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

Patricia Cmielewski, Nigel Farrow, Martin Donnelley, Chantelle McIntyre, Jahan Penny-Dimri, Tim Kuchel, David Parsons

Research output: Contribution to journalArticle

7 Citations (Scopus)

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.

LanguageEnglish
Article number183
JournalBMC Pulmonary Medicine
Volume14
Issue number1
DOIs
Publication statusPublished - 10 Nov 2014

Keywords

  • Cystic fibrosis
  • Ferret
  • Gene therapy
  • Lentivirus
  • Lung

ASJC Scopus subject areas

  • Pulmonary and Respiratory Medicine

Cite this

Cmielewski, P., Farrow, N., Donnelley, M., McIntyre, C., Penny-Dimri, J., Kuchel, T., & Parsons, D. (2014). Transduction of ferret airway epithelia using a pre-treatment and lentiviral gene vector. BMC Pulmonary Medicine, 14(1), [183]. https://doi.org/10.1186/1471-2466-14-183
Cmielewski, Patricia ; Farrow, Nigel ; Donnelley, Martin ; McIntyre, Chantelle ; Penny-Dimri, Jahan ; Kuchel, Tim ; Parsons, David. / Transduction of ferret airway epithelia using a pre-treatment and lentiviral gene vector. In: BMC Pulmonary Medicine. 2014 ; Vol. 14, No. 1.
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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.",
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Cmielewski, P, Farrow, N, Donnelley, M, McIntyre, C, Penny-Dimri, J, Kuchel, T & Parsons, D 2014, 'Transduction of ferret airway epithelia using a pre-treatment and lentiviral gene vector', BMC Pulmonary Medicine, vol. 14, no. 1, 183. https://doi.org/10.1186/1471-2466-14-183

Transduction of ferret airway epithelia using a pre-treatment and lentiviral gene vector. / Cmielewski, Patricia; Farrow, Nigel; Donnelley, Martin; McIntyre, Chantelle; Penny-Dimri, Jahan; Kuchel, Tim; Parsons, David.

In: BMC Pulmonary Medicine, Vol. 14, No. 1, 183, 10.11.2014.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Transduction of ferret airway epithelia using a pre-treatment and lentiviral gene vector

AU - Cmielewski, Patricia

AU - Farrow, Nigel

AU - Donnelley, Martin

AU - McIntyre, Chantelle

AU - Penny-Dimri, Jahan

AU - Kuchel, Tim

AU - Parsons, David

PY - 2014/11/10

Y1 - 2014/11/10

N2 - 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.

AB - 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.

KW - Cystic fibrosis

KW - Ferret

KW - Gene therapy

KW - Lentivirus

KW - Lung

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U2 - 10.1186/1471-2466-14-183

DO - 10.1186/1471-2466-14-183

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JO - BMC Pulmonary Medicine

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