Prime editing functionally corrects cystic fibrosis-causing CFTR mutations in human organoids and airway epithelial cells.

Prime editing is a recent, CRISPR-derived genome editing technology capable of introducing precise nucleotide substitutions, insertions, and deletions. Here, we present prime editing approaches to correct L227R- and N1303K-CFTR, two mutations that cause cystic fibrosis and are not eligible for current market-approved modulator therapies. We show that, upon DNA correction of the CFTR gene, the complex glycosylation, localization, and, most importantly, function of the CFTR protein are restored in HEK293T and 16HBE cell lines. These findings were subsequently validated in patient-derived rectal organoids and human nasal epithelial cells. Through analysis of predicted and experimentally identified candidate off-target sites in primary stem cells, we confirm previous reports on the high prime editor (PE) specificity and its potential for a curative CF gene editing therapy. To facilitate future screening of genetic strategies in a translational CF model, a machine learning algorithm was developed for dynamic quantification of CFTR function in organoids (DETECTOR: "detection of targeted editing of CFTR in organoids").

Nanoblades allow high-level genome editing in murine and human organoids.

Genome engineering has become more accessible thanks to the CRISPR-Cas9 gene-editing system. However, using this technology in synthetic organs called "organoids" is still very inefficient. This is due to the delivery methods for the CRISPR-Cas9 machinery, which include electroporation of CRISPR-Cas9 DNA, mRNA, or ribonucleoproteins containing the Cas9-gRNA complex. However, these procedures are quite toxic for the organoids. Here, we describe the use of the "nanoblade (NB)" technology, which outperformed by far gene-editing levels achieved to date for murine- and human tissue-derived organoids. We reached up to 75% of reporter gene knockout in organoids after treatment with NBs. Indeed, high-level NB-mediated knockout for the androgen receptor encoding gene and the cystic fibrosis transmembrane conductance regulator gene was achieved with single gRNA or dual gRNA containing NBs in murine prostate and colon organoids. Likewise, NBs achieved 20%-50% gene editing in human organoids. Most importantly, in contrast to other gene-editing methods, this was obtained without toxicity for the organoids. Only 4 weeks are required to obtain stable gene knockout in organoids and NBs simplify and allow rapid genome editing in organoids with little to no side effects including unwanted insertion/deletions in off-target sites thanks to transient Cas9/RNP expression.

Functional restoration of a CFTR splicing mutation through RNA delivery of CRISPR adenine base editor.

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The 2789+5G>A CFTR mutation is a quite frequent defect causing an aberrant splicing and a non-functional CFTR protein. Here we used a CRISPR adenine base editing (ABE) approach to correct the mutation in the absence of DNA double-strand breaks (DSB). To select the strategy, we developed a minigene cellular model reproducing the 2789+5G>A splicing defect. We obtained up to 70% editing in the minigene model by adapting the ABE to the PAM sequence optimal for targeting 2789+5G>A with a SpCas9-NG (NG-ABE). Nonetheless, the on-target base correction was accompanied by secondary (bystander) A-to-G conversions in nearby nucleotides, which affected the wild-type CFTR splicing. To decrease the bystander edits, we used a specific ABE (NG-ABEmax), which was delivered as mRNA. The NG-ABEmax RNA approach was validated in patient-derived rectal organoids and bronchial epithelial cells showing sufficient gene correction to recover the CFTR function. Finally, in-depth sequencing revealed high editing precision genome-wide and allele-specific correction. Here we report the development of a base editing strategy to precisely repair the 2789+5G>A mutation resulting in restoration of the CFTR function, while reducing bystander and off-target activities.

Severity of the S1251N allele in cystic fibrosis is affected by the presence of the F508C variant in cis.

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.

One Size Does Not Fit All: The Past, Present and Future of Cystic Fibrosis Causal Therapies.

Cystic fibrosis (CF) is the most common monogenic disorder, caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Over the last 30 years, tremendous progress has been made in understanding the molecular basis of CF and the development of treatments that target the underlying defects in CF. Currently, a highly effective CFTR modulator treatment (Kalydeco™/Trikafta™) is available for 90% of people with CF. In this review, we will give an extensive overview of past and ongoing efforts in the development of therapies targeting the molecular defects in CF. We will discuss strategies targeting the CFTR protein (i.e., CFTR modulators such as correctors and potentiators), its cellular environment (i.e., proteostasis modulation, stabilization at the plasma membrane), the CFTR mRNA (i.e., amplifiers, nonsense mediated mRNA decay suppressors, translational readthrough inducing drugs) or the CFTR gene (gene therapies). Finally, we will focus on how these efforts can be applied to the 15% of people with CF for whom no causal therapy is available yet.

Novel CFTR modulator combinations maximise rescue of G85E and N1303K in rectal organoids.

