Fibrosis Transmembrane Conductance Regulator Gene Research Articles

Recombinant Adeno-Associated Virus-Mediated Editing of the G551D Cystic Fibrosis Transmembrane Conductance Regulator Mutation in Ferret Airway Basal Cells.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), a chronic disease that affects multiple organs, including the lung. We developed a CF ferret model of a scarless G551→D substitution in CFTR (CFTRG551D-KI), enabling approaches to correct this gating mutation in CF airways via gene editing. Homology-directed repair (HDR) was tested in Cas9-expressing CF airway basal cells (Cas9-GKI) from this model, as well as reporter basal cells (Y66S-Cas9-GKI) that express an integrated nonfluorescent Y66S-EGFP (enhanced green fluorescent protein) mutant gene to facilitate rapid assessment of HDR by the restoration of fluorescence. Recombinant adeno-associated virus (rAAV) vectors were used to deliver two DNA templates and sgRNAs for dual-gene editing at the EGFP and CFTR genes, followed by fluorescence-activated cell sorting of EGFPY66S-corrected cells. When gene-edited airway basal cells were polarized at an air-liquid interface, unsorted and EGFPY66S-corrected sorted populations gave rise to 26.0% and 70.4% CFTR-mediated Cl- transport of that observed in non-CF cultures, respectively. The consequences of gene editing at the CFTRG551D locus by HDR and nonhomologous end joining (NHEJ) were assessed by targeted gene next-generation sequencing (NGS) against a specific amplicon. NGS revealed HDR corrections of 3.1% of G551 sequences in the unsorted population of rAAV-infected cells, and 18.4% in the EGFPY66S-corrected cells. However, the largest proportion of sequences had indels surrounding the CRISPR (clustered regularly interspaced short palindromic repeats) cut site, demonstrating that NHEJ was the dominant repair pathway. This approach to simultaneously coedit at two genomic loci using rAAV may have utility as a model system for optimizing gene-editing efficiencies in proliferating airway basal cells through the modulation of DNA repair pathways in favor of HDR.

Spectrum of Cystic Fibrosis Conductance Regulator Gene Mutations Reported in Pakistani Descent Cystic Fibrosis Patients

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. This study aims to determine the genotypic and phenotypic spectrum of the CFTR gene mutations reported in the literature for Pakistani-origin CF patients. Databases were searched for such studies from 1947-2019 for sample size, method of diagnosis, and CFTR gene mutations. The authors identified 12 studies reporting 33 CFTR gene mutations, both intronic as well as exonic in Pakistani origin patients. The most widely tested mutation was D508 with a frequency of 17%-60%. No hotspot zone was identified and not all reported mutations were causing disease. There is a need to identify common mutations in the Pakistani population to develop population-specific CFTR mutations panel. This will enable the researchers to perform phenotype-genotype correlation studies to improve the CF detection rate. Key Words: Cystic fibrosis, Pakistan, Mutations, CFTR.

Emerging Concepts in Defective Macrophage Phagocytosis in Cystic Fibrosis

Cystic fibrosis (CF) is caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Chronic inflammation and decline in lung function are major reasons for morbidity in CF. Mutant CFTR expressed in phagocytic cells such as macrophages contributes to persistent infection, inflammation, and lung disease in CF. Macrophages play a central role in innate immunity by eliminating pathogenic microbes by a process called phagocytosis. Phagocytosis is required for tissue homeostasis, balancing inflammation, and crosstalk with the adaptive immune system for antigen presentation. This review focused on (1) current understandings of the signaling underlying phagocytic mechanisms; (2) existing evidence for phagocytic dysregulation in CF; and (3) the emerging role of CFTR modulators in influencing CF phagocytic function. Alterations in CF macrophages from receptor initiation to phagosome formation are linked to disease progression in CF. A deeper understanding of macrophages in the context of CFTR and phagocytosis proteins at each step of phagosome formation might contribute to the new therapeutic development of dysregulated innate immunity in CF. Therefore, the review also indicates future areas of research in the context of CFTR and macrophages.

