In vitro modulator responsiveness of 655 CFTR variants found in people with cystic fibrosis.

BACKGROUND: In 2017, the US Food and Drug Administration initiated expansion of drug labels for the treatment of cystic fibrosis (CF) to include CF transmembrane conductance regulator (CFTR) gene variants based on in vitro functional studies. This study aims to identify CFTR variants that result in increased chloride (Cl-) transport function by the CFTR protein after treatment with the CFTR modulator combination elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA). These data may benefit people with CF (pwCF) who are not currently eligible for modulator therapies. METHODS: Plasmid DNA encoding 655 CFTR variants and wild-type (WT) CFTR were transfected into Fisher Rat Thyroid cells that do not natively express CFTR. After 24 h of incubation with control or TEZ and ELX, and acute addition of IVA, CFTR function was assessed using the transepithelial current clamp conductance assay. Each variant's forskolin/cAMP-induced baseline Cl- transport activity, responsiveness to IVA alone, and responsiveness to the TEZ/ELX/IVA combination were measured in three different laboratories. Western blots were conducted to evaluate CFTR protein maturation and complement the functional data. RESULTS AND CONCLUSIONS: 253 variants not currently approved for CFTR modulator therapy showed low baseline activity (<10 % of normal CFTR Cl- transport activity). For 152 of these variants, treatment with ELX/TEZ/IVA improved the Cl- transport activity by ≥10 % of normal CFTR function, which is suggestive of clinical benefit. ELX/TEZ/IVA increased CFTR function by ≥10 percentage points for an additional 140 unapproved variants with ≥10 % but <50 % of normal CFTR function at baseline. These findings significantly expand the number of rare CFTR variants for which ELX/TEZ/IVA treatment should result in clinical benefit.

A small molecule that induces translational readthrough of CFTR nonsense mutations by eRF1 depletion.

Premature termination codons (PTCs) prevent translation of a full-length protein and trigger nonsense-mediated mRNA decay (NMD). Nonsense suppression (also termed readthrough) therapy restores protein function by selectively suppressing translation termination at PTCs. Poor efficacy of current readthrough agents prompted us to search for better compounds. An NMD-sensitive NanoLuc readthrough reporter was used to screen 771,345 compounds. Among the 180 compounds identified with readthrough activity, SRI-37240 and its more potent derivative SRI-41315, induce a prolonged pause at stop codons and suppress PTCs associated with cystic fibrosis in immortalized and primary human bronchial epithelial cells, restoring CFTR expression and function. SRI-41315 suppresses PTCs by reducing the abundance of the termination factor eRF1. SRI-41315 also potentiates aminoglycoside-mediated readthrough, leading to synergistic increases in CFTR activity. Combining readthrough agents that target distinct components of the translation machinery is a promising treatment strategy for diseases caused by PTCs.

Chemical modifications of adenine base editor mRNA and guide RNA expand its application scope.

CRISPR-Cas9-associated base editing is a promising tool to correct pathogenic single nucleotide mutations in research or therapeutic settings. Efficient base editing requires cellular exposure to levels of base editors that can be difficult to attain in hard-to-transfect cells or in vivo. Here we engineer a chemically modified mRNA-encoded adenine base editor that mediates robust editing at various cellular genomic sites together with moderately modified guide RNA, and show its therapeutic potential in correcting pathogenic single nucleotide mutations in cell and animal models of diseases. The optimized chemical modifications of adenine base editor mRNA and guide RNA expand the applicability of CRISPR-associated gene editing tools in vitro and in vivo.

Nonsense-mediated RNA Decay Pathway Inhibition Restores Expression and Function of W1282X CFTR.

