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Polaridad Celular , Fibrosis Quística , Factor de Crecimiento Transformador beta , Vía de Señalización Wnt , Fibrosis Quística/metabolismo , Fibrosis Quística/patología , Humanos , Factor de Crecimiento Transformador beta/metabolismo , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Proteínas Wnt/metabolismo , Animales , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genéticaRESUMEN
Trafficking of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein is a complex process that starts with its biosynthesis and folding in the endoplasmic reticulum. Exit from the endoplasmic reticulum (ER) is coupled with the acquisition of a compact structure that can be processed and traffic through the secretory pathway. Once reaching its final destination-the plasma membrane, CFTR stability is regulated through interaction with multiple protein partners that are involved in its post-translation modification, connecting the channel to several signaling pathways. The complexity of the process is further boosted when analyzed in the context of the airway epithelium. Recent advances have characterized in detail the different cell types that compose the surface epithelium and shifted the paradigm on which cells express CFTR and on their individual and combined contribution to the total expression (and function) of this chloride/bicarbonate channel. Here we review CFTR trafficking and its relationship with the knowledge on the different cell types of the airway epithelia. We explore the crosstalk between these two areas and discuss what is still to be clarified and how this can be used to develop more targeted therapies for CF.
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Small molecule drugs known as modulators can treat ~90% of people with cystic fibrosis (CF), but do not work for premature termination codon variants such as W1282X (c.3846G>A). Here we evaluated two gene editing strategies, Adenine Base Editing (ABE) to correct W1282X, and Homology-Independent Targeted Integration (HITI) of a CFTR superexon comprising exons 23-27 (SE23-27) to enable expression of a CFTR mRNA without W1282X. In Flp-In-293 cells stably expressing a CFTR expression minigene bearing W1282X, ABE corrected 24% of W1282X alleles, rescued CFTR mRNA from nonsense mediated decay and restored protein expression. However, bystander editing at the adjacent adenine (c.3847A>G), caused an amino acid change (R1283G) that affects CFTR maturation and ablates ion channel activity. In primary human nasal epithelial cells homozygous for W1282X, ABE corrected 27% of alleles, but with a notably lower level of bystander editing, and CFTR channel function was restored to 16% of wild-type levels. Using the HITI approach, correct integration of a SE23-27 in intron 22 of the CFTR locus in 16HBEge W1282X cells was detected in 5.8% of alleles, resulting in 7.8% of CFTR transcripts containing the SE23-27 sequence. Analysis of a clonal line homozygous for the HITI-SE23-27 produced full-length mature protein and restored CFTR anion channel activity to 10% of wild-type levels, which could be increased three-fold upon treatment with the triple combination of CF modulators. Overall, these data demonstrate two different editing strategies can successfully correct W1282X, the second most common class I variant, with a concomitant restoration of CFTR function.
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Fibrosis Quística , Humanos , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Edición Génica , Codón sin Sentido/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , MutaciónRESUMEN
Most of the 2,100 CFTR gene variants reported to date are still unknown in terms of their disease liability in Cystic Fibrosis (CF) and their molecular and cellular mechanism that leads to CFTR dysfunction. Since some rare variants may respond to currently approved modulators, characterizing their defect and response to these drugs is essential for effective treatment of people with CF (pwCF) not eligible for the current treatment. Here, we assessed how the rare variant, p.Arg334Trp, impacts on CFTR traffic and function and its response to existing CFTR modulators. To this end, we performed the forskolin-induced swelling (FIS) assay on intestinal organoids from 10 pwCF bearing the p.Arg334Trp variant in one or both alleles of the CFTR gene. In parallel, a novel p.Arg334Trp-CFTR expressing CFBE cell line was generated to characterize the variant individually. Results show that p.Arg334Trp-CFTR does not significantly affect the plasma membrane traffic of CFTR and evidences residual CFTR function. This CFTR variant is rescued by currently available CFTR modulators independently of the variant in the second allele. The study, predicting clinical benefit for CFTR modulators in pwCF with at least one p.Arg334Trp variant, demonstrates the high potential of personalized medicine through theranostics to extend the label of approved drugs for pwCF carrying rare CFTR variants. We recommend that this personalized approach should be considered for drug reimbursement policies by health insurance systems/national health services.
