Functional tests for assessment of residual CFTR channel activity and personalized selection of efficacious CFTR-modulators for cystic fibrosis patients with ‘mild’ and ‘severe’ genetic variants

2021 ◽  
Vol 31 (2) ◽  
pp. 167-177
Author(s):  
E. L. Amelina ◽  
A. S. Efremova ◽  
Yu. L. Melyanovskaya ◽  
N. V. Bulatenko ◽  
T. B. Bukharova ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Clinical Data ◽  
Genetic Variant ◽  
Functional Tests ◽  
Channel Activity ◽  
Intestinal Organoids ◽  
Cftr Mutations ◽  
Rectal Biopsies ◽  
Cftr Modulators

Intestinal current measurement (ICM) and forskolin-induced swelling (FIS) assay in human intestinal organoids from rectal biopsies of cystic fibrosis (CF) patients are the new functional tests for assessment of CFTR channel activity that are widely used in the leading laboratories worldwide for scientific and clinical studies.The aim of the study was to assess the use of the new functional tests in adult CF patients with identified N1303K and R334W CFTR gene variants.Methods. Rectal suction biopsies were obtained from the two CF patients aged 36 and 27 years with N1303K/3821delT and R334W/F508del CFTR mutations, respectively. Results of the ICM and FIS assay in intestinal organoids were compared to the clinical data.Results. ICM has demonstrated that R334W is a ‘mild’ genetic variant with high residual CFTR channel activity. At the same time, N1303K is a ‘severe’ genetic variant and leads to a severe loss of CFTR channel function. These findings correlate with the clinical data. CFTR modulators compensate for the reduced activity of the R334W CFTR variant, as shown by the FIS assay. But there was a limited response of the forskolin-stimulated organoids to VX-770 potentiator and VX-809 corrector in the cells with N1303K genetic variant.Conclusion. ICM and FIS assay in human intestinal organoids are reliable methods for quantification of CFTR channel activity. They can also predict the efficacy of the targeted therapy in CF patients in vivo.

scholarly journals Correction of CFTR function in intestinal organoids to guide treatment of cystic fibrosis

2020 ◽  
Vol 57 (1) ◽  
pp. 1902426 ◽  
Author(s):  
Anabela S. Ramalho ◽  
Eva Fürstová ◽  
Annelotte M. Vonk ◽  
Marc Ferrante ◽  
Catherine Verfaillie ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Clinical Data ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Vast Number ◽  
Sweat Chloride ◽  
Intestinal Organoids ◽  
Rectal Biopsies ◽  
Cftr Modulators

RationaleGiven the vast number of cystic fibrosis transmembrane conductance regulator (CFTR) mutations, biomarkers predicting benefit from CFTR modulator therapies are needed for subjects with cystic fibrosis (CF).ObjectivesTo study CFTR function in organoids of subjects with common and rare CFTR mutations and evaluate correlations between CFTR function and clinical data.MethodsIntestinal organoids were grown from rectal biopsies in a cohort of 97 subjects with CF. Residual CFTR function was measured by quantifying organoid swelling induced by forskolin and response to modulators by quantifying organoid swelling induced by CFTR correctors, potentiator and their combination. Organoid data were correlated with clinical data from the literature.ResultsAcross 28 genotypes, residual CFTR function correlated (r2=0.87) with sweat chloride values. When studying the same genotypes, CFTR function rescue by CFTR modulators in organoids correlated tightly with mean improvement in lung function (r2=0.90) and sweat chloride (r2=0.95) reported in clinical trials. We identified candidate genotypes for modulator therapy, such as E92K, Q237E, R334W and L159S. Based on organoid results, two subjects started modulator treatment: one homozygous for complex allele Q359K_T360K, and the second with mutation E60K. Both subjects had major clinical benefit.ConclusionsMeasurements of residual CFTR function and rescue of function by CFTR modulators in intestinal organoids correlate closely with clinical data. Our results for reference genotypes concur with previous results. CFTR function measured in organoids can be used to guide precision medicine in patients with CF, positioning organoids as a potential in vitro model to bring treatment to patients carrying rare CFTR mutations.