The recessive genetic disease cystic fibrosis (CF) is caused by loss-of-function mutations in the CFTR (CF transmembrane conductance regulator) gene. Approximately 10% of patients with CF have at least one allele with a nonsense mutation in CFTR. Nonsense mutations generate premature termination codons that can subject mRNA transcripts to rapid degradation through the nonsense-mediated mRNA decay (NMD) pathway. Currently, there are no approved therapies that specifically target nonsense mutations in CFTR. Here, we identified antisense oligonucleotides (ASOs) that target the NMD factor SMG1 to inhibit the NMD pathway, and determined their effects on the W1282X CFTR mutation. First, we developed and validated two in vitro models of the W1282X CFTR mutation. Next, we treated these cells with antisense oligonucleotides to inhibit NMD and measured the effects of these treatments on W1282X expression and function. SMG1-ASO-mediated NMD inhibition upregulated the RNA, protein, and surface-localized protein expression of the truncated W1282X gene product. Additionally, these ASOs increased the CFTR chloride channel function in cells homozygous for the W1282X mutation. Our approach suggests a new therapeutic strategy for patients harboring nonsense mutations and may be beneficial as a single agent in patients with CF and the W1282X mutation.

CFTR modulator theratyping: Current status, gaps and future directions.

BACKGROUND: New drugs that improve the function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein with discreet disease-causing variants have been successfully developed for cystic fibrosis (CF) patients. Preclinical model systems have played a critical role in this process, and have the potential to inform researchers and CF healthcare providers regarding the nature of defects in rare CFTR variants, and to potentially support use of modulator therapies in new populations. METHODS: The Cystic Fibrosis Foundation (CFF) assembled a workshop of international experts to discuss the use of preclinical model systems to examine the nature of CF-causing variants in CFTR and the role of in vitro CFTR modulator testing to inform in vivo modulator use. The theme of the workshop was centered on CFTR theratyping, a term that encompasses the use of CFTR modulators to define defects in CFTR in vitro, with application to both common and rare CFTR variants. RESULTS: Several preclinical model systems were identified in various stages of maturity, ranging from the expression of CFTR variant cDNA in stable cell lines to examination of cells derived from CF patients, including the gastrointestinal tract, the respiratory tree, and the blood. Common themes included the ongoing need for standardization, validation, and defining the predictive capacity of data derived from model systems to estimate clinical outcomes from modulator-treated CF patients. CONCLUSIONS: CFTR modulator theratyping is a novel and rapidly evolving field that has the potential to identify rare CFTR variants that are responsive to approved drugs or drugs in development.

Isogenic cell models of cystic fibrosis-causing variants in natively expressing pulmonary epithelial cells.

BACKGROUND: Assessment of approved drugs and developmental drug candidates for rare cystic fibrosis (CF)-causing variants of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) requires abundant material from relevant models. METHODS: Isogenic cell lines harboring CFTR variants in the native genomic context were created through the development and utilization of a footprint-less, CRISPR/Cas9 gene editing pipeline in 16HBE14o- immortalized bronchial epithelial cells. RESULTS: Isogenic, homozygous cell lines for three CFTR variants (F508del and the two most common CF-causing nonsense variants, G542X and W1282X) were established and characterized. The F508del model recapitulates the known molecular pathology and pharmacology. The two models of nonsense variants (G542X and W1282X) are sensitive to Nonsense Mediated mRNA Decay (NMD) and responsive to reference compounds that inhibit NMD and promote ribosomal readthrough. CONCLUSIONS: We present a versatile, efficient gene editing pipeline that can be used to create CFTR variants in the native genomic context and the utilization of this pipeline to create homozygous cell models for the CF-causing variants F508del, G542X, and W1282X. The resulting cell lines provide a virtually unlimited source of material with specific pathogenic mutations that can be used in a variety of assays, including functional assays.

A revised airway epithelial hierarchy includes CFTR-expressing ionocytes.

