Journal: Life Science Alliance

Article Title: Validating organoid-derived human intestinal monolayers for personalized therapy in cystic fibrosis

doi: 10.26508/lsa.202201857

Figure Lengend Snippet: (A, B, C) Immunohistochemical imaging of 2D HIO 4 d post-seeding from a non-CF subject in en face (A, B) and cross section view (C). Immunohistochemical stains demonstrate a tight epithelial layer (ZO1 stained for epithelial tight junction), with polarity (ezrin stain for the apical cell membrane) and apical localized CFTR channels. (D) The bar graph shows the comparison of CFTR mRNA expression levels between 2D HIO and HNE from a non-CF subject and an F508del/F508del CF patient. CFTR mRNA expression was normalized to18S rRNA. The normalized mRNA levels are shown as fold levels after setting the HNE mRNA levels of one sample arbitrarily at 1. Mean ± SD were obtained (two biologic replicates for HNE, triplicates for 2D HIO). Statistical comparison: two-sided unpaired t test, * P < 0.05, ** P < 0.01. There was no statistical difference between 2D HIO CFTR mRNA levels between non-CF versus F508del/F508del CF (Tukey’s multiple comparison test, P = 0.11). (E) Transepithelial electrophysiological properties of 2D HIO and HNE measured in one non-CF subject (three technical replicates) and 2 F508del/F508del CF patients (two technical replicates each). Amil-amiloride (100 µM for HIO; 30 µM for HNE), Fsk-Forskolin (10 µM), CFTRinh-CFTR Inh-172 (10 µM). Statistical analysis by one way ANOVA: Tukey’s multiple comparison test, * P < 0.05, ** P < 0.005, **** P < 0.0001. (F) Original traces show transepithelial current traces of the Fsk dose–response experiments assessing CFTR responses to increasing Fsk concentration (0.02–10 μM) and CFTRinh in 2D HIO and HNE from one non-CF subject (three technical replicates) and one F508del/F508del patient after chronic treatment with VX-661 (3 µM) + VX-445 (3 µM) and acute treatment with VX-770 (1 µM), experiments were done in two technical replicates. (G, H) Graphs compare the Fsk dose–response curves generated in non-CF and VX-661+ VX-445+ VX-770-treated F508del/F508del cultures between 2D HIO and HNE cells. Statistical test: Non-linear regression fit of the two dose–response curve and use of extra sum-of-squares F test to determine the statistical difference between the two curves, *** P < 0.0001.

Article Snippet: Furthermore, we used CFTR modulators from Galapagos/AbbVie and from Proteostasis Therapeutics, now governed under FAIR therapeutics, which have been tested preclinically and/or clinically in phase I, II, III clinical trials ( https://www.cff.org/Trials/Pipeline ) ( ; ; ). lists the CFTR modulator drugs used in this study.

Techniques: Immunohistochemical staining, Imaging, Staining, Membrane, Comparison, Expressing, Concentration Assay, Generated

Journal: Life Science Alliance

Article Title: Validating organoid-derived human intestinal monolayers for personalized therapy in cystic fibrosis

doi: 10.26508/lsa.202201857

Figure Lengend Snippet: Representative original Ieq traces from Ussing chamber studies generated from 2D HIO monolayers from a non-CF subject, and a patient with F508del/F508del at baseline and after the treatment of the monolayer with VX-661+VX-445+VX-770. Amiloride (Amil, 100 µM), forskolin (Fsk, 10 µM), ivacaftor (VX-770, 1 µM), CFTR Inh-172 (CFTRinh, 10 µM). The summary of these recordings is presented in .

Article Snippet: Furthermore, we used CFTR modulators from Galapagos/AbbVie and from Proteostasis Therapeutics, now governed under FAIR therapeutics, which have been tested preclinically and/or clinically in phase I, II, III clinical trials ( https://www.cff.org/Trials/Pipeline ) ( ; ; ). lists the CFTR modulator drugs used in this study.

Techniques: Generated

Journal: Life Science Alliance

Article Title: Validating organoid-derived human intestinal monolayers for personalized therapy in cystic fibrosis

doi: 10.26508/lsa.202201857

Figure Lengend Snippet: (A, B) Graphs display Fsk dose–response curves assessing CFTR responses to increasing Fsk concentration (0.02–10 μM) performed in 2D HIO and 3D HIO in (A): a non-CF subject (three technical replicates for 2D HIO and four technical replicates for 3D HIO); (B): 2 F508del/F508del patients after chronic treatment with VX-661 (3 µM) + VX-445 (3 µM) +acute treatment with VX-770 (1 µM for 2D HIO, 3 µM for 3D HIO), (two technical replicates for 2D HIO and four technical replicates for 3D HIO for each patient). Statistical test: Non-linear regression fit of the two dose–response curve and the use of extra sum-of-squares F test to determine the statistical difference between the two curves, *** P < 0.0001.

