FIGURE 3 - available via license: Creative Commons Attribution 4.0 International
Content may be subject to copyright.
CFTR mutation types and CREIPSR-based Gene Editing. CRIPSR/Cas editing approaches rely on the generation of double stranded DNA break
Source publication
Cystic fibrosis (CF) is a life-threatening autosomal-recessive disease caused by mutations in a single gene encoding cystic fibrosis transmembrane conductance regulator (CFTR). CF effects multiple organs, and lung disease is the primary cause of mortality. The median age at death from CF is in the early forties. CF was one of the first diseases to...
Context in source publication
Context 1
... consists of a Cas9 nickase fused with a modified reverse transcriptase and a multifunctional prime editing guide RNA (pegRNA), which directs the fusion complex to the target site for flexible PAM recognition and also encodes an RNA template for editing as a contiguous extension of the gRNA. These CRISPRbased gene editing tools are outlined in Figure 3. CFTR mutation types and CREIPSR-based Gene Editing. ...
Citations
... Ce sont des traitements symptomatiques qui nécessitent une administration à vie, et dont les effets secondaires potentiels à long terme restent incertains. De plus, 10 % des patients ne peuvent pas bénéficier de cette prise en charge thérapeutique de par l'inefficacité sur certaines mutations [7]. La thérapie génique : la solution au problème ? ...
... Cette nanoparticule est composée de polymères de PLGA (poly lactic-co-glycolic acid) contenant une copie d'ADN simple brin donneur corrigée, ainsi qu'une séquence d'acides nucléiques peptidiques PNA (peptide nucleic acid) de structure tail-clamp (tcPNA) ( Figure 1A). Cette molécule tcPNA est composée de bases puriques et pyrimidiques classiques et synthétiques, dont le squelette sucre-phosphate est remplacé par un squelette peptidique, ce qui confère une plus grande stabilité de liaison aux acides nucléiques endo-vecteurs viraux dérivés d'adénovirus a été testée mais n'a permis qu'une correction partielle de l'activité de la protéine CFTR et a engendré une inflammation exacerbée et destructrice [7]. Depuis 2012, l'édition de gènes par la méthode CRISPR/Cas9 est exploitée dans le cadre des maladies génétiques [8]. ...
Le master I ² VB se propose de donner les bases conceptuelles et pratiques des différents aspects de l’infectiologie. Il s’appuie sur une coopération exemplaire entre les équipes de recherche en infectiologie et en immunologie de l’Université de Tours, et celles, entre autres, de l’Unité Infectiologie et Santé Publique (ISP) du Centre INRAE de Tours-Nouzilly, concrétisée par une profonde interaction entre chercheurs et enseignants-chercheurs.
Cette formation aborde aussi bien les aspects fondamentaux et appliqués de l’infectiologie et de l’immunologie allant de l’étude moléculaire des interactions entre le pathogène et son hôte, jusqu’à la conception et la mise sur le marché des produits de la vaccinologie, des biothérapies anti-infectieuses et des anticorps immuno-thérapeutiques.
Le master I ² VB (niveau M1) donne lieu aux parcours ICM, I&B et AcT (niveau M2).
L’option Infectiologie Cellulaire et Moléculaire (ICM) (responsables : Françoise Debierre-Grockiego et Martine Braibant) a pour objectifs de :
former des scientifiques dotés d’une culture générale et technique spécialisée dans les biotechnologies, l’infectiologie, les interactions hôte-pathogène et les mécanismes de la réponse immunitaire anti-infectieuse, contribuant à l’avancée des connaissances scientifiques et à ses applications industrielles, demandes sociétales en forte progression.
former des pharmaciens, médecins, vétérinaires, ingénieurs agronomes aux enjeux actuels de l’infectiologie à la fois dans les domaines fondamentaux et appliqués.
L’option Immunité et biomédicaments (I&B) (responsables : Anne di Tommaso et Isabelle Dimier-Poisson) a pour objectifs de :
former des scientifiques dotés d’une culture générale et technique spécialisée dans les biotechnologies, l’infectiologie, la vaccinologie, les biomédicaments et les biothérapies anti-infectieuses contribuant à l’avancée des connaissances scientifiques et à ses applications industrielles, demandes sociétales en forte progression.
former de jeunes scientifiques, pharmaciens, médecins, vétérinaires, ingénieurs agronomes aux enjeux actuels de l’infectiologie et des biomédicaments à la fois dans les domaines fondamentaux et appliqués.