Molecular mechanisms of cystic fibrosis - how mutations lead to misfunction and guide therapy.

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.

Geographical distribution of cystic fibrosis carriers as population genetic determinant of COVID-19 spread and fatality in 37 countries

COVID-19 has shown a relevant heterogeneity in spread and fatality among countries together with a significant variability in its clinical presentation, indicating that host genetic factors may influence COVID-19 pathogenicity. Indeed, subjects carrying single pathogenic variants of the Cystic Fibrosis (CF) Transmembrane Conductance Regulator (CFTR) gene – i.e. CF carriers – are more susceptible to respiratory tract infections and are more likely to undergo severe COVID-19 with higher risk of 14-day mortality.Given that CF carrier prevalence varies among ethnicities and nations, an ecological study in 37 countries was conducted, in order to determine to what extent the diverse CF carrier geographical distribution may have affected COVID-19 spread and fatality during the first pandemic wave.The CF prevalence in countries, as indicator of the geographical distribution of CF carriers, significantly correlated in a direct manner with both COVID-19 prevalence and its Case Fatality Rate (CFR). In a regression study weighted for the number of tests performed, COVID-19 prevalence positively correlated with CF prevalence, while CFR correlated with population percentage older than 65-year, cancer and CF prevalence. Multivariate regression model also confirmed COVID-19 CFR to be associated with CF prevalence, after adjusting for elderly, cancer prevalence, and weighting for the number of tests performed.This study suggests a putative contribution of population genetics of CFTR in understanding the spatial distribution of COVID-19 spread and fatality.

PD09-04 INCREASED FREQUENCY OF CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR GENE CARRIER MUTATIONS IN NON-CBAVD INFERTILE MALES

You have accessJournal of UrologyCME1 May 2022PD09-04 INCREASED FREQUENCY OF CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR GENE CARRIER MUTATIONS IN NON-CBAVD INFERTILE MALES Thomas Garcia, J. Sam Fisher, Kenny Kim, and Larry Lipshultz Thomas GarciaThomas Garcia More articles by this author , J. Sam FisherJ. Sam Fisher More articles by this author , Kenny KimKenny Kim More articles by this author , and Larry LipshultzLarry Lipshultz More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002535.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Homozygous damaging mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is the primary cause of congenital bilateral absence of the vas deferens (CBAVD). In addition, a high incidence of heterozygous damaging mutations in CFTR have long been implicated in infertility in non-CBAVD males. In the present study we sought to identify novel carrier mutations significantly associated with male infertility from a panel of 62 genes causing common congenital disorders including cystic fibrosis. METHODS: Over the most recent 18-month period, our center performed expanded carrier screening on 165 men receiving a variety of urological treatments including treatment for male infertility, hypogonadism, erectile dysfunction, hematuria, Peyronie's disease, scrotal mass, varicocele, vasectomy, and vasovasostomy. Of these 165 men, 59 were being treated for infertility with full semen analysis reports confirming abnormal semen parameters in 72% of these men (azoospermia, oligozoospermia, asthenozoospermia, or oligoasthenozoospermia). RESULTS: Of the 62 genes tested in 165 men, heterozygous carrier mutations were detected in 12 genes in 57 men (Table 1). No published association between these gene mutations and male infertility could be found for any of the genes except for CFTR. For CFTR, we found a significant 2.3-fold (P <0.05) increase in the number of infertile men with CFTR mutations (9 out of 59 men, or 15.3% of men) relative to the number of other men (men treated for other conditions) with CFTR mutations (7 out of 106 men, or 6.6% of men). None of the azoospermic men with CFTR mutations had congenital bilateral absence of the vas deferens. CONCLUSIONS: Our results are consistent with, but more robust than those of previous studies due to the increased number of mutations tested for in our study. CFTR function has been shown in the epididymis, CFTR protein is present in developing germ cells and sperm, and its function contributes to events required for capacitation in the female reproductive tract. In conclusion, these data demonstrate that mutations in CFTR, even heterozygous mutations, are a likely cause of non-CBAVD male factor infertility in at least a subset of men affected by CFTR mutations. Source of Funding: None © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e178 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Thomas Garcia More articles by this author J. Sam Fisher More articles by this author Kenny Kim More articles by this author Larry Lipshultz More articles by this author Expand All Advertisement PDF DownloadLoading ...