The airways of the lung are the primary sites of disease in asthma and cystic fibrosis. Here we study the cellular composition and hierarchy of the mouse tracheal epithelium by single-cell RNA-sequencing (scRNA-seq) and in vivo lineage tracing. We identify a rare cell type, the Foxi1+ pulmonary ionocyte; functional variations in club cells based on their location; a distinct cell type in high turnover squamous epithelial structures that we term 'hillocks'; and disease-relevant subsets of tuft and goblet cells. We developed 'pulse-seq', combining scRNA-seq and lineage tracing, to show that tuft, neuroendocrine and ionocyte cells are continually and directly replenished by basal progenitor cells. Ionocytes are the major source of transcripts of the cystic fibrosis transmembrane conductance regulator in both mouse (Cftr) and human (CFTR). Knockout of Foxi1 in mouse ionocytes causes loss of Cftr expression and disrupts airway fluid and mucus physiology, phenotypes that are characteristic of cystic fibrosis. By associating cell-type-specific expression programs with key disease genes, we establish a new cellular narrative for airways disease.

Partial rescue of F508del-cystic fibrosis transmembrane conductance regulator channel gating with modest improvement of protein processing, but not stability, by a dual-acting small molecule.

BACKGROUND AND PURPOSE: Rescue of F508del-cystic fibrosis (CF) transmembrane conductance regulator (CFTR), the most common CF mutation, requires small molecules that overcome protein processing, stability and channel gating defects. Here, we investigate F508del-CFTR rescue by CFFT-004, a small molecule designed to independently correct protein processing and channel gating defects. EXPERIMENTAL APPROACH: Using CFTR-expressing recombinant cells and CF patient-derived bronchial epithelial cells, we studied CFTR expression by Western blotting and channel gating and stability with the patch-clamp and Ussing chamber techniques. KEY RESULTS: Chronic treatment with CFFT-004 improved modestly F508del-CFTR processing, but not its plasma membrane stability. By contrast, CFFT-004 rescued F508del-CFTR channel gating better than C18, an analogue of the clinically used CFTR corrector lumacaftor. Subsequent acute addition of CFFT-004, but not C18, potentiated F508del-CFTR channel gating. However, CFFT-004 was without effect on A561E-CFTR, a CF mutation with a comparable mechanism of CFTR dysfunction as F508del-CFTR. To investigate the mechanism of action of CFFT-004, we used F508del-CFTR revertant mutations. Potentiation by CFFT-004 was unaffected by revertant mutations, but correction was abolished by the revertant mutation G550E. These data suggest that correction, but not potentiation, by CFFT-004 might involve nucleotide-binding domain 1 of CFTR. CONCLUSIONS AND IMPLICATIONS: CFFT-004 is a dual-acting small molecule with independent corrector and potentiator activities that partially rescues F508del-CFTR in recombinant cells and native airway epithelia. The limited efficacy and potency of CFFT-004 suggests that combinations of small molecules targeting different defects in F508del-CFTR might be a more effective therapeutic strategy than a single agent.

High-Throughput Screening for Readthrough Modulators of CFTR PTC Mutations.

Cystic fibrosis (CF) is a hereditary disease caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). A large number of nearly 2000 reported mutations, including the premature termination codon (PTC) mutations, urgently require new and personalized medicines. We have developed cell-based assays for readthrough modulators of CFTR PTC mutations (or nonsense mutation suppressors), based on the trafficking and surface expression of CFTR. Approximately 85,000 compounds have been screened for two PTC mutations (Y122X and W1282X). The hit rates at the threshold of 50% greater than vehicle response are 2% and 1.4% for CFTR Y122X and CFTR W1282X, respectively. The overlap of the two hit sets at this stringent hit threshold is relatively small. Only ~28% of the hits from the W1282X screen were also hits in the Y122X screen. The overlap increases to ~50% if compounds are included that in the second screen achieve only a less stringent hit criterion, that is, horseradish peroxidase (HRP) activity greater than three standard deviations above the mean of the vehicle. Our data suggest that personalization may not need to address individual genotypes, but that patients with different CFTR PTC mutations could benefit from the same medicines.