Article Snippet: Furthermore, we used CFTR modulators from Galapagos/AbbVie and from Proteostasis Therapeutics, now governed under FAIR therapeutics, which have been tested preclinically and/or clinically in phase I, II, III clinical trials ( https://www.cff.org/Trials/Pipeline ) ( ; ; ). lists the CFTR modulator drugs used in this study.

Techniques: Concentration Assay

Journal: Life Science Alliance

Article Title: Validating organoid-derived human intestinal monolayers for personalized therapy in cystic fibrosis

doi: 10.26508/lsa.202201857

Figure Lengend Snippet: (A) Graphs show representative original transepithelial current (Ieq) traces from Ussing chamber studies on 2D HIO and HNE and the change of the area under the curve (FIS AUC) over 60 min in 3D HIO derived from CF patients with W1282X/W1282X , N1303K/N1303K , F508del/F508del , and G551D/F508del in response to various CFTR modulator drug treatments. Cultures were incubated with corrector drugs 18–24 h before Ussing experiments, and the potentiator drugs (pot) were applied before forskolin (Fsk) addition. Fsk - 10 µM/basolateral for 2D HIO and HNE, 5 µM for 3D HIO FIS; CFTRinh-172 - 10 µM/apical; amiloride (amil) - 100 µM/apical for 2D HIO, 30 µM/apical for HNE. Potentiators were VX-770, PTI-808, and AP2. (B) Bar graphs summarize assessed CFTR function after the different CFTR modulator treatments comparing the assessment in 2D HIO, HNE, and 3D HIO. Each bar represents one single patient with two technical replicates for class I and III patients and four technical replicates for class II patients. For tissue cultures from patients with class 1 mutations, G418 (read-through agent, 200 µg/ml) and SMGi (inhibitor of nonsense-mediated decay, 0.5 μM) were added to the corrector drugs 18–24 h before assessment. Note: HNE cell cultures of one patient with F508del/F508del failed the re-culturing process repeatedly after a freeze–thaw cycle leading to missing experimental data.

Article Snippet: Furthermore, we used CFTR modulators from Galapagos/AbbVie and from Proteostasis Therapeutics, now governed under FAIR therapeutics, which have been tested preclinically and/or clinically in phase I, II, III clinical trials ( https://www.cff.org/Trials/Pipeline ) ( ; ; ). lists the CFTR modulator drugs used in this study.

Techniques: Derivative Assay, Incubation

Journal: Life Science Alliance

Article Title: Validating organoid-derived human intestinal monolayers for personalized therapy in cystic fibrosis

doi: 10.26508/lsa.202201857

Figure Lengend Snippet: Representative original transepithelial current (Ieq) traces from Ussing chamber studies on 2D HIO and HNE (Fsk, 10 µM/basolateral; CFTRinh-172, 10 µM/apical; amiloride 100 µM for 2D HIO, 30 µM for HNE), and the change of the area under the curve (FIS AUC) over 60 min in 3D HIO derived from the additional CF patients in response to various CFTR modulator drug treatments. Summary graphs are presented in . All corrector drugs were applied for 18–24 h before the experiments, all potentiator drugs were applied acutely following addition of Fsk addition. For tissue cultures from patients with class 1 mutations G418 (read-through agent, 200 µg/ml) and SMGi (inhibitor of nonsense-mediated decay, 0.5 µM) were added to all triple drug combinations.

Article Snippet: Furthermore, we used CFTR modulators from Galapagos/AbbVie and from Proteostasis Therapeutics, now governed under FAIR therapeutics, which have been tested preclinically and/or clinically in phase I, II, III clinical trials ( https://www.cff.org/Trials/Pipeline ) ( ; ; ). lists the CFTR modulator drugs used in this study.

