Organoids are self‐organizing 3D structures grown from stem cells that recapitulate essential aspects of organ structure and function. Here, we describe a method to establish long‐term‐expanding ...human airway organoids from broncho‐alveolar resections or lavage material. The pseudostratified airway organoids consist of basal cells, functional multi‐ciliated cells, mucus‐producing secretory cells, and CC10‐secreting club cells. Airway organoids derived from cystic fibrosis (CF) patients allow assessment of CFTR function in an organoid swelling assay. Organoids established from lung cancer resections and metastasis biopsies retain tumor histopathology as well as cancer gene mutations and are amenable to drug screening. Respiratory syncytial virus (RSV) infection recapitulates central disease features, dramatically increases organoid cell motility via the non‐structural viral NS2 protein, and preferentially recruits neutrophils upon co‐culturing. We conclude that human airway organoids represent versatile models for the in vitro study of hereditary, malignant, and infectious pulmonary disease.
Synopsis
To date, persistent in vitro culture of adult human lung epithelium remains elusive. In this methods resource article, culture conditions to maintain three‐dimensional pulmonary tissue long‐term are reported and applied to recapitulate related diseases.
Culture conditions for long‐term expansion of healthy, hereditary disease and malignant human airway epithelial organoids.
Airway organoids are amenable for medium‐throughput drug screening.
Airway organoids readily allow modelng of viral infection.
Three‐dimensional human pulmonary tissue culture allows for investigation of hereditary diseases.
Single murine and human intestinal stem cells can be expanded in culture over long time periods as genetically and phenotypically stable epithelial organoids. Increased cAMP levels induce rapid ...swelling of such organoids by opening the cystic fibrosis transmembrane conductor receptor (CFTR). This response is lost in organoids derived from cystic fibrosis (CF) patients. Here we use the CRISPR/Cas9 genome editing system to correct the CFTR locus by homologous recombination in cultured intestinal stem cells of CF patients. The corrected allele is expressed and fully functional as measured in clonally expanded organoids. This study provides proof of concept for gene correction by homologous recombination in primary adult stem cells derived from patients with a single-gene hereditary defect.
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•The CRISPR/Cas9 system enables genome editing in intestinal stem cell organoids•cAMP-induced swelling is lost in CFTR mutant organoids of cystic fibrosis patients•CRISPR/Cas9-mediated repair of the CFTR locus restores organoid swelling
Correction of a disease-causing CFTR mutation in cultured intestinal stem cells from cystic fibrosis patients is demonstrated using the CRISPR/Cas9 system.
Combination treatment with the cystic fibrosis transmembrane conductance regulator (CFTR) modulators tezacaftor (VX-661) and ivacaftor (VX-770) was designed to target the underlying cause of disease ...in patients with cystic fibrosis.
In this phase 3, randomized, double-blind, multicenter, placebo-controlled, parallel-group trial, we evaluated combination therapy with tezacaftor and ivacaftor in patients 12 years of age or older who had cystic fibrosis and were homozygous for the CFTR Phe508del mutation. Patients were randomly assigned in a 1:1 ratio to receive either 100 mg of tezacaftor once daily and 150 mg of ivacaftor twice daily or matched placebo for 24 weeks. The primary end point was the absolute change in the percentage of the predicted forced expiratory volume in 1 second (FEV
) through week 24 (calculated in percentage points); relative change in the percentage of the predicted FEV
through week 24 (calculated as a percentage) was a key secondary end point.
Of the 510 patients who underwent randomization, 509 received tezacaftor-ivacaftor or placebo, and 475 completed 24 weeks of the trial regimen. The mean FEV
at baseline was 60.0% of the predicted value. The effects on the absolute and relative changes in the percentage of the predicted FEV
in favor of tezacaftor-ivacaftor over placebo were 4.0 percentage points and 6.8%, respectively (P<0.001 for both comparisons). The rate of pulmonary exacerbation was 35% lower in the tezacaftor-ivacaftor group than in the placebo group (P=0.005). The incidence of adverse events was similar in the two groups. Most adverse events were of mild severity (in 41.8% of patients overall) or moderate severity (in 40.9% overall), and serious adverse events were less frequent with tezacaftor-ivacaftor (12.4%) than with placebo (18.2%). A total of 2.9% of patients discontinued the assigned regimen owing to adverse events. Fewer patients in the tezacaftor-ivacaftor group than in the placebo group had respiratory adverse events, none of which led to discontinuation.
The combination of tezacaftor and ivacaftor was efficacious and safe in patients 12 years of age or older who had cystic fibrosis and were homozygous for the CFTR Phe508del mutation. (Funded by Vertex Pharmaceuticals; EVOLVE ClinicalTrials.gov number, NCT02347657 .).
