Engineered tRNAs suppress nonsense mutations in cells and in vivo.

Nonsense mutations are the underlying cause of approximately 11% of all inherited genetic diseases1. Nonsense mutations convert a sense codon that is decoded by tRNA into a premature termination codon (PTC), resulting in an abrupt termination of translation. One strategy to suppress nonsense mutations is to use natural tRNAs with altered anticodons to base-pair to the newly emerged PTC and promote translation2-7. However, tRNA-based gene therapy has not yielded an optimal combination of clinical efficacy and safety and there is presently no treatment for individuals with nonsense mutations. Here we introduce a strategy based on altering native tRNAs into  efficient suppressor tRNAs (sup-tRNAs) by individually fine-tuning their sequence to the physico-chemical properties of the amino acid that they carry. Intravenous and intratracheal lipid nanoparticle (LNP) administration of sup-tRNA in mice restored the production of functional proteins with nonsense mutations. LNP-sup-tRNA formulations caused no discernible readthrough at endogenous native stop codons, as determined by ribosome profiling. At clinically important PTCs in the cystic fibrosis transmembrane conductance regulator gene (CFTR), the sup-tRNAs re-established expression and function in cell systems and patient-derived nasal epithelia and restored airway volume homeostasis. These results provide a framework for the development of tRNA-based therapies with a high molecular safety profile and high efficacy in targeted PTC suppression.

Elexacaftor/VX-445-mediated CFTR interactome remodeling reveals differential correction driven by mutation-specific translational dynamics.

Cystic fibrosis (CF) is one of the most prevalent lethal genetic diseases with over 2000 identified mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Pharmacological chaperones such as lumacaftor (VX-809), tezacaftor (VX-661), and elexacaftor (VX-445) treat mutation-induced defects by stabilizing CFTR and are called correctors. These correctors improve proper folding and thus facilitate processing and trafficking to increase the amount of functional CFTR on the cell surface. Yet, CFTR variants display differential responses to each corrector. Here, we report that variants P67L and L206W respond similarly to VX-809 but divergently to VX-445 with P67L exhibiting little rescue when treated with VX-445. We investigate the underlying cellular mechanisms of how CFTR biogenesis is altered by correctors in these variants. Affinity purification-mass spectrometry multiplexed with isobaric tandem mass tags was used to quantify CFTR protein-protein interaction changes between variants P67L and L206W. VX-445 facilitates unique proteostasis factor interactions especially in translation, folding, and degradation pathways in a CFTR variant-dependent manner. A number of these interacting proteins knocked down by siRNA, such as ribosomal subunit proteins, moderately rescued fully glycosylated P67L. Importantly, these knockdowns sensitize P67L to VX-445 and further enhance the trafficking correction of this variant. Partial inhibition of protein translation also mildly sensitizes P67L CFTR to VX-445 correction, supporting a role for translational dynamics in the rescue mechanism of VX-445. Our results provide a better understanding of VX-445 biological mechanism of action and reveal cellular targets that may sensitize nonresponsive CFTR variants to known and available correctors.

Mechanisms by which the cystic fibrosis transmembrane conductance regulator may influence SARS-CoV-2 infection and COVID-19 disease severity.

Patients with cystic fibrosis (CF) exhibit pronounced respiratory damage and were initially considered among those at highest risk for serious harm from SARS-CoV-2 infection. Numerous clinical studies have subsequently reported that individuals with CF in North America and Europe-while susceptible to severe COVID-19-are often spared from the highest levels of virus-associated mortality. To understand features that might influence COVID-19 among patients with cystic fibrosis, we studied relationships between SARS-CoV-2 and the gene responsible for CF (i.e., the cystic fibrosis transmembrane conductance regulator, CFTR). In contrast to previous reports, we found no association between CFTR carrier status (mutation heterozygosity) and more severe COVID-19 clinical outcomes. We did observe an unexpected trend toward higher mortality among control individuals compared with silent carriers of the common F508del CFTR variant-a finding that will require further study. We next performed experiments to test the influence of homozygous CFTR deficiency on viral propagation and showed that SARS-CoV-2 production in primary airway cells was not altered by the absence of functional CFTR using two independent protocols. On the contrary, experiments performed in vitro strongly indicated that virus proliferation depended on features of the mucosal fluid layer known to be disrupted by absent CFTR in patients with CF, including both low pH and increased viscosity. These results point to the acidic, viscous, and mucus-obstructed airways in patients with cystic fibrosis as unfavorable for the establishment of coronaviral infection. Our findings provide new and important information concerning relationships between the CF clinical phenotype and severity of COVID-19.

