Detargeting Lentiviral-Mediated CFTR Expression: Comparison
Please note this is a comparison between versions V2 by Rita Xu and V1 by Soon Choi.

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.

  • miRNA
  • airway basal cell
  • CFTR
  • gene therapy
  • lentivirus
Please wait, diff process is still running!


  1. Riordan, J.R.; Rommens, J.M.; Kerem, B.; Alon, N.; Rozmahel, R.; Grzelczak, Z.; Zielenski, J.; Lok, S.; Plavsic, N.; Chou, J.L.; et al. Identification of the cystic fibrosis gene: Cloning and characterization of complementary DNA. Science 1989, 245, 1066–1073.
  2. Xie, Y.; Ostedgaard, L.; Abou Alaiwa, M.H.; Lu, L.; Fischer, A.J.; Stoltz, D.A. Mucociliary Transport in Healthy and Cystic Fibrosis Pig Airways. Ann. Am. Thorac. Soc. 2018, 15, S171–S176.
  3. Tang, Y.; Yan, Z.; Engelhardt, J.F. Viral Vectors, Animal Models, and Cellular Targets for Gene Therapy of Cystic Fibrosis Lung Disease. Hum. Gene Ther. 2020, 31, 524–537.
  4. Rock, J.R.; Randell, S.H.; Hogan, B.L. Airway basal stem cells: A perspective on their roles in epithelial homeostasis and remodeling. Dis. Model. Mech. 2010, 3, 545–556.
  5. Montoro, D.T.; Haber, A.L.; Biton, M.; Vinarsky, V.; Lin, B.; Birket, S.E.; Yuan, F.; Chen, S.; Leung, H.M.; Villoria, J.; et al. A revised airway epithelial hierarchy includes CFTR-expressing ionocytes. Nature 2018, 560, 319–324.
  6. Plasschaert, L.W.; Zilionis, R.; Choo-Wing, R.; Savova, V.; Knehr, J.; Roma, G.; Klein, A.M.; Jaffe, A.B. A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte. Nature 2018, 560, 377–381.
  7. Carraro, G.; Mulay, A.; Yao, C.; Mizuno, T.; Konda, B.; Petrov, M.; Lafkas, D.; Arron, J.R.; Hogaboam, C.M.; Chen, P.; et al. Single Cell Reconstruction of Human Basal Cell Diversity in Normal and IPF Lung. Am. J. Respir. Crit. Care Med. 2020.
  8. Xu, Y.; Mizuno, T.; Sridharan, A.; Du, Y.; Guo, M.; Tang, J.; Wikenheiser-Brokamp, K.A.; Perl, A.T.; Funari, V.A.; Gokey, J.J.; et al. Single-cell RNA sequencing identifies diverse roles of epithelial cells in idiopathic pulmonary fibrosis. JCI Insight 2016, 1, e90558.
  9. Mou, H.; Vinarsky, V.; Tata, P.R.; Brazauskas, K.; Choi, S.H.; Crooke, A.K.; Zhang, B.; Solomon, G.M.; Turner, B.; Bihler, H.; et al. Dual SMAD Signaling Inhibition Enables Long-Term Expansion of Diverse Epithelial Basal Cells. Cell Stem Cell 2016.
  10. Barde, I.; Salmon, P.; Trono, D. Production and titration of lentiviral vectors. Curr. Protoc. Neurosci. 2010, 53, 4–21.
  11. Denning, W.; Das, S.; Guo, S.; Xu, J.; Kappes, J.C.; Hel, Z. Optimization of the transductional efficiency of lentiviral vectors: Effect of sera and polycations. Mol. Biotechnol. 2013, 53, 308–314.
  12. Sun, X.; Olivier, A.K.; Liang, B.; Yi, Y.; Sui, H.; Evans, T.I.; Zhang, Y.; Zhou, W.; Tyler, S.R.; Fisher, J.T.; et al. Lung phenotype of juvenile and adult cystic fibrosis transmembrane conductance regulator-knockout ferrets. Am. J. Respir. Cell Mol. Biol. 2014, 50, 502–512.
  13. Yan, Z.; Sun, X.; Feng, Z.; Li, G.; Fisher, J.T.; Stewart, Z.A.; Engelhardt, J.F. Optimization of Recombinant Adeno-Associated Virus-Mediated Expression for Large Transgenes, Using a Synthetic Promoter and Tandem Array Enhancers. Hum. Gene Ther. 2015, 26, 334–346.