Techniques: Derivative Assay

Journal: Life Science Alliance

Article Title: Validating organoid-derived human intestinal monolayers for personalized therapy in cystic fibrosis

doi: 10.26508/lsa.202201857

Figure Lengend Snippet: (A) Representative original recordings are presented as Ieq traces generated from fresh rectal tissue specimen which were mounted in the Ussing chamber to perform functional studies from pwCF with class I, II, III CFTR mutations (intestinal current measurement studies). *Rectal tissue specimens from patients with CF in class III mutations were obtained after starting treatment with ivacaftor. (B) Summary bar graph of the forskolin/IBMX (3-isobutyl-1-methylxanthine) stimulated change in Ieq. Two to three technical replicates were performed per patient. (C) Graph shows good correlation between the CFTR function measured as Fsk-induced change in Ieq (Fsk-Ieq) in freshly excised rectal tissue specimen and Fsk-Ieq measured in 2D HIO from the same patient.

Article Snippet: Furthermore, we used CFTR modulators from Galapagos/AbbVie and from Proteostasis Therapeutics, now governed under FAIR therapeutics, which have been tested preclinically and/or clinically in phase I, II, III clinical trials ( https://www.cff.org/Trials/Pipeline ) ( ; ; ). lists the CFTR modulator drugs used in this study.

Techniques: Generated, Functional Assay

Journal: Life Science Alliance

Article Title: Validating organoid-derived human intestinal monolayers for personalized therapy in cystic fibrosis

doi: 10.26508/lsa.202201857

Figure Lengend Snippet: (A) Original Ieq traces in 2D HIO from a N1303K/N1303K patient. CFTR modulator drugs were applied acutely at the time of Fsk (10 µM) stimulation to the apical side: VX-770 (1 µM), VX-445 (3 µM). CFTR Inh-172 -Ieq (CFTRinh, 10 µM) was added to confirm CFTR activity; chronic treatment with VX-661 (3 µM) and VX-445 (3 µM) was done over 24 h. Experiments were done in the presence of 100 µM amiloride. (B) Graphs summarize the drug-induced change in Fsk-Ieq and CFTRinh-Ieq after (1) acute addition of CFTR modulators apically after Fsk-induced CFTR activation: acuteVX-770 (aVX-770), acute VX-445 (aVX-445), (2) combination of aVX-770 and aVX-445 in different sequence of addition, and treatment with chronic incubation (18–24 h) of VX-770 (cVX-770)+ VX-445 (cVX-445), (3) triple drug combination chronic incubation of VX-661 and VX-445 with acute addition of VX-770 (cVX-661+cVX-445 +aVX-770). Results were obtained from two patients homozygous for the N1303K variant. Data are means ± SD, statistical analysis: Analysis of variance with Tukey’s multiple comparison testing, * P < 0.05, ** P < 0.005, *** P < 0.0005.

Article Snippet: Furthermore, we used CFTR modulators from Galapagos/AbbVie and from Proteostasis Therapeutics, now governed under FAIR therapeutics, which have been tested preclinically and/or clinically in phase I, II, III clinical trials ( https://www.cff.org/Trials/Pipeline ) ( ; ; ). lists the CFTR modulator drugs used in this study.

Techniques: Activity Assay, Activation Assay, Sequencing, Incubation, Variant Assay, Comparison

Journal: Life Science Alliance

Article Title: Validating organoid-derived human intestinal monolayers for personalized therapy in cystic fibrosis

doi: 10.26508/lsa.202201857

Figure Lengend Snippet: (A, B, C, D) Graphs show the correlation between the in vitro measured change in CFTR function after treatment with CFTR modulator drugs (VX-770 or IVA and VX-880 + VX-770 or LUMA/IVA) in 2D HIO (A, B) and in 3D HIO (C, D) on one side and clinical drug efficacy assessed as change in sweat chloride (Sweat Cl). (A, B, C, D) and change in FEV1%pred on the other (B, D). Fsk concentration for the 2D HIO experiments was 10 µM and 5 µM for the 3D FIS. Changes in the clinical markers are expressed as the difference between an assessment at baseline and 6 mo post treatment start. Each dot represents one pwCF. Open circles represent pwCF carrying at least one G551D allele ( G551D/F508del, G551D/3272-26A>G, G551D/5T ), open square represents one patient with G178R/F508del who started treatment with ivacaftor; closed circles represent pwCF carrying F508del/F508del alleles. This cohort contained 6 pwCF, however, post-treatment sweat chloride measurements were only available for 5 pwCF.

Article Snippet: Furthermore, we used CFTR modulators from Galapagos/AbbVie and from Proteostasis Therapeutics, now governed under FAIR therapeutics, which have been tested preclinically and/or clinically in phase I, II, III clinical trials ( https://www.cff.org/Trials/Pipeline ) ( ; ; ). lists the CFTR modulator drugs used in this study.