Introduction: Cystic fibrosis (CF) is a severe monogenic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Several types of CFTR modulators (correctors/potentiators) have been developed to overcome protein dysfunction associated with these mutations. CFTR modulator therapy is now available for the major CF-causing mutations; however, 10% of people with CF remain without causal treatments. By combining investigational and market-approved CFTR modulators, we aimed to maximise functional rescue of iva-, luma- and tezacaftor refractory mutants G85E and N1303K. Methods: We used the well-established forskolin-induced swelling (FIS) in primary rectal organoids to assess responses to different CFTR corrector and potentiator types. The FIS analysis was performed with brightfield microscopy, allowing both 1-h and 24-h follow-up. Corrector and potentiator activity of elexacaftor was investigated. Results: For G85E, maximal rescue was observed by a combination of elexacaftor and corr4a. For N1303K, the quadruple combination teza-elexa-ivacaftor with apigenin was required to obtain a rescue similar to that of luma-ivacaftor rescued F508del. Elexacaftor rescued G85E and N1303K by different mechanisms, with chronic corrector effects on G85E and acute potentiation of N1303K only in the presence of ivacaftor. Synergy in N1303K rescue for iva-elexacaftor and apigenin suggests at least three potentiator mechanisms for this mutant. 24-h FIS identified ivacaftor as the main CFTR modulator for N1303K and elexacaftor and apigenin as co-potentiators. Conclusions: Novel combinations of CFTR modulators can further improve functional rescue of G85E and N1303K in rectal organoids, although for N1303K, more effective CFTR modulators are still needed.

Rewriting CFTR to cure cystic fibrosis.

Cystic fibrosis (CF) is an autosomal recessive monogenic disease caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. Although F508del is the most frequent mutation, there are in total 360 confirmed disease-causing CFTR mutations, impairing CFTR production, function and stability. Currently, the only causal treatments available are CFTR correctors and potentiators that directly target the mutant protein. While these pharmacological advances and better symptomatic care have improved life expectancy of people with CF, none of these treatments provides a cure. The discovery and development of programmable nucleases, in particular CRISPR nucleases and derived systems, rekindled the field of CF gene therapy, offering the possibility of a permanent correction of the CFTR gene. In this review we will discuss different strategies to restore CFTR function via gene editing correction of CFTR mutations or enhanced CFTR expression, and address how best to deliver these treatments to target cells.

On the Corner of Models and Cure: Gene Editing in Cystic Fibrosis.

Cystic fibrosis (CF) is a severe genetic disease for which curative treatment is still lacking. Next generation biotechnologies and more efficient cell-based and in vivo disease models are accelerating the development of novel therapies for CF. Gene editing tools, like CRISPR-based systems, can be used to make targeted modifications in the genome, allowing to correct mutations directly in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. Alternatively, with these tools more relevant disease models can be generated, which in turn will be invaluable to evaluate novel gene editing-based therapies for CF. This critical review offers a comprehensive description of currently available tools for genome editing, and the cell and animal models which are available to evaluate them. Next, we will give an extensive overview of proof-of-concept applications of gene editing in the field of CF. Finally, we will touch upon the challenges that need to be addressed before these proof-of-concept studies can be translated towards a therapy for people with CF.

Transcriptomic analysis of CFTR-impaired endothelial cells reveals a pro-inflammatory phenotype.

Cystic fibrosis (CF) is a life-threatening disorder characterised by decreased pulmonary mucociliary and pathogen clearance, and an exaggerated inflammatory response leading to progressive lung damage. CF is caused by bi-allelic pathogenic variants of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel. CFTR is expressed in endothelial cells (ECs) and EC dysfunction has been reported in CF patients, but a role for this ion channel in ECs regarding CF disease progression is poorly described.We used an unbiased RNA sequencing approach in complementary models of CFTR silencing and blockade (by the CFTR inhibitor CFTRinh-172) in human ECs to characterise the changes upon CFTR impairment. Key findings were further validated in vitro and in vivo in CFTR-knockout mice and ex vivo in CF patient-derived ECs.Both models of CFTR impairment revealed that EC proliferation, migration and autophagy were downregulated. Remarkably though, defective CFTR function led to EC activation and a persisting pro-inflammatory state of the endothelium with increased leukocyte adhesion. Further validation in CFTR-knockout mice revealed enhanced leukocyte extravasation in lung and liver parenchyma associated with increased levels of EC activation markers. In addition, CF patient-derived ECs displayed increased EC activation markers and leukocyte adhesion, which was partially rescued by the CFTR modulators VX-770 and VX-809.Our integrated analysis thus suggests that ECs are no innocent bystanders in CF pathology, but rather may contribute to the exaggerated inflammatory phenotype, raising the question of whether normalisation of vascular inflammation might be a novel therapeutic strategy to ameliorate the disease severity of CF.

Phenotyping of Rare CFTR Mutations Reveals Distinct Trafficking and Functional Defects.

Background. The most common CFTR mutation, F508del, presents with multiple cellular defects. However, the possible multiple defects caused by many rarer CFTR mutations are not well studied. We investigated four rare CFTR mutations E60K, G85E, E92K and A455E against well-characterized mutations, F508del and G551D, and their responses to corrector VX-809 and/or potentiator VX-770. Methods. Using complementary assays in HEK293T stable cell lines, we determined maturation by Western blotting, trafficking by flow cytometry using extracellular 3HA-tagged CFTR, and function by halide-sensitive YFP quenching. In the forskolin-induced swelling assay in intestinal organoids, we validated the effect of tagged versus endogenous CFTR. Results. Treatment with VX-809 significantly restored maturation, PM localization and function of both E60K and E92K. Mechanistically, VX-809 not only raised the total amount of CFTR, but significantly increased the traffic efficiency, which was not the case for A455E. G85E was refractory to VX-809 and VX-770 treatment. Conclusions. Since no single model or assay allows deciphering all defects at once, we propose a combination of phenotypic assays to collect rapid and early insights into the multiple defects of CFTR variants.