Three complex alleles of CFTR gene identified in Lebanese, Egyptian and French population and their potential impact on splicing

Cystic Fibrosis (CF) in Arab Mediterranean countries has a different CFTR mutational profile if compared either to Caucasians or in the Arabian Peninsula. The c.3909C>G (N1303K, p.Asn1303Lys) mutation of the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR). This mutation represents a higher frequency in the Mediterranean countries in association with different polymorphisms or mutations in cis position constituting various complex alleles. N1303K mutation induces many phenotypes, especially pancreatic insufficiency from mild to severe and it is associated in cis with other polymorphisms. The aim of this investigation is therefore to screen complex alleles carrying N1303K mutation among Lebanese, Egyptian and French patients. All exons of the CFTR and their flanking regions were performed by PCR amplification, followed by automated direct DNA sequencing. Two complex alleles are more frequent corresponding to Wild Type and mutated haplotype. Besides that two other very rare complex alleles have been detected, one in Egyptian and French samples, and then another one in Lebanon samples. We have studied their impact on the CFTR mRNA splicing using a minigene strategy. Constructs containing wild-type and mutant CFTR cloned into the pTBNdeI hybride minigene have been expressed in HeLa, HT29 and HEK293 cells. RT-PCR analysis of mRNA using β-globin-specific primers revealed that N1303K and the polymorphisms associated with cis induce weak abnormal splicing and a modification of the quality and the quantity of CFTR protein. These different associations of identified polymorphisms with N1303K in cis could have an impact on the severity of the disease.

CFTR mRNAs with nonsense codons are degraded by the SMG6-mediated endonucleolytic decay\xa0pathway

Approximately 10% of cystic fibrosis patients harbor nonsense mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene which can generate nonsense codons in the CFTR mRNA and subsequently activate the nonsense-mediated decay (NMD) pathway resulting in rapid mRNA degradation. However, it is not known which NMD branches govern the decay of CFTR mRNAs containing nonsense codons. Here we utilize antisense oligonucleotides targeting NMD factors to evaluate the regulation of nonsense codon-containing CFTR mRNAs by the NMD pathway. We observe that CFTR mRNAs with nonsense codons G542X, R1162X, and W1282X, but not Y122X, require UPF2 and UPF3 for NMD. Furthermore, we demonstrate that all evaluated CFTR mRNAs harboring nonsense codons are degraded by the SMG6-mediated endonucleolytic pathway rather than the SMG5-SMG7-mediated exonucleolytic pathway. Finally, we show that upregulation of all evaluated CFTR mRNAs with nonsense codons by NMD pathway inhibition improves outcomes of translational readthrough therapy.

Alterations of mucosa-attached microbiome and epithelial cell numbers in the cystic fibrosis small intestine with implications for intestinal disease

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Defective CFTR leads to accumulation of dehydrated viscous mucus within the small intestine, luminal acidification and altered intestinal motility, resulting in blockage. These changes promote gut microbial dysbiosis, adversely influencing the normal proliferation and differentiation of intestinal epithelial cells. Using Illumina 16S rRNA gene sequencing and immunohistochemistry, we assessed changes in mucosa-attached microbiome and epithelial cell profile in the small intestine of CF mice and a CF patient compared to wild-type mice and non-CF humans. We found increased abundance of pro-inflammatory Escherichia and depletion of beneficial secondary bile-acid producing bacteria in the ileal mucosa-attached microbiome of CFTR-null mice. The ileal mucosa in a CF patient was dominated by a non-aeruginosa Pseudomonas species and lacked numerous beneficial anti-inflammatory and short-chain fatty acid-producing bacteria. In the ileum of both CF mice and a CF patient, the number of absorptive enterocytes, Paneth and glucagon-like peptide 1 and 2 secreting L-type enteroendocrine cells were decreased, whereas stem and goblet cell numbers were increased. These changes in mucosa-attached microbiome and epithelial cell profile suggest that microbiota-host interactions may contribute to intestinal CF disease development with implications for therapy.