Positive epistasis between disease-causing missense mutations and silent polymorphism with effect on mRNA translation velocity.

Epistasis refers to the dependence of a mutation on other mutation(s) and the genetic context in general. In the context of human disorders, epistasis complicates the spectrum of disease symptoms and has been proposed as a major contributor to variations in disease outcome. The nonadditive relationship between mutations and the lack of complete understanding of the underlying physiological effects limit our ability to predict phenotypic outcome. Here, we report positive epistasis between intragenic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR)-the gene responsible for cystic fibrosis (CF) pathology. We identified a synonymous single-nucleotide polymorphism (sSNP) that is invariant for the CFTR amino acid sequence but inverts translation speed at the affected codon. This sSNP in cis exhibits positive epistatic effects on some CF disease-causing missense mutations. Individually, both mutations alter CFTR structure and function, yet when combined, they lead to enhanced protein expression and activity. The most robust effect was observed when the sSNP was present in combination with missense mutations that, along with the primary amino acid change, also alter the speed of translation at the affected codon. Functional studies revealed that synergistic alteration in ribosomal velocity is the underlying mechanism; alteration of translation speed likely increases the time window for establishing crucial domain-domain interactions that are otherwise perturbed by each individual mutation.

Mutation profiling of the c.1521_1523delCTT (p.Phe508del, F508del) cystic fibrosis transmembrane conductance regulator allele using haplotype-resolved long-read next generation sequencing.

Current approaches to characterize the mutational profile of the cystic fibrosis transmembrane conductance regulator (CFTR) gene are based on targeted mutation analysis or whole gene studies derived from short-read next generation sequencing (NGS). However, these methods lack phasing capability which, in certain scenarios, can provide clinically valuable information. In the present work, we performed near-full length CFTR using Single-Molecule Real-Time Sequencing to produce haplotype-resolved data from both homozygous and heterozygous individuals for mutation c.1521_1523delCTT (p.Phe508del, F508del). This approach utilizes target enrichment of the CFTR gene using biotinylated probes, facilitates multiplexing samples in the same sequencing run, and utilizes fully-automated bioinformatics pipelines for error correction and variant calling. We show a remarkable conservation of F508del haplotype, consistent with the single gene founder effect, as well as diverse mutational profiles in non-F508del alleles. By the same method, 105 single nucleotide polymorphisms (SNPs) exhibiting invariant linkage to F508del CFTR (which better define the founder haplotype) were identified. High level homology between F508del sequences derived from heterozygotes, and those obtained from homozygous individuals, demonstrate accuracy of this method to produce haplotype resolved sequencing. The studies provide a new diagnostic technology for detailed analysis of complex CFTR alleles linked to disease severity.

The CFTR P67L variant reveals a key role for N-terminal lasso helices in channel folding, maturation, and pharmacologic rescue.

Patients with cystic fibrosis (CF) harboring the P67L variant in the cystic fibrosis transmembrane conductance regulator (CFTR) often exhibit a typical CF phenotype, including severe respiratory compromise. This rare mutation (reported in <300 patients worldwide) responds robustly to CFTR correctors, such as lumacaftor and tezacaftor, with rescue in model systems that far exceed what can be achieved for the archetypical CFTR mutant F508del. However, the specific molecular consequences of the P67L mutation are poorly characterized. In this study, we conducted biochemical measurements following low-temperature growth and/or intragenic suppression, which suggest a mechanism underlying P67L that (1) shares key pathogenic features with F508del, including off-pathway (non-native) folding intermediates, (2) is linked to folding stability of nucleotide-binding domains 1 and 2, and (3) demonstrates pharmacologic rescue that requires domains in the carboxyl half of the protein. We also investigated the "lasso" helices 1 and 2, which occur immediately upstream of P67. Based on limited proteolysis, pulse chase, and molecular dynamics analysis of full-length CFTR and a series of deletion constructs, we argue that P67L and other maturational processing (class 2) defects impair the integrity of the lasso motif and confer misfolding of downstream domains. Thus, amino-terminal missense variants elicit a conformational change throughout CFTR that abrogates maturation while providing a robust substrate for pharmacologic repair.