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BACKGROUND: The phenotypic heterogeneity observed in Cystic Fibrosis (CF) patients suggests the involvement of other genes, besides CFTR. Here, we combined transcriptome and proteome analysis to understand the global gene expression patterns associated with five prototypical CFTR mutations. RESULTS: Evaluation of differentially expressed genes and proteins unveiled common and mutation-specific changes revealing functional signatures that are much more associated with the specific molecular defects associated with each mutation than to the CFTR loss-of-function phenotype. The combination of both datasets revealed that mutation-specific detected translated-transcripts (Dtt) have a high level of consistency. CONCLUSIONS: This is the first combined transcriptomic and proteomic study focusing on prototypical CFTR mutations. Analysis of Dtt provides novel insight into the pathophysiology of CF, and the mechanisms through which each mutation class causes disease and will likely contribute to the identification of new therapeutic targets and/or biomarkers for CF.
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Cystic Fibrosis (CF) is a genetic disease caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Currently, more than 2100 variants have been identified in the gene, with a large number being very rare. The approval of modulators that act on mutant CFTR protein, correcting its molecular defect and thus alleviating the burden of the disease, revolutionized the field of CF. However, these drugs do not apply to all patients with CF, especially those with rare mutations-for which there is a lack of knowledge on the molecular mechanisms of the disease and the response to modulators. In this work, we evaluated the impact of several rare putative class II mutations on the expression, processing, and response of CFTR to modulators. Novel cell models consisting of bronchial epithelial cell lines expressing CFTR with 14 rare variants were created. The variants studied are localized at Transmembrane Domain 1 (TMD1) or very close to the signature motif of Nucleotide Binding Domain 1 (NBD1). Our data show that all mutations analyzed significantly decrease CFTR processing and while TMD1 mutations respond to modulators, those localized in NBD1 do not. Molecular modeling calculations confirm that the mutations in NBD1 induce greater destabilization of CFTR structure than those in TMD1. Furthermore, the structural proximity of TMD1 mutants to the reported binding site of CFTR modulators such as VX-809 and VX-661, make them more efficient in stabilizing the CFTR mutants analyzed. Overall, our data suggest a pattern for mutation location and impact in response to modulators that correlates with the global effect of the mutations on CFTR structure.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Humanos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Sitios de Unión , Mutación , Modelos Moleculares , Benzodioxoles/farmacologíaRESUMEN
Clinical treatments for cystic fibrosis (CF) underwent significant changes in the last decade as therapies targeting the basic defect in the CFTR protein were approved. Significant scientific progress has also been made in several other areas that may lead in the future to novel therapeutic approaches that can help fight CF in all individuals living with this disease. Thus, focusing on fundamental research in the CF field has and will continue to be of great importance. This has been one of the aims of the European Cystic Fibrosis Society (ECFS), which has promoted the ECFS Basic Science Conference (BSC) every year since 2004. This special issue covers the topics featured and discussed at the 17th ECFS BSC, held in Albufeira (Portugal) in March 2022, and highlights advances in understanding CFTR, in using personalized medicine, and in developing innovative strategies to identify breakthrough therapies. This introduction highlights the topics presented throughout this special issue, thereby underscoring the relevance of fundamental research in CF.
