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.

The C-terminal domain of A1/Bfl-1 regulates its anti-inflammatory function in human endothelial cells.

A1/Bfl-1 is a NF-κB dependent, anti-apoptotic Bcl-2 family member that contains four Bcl-2 homology domains (BH) and an amphipathic C-terminal domain, and is expressed in endothelial cells (EC). Based on NF-κB reporter assays in bovine aortic EC, we have previously demonstrated that A1, like Bcl-2 and Bcl-xL, inhibits NF-κB activation. These results, however, do not fully translate when evaluating the cell's own NF-κB machinery in human EC overexpressing A1 by means of recombinant adenovirus (rAd.) mediated gene transfer. Indeed, overexpression of full-length A1 in human umbilical vein EC (HUVEC), and human dermal microvascular EC (HDMEC) failed to inhibit NF-κB activation. However, overexpression of a mutant lacking the C-terminal domain of A1 (A1ΔC) demonstrated a potent NF-κB inhibitory effect in these cells. Disparate effects of A1 and A1ΔC on NF-κB inhibition in human EC correlated with mitochondrial (A1) versus non-mitochondrial (A1ΔC) localization. In contrast, both full-length A1 and A1ΔC protected EC from staurosporine (STS)-induced cell death, indicating that mitochondrial localization was not necessary for A1's cytoprotective function in human EC. In conclusion, our data uncover a regulatory role for the C-terminal domain of A1 in human EC: anchoring A1 to the mitochondrion, which conserves but is not necessary for its cytoprotective function, or by its absence freeing A1 from the mitochondrion and uncovering an additional anti-inflammatory effect.

A20 promotes liver regeneration by decreasing SOCS3 expression to enhance IL-6/STAT3 proliferative signals.

UNLABELLED: Liver regeneration is of major clinical importance in the setting of liver injury, resection, and transplantation. A20, a potent antiinflammatory and nuclear factor kappa B (NF-κB) inhibitory protein, has established pro-proliferative properties in hepatocytes, in part through decreasing expression of the cyclin dependent kinase inhibitor, p21. Both C-terminal (7-zinc fingers; 7Zn) and N-terminal (Nter) domains of A20 were required to decrease p21 and inhibit NF-κB. However, both independently increased hepatocyte proliferation, suggesting that additional mechanisms contributed to the pro-proliferative function of A20 in hepatocytes. We ascribed one of A20's pro-proliferative mechanisms to increased and sustained interleukin (IL)-6-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation, as a result of decreased hepatocyte expression of the negative regulator of IL-6 signaling, suppressor of cytokine signaling 3 (SOCS3). This novel A20 function segregates with its 7Zn not Nter domain. Conversely, total and partial loss of A20 in hepatocytes increased SOCS3 expression, hampering IL-6-induced STAT3 phosphorylation. Following liver resection in mice pro-proliferative targets downstream of IL-6/STAT3 signaling were increased by A20 overexpression and decreased by A20 knockdown. In contrast, IL-6/STAT3 proinflammatory targets were increased in A20-deficient livers, and decreased or unchanged in A20 overexpressing livers. Upstream of SOCS3, levels of its microRNA regulator miR203 were significantly decreased in A20-deficient livers. CONCLUSION: A20 enhances IL-6/STAT3 pro-proliferative signals in hepatocytes by down-regulating SOCS3, likely through a miR203-dependent manner. This finding together with A20 reducing the levels of the potent cell cycle brake p21 establishes its pro-proliferative properties in hepatocytes and prompts the pursuit of A20-based therapies to promote liver regeneration and repair.

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.

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.

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.

A20 protects from CD40-CD40 ligand-mediated endothelial cell activation and apoptosis.