Evaluation of CFTR Channel Functions and Responses to Modulators in Patients with Cystic Fibrosis who Have a Pathogenic F508del Variant in Their Genomes

2020 ◽  
Vol 36 (4) ◽  
pp. 69-73
Author(s):  
A.S. Efremova ◽  
Т.В. Bukharova ◽  
N.V. Petrova ◽  
N.Yu. Kashirskaya ◽  
Yu.L. Melyanovskaya ◽  
...  
Keyword(s):  
Higher Education ◽  
Cystic Fibrosis ◽  
Functional Activity ◽  
Channel Function ◽  
Intestinal Organoids ◽  
Highly Sensitive ◽  
Variant Genotype ◽  
Healthy Control ◽  
Rectal Biopsies ◽  
Cftr Modulators

Intestinal organoids derived from rectal biopsies of cystic fibrosis patients are a highly sensitive personalized method for evaluating the functional activity of the CFTR channel and the efficacy of target drugs. We examined four patients whose genotype contains the pathogenic F508del variant (three patients and one healthy heterozygous person). It was shown that F508del/F508del genotype (two patients) could be effectively corrected by CFTR modulators (VX-770 and VX-809). The modulators had no significant effect on the restoration of the chlorine CFTR channel function in a patient with the W361X/F508del genotype. The CFTR functional activity in the heterozygous F508del carrier was as high as in the healthy control. cystic fibrosis, intestinal organoids, genetic F508del variant, genotype, forskolin-induced swelling (FIS) assay, CFTR modulators This study was supported by the Ministry of Science and Higher Education as part of a State Assignment for RCMG.

scholarly journals Personalized Medicine Based on Nasal Epithelial Cells: Comparative Studies with Rectal Biopsies and Intestinal Organoids

2021 ◽  
Vol 11 (5) ◽  
pp. 421
Author(s):  
Iris A. L. Silva ◽  
Violeta Railean ◽  
Aires Duarte ◽  
Margarida D. Amaral
Keyword(s):  
Clinical Trials ◽  
Personalized Medicine ◽  
Unmet Need ◽  
Rare Mutations ◽  
Intestinal Organoids ◽  
Bona Fide ◽  
The Individual ◽  
Rectal Biopsies ◽  
Effective Drugs ◽  
Cftr Modulators

As highly effective CFTR modulator therapies (HEMT) emerge, there is an unmet need to find effective drugs for people with CF (PwCF) with ultra-rare mutations who are too few for classical clinical trials and for whom there are no drug discovery programs. Therefore, biomarkers reliably predicting the benefit from CFTR modulator therapies are essential to find effective drugs for PwCF through personalized approaches termed theranostics. Here, we assess CFTR basal function and the individual responses to CFTR modulators in primary human nasal epithelial (pHNE) cells from PwCF carrying rare mutations and compare these measurements with those in native rectal biopsies and intestinal organoids, respectively, in the same individual. The basal function in pHNEs shows good correlation with CFTR basal function in rectal biopsies. In parallel, CFTR rescue in pHNEs by CFTR modulators correlates to that in intestinal organoids. Altogether, results show that pHNEs are a bona fide theranostic model to assess CFTR rescue by CFTR modulator drugs, in particular for PwCF and rare mutations.

scholarly journals Rare Trafficking CFTR Mutations Involve Distinct Cellular Retention Machineries and Require Different Rescuing Strategies

2021 ◽  
Vol 23 (1) ◽  
pp. 24
Author(s):  
Sofia S. Ramalho ◽  
Iris A. L. Silva ◽  
Margarida D. Amaral ◽  
Carlos M. Farinha
Keyword(s):  
Cystic Fibrosis ◽  
Drug Discovery ◽  
Cell Lines ◽  
Class Ii ◽  
Molecular Defects ◽  
Knock Down ◽  
Intestinal Organoids ◽  
Cftr Mutations ◽  
Cellular Machinery