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Fibrosis Quística , Humanos , Fibrosis Quística/tratamiento farmacológico , Investigación Biomédica Traslacional , Mutación , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Medicina de PrecisiónRESUMEN
The plasma membrane (PM) stability of the cystic fibrosis transmembrane conductance regulator (CFTR), the protein which when mutated causes Cystic Fibrosis (CF), relies on multiple interaction partners that connect CFTR to signaling pathways, including cAMP signaling. It was previously shown that activation of exchange protein directly activated by cAMP 1 (EPAC1) by cAMP promotes an increase in CFTR PM levels in airway epithelial cells. However, the relevance of this pathway in other tissues, particularly the intestinal tissue, remains uncharacterized. Here, we used Western blot and forskolin-induced swelling assay to demonstrate that the EPAC1 protein is not expressed in the intestinal organoid model, and consequently the EPAC1 stabilization pathway is not in place. On the other hand, using cell surface biotinylation, EPAC1-mediated stabilization of PM CFTR is observed in intestinal cell lines. These results indicate that the EPAC1 stabilization pathway also occurs in intestinal cells and is a potential target for the development of novel combinatorial therapies for treatment of CF.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Factores de Intercambio de Guanina Nucleótido , Línea Celular , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Organoides/metabolismo , Transducción de SeñalRESUMEN
Cystic fibrosis, the most common autosomal recessive disorder in Caucasians, is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a cAMP-activated chloride and bicarbonate channel that regulates ion and water transport in secretory epithelia. Although all mutations lead to the lack or reduction in channel function, the mechanisms through which this occurs are diverse - ranging from lack of full-length mRNA, reduced mRNA levels, impaired folding and trafficking, targeting to degradation, decreased gating or conductance, and reduced protein levels to decreased half-life at the plasma membrane. Here, we review the different molecular mechanisms that cause cystic fibrosis and detail how these differences identify theratypes that can inform the use of directed therapies aiming at correcting the basic defect. In summary, we travel through CFTR life cycle from the gene to function, identifying what can go wrong and what can be targeted in terms of the different types of therapeutic approaches.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Fibrosis Quística/terapia , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Humanos , Transporte Iónico , Mutación , ARN Mensajero/metabolismoRESUMEN
BACKGROUND: In cystic fibrosis (CF), genotype-phenotype correlation is complicated by the large number of CFTR variants, the influence of modifier genes, environmental effects, and the existence of complex alleles. We document the importance of complex alleles, in particular the F508C variant present in cis with the S1251N disease-causing variant, by detailed analysis of a patient with CF, with the [S1251N;F508]/G542X genotype and a very mild phenotype, contrasting it to that of four subjects with the [S1251N;F508C]/F508del genotype and classical CF presentation. METHODS: Genetic differences were identified by Sanger sequencing and CFTR function was quantified using rectal organoids in rectal organoid morphology analysis (ROMA) and forskolin-induced swelling (FIS) assays. CFTR variants were further characterised in CF bronchial epithelial (CFBE) cell lines. The impact of involved amino acid changes in the CFTR 3D protein structure was evaluated. RESULTS: Organoids of the patient [S1251N;F508] with mild CF phenotype confirmed the CF diagnosis but showed higher residual CFTR function compared to the four others [S1251N;F508C]. CFBE cell lines showed a decrease in [S1251N;F508C]-CFTR function but not in processing when compared to [S1251N;F508]-CFTR. Analysis of the 3D CFTR structure suggested an additive deleterious effect of the combined presence of S1251N and F508C with respect to NBD1-2 dimerisation. CONCLUSIONS: In vitro and in silico data show that the presence of F508C in cis with S1251N decreases CFTR function without affecting processing. Complex CFTR alleles play a role in clinical phenotype and their identification is relevant in the context of personalised medicine for each patient with CF.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Alelos , Fibrosis Quística/diagnóstico , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Genotipo , Humanos , Mutación , FenotipoRESUMEN
Although some therapeutic progress has been achieved in developing small molecules that correct F508del-CFTR defects, the mechanism of action (MoA) of these compounds remain poorly elucidated. Here, we investigated the effects and MoA of MCG1516A, a newly developed F508del-CFTR corrector. MCG1516A effects on wild-type (WT) and F508del-CFTR were assessed by immunofluorescence microscopy, and biochemical and functional assays both in cell lines and in intestinal organoids. To shed light on the MoA of MCG1516A, we evaluated its additivity to the FDA-approved corrector VX-661, low temperature, genetic revertants of F508del-CFTR (G550E, R1070W, and 4RK), and the traffic-null variant DD/AA. Finally, we explored the ability of MCG1516A to rescue trafficking and function of other CF-causing mutations. We found that MCG1516A rescues F508del-CFTR with additive effects to VX-661. A similar behavior was observed for WT-CFTR. Under