BACKGROUND: CD40/CD40 ligand (CD40L) signaling is a potent activator of endothelial cells (ECs) and promoter of atherosclerosis. In this study, we investigate whether A20 (a gene we have shown to be antiinflammatory and antiapoptotic in ECs) can protect from CD40/CD40L-mediated EC activation. METHODS AND RESULTS: Overexpression of CD40, in a transient transfection system, activates the transcription factor NF-kappaB and upregulates IkappaBalpha, E-selectin, and tissue factor (TF) reporter activity. Coexpression of A20 inhibits NF-kappaB and upregulation of IkappaBalpha and E-Selectin but not TF, suggesting that CD40 induces TF in a non-NF-kappaB-dependent manner. In human coronary artery ECs (HCAECs), adenovirus-mediated overexpression of A20 blocks physiological, CD40-induced activation of NF-kappaB, upstream of IkappaBalpha degradation (Western blot) and subsequently upregulation of ICAM-1, VCAM-1, and E-selectin (flow cytometry). Although A20 does not block TF transcription its expression in HCAECs inhibits TF induction (colorimetric assay and RT-PCR) by blunting CD40 upregulation. We demonstrate that CD40 signaling induces apoptosis in a proinflammatory microenvironment. A20 overexpression protects from CD40-mediated EC apoptosis (DNA content analysis and trypan blue exclusion). We also demonstrate that signaling through CD40L activates NF-kappaB and induces apoptosis in ECs, both of which are inhibited by A20 overexpression. CONCLUSIONS: A20 works at multiple levels to protect ECs from CD40/CD40L mediated activation and apoptosis. A20-based therapy could be beneficial for the treatment of vascular diseases such as atherosclerosis and transplant-associated vasculopathy.

A20 protects mice from D-galactosamine/lipopolysaccharide acute toxic lethal hepatitis.

Apoptosis of hepatocytes is a seminal feature of fulminant hepatic failure. We show that the anti-apoptotic protein A20 is upregulated in hepatocytes by pro-inflammatory stimuli and functions to protect from apoptosis and limit inflammation by inhibiting NF-kappaB. Adenoviral mediated hepatic expression of A20 in BALB/c mice yields an 85% survival rate in the D-galactosamine (D-gal)/lipolysaccharide (LPS) model of acute toxic hepatitis compared with 15% to 20 % in control mice. Expression of A20 preserves normal liver function as assessed by prothrombin time. The protective effect of A20 is independent of tumor necrosis factor (TNF) inhibition. Maintaining high circulating TNF levels may be advantageous for liver regeneration. Our data supports this hypothesis as evidenced by increased proliferating cell nuclear antigen (PCNA) expression in the livers of mice expressing A20 compared with a dominant negative mutant of the TNF receptor (TNF-R), 6 hours following D-gal/LPS administration. In conclusion, these results qualify A20 as part of a physiologic, protective response of hepatocytes to injury and a promising gene therapy candidate for clinical applications aimed at preventing and treating viral and toxic fulminant hepatic failure.

A20 protects endothelial cells from TNF-, Fas-, and NK-mediated cell death by inhibiting caspase 8 activation.

A20 is a stress response gene in endothelial cells (ECs). A20 serves a dual cytoprotective function, protecting from tumor necrosis factor (TNF)-mediated apoptosis and inhibiting inflammation via blockade of the transcription factor nuclear factor-kappaB (NF-kappaB). In this study, we evaluated the molecular basis of the cytoprotective function of A20 in EC cultures and questioned whether its protective effect extends beyond TNF to other apoptotic and necrotic stimuli. Our data demonstrate that A20 targets the TNF apoptotic pathway by inhibiting proteolytic cleavage of apical caspases 8 and 2, executioner caspases 3 and 6, Bid cleavage, and release of cytochrome c, thus preserving mitochondrion integrity. A20 also protects from Fas/CD95 and significantly blunts natural killer cell-mediated EC apoptosis by inhibiting caspase 8 activation. In addition to protecting ECs from apoptotic stimuli, A20 safeguards ECs from complement-mediated necrosis. These data demonstrate, for the first time, that the cytoprotective effect of A20 in ECs is not limited to TNF-triggered apoptosis. Rather, A20 affords broad EC protective functions by effectively shutting down cell death pathways initiated by inflammatory and immune offenders.