In vitro drug tests using patient-derived stem cell cultures offer opportunities to individually select efficacious treatments. Here, we provide a study that demonstrates that in vitro drug responses ...in rectal organoids from individual patients with cystic fibrosis (CF) correlate with changes in two in vivo therapeutic endpoints. We measured individual in vitro efficaciousness using a functional assay in rectum-derived organoids based on forskolin-induced swelling and studied the correlation with in vivo effects. The in vitro organoid responses correlated with both change in pulmonary response and change in sweat chloride concentration. Receiver operating characteristic curves indicated good-to-excellent accuracy of the organoid-based test for defining clinical responses. This study indicates that an in vitro assay using stem cell cultures can prospectively select efficacious treatments for patients and suggests that biobanked stem cell resources can be used to tailor individual treatments in a cost-effective and patient-friendly manner.
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•Organoids of CF patients were used to quantitate individual drug response in vitro•Organoid responses correlate with two clinical response parameters ppFEV1 and SCC•In vivo (non)responders were identified with a PPV of 100% and a NPV of 80%•Organoids may be used for personalized medicine in cystic fibrosis
Berkers et al. demonstrate that stem cell cultures (organoids) can be a tool for personalized medicine. They show a high correlation between in vitro and in vivo effects of drugs and demonstrate good-to-excellent predictive values of the organoid test for preclinical identification of responders to CFTR modulators.
Background
Food allergy significantly impairs health‐related quality of life (HRQL). Currently, it is still unknown whether diagnostic interventions for food allergy improve HRQL. We aim to assess ...the impact of diagnostic interventions for food allergy on HRQL.
Methods
A systematic search was performed in MEDLINE, Embase, Cochrane Library, and CINAHL focused on patients with a (suspected) food allergy who underwent diagnostic interventions (ie, skin prick test, specific IgE, or oral food challenges OFC) and in whom HRQL was assessed. The mean difference between HRQL before and after the diagnostic intervention was calculated. A minimal clinically important difference of 0.5 was considered clinically relevant for the food allergy quality of life questionnaire.
Results
Seven of 1465 original identified publications were included in which the impact of an OFC on HRQL was investigated (total patients n = 1370). No other diagnostic interventions were investigated. Food allergy‐specific parent‐reported HRQL improved significantly after an OFC irrespective of the outcome in children with a suspected food allergy in two publications. The change was considered clinically relevant in one of two publications. In addition, parent‐reported HRQL improved after an OFC to assess the eliciting dose in children with a confirmed food allergy. The parental burden was significantly reduced after an OFC to assess resolution of food allergy. A meta‐analysis could not be performed due to the limited numbers of, and considerable heterogeneity between, eligible publications.
Conclusion
An OFC is associated with an improved food allergy‐specific HRQL and a reduced parental burden of food allergy.
A new era for people with cystic fibrosis Bierlaagh, Marlou C.; Muilwijk, Danya; Beekman, Jeffrey M. ...
European journal of pediatrics,
09/2021, Volume:
180, Issue:
9
Journal Article
Peer reviewed
Open access
Cystic fibrosis is the most prevalent inherited disease caused by a defect in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The impaired electrolyte homeostasis caused by the ...mutated or absent protein leads to symptoms in multiple organ systems. However, the pulmonary manifestation with chronic infections and eventually respiratory failure remains the most important threat. Until one decade ago, only symptomatic treatment was available. However, since 2012, different combinations of CFTR modulators are available for people with cystic fibrosis (pwCF) that carry different mutations. The advent of these drugs has impressively changed life expectancy and quality of life in people with cystic fibrosis and raised new challenges regarding long-term complications and tapering of conventional therapies.
Conclusion
: In this review, we provide an update on the latest developments around diagnostics, treatment, and prognosis of pwCF.
What is Known:
• Cystic fibrosis is an incurable and life-shortening disease asking for life-long symptomatic treatment.
• Three combination CFTR modulating drugs has gained marked approval over the last 10 years.
What is New:
• The emerge of new (modulating) therapies contribute to the increasing life expectancy.
• A high unmet need to develop new therapies for people with CF who cannot access or benefit from these drugs remains. This review gives an update on the current status.
Adenine base editing (ABE) enables enzymatic conversion from A-T into G-C base pairs. ABE holds promise for clinical application, as it does not depend on the introduction of double-strand breaks, ...contrary to conventional CRISPR/Cas9-mediated genome engineering. Here, we describe a cystic fibrosis (CF) intestinal organoid biobank, representing 664 patients, of which ~20% can theoretically be repaired by ABE. We apply SpCas9-ABE (PAM recognition sequence: NGG) and xCas9-ABE (PAM recognition sequence: NGN) on four selected CF organoid samples. Genetic and functional repair was obtained in all four cases, while whole-genome sequencing (WGS) of corrected lines of two patients did not detect off-target mutations. These observations exemplify the value of large, patient-derived organoid biobanks representing hereditary disease and indicate that ABE may be safely applied in human cells.