Analysis Variants of the CFTR Gene in Iranian Cystic Fibrosis Patients

Background: Cystic fibrosis (CF) is known as one of the most common autosomal recessive disorders, which is caused by mucosal glands. A deficiency in the Cystic fibrosis transmembrane conductance regulator gene (CFTR), which encodes a chloride channel, triggers damage to epithelial cells in respiratory ducts, pancreas, intestine, genital ducts in males, liver system, and sweat glands. Objective: This study aimed to conduct the sequencing of 27 exons of the CFTR gene to screen the spectrum of the variants in patients from all over Iran from different ethnicities. Methods: This study is a descriptive-analytical, that was performed for eleven years from 2010 to 2021. Totally 345 patients were referred to Tehran Medical Genetics Laboratory by specialists. These patients were categorized into four groups. The first group included clinically confirmed patients of CF having clinical features and biochemical abnormalities, plus a positive sweat chloride test. The second group included couples with an alive or deceased child affected. The third group included CBAVD (Congenital bilateral absence of the vas deferens) cases, and the fourth group included prenatal diagnoses who were looking for carrier detection, or her spouse is affected with CBAVD. Results: Fifty-four variants and five deletions were found from 345 patients, the most common frequent variant were c.1521_1523delCTT ([delta]F508) (47 (6.81%)), c.1000C&gt;T (R334W, 31 (4.45%)), c.1911delG (2043delG, 25 (3.62%)), c.2051_2052delAAinsG (2183AA-&gt;G, 15 (2.17%)), c.1624G&gt;T (G542X, 12 (1.74%)), c.1697C&gt;A (A566D, 12 (1.74%)), c.1210-12T [5] (9(1.30%)) and c.3196C&gt;T (R1066C,7 (1.01%)) respectively and frequency of other variants were less than 1%. Deletion in some exons was established by MLPA assay, the most common deletion was c. (53+1_54-1) _ (164+1_165-1) del (CFTRdele2, 7 (1.01%)). Conclusions: This study improves our knowledge concerning carrier analysis and genetic counseling. Also, it helps to develop a cost-effective newborn screening program.

Occurrence of Cystic Fibrosis Transmembrane Conductance Regulator Gene Mutations in Patients with Allergic Bronchopulmonary Aspergillosis Complicating Asthma.

Whether cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations contribute to the high prevalence of allergic bronchopulmonary aspergillosis (ABPA) in India remains unknown. We aimed to evaluate the occurrence of CFTR mutations in subjects with ABPA complicating asthma. We sequenced the CFTR gene using genomic DNA from blood on the Illumina NextSeq500 platform. Before undertaking zygosity analysis by genome analysis toolkit, the known or novel single nucleotide polymorphisms (SNPs) and indels were called. For rigorous analysis, we included only high-quality SNPs (scores > 500) and coverage ranging from 30 × 150x. We included 18, 12, and eight adult participants of ABPA, asthma, and healthy controls, respectively. The frequency of SNPs was higher in asthmatic subjects than ABPA or healthy controls, albeit not statistically significant (9/12 [75%] vs. 11/18 [61.1%] vs. 3/8 [37.5%], p = 0.24). Of the 38 subjects, 23 yielded 50 variants (healthy controls [n = 5], ABPA [n = 22], asthma [n = 23]) corresponding to six SNPs not previously linked with ABPA. Of these, four SNPs (rs213950, rs200735475, rs1800113, and rs1800136) were catalogued in the NCBI database. We identified two novel SNPs (chr7:117250703, chr7:117282655) in four (ABPA [n = 1], asthma [n = 3]) subjects without corresponding reference SNP. Most SNPs (85.5%) were heterozygous. The frequency of SNPs was higher in ABPA subjects with high-attenuation mucus (52.2%) and bronchiectasis (39.1%) than serological ABPA (8.7%). Our study suggests the role of CFTR mutations in the pathogenesis of ABPA. The SNPs in the CFTR gene may contribute to disease severity in ABPA. Larger studies are required to confirm our findings.

