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  1. Article ; Online: Genomic characterization and therapeutic utilization of IL-13-responsive sequences in asthma

    Kyung Duk Koh / Luke R. Bonser / Walter L. Eckalbar / Ofer Yizhar-Barnea / Jiangshan Shen / Xiaoning Zeng / Kirsten L. Hargett / Dingyuan I. Sun / Lorna T. Zlock / Walter E. Finkbeiner / Nadav Ahituv / David J. Erle

    Cell Genomics, Vol 3, Iss 1, Pp 100229- (2023)

    2023  

    Abstract: Summary: Epithelial responses to the cytokine interleukin-13 (IL-13) cause airway obstruction in asthma. Here we utilized multiple genomic techniques to identify IL-13-responsive regulatory elements in bronchial epithelial cells and used these data to ... ...

    Abstract Summary: Epithelial responses to the cytokine interleukin-13 (IL-13) cause airway obstruction in asthma. Here we utilized multiple genomic techniques to identify IL-13-responsive regulatory elements in bronchial epithelial cells and used these data to develop a CRISPR interference (CRISPRi)-based therapeutic approach to downregulate airway obstruction-inducing genes in a cell type- and IL-13-specific manner. Using single-cell RNA sequencing (scRNA-seq) and acetylated lysine 27 on histone 3 (H3K27ac) chromatin immunoprecipitation sequencing (ChIP-seq) in primary human bronchial epithelial cells, we identified IL-13-responsive genes and regulatory elements. These sequences were functionally validated and optimized via massively parallel reporter assays (MPRAs) for IL-13-inducible activity. The top secretory cell-selective sequence from the MPRA, a novel, distal enhancer of the sterile alpha motif pointed domain containing E-26 transformation-specific transcription factor (SPDEF) gene, was utilized to drive CRISPRi and knock down SPDEF or mucin 5AC (MUC5AC), both involved in pathologic mucus production in asthma. Our work provides a catalog of cell type-specific genes and regulatory elements involved in IL-13 bronchial epithelial response and showcases their use for therapeutic purposes.
    Keywords enhancer ; IL-13 ; cell-specific ; CRISPRi ; HBEC ; SPDEF ; Genetics ; QH426-470 ; Internal medicine ; RC31-1245
    Subject code 572
    Language English
    Publishing date 2023-01-01T00:00:00Z
    Publisher Elsevier
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  2. Article ; Online: Nanomolar-potency ‘co-potentiator’ therapy for cystic fibrosis caused by a defined subset of minimal function CFTR mutants

    Puay-Wah Phuan / Joseph-Anthony Tan / Amber A. Rivera / Lorna Zlock / Dennis W. Nielson / Walter E. Finkbeiner / Peter M. Haggie / Alan S. Verkman

    Scientific Reports, Vol 9, Iss 1, Pp 1-

    2019  Volume 12

    Abstract: Abstract Available CFTR modulators provide no therapeutic benefit for cystic fibrosis (CF) caused by many loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, including N1303K. We previously ... ...

    Abstract Abstract Available CFTR modulators provide no therapeutic benefit for cystic fibrosis (CF) caused by many loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, including N1303K. We previously introduced the concept of ‘co-potentiators’ (combination-potentiators) to rescue CFTR function in some minimal function CFTR mutants. Herein, a screen of ~120,000 drug-like synthetic small molecules identified active co-potentiators of pyrazoloquinoline, piperidine-pyridoindole, tetrahydroquinoline and phenylazepine classes, with EC50 down to ~300 nM following initial structure-activity studies. Increased CFTR chloride conductance by up to 8-fold was observed when a co-potentiator (termed ‘Class II potentiator’) was used with a classical potentiator (‘Class I potentiator’) such as VX-770 or GLPG1837. To investigate the range of CFTR mutations benefitted by co-potentiators, 14 CF-associated CFTR mutations were studied in transfected cell models. Co-potentiator efficacy was found for CFTR missense, deletion and nonsense mutations in nucleotide binding domain-2 (NBD2), including W1282X, N1303K, c.3700A > G and Q1313X (with corrector for some mutations). In contrast, CFTR mutations G85E, R334W, R347P, V520F, R560T, A561E, M1101K and R1162X showed no co-potentiator activity, even with corrector. Co-potentiator efficacy was confirmed in primary human bronchial epithelial cell cultures generated from a N1303K homozygous CF subject. The Class II potentiators identified here may have clinical benefit for CF caused by mutations in the NBD2 domain of CFTR.
    Keywords Medicine ; R ; Science ; Q
    Language English
    Publishing date 2019-11-01T00:00:00Z
    Publisher Nature Publishing Group
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  3. Article ; Online: Epithelial miR-141 regulates IL-13–induced airway mucus production