Marine Natural Products as Leads against SARS-CoV-2 Infection.

Since early 2020, disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic, causing millions of infections and deaths worldwide. Despite rapid deployment of effective vaccines, it is apparent that the global community lacks multipronged interventions to combat viral infection and disease. A major limitation is the paucity of antiviral drug options representing diverse molecular scaffolds and mechanisms of action. Here we report the antiviral activities of three distinct marine natural products─homofascaplysin A (1), (+)-aureol (2), and bromophycolide A (3)─evidenced by their ability to inhibit SARS-CoV-2 replication at concentrations that are nontoxic toward human airway epithelial cells. These compounds stand as promising candidates for further exploration toward the discovery of novel drug leads against SARS-CoV-2.

Association of cystic fibrosis transmembrane conductance regulator with epithelial sodium channel subunits carrying Liddle's syndrome mutations.

The association of the cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) in the pathophysiology of cystic fibrosis (CF) is controversial. Previously, we demonstrated a close physical association between wild-type (WT) CFTR and WT ENaC. We have also shown that the F508del CFTR fails to associate with ENaC unless the mutant protein is rescued pharmacologically or by low temperature. In this study, we present the evidence for a direct physical association between WT CFTR and ENaC subunits carrying Liddle's syndrome mutations. We show that all three ENaC subunits bearing Liddle's syndrome mutations (both point mutations and the complete truncation of the carboxy terminus), could be coimmunoprecipitated with WT CFTR. The biochemical studies were complemented by fluorescence lifetime imaging microscopy (FLIM), a distance-dependent approach that monitors protein-protein interactions between fluorescently labeled molecules. Our measurements revealed significantly increased fluorescence resonance energy transfer between CFTR and all tested ENaC combinations as compared with controls (ECFP and EYFP cotransfected cells). Our findings are consistent with the notion that CFTR and ENaC are within reach of each other even in the setting of Liddle's syndrome mutations, suggestive of a direct intermolecular interaction between these two proteins.

Type I and Type III Interferons Restrict SARS-CoV-2 Infection of Human Airway Epithelial Cultures.

The newly emerged human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a pandemic of respiratory illness. Current evidence suggests that severe cases of SARS-CoV-2 are associated with a dysregulated immune response. However, little is known about how the innate immune system responds to SARS-CoV-2. In this study, we modeled SARS-CoV-2 infection using primary human airway epithelial (pHAE) cultures, which are maintained in an air-liquid interface. We found that SARS-CoV-2 infects and replicates in pHAE cultures and is directionally released on the apical, but not basolateral, surface. Transcriptional profiling studies found that infected pHAE cultures had a molecular signature dominated by proinflammatory cytokines and chemokine induction, including interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), and CXCL8, and identified NF-κB and ATF-4 as key drivers of this proinflammatory cytokine response. Surprisingly, we observed a complete lack of a type I or III interferon (IFN) response to SARS-CoV-2 infection. However, pretreatment and posttreatment with type I and III IFNs significantly reduced virus replication in pHAE cultures that correlated with upregulation of antiviral effector genes. Combined, our findings demonstrate that SARS-CoV-2 does not trigger an IFN response but is sensitive to the effects of type I and III IFNs. Our studies demonstrate the utility of pHAE cultures to model SARS-CoV-2 infection and that both type I and III IFNs can serve as therapeutic options to treat COVID-19 patients.IMPORTANCE The current pandemic of respiratory illness, COVID-19, is caused by a recently emerged coronavirus named SARS-CoV-2. This virus infects airway and lung cells causing fever, dry cough, and shortness of breath. Severe cases of COVID-19 can result in lung damage, low blood oxygen levels, and even death. As there are currently no vaccines approved for use in humans, studies of the mechanisms of SARS-CoV-2 infection are urgently needed. Our research identifies an excellent system to model SARS-CoV-2 infection of the human airways that can be used to test various treatments. Analysis of infection in this model system found that human airway epithelial cell cultures induce a strong proinflammatory cytokine response yet block the production of type I and III IFNs to SARS-CoV-2. However, treatment of airway cultures with the immune molecules type I or type III interferon (IFN) was able to inhibit SARS-CoV-2 infection. Thus, our model system identified type I or type III IFN as potential antiviral treatments for COVID-19 patients.

