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

Soon H Choi,Eric J Devor,John F Engelhardt,Rosie E Reeves,Guillermo S Romano Ibarra,Yulong Zhang,David A Stoltz,Zehua Feng,Grace N Gasser,Meihui Luo,Xiaoming Liu,Michael C Winter,Ziying Yan,Weam S Shahin,Thomas J Lynch,T Idil Apak Evans

doi:10.3390/genes11101169

Abstract

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.

Highlights

Cystic fibrosis (CF) is an inherited disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene [1]
NCBI Gene Expression Omnibus (GEO) under serial number GSE22145 that compared basal cells vs. columnar cells in nasal airway [14] and found seven miRNAs that were consistently expressed in basal cells but not columnar cells from the nasal epithelia of three donors (Figure 1A)
While we had initial chosen miR-106b as a candidate based on its lack of expression in nasal columnar cells, our reporter gene expression studies suggest that it may be expressed in a subset of columnar cells

Summary

Introduction

Cystic fibrosis (CF) is an inherited disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene [1]. Gene and cell-based therapies for CF lung disease are gaining momentum, but knowledge gaps do remain regarding the target airway cell types that can prevent or reverse lung disease once a functional CFTR gene is expressed [3]. Both the proximal and distal airways express CFTR, but the landscape of cell types and CFTR expression patterns differ in these two levels of the airway. CFTR is most abundantly expressed in club secretory cells of bronchioles and alveolar type II cells [3,7,8]

Methods

Results

Discussion

Conclusion