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Molecular Diagnosis and Genetic Counseling of Cystic Fibrosis and Related Disorders: New Challenges
Open AccessArticle

Detargeting Lentiviral-Mediated CFTR Expression in Airway Basal Cells Using miR-106b

1
Department of Anatomy and Cell Biology, University of Iowa, Carver College of Medicine, Iowa City, IA 52242, USA
2
Molecular Medicine Program, University of Iowa, Carver College of Medicine, Iowa City, IA 52246, USA
3
Department of Internal Medicine, University of Iowa, Carver College of Medicine, Iowa City, IA 52246, USA
4
Department of Obstetrics and Gynecology, University of Iowa, Carver College of Medicine, Iowa City, IA 52246, USA
*
Author to whom correspondence should be addressed.
Genes 2020, 11(10), 1169; https://doi.org/10.3390/genes11101169
Received: 12 September 2020 / Revised: 29 September 2020 / Accepted: 2 October 2020 / Published: 6 October 2020
(This article belongs to the Special Issue Molecular Basis and Gene Therapies of Cystic Fibrosis)
Lentiviral-mediated integration of a CFTR transgene cassette into airway basal cells is a strategy being considered for cystic fibrosis (CF) cell-based therapies. However, CFTR expression is highly regulated in differentiated airway cell types and a subset of intermediate basal cells destined to differentiate. Since basal stem cells typically do not express CFTR, suppressing the CFTR expression from the lentiviral vector in airway basal cells may be beneficial for maintaining their proliferative capacity and multipotency. We identified miR-106b as highly expressed in proliferating airway basal cells and extinguished in differentiated columnar cells. Herein, we developed lentiviral vectors with the miR-106b-target sequence (miRT) to both study miR-106b regulation during basal cell differentiation and detarget CFTR expression in basal cells. Given that miR-106b is expressed in the 293T cells used for viral production, obstacles of viral genome integrity and titers were overcome by creating a 293T-B2 cell line that inducibly expresses the RNAi suppressor B2 protein from flock house virus. While miR-106b vectors effectively detargeted reporter gene expression in proliferating basal cells and following differentiation in the air–liquid interface and organoid cultures, the CFTR-miRT vector produced significantly less CFTR-mediated current than the non-miR-targeted CFTR vector following transduction and differentiation of CF basal cells. These findings suggest that miR-106b is expressed in certain airway cell types that contribute to the majority of CFTR anion transport in airway epithelium. View Full-Text
Keywords: miRNA; airway basal cell; CFTR; gene therapy; lentivirus miRNA; airway basal cell; CFTR; gene therapy; lentivirus
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Choi, S.H.; Reeves, R.E.; Romano Ibarra, G.S.; Lynch, T.J.; Shahin, W.S.; Feng, Z.; Gasser, G.N.; Winter, M.C.; Evans, T.I.A.; Liu, X.; Luo, M.; Zhang, Y.; Stoltz, D.A.; Devor, E.J.; Yan, Z.; Engelhardt, J.F. Detargeting Lentiviral-Mediated CFTR Expression in Airway Basal Cells Using miR-106b. Genes 2020, 11, 1169.

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