Dual SMAD Signaling Inhibition Enables Long-Term Expansion of Diverse Epithelial Basal Cells.

Functional modeling of many adult epithelia is limited by the difficulty in maintaining relevant stem cell populations in culture. Here, we show that dual inhibition of SMAD signaling pathways enables robust expansion of primary epithelial basal cell populations. We find that TGFß/BMP/SMAD pathway signaling is strongly activated in luminal and suprabasal cells of several epithelia, but suppressed in p63+ basal cells. In airway epithelium, SMAD signaling promotes differentiation, and its inhibition leads to stem cell hyperplasia. Using dual SMAD signaling inhibition in a feeder-free culture system, we have been able to expand airway basal stem cells from multiple species. Expanded cells can produce functional airway epithelium physiologically responsive to clinically relevant drugs, such as CFTR modulators. This approach is effective for the clonal expansion of single human cells and for basal cell populations from epithelial tissues from all three germ layers and therefore may be broadly applicable for modeling of epithelia.

A novel approach to recovery of function of mutant proteins by slowing down translation.

Protein homeostasis depends on a balance of translation, folding, and degradation. Here, we demonstrate that mild inhibition of translation results in a dramatic and disproportional reduction in production of misfolded polypeptides in mammalian cells, suggesting an improved folding of newly synthesized proteins. Indeed, inhibition of translation elongation, which slightly attenuated levels of a copepod GFP mutant protein, significantly enhanced its function. In contrast, inhibition of translation initiation had minimal effects on copepod GFP folding. On the other hand, mild suppression of either translation elongation or initiation corrected folding defects of the disease-associated cystic fibrosis transmembrane conductance regulator mutant F508del. We propose that modulation of translation can be used as a novel approach to improve overall proteostasis in mammalian cells, as well as functions of disease-associated mutant proteins with folding deficiencies.

Conformational changes relevant to channel activity and folding within the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator.

Deletion of Phe-508 (F508del) in the first nucleotide binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to defects in folding and channel gating. NMR data on human F508del NBD1 indicate that an H620Q mutant, shown to increase channel open probability, and the dual corrector/potentiator CFFT-001 similarly disrupt interactions between ß-strands S3, S9, and S10 and the C-terminal helices H8 and H9, shifting a preexisting conformational equilibrium from helix to coil. CFFT-001 appears to interact with ß-strands S3/S9/S10, consistent with docking simulations. Decreases in T(m) from differential scanning calorimetry with H620Q or CFFT-001 suggest direct compound binding to a less thermostable state of NBD1. We hypothesize that, in full-length CFTR, shifting the conformational equilibrium to reduce H8/H9 interactions with the uniquely conserved strands S9/S10 facilitates release of the regulatory region from the NBD dimerization interface to promote dimerization and thereby increase channel open probability. These studies enabled by our NMR assignments for F508del NBD1 provide a window into the conformational fluctuations within CFTR that may regulate function and contribute to folding energetics.

Small molecule correctors of F508del-CFTR discovered by structure-based virtual screening.

Folding correctors of F508del-CFTR were discovered by in silico structure-based screening utilizing homology models of CFTR. The intracellular segment of CFTR was modeled and three cavities were identified at inter-domain interfaces: (1) Interface between the two Nucleotide Binding Domains (NBDs); (2) Interface between NBD1 and Intracellular Loop (ICL) 4, in the region of the F508 deletion; (3) multi-domain interface between NBD1:2:ICL1:2:4. We hypothesized that compounds binding at these interfaces may improve the stability of the protein, potentially affecting the folding yield or surface stability. In silico structure-based screening was performed at the putative binding-sites and a total of 496 candidate compounds from all three sites were tested in functional assays. A total of 15 compounds, representing diverse chemotypes, were identified as F508del folding correctors. This corresponds to a 3% hit rate, ~tenfold higher than hit rates obtained in corresponding high-throughput screening campaigns. The same binding sites also yielded potentiators and, most notably, compounds with a dual corrector-potentiator activity (dual-acting). Compounds harboring both activity types may prove to be better leads for the development of CF therapeutics than either pure correctors or pure potentiators. To the best of our knowledge this is the first report of structure-based discovery of CFTR modulators.