Most of the ~2100 CFTR variants so far reported are very rare and still uncharacterized regarding their cystic fibrosis (CF) disease liability. Since some may respond to currently approved modulators, characterizing their defect and response to these drugs is essential. Here we aimed characterizing the defect associated with four rare missense (likely Class II) CFTR variants and assess their rescue by corrector drugs. We produced CFBE cell lines stably expressing CFTR with W57G, R560S, H1079P and Q1100P, assessed their effect upon CFTR expression and maturation and their rescue by VX-661/VX-445 correctors. Results were validated by forskolin-induced swelling assay (FIS) using intestinal organoids from individuals bearing these variants. Finally, knock-down (KD) of genes previously shown to rescue F508del-CFTR was assessed on these mutants. Results show that all the variants preclude the production of mature CFTR, confirming them as Class II mutations. None of the variants responded to VX-661 but the combination rescued H1079P- and Q1100P-CFTR. The KD of factors that correct F508del-CFTR retention only marginally rescued R560S- and H1079P-CFTR. Overall, data evidence that Class II mutations induce distinct molecular defects that are neither rescued by the same corrector compounds nor recognized by the same cellular machinery, thus requiring personalized drug discovery initiatives.

scholarly journals Use of a High-Throughput Phenotypic Screening Strategy to Identify Amplifiers, a Novel Pharmacological Class of Small Molecules That Exhibit Functional Synergy with Potentiators and Correctors

2017 ◽  
Vol 23 (2) ◽  
pp. 111-121 ◽  
Author(s):  
Kenneth A. Giuliano ◽  
Shinichiro Wachi ◽  
Lawrence Drew ◽  
Danijela Dukovski ◽  
Olivia Green ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Small Molecules ◽  
High Throughput ◽  
Stress Responses ◽  
Genetic Disorder ◽  
Screening Strategy ◽  
New Class ◽  
Cftr Mutations ◽  
Cellular Stress Responses ◽  
Cftr Modulators

Cystic fibrosis (CF) is a lethal genetic disorder caused by mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Despite recent groundbreaking approval of genotype-specific small-molecule drugs, a significant portion of CF patients still lack effective therapeutic options that address the underlying cause of the disease. Through a phenotypic high-throughput screen of approximately 54,000 small molecules, we identified a novel class of CFTR modulators called amplifiers. The identified compound, the characteristics of which are represented here by PTI-CH, selectively increases the expression of immature CFTR protein across different CFTR mutations, including F508del-CFTR, by targeting the inefficiencies of early CFTR biosynthesis. PTI-CH also augments the activity of other CFTR modulators and was found to possess novel characteristics that distinguish it from CFTR potentiator and corrector moieties. The PTI-CH–mediated increase in F508del-CFTR did not elicit cytosolic or endoplasmic reticulum–associated cellular stress responses. Based on these data, amplifiers represent a promising new class of CFTR modulators for the treatment of CF that can be used synergistically with other CFTR modulators.

scholarly journals β2-Adrenergic receptor agonists activate CFTR in intestinal organoids and subjects with cystic fibrosis

2016 ◽  
Vol 48 (3) ◽  
pp. 768-779 ◽  
Author(s):  
Lodewijk A.W. Vijftigschild ◽  
Gitte Berkers ◽  
Johanna F. Dekkers ◽  
Domenique D. Zomer-van Ommen ◽  
Elizabeth Matthes ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Adrenergic Receptor ◽  
Ex Vivo ◽  
Gene Mutations ◽  
Airway Epithelial Cells ◽  
Potential Difference ◽  
Response To Treatment ◽  
Receptor Agonists ◽  
Intestinal Organoids

