcome Trust Centre for Research in Comparative Respiratory Medicine, The Roslin Institute & R(D)SVS, University of Edinburgh, UK; 3 DNAVEC Corporation, Tsukuba, Japan and 4 UK CF Gene Therapy Consortium, UK Correspondence: Dr U Griesenbach, Department of Gene Therapy, Imperial College at the National Heart and Lung Institute, Manresa Road, London SW3 6LR, UK. E-mail: u.griesenbach@imperial.ac.uk 5 These authors contributed equally to this work. Gene Therapy (2011) 18, 182–188 & 2011 Macmillan Publishers Limited All rights reserved 0969-7128/11 www.nature.com/gt