High-Throughput Screening for Modulators of CFTR Activity Based on Genetically Engineered Cystic Fibrosis Disease-Specific iPSCs

Merkert, Sylvia; Schubert, Madline; Olmer, Ruth; Engels, Lena; Radetzki, Silke; Veltman, Mieke; Scholte, Bob J.; Zöllner, Janina; Pedemonte, Nicoletta; Galietta, Luis J V; Kries, Jens Peter von; Martin, Ulrich

doi:10.1016/j.stemcr.2019.04.014

Cited by 44 publications

(44 citation statements)

References 43 publications

(50 reference statements)

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“…In general, this alternative ASO based approach for rescuing exon skipping defects is based on the identification of appropriate splicing regulatory elements and thus more time consuming and expensive and not easy testable on several splicing defects. Similarly, a pharmacological approach to correct splicing requires high-throughput screening platforms and may lack of specificity (Berg et al, 2019;Giuliano et al, 2018;Liang et al, 2017;Merkert et al, 2019;Pereira et al, 2019). The positive effect we observed here on different CFTR mutations, along with those previously reported for other diseases in several cellular and mouse models (Dario Balestra et al, 2020;Dal Mas, Fortugno, et al, 2015;Donadon et al, 2018;Tajnik et al, 2016), indicates a general applicability and translatability of the ExSpeU1 approach in rescuing exon skipping defects.…”

Section: Discussionsupporting

confidence: 64%

Rescue of common exon skipping mutations in Cystic Fibrosis with modified U1 snRNAs

Donegà¹,

Rogalska²,

Pianigiani³

et al. 2020

Preprint

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In Cystic Fibrosis (CF), correction of splicing defects represents an interesting therapeutic approach to restore normal CFTR function. In this study, we focused on ten common mutations/variants, 711+3A>G/C, 711+5G>A, 1863C>T, 1898+3A>G, 2789+5G>A, TG13T3, TG13T5, TG12T5 and 3120G>A that induce skipping of the corresponding CFTR exons 5, 9, 13, 16 and 18. To rescue the splicing defects we tested, in a minigene assay, a panel of modified U1 snRNAs, named Exon Specific U1s (ExSpeU1) that were engineered to bind to intronic sequences downstream of each defective exon. Using this approach, we show that all ten splicing mutations analysed are efficiently corrected by specific ExSpeU1s. Using cDNA-splicing competent minigenes, we also show that the ExspeU1-mediated splicing correction at the RNA level recovered the full-length CFTR protein for 1863C>T, 1898+3A>G, 2789+5G>A variants. In addition, detailed mutagenesis experiments performed on exon 13 led us to identify a novel intronic regulatory element involved in the ExSpeU1-mediated splicing rescue. These results provide a common strategy based on modified U1 snRNAs to correct exon skipping in a group of disease-causing CFTR mutations.

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Section: Discussionsupporting

confidence: 64%

Rescue of common exon skipping mutations in Cystic Fibrosis with modified U1 snRNAs

Donegà¹,

Rogalska²,

Pianigiani³

et al. 2020

Preprint

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“…Quantitative differences in mean vesicle diameter and immunoflourescent colocalization may be useful in pathogenicity screening of uncharacterized ABCA3 variants. Future automation of these measurements could permit high‐throughput screening of pharmacologic correctors similar to strategies used to identify compounds that rescue CFTR function (Merkert et al, 2019; Pedemonte et al, 2005; Veit et al, 2018).…”

Section: Discussionmentioning

confidence: 99%

Functional characterization of four ATP‐binding cassette transporter A3 gene (ABCA3) variants

Yang

Wegner

et al. 2020

Human Mutation

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ABCA3 transports phospholipids across lamellar body membranes in pulmonary alveolar type II cells and is required for surfactant assembly. Rare, biallelic, pathogenic ABCA3 variants result in lethal neonatal respiratory distress syndrome and childhood interstitial lung disease. Qualitative functional characterization of ABCA3 missense variants suggests two pathogenic classes: disrupted intracellular trafficking (type I mutant) or impaired ATPase‐mediated phospholipid transport into the lamellar bodies (type II mutant). We qualitatively compared wild‐type (WT‐ABCA3) with four uncharacterized ABCA3 variants (c.418A>C;p.Asn140His, c.3609_3611delCTT;p.Phe1203del, c.3784A>G;p.Ser1262Gly, and c.4195G>A;p.Val1399Met) in A549 cells using protein processing, colocalization with intracellular organelles, lamellar body ultrastructure, and ATPase activity. We quantitatively measured lamellar body‐like vesicle diameter and intracellular ABCA3 trafficking using fluorescence‐based colocalization. Three ABCA3 variants (p.Asn140His, p.Ser1262Gly, and p.Val1399Met) were processed and trafficked normally and demonstrated well‐organized lamellar body‐like vesicles, but had reduced ATPase activity consistent with type II mutants. P.Phe1203del was processed normally, had reduced ATPase activity, and well‐organized lamellar body‐like vesicles, but quantitatively colocalized with both endoplasmic reticulum and lysosomal markers, an intermediate phenotype suggesting disruption of both intracellular trafficking and phospholipid transport. All ABCA3 mutants demonstrated mean vesicle diameters smaller than WT‐ABCA3. Qualitative and quantitative functional characterization of ABCA3 variants informs mechanisms of pathogenicity.