Improved correction of F508del-CFTR biogenesis with a folding facilitator and an inhibitor of protein ubiquitination.

A growing number of diseases are linked to the misfolding of integral membrane proteins, and many of these proteins are targeted for ubiquitin-proteasome-dependent degradation. One such substrate is a mutant form of the Cystic Fibrosis Transmembrane Conductance Regulator (F508del-CFTR). Protein folding "correctors" that repair the F508del-CFTR folding defect have entered the clinic, but they are unlikely to protect the entire protein from degradation. To increase the pool of F508del-CFTR protein that is available for correction by existing treatments, we determined a structure-activity relationship to improve the efficacy and reduce the toxicity of an inhibitor of the E1 ubiquitin activating enzyme that facilitates F508del-CFTR maturation. A resulting lead compound lacked measurable toxicity and improved the ability of an FDA-approved corrector to augment F508del-CFTR folding, transport the protein to the plasma membrane, and maintain its activity. These data support a proof-of-concept that modest inhibition of substrate ubiquitination improves the activity of small molecule correctors to treat CF and potentially other protein conformational disorders.

Stability Prediction for Mutations in the Cytosolic Domains of Cystic Fibrosis Transmembrane Conductance Regulator.

Cystic Fibrosis (CF) is caused by mutations to the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) chloride channel. CFTR is composed of two membrane spanning domains, two cytosolic nucleotide-binding domains (NBD1 and NBD2) and a largely unstructured R-domain. Multiple CF-causing mutations reside in the NBDs and some are known to compromise the stability of these domains. The ability to predict the effect of mutations on the stability of the cytosolic domains of CFTR and to shed light on the mechanisms by which they exert their effect is therefore important in CF research. With this in mind, we have predicted the effect on domain stability of 59 mutations in NBD1 and NBD2 using 15 different algorithms and evaluated their performances via comparison to experimental data using several metrics including the correct classification rate (CCR), and the squared Pearson correlation (R2) and Spearman's correlation (ρ) calculated between the experimental ΔTm values and the computationally predicted ΔΔG values. Overall, the best results were obtained with FoldX and Rosetta. For NBD1 (35 mutations), FoldX provided R2 and ρ values of 0.64 and -0.71, respectively, with an 86% correct classification rate (CCR). For NBD2 (24 mutations), FoldX R2, ρ, and CCR were 0.51, -0.73, and 75%, respectively. Application of the Rosetta high-resolution protocol (Rosetta_hrp) to NBD1 yielded R2, ρ, and CCR of 0.64, -0.75, and 69%, respectively, and for NBD2 yielded R2, ρ, and CCR of 0.29, -0.27, and 50%, respectively. The corresponding numbers for the Rosetta's low-resolution protocol (Rosetta_lrp) were R2 = 0.47, ρ = -0.69, and CCR = 69% for NBD1 and R2 = 0.27, ρ = -0.24, and CCR = 63% for NBD2. For NBD1, both algorithms suggest that destabilizing mutations suffer from destabilizing vdW clashes, whereas stabilizing mutations benefit from favorable H-bond interactions. Two triple consensus approaches based on FoldX, Rosetta_lpr, and Rosetta_hpr were attempted using either "majority-voting" or "all-voting". The all-voting consensus outperformed the individual predictors, albeit on a smaller data set. In summary, our results suggest that the effect of mutations on the stability of CFTR's NBDs could be largely predicted. Since NBDs are common to all ABC transporters, these results may find use in predicting the effect and mechanism of the action of multiple disease-causing mutations in other proteins.