low temperature incubation, F508del-CFTR demonstrated an additivity in processing and function with VX-661, but not with MCG1516A. In contrast, both compounds promoted additional effects to low temperature to WT-CFTR. MCG1516A demonstrated additivity to genetic revertant R1070W, while VX-661 was additive to G550E and 4RK. Nevertheless, none of these compounds rescued DD/AA trafficking. Both MCG1516A and VX-661 rescued CFTR processing of L206W- and R334W-CFTR with greater effects when these compounds were combined. In summary, the absence of additivity of MCG1516A to genetic revertant G550E suggests a putative binding site for this compound on NBD1:NBD2 interface. Therefore, a combination of MCG1516A with compounds able to rescue DD/AA traffic, or mimicking the actions of revertant R1070W (e.g., VX-661), could enhance correction of F508del-CFTR defects.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/genética , Descubrimiento de Drogas/métodos , Humanos , Mutación , Pliegue de ProteínaRESUMEN
BACKGROUND: W1282X-CFTR variant (c.3846G>A) is the second most common nonsense cystic fibrosis (CF)-causing mutation in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Even though remarkable breakthroughs have been done towards CF treatment with the approval of four CFTR protein modulators, none of these are approved for patients with nonsense mutations. CRISPR gene editing tools can be of great value to permanently correct the genetic defects caused by these mutations. METHODS: We compared the capacity of homology-directed repair (HDR) mediated by Cas9 or Cas12a to correct W1282X CFTR mutation in the CFF-16HBEge W1282X CFTR cell line (obtained from CFF), using Cas9/gRNA and Cas12a/gRNA ribonucleoproteins (RNPs) and single strand DNA (ssODN) oligonucleotide donors. RESULTS: Cas9 shows higher levels of correction than Cas12a as, by electroporating cells with Cas9 RNPs and ssODN donor, nearly 18% of precise editing was achieved compared to just 8% for Cas12a. Such levels of correction increase the abundance of CFTR mRNA and protein, and partially restore CFTR function in the pool of edited cells to 18% of WT CFTR function. Moreover, homozygous corrected clones produced levels of mRNA, protein, and function comparable to those of cells expressing WT CFTR. CONCLUSION: Altogether, this work demonstrates the potential of gene editing as a therapeutic strategy for CF directly correcting the root cause of the disease.
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Proteínas Bacterianas/genética , Proteína 9 Asociada a CRISPR/genética , Proteínas Asociadas a CRISPR/genética , Sistemas CRISPR-Cas/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Endodesoxirribonucleasas/genética , Edición Génica/métodos , Línea Celular , Humanos , MutaciónRESUMEN
Remarkable progress in CFTR research has led to the therapeutic development of modulators that rescue the basic defect in cystic fibrosis. There is continuous interest in studying CFTR molecular disease mechanisms as not all cystic fibrosis patients have a therapeutic option available. Addressing the basis of the problem by comprehensively understanding the critical molecular associations of CFTR interactions remains key. With the availability of CFTR modulators, there is interest in comprehending which interactions are critical to rescue CFTR and which are altered by modulators or CFTR mutations. Here, the current knowledge on interactions that govern CFTR folding, processing, and stability is summarized. Furthermore, we describe protein complexes and signal pathways that modulate the CFTR function. Primary epithelial cells display a spatial control of the CFTR interactions and have become a common system for preclinical and personalized medicine studies. Strikingly, the novel roles of CFTR in development and differentiation have been recently uncovered and it has been revealed that specific CFTR gene interactions also play an important role in transcriptional regulation. For a comprehensive understanding of the molecular environment of CFTR, it is important to consider CFTR mutation-dependent interactions as well as factors affecting the CFTR interactome on the cell type, tissue-specific, and transcriptional levels.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/metabolismo , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Humanos , Mutación , Medicina de Precisión , Unión Proteica , Pliegue de Proteína , Estabilidad Proteica , Transducción de SeñalRESUMEN
Cystic fibrosis (CF) is a life-threatening autosomal recessive disease caused by more than 2100 mutations in the CF transmembrane conductance regulator (CFTR) gene, generating variability in disease severity among individuals with CF sharing the same CFTR genotype. Systems biology can assist in the collection and visualization of CF data to extract additional biological significance and find novel therapeutic targets. Here, we present the CyFi-MAP-a disease map repository of CFTR molecular mechanisms and pathways involved in CF. Specifically, we represented the wild-type (wt-CFTR) and the F508del associated processes (F508del-CFTR) in separate submaps, with pathways related to protein biosynthesis, endoplasmic reticulum retention, export, activation/inactivation of channel function, and recycling/degradation after endocytosis. CyFi-MAP is an open-access resource with specific, curated and continuously updated information on CFTR-related pathways available online at https://cysticfibrosismap.github.io/ . This tool was developed as a reference CF pathway data repository to be continuously updated and used worldwide in CF research.