L’option Anticorps thérapeutiques (AcT) (responsables : Laurie Lajoie et Isabelle Dimier-Poisson) a pour objectifs de :
former des scientifiques dotés d’une culture générale et technique spécialisée dans les biotechnologies, l’immunologie, la cancérologie et les biomédicaments dont les anticorps thérapeutiques, contribuant à l’avancée des connaissances scientifiques et à ses application industrielles et juridiques, demandes sociétales en forte progression.
former de jeunes scientifiques, pharmaciens, médecins, vétérinaires, ingénieurs agronomes aux enjeux actuels de l’infectiologie et des biomédicaments à la fois dans les domaines fondamentaux et appliqués.
... Overview of Balanced Diet for CF infected patient[46][47][48][49][50]. ment in forced expiratory volume in 1 second (FEV1) following treatment with tgAAVCF[39].CF gene therapy has undergone significant advancements, transitioning from initial attempts with rAd vectors to promising trials utilizing rAAV vectors. While Phase II trials with tgAAVCF showed partial success in improving lung function for some patients, challenges persist in achieving consistent therapeutic outcomes. ...
Cystic fibrosis (CF) is a genetic disorder inherited in an autosomal recessive pattern, arising from mutations in the gene responsible for encoding the cystic fibrosis transmembrane conductance regulator (CFTR). While CF primarily manifests with lung disease, it also affects other organs such as the pancreas, intestines, and skin, often utilized for early diagnostic testing. In CF, lung disease represents the primary cause of morbidity and mortality. Despite being largely infectious in nature, the associated inflammation is severe and ineffective in clearing pathogens. This persistent, high-intensity inflammation leads to structural damage in the airways and compromised lung function, ultimately culminating in respiratory failure and death. Autopsy cases of CF reveal multiorgan involvement, with some rarely observed changes. Defective inflammatory responses associated with CFTR deficiency include dysregulation of both innate and acquired immunity, abnormalities in cell membrane lipids, signaling defects in various transcription factors, and altered responses of kinases and toll-like receptors. Recent advancements in understanding the molecular mechanisms underlying CF have facilitated the development of CFTR modulator therapies, marking significant progress in CF treatment. These targeted therapies represent a shift towards precision medicine and are anticipated to further enhance survival rates in the foreseeable future.
... Despite a lack of success in the gene therapy clinical trials performed to date, recombinant adeno-associated virus (rAAV) appears to be the most promising human gene therapy vector. 10 However, a significant limitation remains potential immune-related adverse events such as acute respiratory distress syndrome (ARDS) when given at higher doses. 11 The emergence of cell-based gene therapies opens a whole new raft of possibilities. ...
... For example, a gene therapy to treat cystic fibrosis would optimally be designed to deliver the protein replacement mRNA or gene editor, required to restore function, to multiple cell types in the lung. This list of cells lining the human superficial airway epithelium would include secretory cells, and particularly goblet cells and club cells that are located in the tracheobronchial region, basal cells, and ionocytes ( Fig. 2b) (Sui, 2022;Carraro, 2021). Ideally, thus, an optimized nanocarrier would maximize the deposited RNA dose to these cells (Martin, 2021). ...
... The advent of CFTR modulators has undoubtedly changed the clinical course of CF, leading to significant improvements in the lives of a large proportion of people with CF, whose life expectancy has increased from 5 years in 1960 to over 50 years. However, the use of these CFTR drugs is limited to certain mutations, and almost 15% of people with CF remain without any CFTR-targeted therapy [33,34]. The encouraging results obtained so far have motivated further efforts to discover new correctors to be used alone or in combination with already existing therapeutics to treat a wider cohort of people with CF. ...
... To guide a future feasible and reliable rational design process of new derivatives, in this study, we combined computational analyses with different biochemical approaches to identify the putative mechanism of action of 2a, 7a, and 7m, the most promising hybrids The advent of CFTR modulators has undoubtedly changed the clinical course of CF, leading to significant improvements in the lives of a large proportion of people with CF, whose life expectancy has increased from 5 years in 1960 to over 50 years. However, the use of these CFTR drugs is limited to certain mutations, and almost 15% of people with CF remain without any CFTR-targeted therapy [33,34]. The encouraging results obtained so far have motivated further efforts to discover new correctors to be used alone or in combination with already existing therapeutics to treat a wider cohort of people with CF. ...
