Transduction of ferret airway epithelia using a pre-treatment and lentiviral gene vector

BMC Pulmonary Medicine

Research Article

Patricia Cmielewski¹  Nigel Farrow²  David Parsons²  Martin Donnelley²  Chantelle McIntyre³  Jahan Penny-Dimri⁴  Tim Kuchel⁵ 

[1] Respiratory and Sleep Medicine, Women’s and Children’s Hospital, 72 King William Road, 5006, North Adelaide, SA, Australia;Centre for Stem Cell Research, University of Adelaide, 5001, Adelaide, SA, Australia;Respiratory and Sleep Medicine, Women’s and Children’s Hospital, 72 King William Road, 5006, North Adelaide, SA, Australia;Centre for Stem Cell Research, University of Adelaide, 5001, Adelaide, SA, Australia;School of Paediatrics and Reproductive Health, University of Adelaide, 5001, Adelaide, SA, Australia;Respiratory and Sleep Medicine, Women’s and Children’s Hospital, 72 King William Road, 5006, North Adelaide, SA, Australia;School of Paediatrics and Reproductive Health, University of Adelaide, 5001, Adelaide, SA, Australia;School of Paediatrics and Reproductive Health, University of Adelaide, 5001, Adelaide, SA, Australia;South Australian Health and Medical Research Institute, 5086, Gilles Plains, SA, Australia;

关键词: Ferret;    Lung;    Lentivirus;    Cystic fibrosis;    Gene therapy;   

DOI  :  10.1186/1471-2466-14-183

 received in 2014-09-19, accepted in 2014-11-10,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundThe safety and efficiency of gene therapies for cystic fibrosis (CF) need to be assessed in pre-clinical models. Using the normal ferret, this study sought to determine whether ferret airway epithelia could be transduced with a lysophosphatidylcholine (LPC) pre-treatment followed by a VSV-G pseudotyped HIV-1 based lentiviral (LV) vector, in preparation for future studies in CF ferrets.MethodsSix normal ferrets (7 -8 weeks old) were treated with a 150 μL LPC pre-treatment, followed one hour later by a 500 μL LV vector dose containing the LacZ transgene. LacZ gene expression in the conducting airways and lung was assessed by X-gal staining after 7 days. The presence of transduction in the lung, as well as off-target transduction in the liver, spleen and gonads, were assessed by qPCR. The levels of LV vector p24 protein bio-distribution in blood sera were assessed by ELISA at 0, 1, 3, 5 and 7 days.ResultsThe dosing protocol was well tolerated. LacZ gene expression was observed en face in the trachea of all animals. Histology showed that ciliated and basal cells were transduced in the trachea, with rare LacZ transduced single cells noted in lung. p24 levels was not detectable in the sera of 5 of the 6 animals. The LacZ gene was not detected in the lung tissue and no off-target transduction was detected by qPCR.ConclusionsThis study shows that ferret airway epithelia are transducible using our unique two-step pre-treatment and LV vector dosing protocol. We have identified a number of unusual anatomical factors that are likely to influence the level of transduction that can be achieved in ferret airways. The ability to transduce ferret airway epithelium is a promising step towards therapeutic LV-CFTR testing in a CF ferret model.

【 授权许可】

CC BY   
© Cmielewski et al.; licensee BioMed Central Ltd. 2014