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Biomarcadores , Fibrosis Quística/etiología , Fibrosis Quística/metabolismo , Bases de Datos Genéticas , Susceptibilidad a Enfermedades , Transducción de Señal , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Humanos , Programas Informáticos , Navegador WebRESUMEN
Major advances have recently been made in the development and application of CFTR (cystic fibrosis transmembrane conductance regulator) mutation class-specific modulator therapies, but to date, there are no approved modulators for Class I mutations, i.e., those introducing a premature termination codon (PTC) into the CFTR mRNA. Such mutations induce nonsense-mediated decay (NMD), a cellular quality control mechanism that reduces the quantity of PTC bearing mRNAs, presumably to avoid translation of potentially deleterious truncated CFTR proteins. The NMD-mediated reduction of PTC-CFTR mRNA molecules reduces the efficacy of one of the most promising approaches to treatment of such mutations, namely, PTC readthrough therapy, using molecules that induce the incorporation of near-cognate amino acids at the PTC codon, thereby enabling translation of a full-length protein. In this study, we measure the effect of three different PTC mutations on the abundance, integrity, and stability of respective CFTR mRNAs, using CFTR specific RT-qPCR-based assays. Altogether, our data suggest that optimized rescue of PTC mutations has to take into account (1) the different steady-state levels of the CFTR mRNA associated with each specific PTC mutation; (2) differences in abundance between the 3' and 5' regions of CFTR mRNA, even following PTC readthrough or NMD inhibition; and (3) variable effects on CFTR mRNA stability for each specific PTC mutation.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Degradación de ARNm Mediada por Codón sin Sentido , Línea Celular , Codón sin Sentido , Codón de Terminación , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Humanos , Mucosa Respiratoria/metabolismoRESUMEN
Lemur tyrosine kinase 2 (LMTK2) is a transmembrane Ser/Thr kinase whose role has been increasingly recognized; however, when compared to other kinases, understanding of the LMTK2 networks and biological functions is still limited. Recent data have shown that transforming growth factor (TGF)-ß1 plays a role in modulating LMTK2 function by controlling its endocytic trafficking in human bronchial epithelial cells. Here, we aimed to unveil the LMTK2 regulatory network and elucidate how it affects cellular functions and disease pathways in either TGF-ß1 dependent or independent manner. To understand how the LMTK2 and TGF-ß1 pathways interconnect, we knocked down (KD) LMTK2 using small(si)RNA-mediated silencing in human bronchial epithelial CFBE41o- cells, treated cells with TGF-ß1 or vehicle control, and performed differential gene expression analysis by RNA sequencing (RNAseq). In vehicle-treated cells, LMTK2 KD affected expression of 2,506 genes while it affected 4,162 genes after TGF-ß1 stimulation. Bioinformatics analysis shows that LMTK2 is involved in diverse cellular functions and disease pathways, such as cell death and survival, cellular development, and cancer susceptibility. In summary, our study increases current knowledge about the LMTK2 network and its intersection with the TGF-ß1 signaling pathway. These findings will serve as basis for future exploration of the predicted LMTK2 interactions and signaling pathways.