    Sana Siddiqui / Kristina Johansson / Alex Joo / Luke R. Bonser / Kyung Duk Koh / Olivier Le Tonqueze / Samaneh Bolourchi / Rodriel A. Bautista / Lorna Zlock / Theodore L. Roth / Alexander Marson / Nirav R. Bhakta / K. Mark Ansel / Walter E. Finkbeiner / David J. Erle / Prescott G. Woodruff

    JCI Insight, Vol 6, Iss

    2021  Volume 5

    Abstract: IL-13–induced goblet cell metaplasia contributes to airway remodeling and pathological mucus hypersecretion in asthma. miRNAs are potent modulators of cellular responses, but their role in mucus regulation is largely unexplored. We hypothesized that ... ...

    Abstract IL-13–induced goblet cell metaplasia contributes to airway remodeling and pathological mucus hypersecretion in asthma. miRNAs are potent modulators of cellular responses, but their role in mucus regulation is largely unexplored. We hypothesized that airway epithelial miRNAs play roles in IL-13–induced mucus regulation. miR-141 is highly expressed in human and mouse airway epithelium, is altered in bronchial brushings from asthmatic subjects at baseline, and is induced shortly after airway allergen exposure. We established a CRISPR/Cas9-based protocol to target miR-141 in primary human bronchial epithelial cells that were differentiated at air-liquid-interface, and goblet cell hyperplasia was induced by IL-13 stimulation. miR-141 disruption resulted in decreased goblet cell frequency, intracellular MUC5AC, and total secreted mucus. These effects correlated with a reduction in a goblet cell gene expression signature and enrichment of a basal cell gene expression signature defined by single cell RNA sequencing. Furthermore, intranasal administration of a sequence-specific mmu-miR-141-3p inhibitor in mice decreased Aspergillus-induced secreted mucus and mucus-producing cells in the lung and reduced airway hyperresponsiveness without affecting cellular inflammation. In conclusion, we have identified a miRNA that regulates pathological airway mucus production and is amenable to therapeutic manipulation through an inhaled route.
    Keywords Pulmonology ; Medicine ; R
    Language English
    Publishing date 2021-03-01T00:00:00Z
    Publisher American Society for Clinical investigation
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  4. Article ; Online: Expansion of cultures of human tracheal epithelium with maintenance of differentiated structure and function

    Jonathan H. Widdicombe / Lorne A. Sachs / Joby L. Morrow / Walter E. Finkbeiner

    BioTechniques, Vol 39, Iss 2, Pp 249-

    2005  Volume 255

    Abstract: We have developed a technique for expanding primary cultures of human tracheal epithelium while minimizing loss of differentiated structure and function. Cells were seeded at 2 × 104 cells/cm2 into T75 flasks and trypsinized when approximately 80% ... ...

    Abstract We have developed a technique for expanding primary cultures of human tracheal epithelium while minimizing loss of differentiated structure and function. Cells were seeded at 2 × 104 cells/cm2 into T75 flasks and trypsinized when approximately 80% confluent. The dispersed cells were then passaged at the same plating density into further T75 flasks or seeded at 5 × 105 cells/cm2 on porous-bottomed inserts and maintained with an air-interface. Differentiation of cells on inserts was assessed from transepithelial electrical resistance (an index of tight junction formation), short-circuit current (an index of transepithelial salt transport), cell numbers, total cell protein, and histology. Unpassaged cells (P0) and cells passaged once (P1) took about a week to become 80% confluent on T75 flasks, with 10-fold and 5-fold increases in cell numbers, respectively. Confluence was achieved in approximately 3 days following plating to inserts. Functionally and structurally, P1 and P2 cells (cells passaged twice) were little different from P0 cells. Thus, within a little over 2 weeks, the numbers of confluent cell sheets can be increased 50-fold with minimal change in function. However, there was a marked decline in differentiation by cells passaged three times (P3), and not all cell preparations could be taken to P4 (cells passaged four times).
    Keywords Biology (General) ; QH301-705.5
    Subject code 610 ; 571
    Language English
    Publishing date 2005-08-01T00:00:00Z
    Publisher Future Science Ltd
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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