Techniques: In Vitro, Concentration Assay

Journal: Life Science Alliance

Article Title: Validating organoid-derived human intestinal monolayers for personalized therapy in cystic fibrosis

doi: 10.26508/lsa.202201857

Figure Lengend Snippet: CFTR modifying drugs.

Article Snippet: Furthermore, we used CFTR modulators from Galapagos/AbbVie and from Proteostasis Therapeutics, now governed under FAIR therapeutics, which have been tested preclinically and/or clinically in phase I, II, III clinical trials ( https://www.cff.org/Trials/Pipeline ) ( ; ; ). lists the CFTR modulator drugs used in this study.

Techniques:

Journal: Therapeutic Advances in Respiratory Disease

Article Title: The combination of tezacaftor and ivacaftor in the treatment of patients with cystic fibrosis: clinical evidence and future prospects in cystic fibrosis therapy

doi: 10.1177/1753466619844424

Figure Lengend Snippet: Brief description of referenced CFTR protein modulator studies.

Article Snippet: This year, the Institute for Clinical and Economic Review (ICER; an independent, nonprofit research institute) evaluated the addition of CFTR protein modulators to standard CF care.

Techniques: Mutagenesis, Control, Drug discovery

Journal: Therapeutic Advances in Respiratory Disease

Article Title: The combination of tezacaftor and ivacaftor in the treatment of patients with cystic fibrosis: clinical evidence and future prospects in cystic fibrosis therapy

doi: 10.1177/1753466619844424

Figure Lengend Snippet: CFTR mutations beyond F508del homozygous approved for tezacaftor/ivacaftor use. *

Article Snippet: This year, the Institute for Clinical and Economic Review (ICER; an independent, nonprofit research institute) evaluated the addition of CFTR protein modulators to standard CF care.

Techniques: Mutagenesis

Journal: ERJ Open Research

Article Title: BOS-318 treatment enhances elexacaftor–tezacaftor–ivacaftor-mediated improvements in airway hydration and mucociliary transport

doi: 10.1183/23120541.00445-2024

Figure Lengend Snippet: Effect of long-term BOS-318 treatment in combination with ETI on ion channel function. Cystic fibrosis human bronchial epithelial cells were treated with either vehicle or BOS-318 in the presence or absence of elexacaftor–tezacaftor–ivacaftor (ETI) for 48 h. After the treatment period, baseline I eq was established, followed by the sequential addition of 10 µM amiloride, 20 µM forskolin, 20 µM CFTRinh-172 and 100 µM UTP. (a) Representative equivalent current (I eq ) recordings. (b) CFTRinh-172-sensitive I eq representing CFTR activity. (c) Amiloride-sensitive I eq , representing epithelial sodium channel (ENaC) activity. n≥5 individual inserts from three donors. Statistical analyses were performed using a Kruskal–Wallis test and Dunn's multiple comparisons test in comparison to the relevant dimethylsulfoxide control. *: p≤0.05; **: p≤0.01. CFTR: cystic fibrosis transmembrane conductance regulator.

Article Snippet: CFTR modulators (VX-661 and VX-770) were from Adooq Biosciences (Irvine, CA, USA).

Techniques: Activity Assay, Comparison, Control

Journal: ERJ Open Research

Article Title: BOS-318 treatment enhances elexacaftor–tezacaftor–ivacaftor-mediated improvements in airway hydration and mucociliary transport

doi: 10.1183/23120541.00445-2024

Figure Lengend Snippet: BOS-318 treatment enhances ETI-mediated improvements in MCT. Cystic fibrosis human bronchial epithelial cells were treated with vehicle, BOS-318, elexacaftor–tezacaftor–ivacaftor (ETI) or a combination of ETI and BOS-318, in the presence of 30 nM vasoactive intestinal peptide, for 48 h. The mucociliary transport (MCT) rate was determined by tracking fluorescently labelled microspheres on the apical surface of the cells. (a) MCT rate shown as μm·s −1 and (b) MCT rate shown as a % of triple CFTR modulator therapy rate. n=7 individual inserts from four cystic fibrosis donors. Statistical analyses were performed using a Mann–Whitney U-test. **: p≤0.01. CFTR: CF transmembrane conductance regulator; DMSO: dimethylsulfoxide.

Article Snippet: CFTR modulators (VX-661 and VX-770) were from Adooq Biosciences (Irvine, CA, USA).

Techniques: MANN-WHITNEY