Systemic bis-phosphinic acid derivative restores chloride transport in Cystic Fibrosis mice

Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) are responsible for Cystic Fibrosis (CF). The most common CF-causing mutation is the deletion of the 508th amino-acid of CFTR (F508del), leading to dysregulation of the epithelial fluid transport in the airway’s epithelium and the production of a thickened mucus favoring chronic bacterial colonization, sustained inflammation and ultimately respiratory failure. c407 is a bis-phosphinic acid derivative which corrects CFTR dysfunction in epithelial cells carrying the F508del mutation. This study aimed to investigate c407 in vivo activity in the F508del Cftrtm1Eur murine model of CF. Using nasal potential difference measurement, we showed that in vivo administration of c407 by topical, short-term intraperitoneal and long-term subcutaneous route significantly increased the CFTR dependent chloride (Cl−) conductance in F508del Cftrtm1Eur mice. This functional improvement was correlated with a relocalization of F508del-cftr to the apical membrane in nasal epithelial cells. Importantly, c407 long-term administration was well tolerated and in vitro ADME toxicologic studies did not evidence any obvious issue. Our data provide the first in vivo preclinical evidence of c407 efficacy and absence of toxicity after systemic administration for the treatment of Cystic Fibrosis.

Safety of chronic hypertonic bicarbonate inhalation in a cigarette smoke-induced airway irritation guinea pig model

BackgroundCystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) are often associated with airway fluid acidification. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to impaired bicarbonate secretion contributing to CF airway pathology. Chronic cigarette smoke (CS) -the major cause of COPD- is reported to induce acquired CFTR dysfunction underlying airway acidification and inflammation. We hypothesize that bicarbonate-containing aerosols could be beneficial for patients with CFTR dysfunctions. Thus, we investigated the safety of hypertonic sodium bicarbonate (NaHCO3) inhalation in CS-exposed guinea pigs.MethodsAnimals were divided into groups inhaling hypertonic NaCl (8.4%) or hypertonic NaHCO3 (8.4%) aerosol for 8 weeks. Subgroups from each treatment groups were further exposed to CS. Respiratory functions were measured at 0 and after 2, 4, 6 and 8 weeks. After 8 weeks blood tests and pulmonary histopathological assessment were performed.ResultsNeither smoking nor NaHCO3-inhalation affected body weight, arterial and urine pH, or histopathology significantly. NaHCO3-inhalation did not worsen respiratory parameters. Moreover, it normalized the CS-induced transient alterations in frequency, peak inspiratory flow, inspiratory and expiratory times.ConclusionLong-term NaHCO3-inhalation is safe in chronic CS-exposed guinea pigs. Our data suggest that bicarbonate-containing aerosols might be carefully applied to CF patients.

Mutation profiling of the c.1521_1523delCTT (p.Phe508del, F508del) cystic fibrosis transmembrane conductance regulator allele using haplotype-resolved long-read next generation sequencing.

Current approaches to characterize the mutational profile of the cystic fibrosis transmembrane conductance regulator (CFTR) gene are based on targeted mutation analysis or whole gene studies derived from short-read next generation sequencing (NGS). However, these methods lack phasing capability which, in certain scenarios, can provide clinically valuable information. In the present work, we performed near-full length CFTR using Single-Molecule Real-Time Sequencing to produce haplotype-resolved data from both homozygous and heterozygous individuals for mutation c.1521_1523delCTT (p.Phe508del, F508del). This approach utilizes target enrichment of the CFTR gene using biotinylated probes, facilitates multiplexing samples in the same sequencing run, and utilizes fully-automated bioinformatics pipelines for error correction and variant calling. We show a remarkable conservation of F508del haplotype, consistent with the single gene founder effect, as well as diverse mutational profiles in non-F508del alleles. By the same method, 105 single nucleotide polymorphisms (SNPs) exhibiting invariant linkage to F508del CFTR (which better define the founder haplotype) were identified. High level homology between F508del sequences derived from heterozygotes, and those obtained from homozygous individuals, demonstrate accuracy of this method to produce haplotype resolved sequencing. The studies provide a new diagnostic technology for detailed analysis of complex CFTR alleles linked to disease severity.