  14. Marcet, B.; Chevalier, B.; Luxardi, G.; Coraux, C.; Zaragosi, L.E.; Cibois, M.; Robbe-Sermesant, K.; Jolly, T.; Cardinaud, B.; Moreilhon, C.; et al. Control of vertebrate multiciliogenesis by miR-449 through direct repression of the Delta/Notch pathway. Nat. Cell Biol. 2011, 13, 693–699.
  15. Martinez-Anton, A.; Sokolowska, M.; Kern, S.; Davis, A.S.; Alsaaty, S.; Taubenberger, J.K.; Sun, J.; Cai, R.; Danner, R.L.; Eberlein, M.; et al. Changes in microRNA and mRNA expression with differentiation of human bronchial epithelial cells. Am. J. Respir. Cell Mol. Biol. 2013, 49, 384–395.
  16. Mehlich, D.; Garbicz, F.; Wlodarski, P.K. The emerging roles of the polycistronic miR-106b approximately 25 cluster in cancer-A comprehensive review. Biomed Pharmacother. 2018, 107, 1183–1195.
  17. Kim, Y.K.; Kim, V.N. Processing of intronic microRNAs. EMBO J. 2007, 26, 775–783.
  18. Zhou, Y.; Hu, Y.; Yang, M.; Jat, P.; Li, K.; Lombardo, Y.; Xiong, D.; Coombes, R.C.; Raguz, S.; Yague, E. The miR-106b~25 cluster promotes bypass of doxorubicin-induced senescence and increase in motility and invasion by targeting the E-cadherin transcriptional activator EP300. Cell Death Differ. 2014, 21, 462–474.
  19. Chuang, T.D.; Luo, X.; Panda, H.; Chegini, N. miR-93/106b and their host gene, MCM7, are differentially expressed in leiomyomas and functionally target F3 and IL-8. Mol. Endocrinol. 2012, 26, 1028–1042.
  20. Haldar, S.; Roy, A.; Banerjee, S. Differential regulation of MCM7 and its intronic miRNA cluster miR-106b-25 during megakaryopoiesis induced polyploidy. RNA Biol. 2014, 11, 1137–1147.
  21. Kan, T.; Sato, F.; Ito, T.; Matsumura, N.; David, S.; Cheng, Y.; Agarwal, R.; Paun, B.C.; Jin, Z.; Olaru, A.V.; et al. The miR-106b-25 Polycistron, Activated by Genomic Amplification, Functions as an Oncogene by Suppressing p21 and Bim. Gastroenterology 2009, 136, 1689–1700.
  22. Smith, A.L.; Iwanaga, R.; Drasin, D.J.; Micalizzi, D.S.; Vartuli, R.L.; Tan, A.C.; Ford, H.L. The miR-106b-25 cluster targets Smad7, activates TGF-beta signaling, and induces EMT and tumor initiating cell characteristics downstream of Six1 in human breast cancer. Oncogene 2012, 31, 5162–5171.
  23. Chao, J.A.; Lee, J.H.; Chapados, B.R.; Debler, E.W.; Schneemann, A.; Williamson, J.R. Dual modes of RNA-silencing suppression by Flock House virus protein B2. Nat. Struct. Mol. Biol. 2005, 12, 952–957.
  24. Li, H.; Li, W.X.; Ding, S.W. Induction and suppression of RNA silencing by an animal virus. Science 2002, 296, 1319–1321.
  25. Lingel, A.; Simon, B.; Izaurralde, E.; Sattler, M. The structure of the flock house virus B2 protein, a viral suppressor of RNA interference, shows a novel mode of double-stranded RNA recognition. EMBO Rep. 2005, 6, 1149–1155.
  26. Rawson, J.M.O.; Nikolaitchik, O.A.; Keele, B.F.; Pathak, V.K.; Hu, W.S. Recombination is required for efficient HIV-1 replication and the maintenance of viral genome integrity. Nucleic. Acids Res. 2018, 46, 10535–10545.
  27. Rock, J.R.; Onaitis, M.W.; Rawlins, E.L.; Lu, Y.; Clark, C.P.; Xue, Y.; Randell, S.H.; Hogan, B.L. Basal cells as stem cells of the mouse trachea and human airway epithelium. Proc. Natl. Acad. Sci. USA 2009, 106, 12771–12775.