Functional consequences of leucine and tyrosine mutations in the dual pore motifs of the yeast K(+) channel, Tok1p.

Tandem pore-loop potassium channels differ from the majority of K(+) channels in that a single polypeptide chain carries two K(+)-specific segments (P) each sandwiched between two transmembrane helices (M) to form an MP(1)M-MP(2)M series. Two of these peptide molecules assemble to form one functional potassium channel, which is expected to have biaxial symmetry (commonly described as asymmetric) due to independent mutation in the two MPM units. The resulting intrinsic asymmetry is exaggerated in fungal 2P channels, especially in Tok1p of Saccharomyces, by the N-terminal presence of four more transmembrane helices. Functional implications of such structural asymmetry have been investigated via mutagenesis of residues (L290 in P(1) and Y424 in P(2)) that are believed to provide the outermost ring of carbonyl oxygen atoms for coordination with potassium ions. Both complementary mutations (L290Y and Y424L) yield functional potassium channels having quasi-normal conductance when expressed in Saccharomyces itself, but the P(1) mutation (only) accelerates channel opening about threefold in response to depolarizing voltage shifts. The more pronounced effect at P(1) than at P(2) appears paradoxical in relation to evolution, because a comparison of fungal Tok1p sequences (from 28 ascomycetes) shows the filter sequence of P(2) (overwhelmingly TIGYGD) to be much stabler than that of P(1) (mostly TIGLGD). Profound functional asymmetry is revealed by the fact that combining mutations (L290Y + Y424L)-which inverts the order of residues from the wild-type channel-reduces the expressed channel conductance by a large factor (20-fold, cf.

TPK1 is a vacuolar ion channel different from the slow-vacuolar cation channel.

TPK1 (formerly KCO1) is the founding member of the family of two-pore domain K(+) channels in Arabidopsis (Arabidopsis thaliana), which originally was described following expression in Sf9 insect cells as a Ca(2+)- and voltage-dependent outwardly rectifying plasma membrane K(+) channel. In plants, this channel has been shown by green fluorescent protein fusion to localize to the vacuolar membrane, which led to speculations that the TPK1 gene product would be a component of the nonselective, Ca(2+) and voltage-dependent slow-vacuolar (SV) cation channel found in many plants species. Using yeast (Saccharomyces cerevisiae) as an expression system for TPK1, we show functional expression of the channel in the vacuolar membrane. In isolated vacuoles of yeast yvc1 disruption mutants, the TPK1 gene product shows ion channel activity with some characteristics very similar to the SV-type channel. The open channel conductance of TPK1 in symmetrically 100 mM KCl is slightly asymmetric with roughly 40 pS at positive membrane voltages and 75 pS at negative voltages. Similar to the SV-type channel, TPK1 is activated by cytosolic Ca(2+), requiring micromolar concentration for activation. However, in contrast to the SV-type channel, TPK1 exhibits strong selectivity for K(+) over Na(+), and its activity turned out to be independent of the membrane voltage over the range of +/-80 mV. Our data clearly demonstrate that TPK1 is a voltage-independent, Ca(2+)-activated, K(+)-selective ion channel in the vacuolar membrane that does not mediate SV-type ionic currents.

Electrophysiological analysis of the yeast V-type proton pump: variable coupling ratio and proton shunt.