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•664 patients and 154 CFTR mutations represented in an organoid biobank•Adenine base editors enable efficient repair of nonsense mutations in CFTR•xCas9 increases the target scope of CFTR repair in our biobank•Adenine base editors cause no detectable off-target effects during repair
Here, we show the generation of an extensive cystic fibrosis patient-derived intestinal organoid biobank. We use this biobank to study gene correction by adenine base editors and show genetic repair of four selected nonsense mutations in CFTR without any genome-wide off-target effects on canonical and non-canonical PAMs.
We recently established conditions allowing for long-term expansion of epithelial organoids from intestine, recapitulating essential features of the in vivo tissue architecture. Here we apply this ...technology to study primary intestinal organoids of people suffering from cystic fibrosis, a disease caused by mutations in CFTR, encoding cystic fibrosis transmembrane conductance regulator. Forskolin induces rapid swelling of organoids derived from healthy controls or wild-type mice, but this effect is strongly reduced in organoids of subjects with cystic fibrosis or in mice carrying the Cftr F508del mutation and is absent in Cftr-deficient organoids. This pattern is phenocopied by CFTR-specific inhibitors. Forskolin-induced swelling of in vitro-expanded human control and cystic fibrosis organoids corresponds quantitatively with forskolin-induced anion currents in freshly excised ex vivo rectal biopsies. Function of the CFTR F508del mutant protein is restored by incubation at low temperature, as well as by CFTR-restoring compounds. This relatively simple and robust assay will facilitate diagnosis, functional studies, drug development and personalized medicine approaches in cystic fibrosis.
Introduction
The simultaneously increased prevalence of atopic diseases and decreased prevalence of infectious diseases might point to a link between the two entities. Past work mainly focused on ...either atopic diseases or recurrent infections. We aim to investigate whether risk factors for atopic diseases (ie, asthma, allergic rhinitis, atopic dermatitis, and/or food allergy) differ from risk factors for recurrent respiratory tract infections (RRTIs) in children.
Methods
Cross‐sectional data were used from 5517 children aged 1 to 18 years who participated in an Electronic Portal for children between 2011 and 2019. Univariable/multivariable logistic regression analyses were performed to determine risk factors for any atopic disease and RRTIs.
Results
Children aged ≥5 years were more likely to have any atopic disease (adjusted odds ratio OR: 1.50‐2.77) and less likely to have RRTIs (OR: 0.68‐0.84) compared to children aged less than 5 years. Female sex (OR: 0.72; 95% confidence interval CI: 0.63‐0.81), low birth weight (OR: 0.74; 95% CI: 0.57‐0.97) and dog ownership (OR: 0.79; 95% CI: 0.66‐0.95) reduced the odds of any atopic disease, but not of RRTIs. Daycare attendance (OR: 1.22; 95% CI: 1.02‐1.47) was associated with RRTIs, but not with atopic diseases. A family history of asthma, allergic rhinitis, atopic dermatitis, and RRTIs was significantly associated with the same entity in children, with OR varying from 1.58 (95% CI: 1.35‐1.85) in allergic rhinitis to 2.20 (95% CI: 1.85‐2.61) in asthma.
Conclusion
Risk factors for atopic diseases are distinct from risk factors for RRTIs, suggesting that the changing prevalence of both entities is not related to shared risk factors.
Approximately 10% of all pathological mutations are nonsense mutations which are responsible for the most severe cases of genetic diseases but for which no treatment regimens are available.The main ...strategy for treating nonsense mutations is by enhancing ribosomal readthrough of premature stop codons through the action of readthrough compounds, thus restoring production of full-length protein.Most preclinical studies on readthrough compounds have yielded positive results, but have been performed in reporter-based assays despite recent scientific developments that have significantly improved disease models, such as patient-derived cells in functional assays.Clinical trials have yielded disappointing results, and further interpretation of readthrough compound efficacy across clinical trials is challenging because of differences in research design, treatment length, and clinical endpoints.Characterization of factors that contribute to the translational gap between readthrough compounds in preclinical studies versus clinical trial results is necessary to make sense of nonsense mutation therapy.
Approximately 10% of all pathological mutations are nonsense mutations that are responsible for several severe genetic diseases for which no treatment regimens are currently available. The most widespread strategy for treating nonsense mutations is by enhancing ribosomal readthrough of premature termination codons (PTCs) to restore the production of the full-length protein. In the past decade several compounds with readthrough potential have been identified. However, although preclinical results on these compounds are promising, clinical studies have not yielded positive outcomes. We review preclinical and clinical research related to readthrough compounds and characterize factors that contribute to the observed translational gap.
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