Abstract
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.
Highlights
The safety and efficiency of gene therapies for cystic fibrosis (CF) need to be assessed in pre-clinical models
Cystic Fibrosis (CF) is an autosomal recessive disorder caused by a mutational error in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene and its associated protein [1]
Lentiviral gene vectors have a number of potential benefits, including the ability to transduce both dividing and non-dividing cells, be pseudotyped with an envelope glycoprotein to allow for broad tissue tropism [4] and have the ability to carry large genes such as Cystic fibrosis transmembrane conductance regulator (CFTR) [5]
Summary
The safety and efficiency of gene therapies for cystic fibrosis (CF) need to be assessed in pre-clinical models. 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. Gene therapy for a loss-of-function disease like CF involves delivering the appropriate corrective DNA into the cells of an organism to produce adequate functional protein to ameliorate the symptoms of that disease. Lentiviral gene vectors have a number of potential benefits, including the ability to transduce both dividing and non-dividing cells, be pseudotyped with an envelope glycoprotein to allow for broad tissue tropism [4] and have the ability to carry large genes such as CFTR [5]. The similarities between CF humans and CF ferrets in their respiratory pathophysiology include defective chloride transport, submucosal gland fluid secretion, and a propensity towards lung infection in the early post natal period [6]
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