We hypothesized that people with cystic fibrosis (CF) who expressCFTR(cystic fibrosis transmembrane conductance regulator) gene mutations associated with residual function may benefit from G-protein coupled receptor (GPCR)-targeting drugs that can activate and enhance CFTR function.We used intestinal organoids to screen a GPCR-modulating compound library and identified β2-adrenergic receptor agonists as the most potent inducers of CFTR function.β2-Agonist-induced organoid swelling correlated with theCFTRgenotype, and could be induced in homozygous CFTR-F508del organoids and highly differentiated primary CF airway epithelial cells after rescue of CFTR trafficking by small molecules. Thein vivoresponse to treatment with an oral or inhaled β2-agonist (salbutamol) in CF patients with residual CFTR function was evaluated in a pilot study. 10 subjects with a R117H or A455E mutation were included and showed changes in the nasal potential difference measurement after treatment with oral salbutamol, including a significant improvement of the baseline potential difference of the nasal mucosa (+6.35 mV, p<0.05), suggesting that this treatment might be effectivein vivo. Furthermore, plasma that was collected after oral salbutamol treatment induced CFTR activation when administeredex vivoto organoids.This proof-of-concept study suggests that organoids can be used to identify drugs that activate CFTR functionin vivoand to select route of administration.

scholarly journals Therapeutic Approaches for Patients with Cystic Fibrosis Not Eligible for Current CFTR Modulators

Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2793
Author(s):  
Isabelle Fajac ◽  
Isabelle Sermet
Keyword(s):  
Cystic Fibrosis ◽  
Protein Function ◽  
Protein A ◽  
Autosomal Recessive Disorder ◽  
New Drugs ◽  
Gene Encoding ◽  
Disease Modifying Drugs ◽  
Cftr Mutations ◽  
Cftr Protein ◽  
Cftr Modulators

Cystic fibrosis is a severe autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding the CFTR protein, a chloride channel expressed in many epithelial cells. New drugs called CFTR modulators aim at restoring the CFTR protein function, and they will benefit many patients with cystic fibrosis in the near future. However, some patients bear rare mutations that are not yet eligible for CFTR modulators, although they might be amenable to these new disease-modifying drugs. Moreover, more than 10% of CFTR mutations do not produce any CFTR protein for CFTR modulators to act upon. The purpose of this review is to provide an overview of different approaches pursued to treat patients bearing mutations ineligible for CFTR modulators. One approach is to broaden the numbers of mutations eligible for CFTR modulators. This requires developing strategies to evaluate drugs in populations bearing very rare genotypes. Other approaches aiming at correcting the CFTR defect develop new mutation-specific or mutation-agnostic therapies for mutations that do not produce a CFTR protein: readthrough agents for nonsense mutations, nucleic acid-based therapies, RNA- or DNA-based, and cell-based therapies. Most of these approaches are in pre-clinical development or, for some of them, early clinical phases. Many hurdles and challenges will have to be solved before they can be safely translated to patients.

scholarly journals Clinical and genetic features of cystic fibrosis patients with novel pathogenic variant CFTR c.1083G> A (p.Trp361*) and functional assessment of the activity of the chloride channel

2019 ◽  
pp. 9-18
Author(s):  
Е.И. Кондратьева ◽  
Ю.Л. Мельяновская ◽  
А.С. Ефремова ◽  
Н.В. Булатенко ◽  
Т.Б. Бухарова ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Russian Federation ◽  
Genetic Variant ◽  
Clinical Manifestations ◽  
Pathogenic Variant ◽  
Cftr Gene ◽  
Genetic Characteristics ◽  
Intestinal Organoids ◽  
The Russian Federation ◽  
First Time

