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.
The derivation of tissue-specific stem cells from human induced pluripotent stem cells (iPSCs) would have broad reaching implications for regenerative medicine. Here, we report the directed ...differentiation of human iPSCs into airway basal cells (“iBCs”), a population resembling the stem cell of the airway epithelium. Using a dual fluorescent reporter system (NKX2-1GFP;TP63tdTomato), we track and purify these cells as they first emerge as developmentally immature NKX2-1GFP+ lung progenitors and subsequently augment a TP63 program during proximal airway epithelial patterning. In response to primary basal cell medium, NKX2-1GFP+/TP63tdTomato+ cells display the molecular and functional phenotype of airway basal cells, including the capacity to self-renew or undergo multi-lineage differentiation in vitro and in tracheal xenografts in vivo. iBCs and their differentiated progeny model perturbations that characterize acquired and genetic airway diseases, including the mucus metaplasia of asthma, chloride channel dysfunction of cystic fibrosis, and ciliary defects of primary ciliary dyskinesia.
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•Directed differentiation of human iPSCs generates airway basal cells (“iBCs”)•iBCs self-renew and display multipotent differentiation in vitro and in vivo•By single-cell RNA-seq, iBCs are highly similar to adult primary airway basal cells•iBCs enable modeling of acquired and genetic airway diseases
Hawkins and colleagues report a directed differentiation protocol enabling the derivation of airway basal cells (“iBCs”) from human iPSCs. iBCs recapitulate hallmark stem cell properties of primary basal cells, including self-renewal and multi-lineage differentiation, thus enabling modeling of airway diseases in vitro and repopulation of tracheal xenografts in vivo.
Chronic obstructive pulmonary disease (COPD) is a progressive respiratory disorder consisting of chronic bronchitis and/or emphysema. COPD patients suffer from chronic infections and display ...exaggerated inflammatory responses and a progressive decline in respiratory function. The respiratory symptoms of COPD are similar to those seen in cystic fibrosis (CF), although the molecular basis of the two disorders differs. CF is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding a chloride and bicarbonate channel (CFTR), leading to CFTR dysfunction. The majority of COPD cases result from chronic oxidative insults such as cigarette smoke. Interestingly, environmental stresses including cigarette smoke, hypoxia, and chronic inflammation have also been implicated in reduced CFTR function, and this suggests a common mechanism that may contribute to both the CF and COPD. Therefore, improving CFTR function may offer an excellent opportunity for the development of a common treatment for CF and COPD. In this article, we review what is known about the CF respiratory phenotype and discuss how diminished CFTR expression-associated ion transport defects may contribute to some of the pathological changes seen in COPD.
Nonsense mutations - the underlying cause of approximately 11% of all genetic diseases - prematurely terminate protein synthesis by mutating a sense codon to a premature stop or termination codon ...(PTC). An emerging therapeutic strategy to suppress nonsense defects is to engineer sense-codon decoding tRNAs to readthrough and restore translation at PTCs. However, the readthrough efficiency of the engineered suppressor tRNAs (sup-tRNAs) largely varies in a tissue- and sequence context-dependent manner and has not yet yielded optimal clinical efficacy for many nonsense mutations. Here, we systematically analyze the suppression efficacy at various pathogenic nonsense mutations. We discover that the translation velocity of the sequence upstream of PTCs modulates the sup-tRNA readthrough efficacy. The PTCs most refractory to suppression are embedded in a sequence context translated with an abrupt reversal of the translation speed leading to ribosomal collisions. Moreover, modeling translation velocity using Ribo-seq data can accurately predict the suppression efficacy at PTCs. These results reveal previously unknown molecular signatures contributing to genotype-phenotype relationships and treatment-response heterogeneity, and provide the framework for the development of personalized tRNA-based gene therapies.