Isolated vacuoles from the yeast Saccharomyces cerevisiae were examined in the whole-vacuole mode of patch recording, to get a detailed functional description of the vacuolar proton pump, the V-ATPase. Functioning of the V-ATPase was characterized by its current-voltage (I-V) relationship, obtained for various levels of vacuolar and cytosolic pH. I-V curves for the V-ATPase were computed as the difference between I-V curves obtained with the pump switched on (ATP, ADP, and Pi present) or off (no ATP). These difference current-voltage relationships usually crossed the voltage axis within the experimental range (from -80 to +80 mV), thus measuring the reversal voltage (ER) for the V-ATPase, which could be compared with the standing ion gradients and free energy of ATP hydrolysis, to calculate the apparent pump stoichiometry or coupling ratio: the number of protons transported for each ATP molecule hydrolyzed. This ratio was found to depend strongly upon the pH difference (DeltapH) across the vacuolar membrane, being approximately 2H+/ATP at high DeltapH (4 pH units) and increasing to >4H+/ATP for small or zero DeltapH. That result is in quantitative agreement with previous determinations on plant vacuoles. Considerations of purely electrical behavior, together with the physical properties of a recent detailed structural model for V-ATPases, led to a linear equivalent circuit--which quantitatively accounts for all observations of variable coupling ratios in fungal and plant V-ATPases by variations of the conductance for bona fide proton pumping (GP) through the ATPase relative to independent proton shunting (GS) through the same protein.

Characterization of potassium transport in wild-type and isogenic yeast strains carrying all combinations of trk1, trk2 and tok1 null mutations.

Saccharomyces cerevisiae cells express three defined potassium-specific transport systems en-coded by TRK1, TRK2 and TOK1. To gain a more complete understanding of the physiological function of these transport proteins, we have constructed a set of isogenic yeast strains carrying all combinations of trk1delta, trk2delta and tok1delta null mutations. The in vivo K+ transport characteristics of each strain have been documented using growth-based assays, and the in vitro biochemical and electrophysiological properties associated with K+ transport have been determined. As has been reported previously, Trk1p and Trk2p facilitate high-affinity potassium uptake and appear to be functionally redundant under a wide range of environmental conditions. In the absence of TRK1 and TRK2, strains lack the ability specifically to take up K+, and trk1deltatrk2delta double mutant cells depend upon poorly understood non-specific cation uptake mechanisms for growth. Under conditions that impair the activity of the non-specific uptake system, termed NSC1, we have found that the presence of functional Tok1p renders cells sensitive to Cs+. Based on this finding, we have established a growth-based assay that monitors the in vivo activity of Tok1p.

Low-affinity potassium uptake by Saccharomyces cerevisiae is mediated by NSC1, a calcium-blocked non-specific cation channel.

Previous descriptions by whole-cell patch clamping of the calcium-inhibited non-selective cation channel (NSC1) in the plasma membrane of Saccharomyces cerevisiae (H. Bihler, C.L. Slayman, A. Bertl, FEBS Lett. 432 (1998); S.K. Roberts, M. Fischer, G.K. Dixon, D.Sanders, J. Bacteriol. 181 (1999)) suggested that this inwardly rectifying pathway could relieve the growth inhibition normally imposed on yeast by disruption of its potassium transporters, Trk1p and Trk2p. Now, demonstration of multiple parallel effects produced by various agonists and antagonists on both NSC1 currents and growth (of trk1 Delta trk2 Delta strains), has identified this non-selective cation pathway as the primary low-affinity uptake route for potassium ions in yeast. Factors which suppress NSC1-mediated inward currents and inhibit growth of trk1 Delta trk2 Delta cells include (i) elevating extracellular calcium over the range of 10 microM-10 mM, (ii) lowering extracellular pH over the range 7.5-4, (iii) blockade of NSC1 by hygromycin B, and (iv) to a lesser extent by TEA(+). Growth of trk1 Delta trk2 Delta cells is also inhibited by lithium and ammonium; however, these ions do not inhibit NSC1, but instead enter yeast cells via NSC1. Growth inhibition by lithium ions is probably a toxic effect, whereas growth inhibition by ammonium ions probably results from competitive inhibition, i.e. displacement of intracellular potassium by entering ammonium.