Fluorescence Quenching of Graphene Quantum Dots by Chloride Ions: A Potential Optical Biosensor for Cystic Fibrosis

Cystic fibrosis is a genetic disorder caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes an ABC transporter-class ion channel protein, resulting in sticky and thick mucus secretion which clogs the airways and leads to respiratory failure and other complications. It also results in higher chloride ions (Clˉ) in body fluids. Cystic fibrosis is generally detected using the sweat chloride test and ion exchange chromatography, which are lab restricted. Therefore, there is a dire need to develop portable assays to monitor circulatory changes (Clˉ ion detection) to detect CF at the point of care. In this work, fluorescence quenching of graphene quantum dots (GQDs) was used as a property of the optical sensor for chloride ion detection. GQDs were synthesized by varying the carbonizing temperature and time, and then their optical and fluorescence (FL) quenching was investigated upon exposure to chloride ions in comparison with different ionic species. GQDs synthesized at 160°C for 50 min were chosen as they displayed the highest fluorescence. The morphological and optical characterization confirmed the preparation of 12–15 nm GQDs, which were amorphous in nature with the peak emission observed at 462 nm when excited at 370 nm. The fluorescence quenching response of GQDs with Clˉ ions displayed linearity up to 100 mM with a correlation coefficient of 0.98 and the lowest detection limit of approximately 10 mM Clˉ ions.

Cross-talk between enhancers, structural elements and activating transcription factors maintains the 3D architecture and expression of the CFTR gene

Robust protocols to examine 3D chromatin structure have greatly advanced knowledge of gene regulatory mechanisms. Here we focus on the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which provides a paradigm for validating models of gene regulation built upon genome-wide analysis. We examine the mechanisms by which multiple cis-regulatory elements (CREs) at the CFTR gene coordinate its expression in intestinal epithelial cells. Using CRISPR/Cas9 to remove CREs, individually and in tandem, followed by assays of gene expression and higher-order chromatin structure (4C-seq), we reveal the cross-talk and dependency of two cell-specific intronic enhancers. The results suggest a mechanism whereby the locus responds when CREs are lost, which may involve activating transcription factors such as FOXA2. Also, by removing the 5′ topologically-associating domain (TAD) boundary, we illustrate its impact on CFTR gene expression and architecture. These data suggest a multi-layered regulatory hierarchy that is highly sensitive to perturbations.

Krüppel-Like Factor 5 Regulates CFTR Expression Through Repression by Maintaining Chromatin Architecture Coupled with Direct Enhancer Activation

Single cell RNA-sequencing has accurately identified cell types within the human airway that express the Cystic Fibrosis Transmembrane Conductance regulator (CFTR) gene. Low abundance CFTR transcripts are seen in many secretory cells, while high levels are restricted to rare pulmonary ionocytes. Here we focus on the mechanisms coordinating basal CFTR expression in the secretory compartment. Cell-selective regulation of CFTR is achieved within its invariant topologically associating domain by the recruitment of cis-regulatory elements (CREs). CRE activity is coordinated by cell-type-selective transcription factors. One such factor, Krüppel-Like Factor 5 (KLF5), profoundly represses CFTR transcript and protein in primary human airway epithelial cells and airway cell lines. Here we reveal the mechanism of action of KLF5 upon the CFTR gene. We find that depletion or ablation of KLF5 from airway epithelial cells changes higher order chromatin structure at the CFTR locus. Critical looping interactions that are required for normal gene expression are altered, the H3K27ac active chromatin mark is redistributed, and CTCF occupancy is modified. However, mutation of a single KLF5 binding site within a pivotal airway cell CRE abolishes CFTR expression. Hence, KLF5 has both direct activating and indirect repressive effects, which together coordinate CFTR expression in the airway.

