title: Transduction of ferret airway epithelia using a pre-treatment and lentiviral gene vector.
abstract: BACKGROUND
  The 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.
  
  
  METHODS
  Six 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.
  
  
  RESULTS
  The 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.
  
  
  CONCLUSIONS
  This 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.
authors.affiliation: Respiratory and Sleep Medicine, Women's and Children's Hospital, 72 King William Road, North Adelaide, SA 5006, Australia. patricia.cmielewski@health.sa.gov.au.
authors.forename: Patricia
authors.initials: P
authors.lastname: Cmielewski
authors.forename: Nigel
authors.initials: N
authors.lastname: Farrow
authors.forename: Martin
authors.initials: M
authors.lastname: Donnelley
authors.forename: Chantelle
authors.initials: C
authors.lastname: McIntyre
authors.forename: Jahan
authors.initials: J
authors.lastname: Penny-Dimri
authors.forename: Tim
authors.initials: T
authors.lastname: Kuchel
authors.forename: David
authors.initials: D
authors.lastname: Parsons
mesh_terms: D000818:Animals; D005260:Female; D005289:Ferrets; D015870:Gene Expression / Q000187:drug effects*; D005822:Genetic Vectors; D016655:HIV Core Protein p24 / Q000097:blood; D007763:Lac Operon / Q000235:genetics*; D016086:Lentivirus / Q000235:genetics*; D008168:Lung; D008244:Lysophosphatidylcholines / Q000494:pharmacology*; D008297:Male; D020545:Respiratory Mucosa / Q000187:drug effects* / Q000473:pathology; D014132:Trachea; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: D016655:HIV Core Protein p24; D008244:Lysophosphatidylcholines; C514799:p24 protein, Human Immunodeficiency Virus Type 1