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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.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Mutación/genética , Benzodioxoles/farmacología , Línea Celular , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Femenino , Humanos , Indoles/farmacología , Masculino , Pirazoles/farmacología , Piridinas/farmacología , Pirrolidinas/farmacologíaRESUMEN
Pretargeted imaging has emerged as an effective multistep strategy aiming to improve imaging contrast and reduce patient radiation exposure through decoupling of the radioactivity from the targeting vector. The inverse electron-demand Diels-Alder (IEDDA) reaction between a trans-cyclooctene (TCO)-conjugated antibody and a labeled tetrazine holds great promise for pretargeted imaging applications due to its bioorthogonality, rapid kinetics under mild conditions, and formation of stable products. Herein, we describe the use of functionalized carbonylacrylic reagents for site-specific incorporation of TCO onto a human epidermal growth factor receptor 2 (HER2) antibody (THIOMAB) containing an engineered unpaired cysteine residue, generating homogeneous conjugates. Precise labeling of THIOMAB-TCO with a fluorescent or radiolabeled tetrazine revealed the potential of the TCO-functionalized antibody for imaging the HER2 after pretargeting in a cellular context in a HER2 positive breast cancer cell line. Control studies with MDA-MD-231 cells, which do not express HER2, further confirmed the target specificity of the modified antibody. THIOMAB-TCO was also evaluated in vivo after pretargeting and subsequent administration of an 111In-labeled tetrazine. Biodistribution studies in breast cancer tumor-bearing mice showed a significant activity accumulation on HER2+ tumors, which was 2.6-fold higher than in HER2- tumors. Additionally, biodistribution studies with THIOMAB without the TCO handle also resulted in a decreased uptake of 111In-DOTA-Tz on HER2+ tumors. Altogether, these results clearly indicate the occurrence of the click reaction at the tumor site, i.e., pretargeting of SK-BR-3 HER2-expressing cells with THIOMAB-TCO and reaction through the TCO moiety present in the antibody. The combined advantages of site-selectivity and stability of TCO tagged-antibodies could allow application of biorthogonal chemistry strategies for pretargeting imaging with minimal side-reactions and background.
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Anticuerpos/química , Química Clic , Cisteína/química , Animales , Línea Celular Tumoral , Colorantes Fluorescentes/química , Humanos , Ratones , Radiofármacos/químicaRESUMEN
Cystic Fibrosis (CF) is caused by mutations in the CF Transmembrane conductance Regulator (CFTR), the only ATP-binding cassette (ABC) transporter functioning as a channel. Unique to CFTR is a regulatory domain which includes a highly conformationally dynamic region-the regulatory extension (RE). The first nucleotide-binding domain of CFTR contains another dynamic region-regulatory insertion (RI). Removal of RI rescues the trafficking defect of CFTR with F508del, the most common CF-causing mutation. Here we aimed to assess the impact of RE removal (with/without RI or genetic revertants) on F508del-CFTR trafficking and how CFTR modulator drugs VX-809/lumacaftor and VX-770/ivacaftor rescue these variants. We generated cell lines expressing ΔRE and ΔRI CFTR (with/without genetic revertants) and assessed CFTR expression, stability, plasma membrane levels, and channel activity. Our data demonstrated that ΔRI significantly enhanced rescue of F508del-CFTR by VX-809. While the presence of the RI seems to be precluding full rescue of F508del-CFTR processing by VX-809, this region appears essential to rescue its function by VX-770, suggesting some contradictory role in rescue of F508del-CFTR by these two modulators. This negative impact of RI removal on VX-770-stimulated currents on F508del-CFTR can be compensated by deletion of the RE which also leads to the stabilization of this mutant. Despite both regions being conformationally dynamic, RI precludes F508del-CFTR processing while RE affects mostly its stability and channel opening.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Aminofenoles/farmacología , Aminopiridinas/farmacología , Benzodioxoles/farmacología , Línea Celular , Membrana Celular/metabolismo , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Humanos , Mutación , Dominios Proteicos/genética , Quinolonas/farmacología , Secuencias Reguladoras de Ácidos Nucleicos/genética , Transducción de Señal/genéticaRESUMEN
BACKGROUND: For most of the >2000 CFTR gene variants reported, neither the associated disease liability nor the underlying basic defect are known, and yet these are essential for disease prognosis and CFTR-based therapeutics. Here we aimed to characterize two ultra-rare mutations - 1717-2A > G (c.1585-2A > G) and S955P (p.Ser955Pro) - as case studies for personalized medicine. METHODS: Patient-derived rectal biopsies and intestinal organoids from two individuals with each of these mutations and F508del (p.Phe508del) in the other allele were used to assess CFTR function, response to modulators and RNA splicing pattern. In parallel, we used cellular models to further characterize S955P independently of F508del and to assess its response to CFTR modulators. RESULTS: Results in both rectal biopsies and intestinal organoids from both patients evidence residual CFTR function. Further characterization shows that 1717-2A > G leads to alternative splicing generating <1% normal CFTR mRNA and that S955P affects CFTR gating. Finally, studies in organoids predict that both patients are responders to VX-770 alone and even more to VX-770 combined with VX-809 or VX-661, although to different levels. CONCLUSION: This study demonstrates the high potential of personalized medicine through theranostics to extend the label of approved drugs to patients with rare mutations.