W1282X is a common nonsense mutation among cystic fibrosis patients that results in the production of a truncated Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Here we show that ...the channel activity of the W1282X-CFTR polypeptide is exceptionally low in excised membrane patches at normally saturating doses of ATP and PKA (single channel open probability (PO) < 0.01). However, W1282X-CFTR channels were stimulated by two CFTR modulators, the FDA-approved VX-770 and the dietary compound curcumin. Each of these compounds is an allosteric modulator of CFTR gating that promotes channel activity in the absence of the native ligand, ATP. Although W1282X-CFTR channels were stimulated by VX-770 in the absence of ATP their activities remained dependent on PKA phosphorylation. Thus, activated W1282X-CFTR channels should remain under physiologic control by cyclic nucleotide signaling pathways in vivo. VX-770 and curcumin exerted additive effects on W1282X-CFTR channel gating (opening/closing) in excised patches such that the Po of the truncated channel approached unity (> 0.9) when treated with both modulators. VX-770 and curcumin also additively stimulated W1282X-CFTR mediated currents in polarized FRT epithelial monolayers. In this setting, however, the stimulated W1282X-CFTR currents were smaller than those mediated by wild type CFTR (3-5%) due presumably to lower expression levels or cell surface targeting of the truncated protein. Combining allosteric modulators of different mechanistic classes is worth considering as a treatment option for W1282X CF patients perhaps when coupled with maneuvers to increase expression of the truncated protein.
The ΔF508 mutant form of the cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR) that is normally degraded by the ER-associated degradative pathway can be rescued to the cell surface ...through low-temperature (27°C) culture or small molecular corrector treatment. However, it is unstable on the cell surface, and rapidly internalized and targeted to the lysosomal compartment for degradation. To understand the mechanism of this rapid turnover, we examined the role of two adaptor complexes (AP-2 and Dab2) and three E3 ubiquitin ligases (c-Cbl, CHIP, and Nedd4-2) on low-temperature rescued ΔF508 CFTR endocytosis and degradation in human airway epithelial cells. Our results demonstrate that siRNA depletion of either AP-2 or Dab2 inhibits ΔF508 CFTR endocytosis by 69% and 83%, respectively. AP-2 or Dab2 depletion also increases the rescued protein half-life of ΔF508 CFTR by ~18% and ~91%, respectively. In contrast, the depletion of each of the E3 ligases had no effect on ΔF508 CFTR endocytosis, whereas CHIP depletion significantly increased the surface half-life of ΔF508 CFTR. To determine where and when the ubiquitination occurs during ΔF508 CFTR turnover, we monitored the ubiquitination of rescued ΔF508 CFTR during the time course of CFTR endocytosis. Our results indicate that ubiquitination of the surface pool of ΔF508 CFTR begins to increase 15 min after internalization, suggesting that CFTR is ubiquitinated in a post-endocytic compartment. This post-endocytic ubiquination of ΔF508 CFTR could be blocked by either inhibiting endocytosis, by siRNA knockdown of CHIP, or by treating cells with the CFTR corrector, VX-809. Our results indicate that the post-endocytic ubiquitination of CFTR by CHIP is a critical step in the peripheral quality control of cell surface ΔF508 CFTR.
Cardiac ischemia and reperfusion (I/R) injury occurs because the acute increase in oxidative/inflammatory stress during reperfusion culminates in the death of cardiomyocytes. Currently, there is no ...drug utilized clinically that attenuates I/R injury in patients. Previous studies have demonstrated degranulation of mast cell contents into the interstitium after I/R. Using a dog model of I/R, we tested the role of chymase, a mast cell protease, in cardiomyocyte injury using a specific oral chymase inhibitor (CI). 15 adult mongrel dogs had left anterior descending artery occlusion for 60 min and reperfusion for 100 minutes. 9 dogs received vehicle and 6 were pretreated with a specific CI. In vivo cardiac microdialysis demonstrated a 3-fold increase in interstitial fluid chymase activity in I/R region that was significantly decreased by CI. CI pretreatment significantly attenuated loss of laminin, focal adhesion complex disruption, and release of troponin I into the circulation. Microarray analysis identified an I/R induced 17-fold increase in nuclear receptor subfamily 4A1 (NR4A1) and significantly decreased by CI. NR4A1 normally resides in the nucleus but can induce cell death on migration to the cytoplasm. I/R caused significant increase in NR4A1 protein expression and cytoplasmic translocation, and mitochondrial degradation, which were decreased by CI. Immunohistochemistry also revealed a high concentration of chymase within cardiomyocytes after I/R. In vitro, chymase added to culture HL-1 cardiomyocytes entered the cytoplasm and nucleus in a dynamin-dependent fashion, and promoted cytoplasmic translocation of NR4A1 protein. shRNA knockdown of NR4A1 on pre-treatment of HL-1 cells with CI significantly decreased chymase-induced cell death and mitochondrial damage. These results suggest that the beneficial effects of an orally active CI during I/R are mediated in the cardiac interstitium as well as within the cardiomyocyte due to a heretofore-unrecognized chymase entry into cardiomyocytes.