В статье впервые представляется клинико-генетическая характеристика мутации c.1083G>A (p.Trp361*) в гене CFTR. Патогенный генетический вариант c.1083G>A (p.Trp361*) гена CFTR относится к нонсенс-мутациям (I класс) и впервые был внесён в базу данных CFTR1 (http://www.genet.sickkids.on.ca) в середине 2019 г. без описания клинической картины муковисцидоза. Методы. Проведен анализ амбулаторных карт и историй болезни двух пациентов из неродственных семей с редким генетическим вариантом c.1083G>A (p.Trp361*). Для определения разности кишечных потенциалов (ОРКП) и проведения форсколинового теста на кишечных органоидах использовали биопсийный материал слизистой прямой кишки пациентов. ДНК для секвенирования выделяли из лейкоцитов венозной крови пациентов. Результаты. Анализ клинических проявлений заболевания у детей 6 и 9 лет показал наличие хронической панкреатической недостаточности, более выраженной у одного ребенка с синдромом дистальной интестинальной обструкции кишечника в анамнезе. Клиническая картина второго пациента характеризовалась развитием в раннем возрасте транзиторной гипербилирубинемии, синдрома псевдо-Барттера, а в дальнейшем - повторными эпизодами бронхиальной обструкции и развитием полипозного риносинусита. ОРКП и форсколиновый тест на кишечных органоидах показали, что генетический вариант c.1083G>A (p.Trp361*) относится к вариантам гена CFTR с отсутствием функции хлорного канала. Выводы. Впервые представлены описание клинической картины муковисцидоза у двух пациентов из неродственных семей с патогенным вариантом c.1083G>A (p.Trp361*) в компаунде с вариантом c.1521_1523delCTT (p.Phe508del) (ранее называемом F508del) и результаты оценки функции белка CFTR методом ОРКП и форсколиновым тестом на кишечных органоидах. In this article we continue to describe the pathogenic variants of the CFTR gene identified among Russian cystic fibrosis (CF) patients. For the first time the clinical and genetic characteristics of the mutation c.1083G> A (p.Trp361 *) are presented. The pathogenic genetic variant c.1083G> A (p.Trp361 *) of the CFTR gene belongs to the nonsense mutations (class I) and was listed for the first time in the CFTR1 database (http://www.genet.sickkids.on.ca) by Professor Milan Macek et al. in mid-2019 without any description of clinical manifestations of cystic fibrosis. Methods. The data of the National Register of Patients with Cystic Fibrosis of the Russian Federation 2017 were analyzed. Outpatient records and case histories of two patients from unrelated families carrying a rare genetic variant c.1083G> A (p.Trp361 *) were analyzed. To determine the Intestinal current measurement (ICM) and Forskolin-induced swelling (FIS) in intestinal organoids, rectal biopsy material of CF patients was used. DNA for sequencing was isolated from leukocytes of venous blood of the patients. Results. Variant c.1083G> A (p.Trp361 *) was found in two patients from unrelated families from different regions of the Russian Federation, according to the Register of Cystic Fibrosis Patients in the Russian Federation 2017. Analysis of clinical manifestations of the disease in children 6 and 9 years old showed the presence of chronic pancreatic insufficiency, more expressed in one child with a history of distal intestinal obstruction syndrome. The clinical manifestation of the second patient was characterized by the development of transient hyperbilirubinemia, Pseudo-Bartter’s syndrome at an early age, and subsequently repeated episodes of bronchial obstruction and the development of polypoid rhinosinusitis. The ICM method and the FIS in intestinal organoids showed that the genetic variant c.1083G> A (p.Trp361 *) refers to the variants of the CFTR gene with the absence of chlorine channel function. Conclusion. The clinical picture of cystic fibrosis in two patients from unrelated families with the pathogenic variant c.1083G> A (p.Trp361 *) in the compound with variant c.1521_1523delCTT (p.Phe508del) (variant legacy name F508del) and results of the evaluation of the CFTR protein functions, obtained by the method of ICM and using the FIS assay in intestinal organoids, are presented for the first time. Patients continue to be under the control in Russian CF centers.

scholarly journals Molecular and Functional Basis of Cystic Fibrosis in Indian Patients: Genetic, Diagnostic and Therapeutic Implications

2018 ◽  
Vol 54 (04) ◽  
pp. 216-230
Author(s):  
Rajendra Prasad
Keyword(s):  
Cystic Fibrosis ◽  
Indian Population ◽  
Transforming Growth Factor ◽  
Functional Characterization ◽  
Gene Mutations ◽  
Cftr Gene ◽  
Channel Activity ◽  
Novel Mutations ◽  
Cftr Mutations ◽  
Cftr Gene Mutations