Cystic Fibrosis: A Review Article

Cystic Fibrosis is considered one of the most common autosomal recessive diseases that is associated with a decrease in the length of age in a Caucasian population. Also, it is considered as one of the most common life-shortening diseases in the white population in the United States. Cystic fibrosis affects around 30,000 people in the United States and more than 80,000 people worldwide. The incidence rate of this disease is 1 out of 3,500 births per year in the white population in the United States, while the incidence rate of the person becoming a carrier is 1:25 in the Caucasian population, the incidence of the disease is 1:2,500. The main cause for this disease is the mutation in Fibrosis Transmembrane Conductance Regulator (CFTR) gene. This disease is considered a life-threatening genetic disease that causes a buildup of thick, viscous mucus secretions in organ systems. Cystic Fibrosis is considered a multiple system disease, but in most cases, the disease gets worse and mortality increases because of respiratory manifestations such as bronchiectasis. Also, pancreatic damage in children is followed by severe wasting, malabsorption, and mortality is one of the recorded observations in children. This article aims to provide a brief introduction and the clinical picture of the disease, Etiology, Pathophysiology, Epidemiology, Nutrition, Prevention, and good practice management advice. Keywords: Cystic Fibrosis, Bronchiectasis, Review Article, Airway Clearance Therapy, Fibrosis Transmembrane Conductance Regulator gene.

Cystic Fibrosis and Sleep Circadian Rhythms.

Cystic fibrosis (CF) is due to a mutation in the cystic fibrosis transmembrane conductance regulator gene (CFTR), which leads to unusual water and chloride secretion across epithelial surfaces. The lungs are responsible for most morbidity, though other organs are frequently affected. Sleep abnormalities have long been recognized in CF. Abnormal ventilation and oxygenation, sinus disease, deconditioning due to muscle weakness and recurrent infections, and inflammation have been thought to play a role in sleep disorders in CF. However, there is evidence that CFTR gene dysregulation can affect circadian rhythms in CF. Early recognition and treatment of circadian rhythms may improve outcomes in CF.

Using Prime Editing and Mesenchymal Stem Cell-Derived Exosomes to Treat Cystic Fibrosis: A Research Protocol

Introduction: Cystic Fibrosis (CF) is a progressive genetic disease that causes the production of thick mucus in the lungs. A mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to a malfunction of the CFTR protein. Current therapies for cystic fibrosis treat the symptoms rather but not the disease source. This study proposes using prime editing in combination with mesenchymal stem cell-derived exosomes (MSCEs) as an alternative treatment for CF. This treatment could potentially (1) correct the CFTR mutation in lung epithelial cells, and (2) regenerate tissue function after damage caused by cystic fibrosis in the lungs. Methods: The MSCEs are obtained from adipose tissue through differential centrifugation and ultracentrifugation. They will then be surface engineered with a low molecular weight polyethylene glycol to help better penetrate the mucus layer and after incubated with the prime editor and liposomes to create the hybrid liposome MSCEs and encapsulate the prime editor. Both in vivo and in vitro experiments with mice models and human lung organoids will be used to test the MSCEs and prime editor. After administration, efficiency of the treatment will be measured through the recombinant protein, FVII (rFVII) and microscopy. Results: The MSCEs will be delivered through nebulization to reach the lung epithelial cells to correct the mutation, leading to the proper expression of the protein in the lungs. The MSCEs will further regenerate damage already caused by the disease. Discussion: If the treatment is effective, we expect to see the production of thinner mucus in the lungs and an increased ability to breathe over time due to the MSCEs. Conclusion: Currently, there is no cure for CF and the lifespan of CF patients is around 44 years. MSCEs and prime editing could be a safe and viable option for treating CF.