Cystic fibrosis (CF), the most common autosomal recessive fatal genetic disease in the Caucasian population, is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel that regulates salt and water transport across a variety of secretory epithelia. Deletion of phenylalanine at position 508, F508del, the most common CF-causing mutation, destabilises the CFTR protein, causing folding and trafficking defects that lead to a dramatic reduction in its functional expression. Small molecules called correctors have been developed to rescue processing-defective F508del CFTR. We have combined in silico and in vitro approaches to investigate the mechanism of action and potential as CFTR correctors of three hybrid derivatives (2a, 7a, and 7m) obtained by merging the amino-arylthiazole core with the benzodioxole carboxamide moiety characterising the corrector lumacaftor. Molecular modelling analyses suggested that the three hybrids interact with a putative region located at the MSD1/NBD1 interface. Biochemical analyses confirmed these results, showing that the three molecules affect the expression and stability of the F508del NBD1. Finally, the YFP assay was used to evaluate the influence of the three hybrid derivatives on F508del CFTR function, assessing that their effect is additive to that of the correctors VX661 and VX445. Our study shows that the development and testing of optimised compounds targeting different structural and functional defects of mutant CFTR is the best strategy to provide more effective correctors that could be used alone or in combination as a valuable therapeutic option to treat an even larger cohort of people affected by CF.
... However, despite the great potential and the enormous advances made in RNA-based and CRISPR/Cas9-based therapeutic strategies, there are still several issues to overcome, particularly in the field of airway diseases. Among these are the following: being able to find an efficient transgene vector that does not create strong immunogenicity and must be able to penetrate the airways mucus layer, targeting the specific cell type in the complex respiratory system, and maintaining an effect over time that does not cause toxicity [48][49][50]. To date, many trials using RNA-based and DNA-based therapeutics have been done or are in progress. ...
ATP12A encodes the catalytic subunit of the non-gastric proton pump, which is expressed in many epithelial tissues and mediates the secretion of protons in exchange for potassium ions. In the airways, ATP12A-dependent proton secretion contributes to complex mechanisms regulating the composition and properties of the fluid and mucus lining the respiratory epithelia, which are essential to maintain the airway host defense and the respiratory health. Increased expression and activity of ATP12A in combination with the loss of other balancing activities, such as the bicarbonate secretion mediated by CFTR, leads to excessive acidification of the airway surface liquid and mucus dysfunction, processes that play relevant roles in the pathogenesis of cystic fibrosis and other chronic inflammatory respiratory disorders. In this review, we summarize the findings dealing with ATP12A expression, function, and modulation in the airways, which led to the consideration of ATP12A as a potential therapeutic target for the treatment of cystic fibrosis and other airway diseases; we also highlight the current advances and gaps regarding the development of therapeutic strategies aimed at ATP12A inhibition.
... This limitation can be minimized by transient transfection and optimizing the effector molecule concentration. Indeed, a recent CRISPR-meditated approach to cystic fibrosis therapy approach was based on transient application of a modified CRISPR system to edit the gene encoding cystic fibrosis transmembrane conductance regulator [28]. dCas13-ADAR fusions for site directed mutagenesis is a powerful technique with higher RNA editing efficiency [7]. ...
Adenosine deaminases acting on RNA (ADARs) have double-stranded RNA binding domains and a deaminase domain (DD). We used the MS2 system and specific guide RNAs to direct ADAR1-DD to target adenosines in the mRNA encoding-enhanced green fluorescence protein. Using this system in transfected HEK-293 cells, we evaluated the effects of changing the length and position of the guide RNA on the efficiency of conversion of amber (TAG) and ochre (TAA) stop codons to tryptophan (TGG) in the target. Guide RNAs of 19, 21 and 23 nt were positioned upstream and downstream of the MS2-RNA, providing a total of six guide RNAs. The upstream guide RNAs were more functionally effective than the downstream guide RNAs, with the following hierarchy of efficiency: 21 nt > 23 nt > 19 nt. The highest editing efficiency was 16.6%. Off-target editing was not detected in the guide RNA complementary region but was detected 50 nt downstream of the target. The editing efficiency was proportional to the amount of transfected deaminase but inversely proportional to the amount of the transfected guide RNA. Our results suggest that specific RNA editing requires precise optimization of the ratio of enzyme, guide RNA, and target RNA.