ABSTRACTCystic fibrosis (CF, MIM#219700) is a common autosomal recessive disorder among Caucasians, which was considered as rare disease for Indian population. CF is caused due to presence of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In this study, we established a spectrum of mutations from both classical CF as well as from infertile male patients with congenital absence of vas deferens (CAVD). In Indian classical CF patients, we reported 14 previously known and eight novel mutations, viz. 3986-3987 delC, 876-6 del4, 1792 InsA, L69H, S158N, Q493L, 1530L and E1329Q. The frequency of delta 508 was found to be 27%. Absolute linkage between delta 508 and KM19-GATT TUB9-M470V-T854T haplotype predicts a relatively recent appearance of delta 508 mutations in Indian population. The CFTR gene analysis in CAVD infertile males documented 13 different CFTR gene mutations and 1 intronic variant that led to aberrant splicing. P.Phe 508 del (n= 16) and p.Arg 117 His (n=4) were among the common severe forms of CFTR mutations identified. The IVS-8-T5 allele (mild form of mutations) was formed with an allele frequency of 28.3%. Eight novel mutations were also found in the CFTR gene from our patient cohort. We also investigated whether genetic modifiers, viz. transforming growth factor (TGF-β) and endothelial receptor type A (EDNRA) of CF lung disease also predispose to CAVD in association with CFTR mutations, which were associated with the CAVD phenotype.Functional characterization of identified 11 novel CFTR gene mutations disclosed that a significant reduction in channel activity for L69H and S549N mutants in CFTR expressing cells was observed whereas impaired CFTR protein maturation was noticed only in L69H substitute CFTR. CFTR correctors (VX809) rescued the defect due to L69H mutation, which is evidenced from detection of C band in L69H mutant expressing cells pre-treated with VX809. The chloride channel activity in S549N and L69H mutant CFTR was also restored in presence of CFTR potentiators VX770.Above findings confirms heterogeneity of CFTR mutations in Indian classical and non-classical CF patients. They may help in developing a strategy to develop counseling and therapeutic approach for CF patients in India.

Effect of IBMX and alkaline phosphatase inhibitors on Cl− secretion in G551D cystic fibrosis mutant mice

1998 ◽  
Vol 274 (2) ◽  
pp. C492-C499 ◽  
Author(s):  
Stephen N. Smith ◽  
Stephen J. Delaney ◽  
Julia R. Dorin ◽  
Raymond Farley ◽  
Duncan M. Geddes ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Alkaline Phosphatase ◽  
Secretory Response ◽  
Mutant Mice ◽  
Mutant Protein ◽  
Wild Type ◽  
Phosphatase Inhibitors ◽  
Order Of Magnitude ◽  
Cftr Mutations

Some cystic fibrosis transmembrane conductance regulator (CFTR) mutations, such as G551D, result in a correctly localized Cl− channel at the cell apical membrane, albeit with markedly reduced function. Patch-clamp studies have indicated that both phosphatase inhibitors and 3-isobutyl-1-methylxanthine (IBMX) can induce Cl− secretion through the G551D mutant protein. We have now assessed whether these agents can induce Cl− secretion in cftrG551D mutant mice. No induction of Cl−secretion was seen with the alkaline phosphatase inhibitors bromotetramisole or levamisole in either the respiratory or intestinal tracts of wild-type or cftrG551D mice. In contrast, in G551D intestinal tissues, IBMX was able to produce a small CFTR-related secretory response [means ± SE: jejunum, 1.8 ± 0.9 μA/cm2, n = 7; cecum, 3.7 ± 0.8 μA/cm2, n = 7; rectum (in vivo), 1.9 ± 0.9 mV, n = 5]. This was approximately one order of magnitude less than the wild-type response to this agent and, in the cecum, was significantly greater than that seen in null mice ( cftrUNC ). In the trachea, IBMX produced a transient Cl− secretory response (37.3 ± 14.7 μA/cm2, n = 6) of a magnitude similar to that seen in wild-type mice (33.7 ± 4.7 μA/cm2, n = 9). This response was also present in null mice and therefore is likely to be independent of CFTR. No effect of IBMX on Cl−secretion was seen in the nasal epithelium of cftrG551D mice. We conclude that IBMX is able to induce detectable levels of CFTR-related Cl− secretion in the intestinal tract but not the respiratory tract through the G551D mutant protein.

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