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“…Primary, reprogrammed and engineered human cell models have become important tools to identify novel pharmacotherapies. The effects of certain therapies may also be exploited at an individual level in ex vivo patient-derived specimens, such as primary bronchial/nasal epithelial cells, and intestinal/respiratory organoids (Fulcher and Randell, 2013;Dekkers et al, 2016a;Dekkers et al, 2016b;Pranke et al, 2017;Awatade et al, 2018;Brewington et al, 2018;Chen et al, 2018;Berkers et al, 2019;Merket et al, 2019). As these cell models recapitulate several features of the parental organ, they are useful to understand the impact of genetic factors on individual disease and predict clinical efficacy of therapies.…”

Section: Cf-causing Mutations and Progress In Precision Medicinementioning

confidence: 99%

“…Numerous libraries of compounds have been screened by distinct high-throughput screening (HTS) methods and using several cell models. These experimental approaches have been contributing to the identification of small-molecules from different chemical series (Pedemonte et al, 2005a;Van Goor et al, 2009;Van Goor et al, 2011;Phuan et al, 2014;Phuan et al, 2015;Liang et al, 2017;Giuliano et al, 2018;Van der Plas et al, 2018;Veit et al, 2018;Wang et al, 2018;Berg et al, 2019;De Wilde et al, 2019;Merket et al, 2019) (Figure 6). Notably, as cell background may significantly influence the pharmacological rescue of CFTR mutants (Pedemonte et al, 2010), most promising hits should be further validated in well-differentiated cells in order to identify non-cytotoxic and effective compounds that might result in optimal therapeutic benefits in the clinical scenario.…”

Section: Cf-causing Mutations and Progress In Precision Medicinementioning

confidence: 99%

CFTR Modulators: The Changing Face of Cystic Fibrosis in the Era of Precision Medicine

2020

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Cystic fibrosis (CF) is a lethal inherited disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which result in impairment of CFTR mRNA and protein expression, function, stability or a combination of these. Although CF leads to multifaceted clinical manifestations, the respiratory disorder represents the major cause of morbidity and mortality of these patients. The life expectancy of CF patients has substantially lengthened due to early diagnosis and improvements in symptomatic therapeutic regimens. Quality of life remains nevertheless limited, as these individuals are subjected to considerable clinical, psychosocial and economic burdens. Since the discovery of the CFTR gene in 1989, tremendous efforts have been made to develop therapies acting more upstream on the pathogenesis cascade, thereby overcoming the underlying dysfunctions caused by CFTR mutations. In this line, the advances in cell-based high-throughput screenings have been facilitating the fast-tracking of CFTR modulators. These modulator drugs have the ability to enhance or even restore the functional expression of specific CF-causing mutations, and they have been classified into five main groups depending on their effects on CFTR mutations: potentiators, correctors, stabilizers, read-through agents, and amplifiers. To date, four CFTR modulators have reached the market, and these pharmaceutical therapies are transforming patients' lives with short-and long-term improvements in clinical outcomes. Such breakthroughs have paved the way for the development of novel CFTR modulators, which are currently under experimental and clinical investigations. Furthermore, recent insights into the CFTR structure will be useful for the rational design of next-generation modulator drugs. This review aims to provide a summary of recent developments in CFTR-directed therapeutics. Barriers and future directions are also discussed in order to optimize treatment adherence, identify feasible and sustainable solutions for equitable access to these therapies, and continue to expand the pipeline of novel modulators that may result in effective precision medicine for all individuals with CF.

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High-Throughput Screening for Modulators of CFTR Activity Based on Genetically Engineered Cystic Fibrosis Disease-Specific iPSCs

Cited by 44 publications

References 43 publications

Rescue of common exon skipping mutations in Cystic Fibrosis with modified U1 snRNAs

Rescue of common exon skipping mutations in Cystic Fibrosis with modified U1 snRNAs

Functional characterization of four ATP‐binding cassette transporter A3 gene (ABCA3) variants

CFTR Modulators: The Changing Face of Cystic Fibrosis in the Era of Precision Medicine

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