... These variants are largely insensitive to (and ineligible for) clinically approved CFTR modulators, thus representing a significant unmet medical need. Recent emphasis has been placed on development of gene transfer, gene editing, and mRNA therapies (reviewed in (Da Silva Sanchez et al., 2020;Egan, 2021;Harrison, 2022;Sui et al., 2022)). Here, we briefly summarize small molecule and short nucleotide-based approaches that directly interact with translation machinery to circumvent cellular disposal of aberrant CFTR transcripts and associated polypeptides ( Table 1). ...
Cystic fibrosis (CF) is an autosomal recessive disease impacting ∼100,000 people worldwide. This lethal disorder is caused by mutation of the CF transmembrane conductance regulator (CFTR) gene, which encodes an ATP-binding cassette-class C protein. More than 2,100 variants have been identified throughout the length of CFTR. These defects confer differing levels of severity in mRNA and/or protein synthesis, folding, gating, and turnover. Drug discovery efforts have resulted in recent development of modulator therapies that improve clinical outcomes for people living with CF. However, a significant portion of the CF population has demonstrated either no response and/or adverse reactions to small molecules. Additional therapeutic options are needed to restore underlying genetic defects for all patients, particularly individuals carrying rare or refractory CFTR variants. Concerted focus has been placed on rescuing variants that encode truncated CFTR protein, which also harbor abnormalities in mRNA synthesis and stability. The current mini-review provides an overview of CFTR mRNA features known to elicit functional consequences on final protein conformation and function, including considerations for RNA-directed therapies under investigation. Alternative exon usage in the 5′-untranslated region, polypyrimidine tracts, and other sequence elements that influence splicing are discussed. Additionally, we describe mechanisms of CFTR mRNA decay and post-transcriptional regulation mediated through interactions with the 3′-untranslated region (e.g. poly-uracil sequences, microRNAs). Contributions of synonymous single nucleotide polymorphisms to CFTR transcript utilization are also examined. Comprehensive understanding of CFTR RNA biology will be imperative for optimizing future therapeutic endeavors intended to address presently untreatable forms of CF.
... Overall, gene-editing tools could represent a strategy to achieve permanent correction by replacing the CFTR faulty gene with a healthy gene. In this scenario, several other strategies are being developed including RNA-based strategies such as mRNA, t-RNA, and antisense oligonucleotides [73][74][75][76]; however, fewer clinical trials for CF RNA-based therapy exist. Nevertheless, the introduction of the correct version of CFTR mRNA by the delivery system ensures its use not only for nonsense mutations, but for all types of variants. ...
Cystic fibrosis (CF) is a high-prevalence disease characterized by significant lung remodeling, responsible for high morbidity and mortality worldwide. The lung structural changes are partly due to proteolytic activity associated with inflammatory cells such as neutrophils and macrophages. Matrix metalloproteases (MMPs) are the major proteases involved in CF, and recent literature data focused on their potential role in the pathogenesis of the disease. In fact, an imbalance of proteases and antiproteases was observed in CF patients, resulting in dysfunction of protease activity and loss of lung homeostasis. Currently, many steps forward have been moved in the field of pharmacological treatment with the recent introduction of triple-combination therapy targeting the CFTR channel. Despite CFTR modulator therapy potentially being effective in up to 90% of patients with CF, there are still patients who are not eligible for the available therapies. Here, we introduce experimental drugs to provide updates on therapy evolution regarding a proportion of CF non-responder patients to current treatment, and we summarize the role of MMPs in pathogenesis and as future therapeutic targets of CF.
A BSTRACT
Cystic fibrosis (CF) is a life-threatening genetic disorder caused by mutations in the CFTR gene. This leads to a defective protein that impairs chloride transport, resulting in thick mucus buildup and chronic inflammation in the airways. The review discusses current and future therapeutic approaches for CFTR dysfunction and airway dysbiosis in the era of personalized medicine. Personalized medicine has revolutionized CF treatment with the advent of CFTR modulator therapies that target specific genetic mutations. These therapies have significantly improved patient outcomes, slowing disease progression, and enhancing quality of life. It also highlights the growing recognition of the airway microbiome’s role in CF pathogenesis and discusses strategies to modulate the microbiome to further improve patient outcomes. This review discusses various therapeutic approaches for cystic fibrosis (CFTR) mutations, including adenovirus gene treatments, nonviral vectors, CRISPR/cas9 methods, RNA replacement, antisense-oligonucleotide-mediated DNA-based therapies, and cell-based therapies. It also introduces airway dysbiosis with CF and how microbes influence the lungs. The review highlights the importance of understanding the cellular and molecular causes of CF and the development of personalized medicine to improve quality of life and health outcomes.