Cystic Fibrosis (CF) is caused by a diverse set of mutations distributed across the approximately 250 thousand base pairs of the
gene locus, of which at least 382 are disease-causing (CFTR2.org). ...Although a variety of editing tools are now available for correction of individual mutations, a strong justification can be made for a more universal gene insertion approach, in principle capable of correcting virtually all
mutations. Provided that such a methodology is capable of efficiently correcting relevant stem cells of the airway epithelium, this could potentially provide life-long correction for the lung. In this Perspective we highlight several requirements for efficient gene insertion into airway epithelial stem cells. In addition, we focus on specific features of the transgene construct and the endogenous
locus that influence whether the inserted gene sequences will give rise to robust and physiologically relevant levels of CFTR function in airway epithelium. Finally, we consider how
gene insertion methodologies may be adapted for direct
editing.
MRI contrast agents (CA) whose contrast enhancement remains relatively high even at the higher end of the magnetic field strength range would be desirable. The purpose of this work was to demonstrate ...such a desired magnetic field dependency of the longitudinal relaxivity for an experimental MRI CA, Gd(ABE-DTTA).
The relaxivity of 0.5mM and 1mM Gd(ABE-DTTA) was measured by Nuclear Magnetic Relaxation Dispersion (NMRD) in the range of 0.0002 to 1T. Two MRI and five NMR instruments were used to cover the range between 1.5 to 20T. Parallel measurement of a Gd-DTPA sample was performed throughout as reference. All measurements were carried out at 37°C and pH 7.4.
The relaxivity values of 0.5mM and 1mM Gd(ABE-DTTA) measured at 1.5, 3, and 7T, within the presently clinically relevant magnetic field range, were 15.3, 11.8, 12.4 s-1mM-1 and 18.1, 16.7, and 13.5 s-1mM-1, respectively. The control 4 mM Gd-DTPA relaxivities at the same magnetic fields were 3.6, 3.3, and 3.0 s-1mM-1, respectively.
The longitudinal relaxivity of Gd(ABE-DTTA) measured within the presently clinically relevant field range is three to five times higher than that of most commercially available agents. Thus, Gd(ABE-DTTA) could be a practical choice at any field strength currently used in clinical imaging including those at the higher end.
CFTR (cystic fibrosis transmembrane conductance regulator) is expressed in the apical membrane of epithelial cells. Cell-surface CFTR levels are regulated by endocytosis and recycling. A number of ...adaptor proteins including AP-2 (μ2 subunit) and Dab2 (Disabled-2) have been proposed to modulate CFTR internalization. In the present study we have used siRNA (small interfering RNA)-mediated silencing of these adaptors to test their roles in the regulation of CFTR cell-surface trafficking and stability in human airway epithelial cells. The results indicate that μ2 and Dab2 performed partially overlapping, but divergent, functions. While μ2 depletion dramatically decreased CFTR endocytosis with little effect on the half-life of the CFTR protein, Dab2 depletion increased the CFTR half-life ~3-fold, in addition to inhibiting CFTR endocytosis. Furthermore, Dab2 depletion inhibited CFTR trafficking from the sorting endosome to the recycling compartment, as well as delivery of CFTR to the late endosome, thus providing a mechanistic explanation for increased CFTR expression and half-life. To test whether two E3 ligases were required for the endocytosis and/or down-regulation of surface CFTR, we siRNA-depleted CHIP C-terminus of the Hsc (heat-shock cognate) 70-interacting protein and c-Cbl (casitas B-lineage lymphoma). We demonstrate that CHIP and c-Cbl depletion have no effect on CFTR endocytosis, but c-Cbl depletion modestly enhanced the half-life of CFTR. The results of the present study define a significant role for Dab2 both in the endocytosis and post-endocytic fate of CFTR.
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