Highly Efficient Gene Editing of Cystic Fibrosis Patient-Derived Airway Basal Cells Results in Functional CFTR Correction.

There is a strong rationale to consider future cell therapeutic approaches for cystic fibrosis (CF) in which autologous proximal airway basal stem cells, corrected for CFTR mutations, are transplanted into the patient's lungs. We assessed the possibility of editing the CFTR locus in these cells using zinc-finger nucleases and have pursued two approaches. The first, mutation-specific correction, is a footprint-free method replacing the CFTR mutation with corrected sequences. We have applied this approach for correction of ΔF508, demonstrating restoration of mature CFTR protein and function in air-liquid interface cultures established from bulk edited basal cells. The second is targeting integration of a partial CFTR cDNA within an intron of the endogenous CFTR gene, providing correction for all CFTR mutations downstream of the integration and exploiting the native CFTR promoter and chromatin architecture for physiologically relevant expression. Without selection, we observed highly efficient, site-specific targeted integration in basal cells carrying various CFTR mutations and demonstrated restored CFTR function at therapeutically relevant levels. Significantly, Omni-ATAC-seq analysis revealed minimal impact on the positions of open chromatin within the native CFTR locus. These results demonstrate efficient functional correction of CFTR and provide a platform for further ex vivo and in vivo editing.

Ivacaftor Reverses Airway Mucus Abnormalities in a Rat Model Harboring a Humanized G551D-CFTR.

Rationale: Animal models have been highly informative for understanding the characteristics, onset, and progression of cystic fibrosis (CF) lung disease. In particular, the CFTR-/- rat has revealed insights into the airway mucus defect characteristic of CF but does not replicate a human-relevant CFTR (cystic fibrosis transmembrane conductance regulator) variant.Objectives: We hypothesized that a rat expressing a humanized version of CFTR and harboring the ivacaftor-sensitive variant G551D could be used to test the impact of CFTR modulators on pathophysiologic development and correction.Methods: In this study, we describe a humanized-CFTR rat expressing the G551D variant obtained by zinc finger nuclease editing of a human complementary DNA superexon, spanning exon 2-27, with a 5' insertion site into the rat gene just beyond intron 1. This targeted insertion takes advantage of the endogenous rat promoter, resulting in appropriate expression compared with wild-type animals.Measurements and Main Results: The bioelectric phenotype of the epithelia recapitulates the expected absence of CFTR activity, which was restored with ivacaftor. Large airway defects, including depleted airway surface liquid and periciliary layers, delayed mucus transport rates, and increased mucus viscosity, were normalized after the administration of ivacaftor.Conclusions: This model is useful to understand the mechanisms of disease and the extent of pathology reversal with CFTR modulators.

Assessing cell-specific effects of genetic variations using tRNA microarrays.

BACKGROUND: By definition, effect of synonymous single-nucleotide variants (SNVs) on protein folding and function are neutral, as they alter the codon and not the encoded amino acid. Recent examples indicate tissue-specific and transfer RNA (tRNA)-dependent effects of some genetic variations arguing against neutrality of synonymous SNVs for protein biogenesis. RESULTS: We performed systematic analysis of tRNA abunandance across in various models used in cystic fibrosis (CF) research and drug development, including Fischer rat thyroid (FRT) cells, patient-derived primary human bronchial epithelia (HBE) from lung biopsies, primary human nasal epithelia (HNE) from nasal curettage, intestinal organoids, and airway progenitor-directed differentiation of human induced pluripotent stem cells (iPSCs). These were compared to an immortalized CF bronchial cell model (CFBE41o-) and two widely used laboratory cell lines, HeLa and HEK293. We discovered that specific synonymous SNVs exhibited differential effects which correlated with variable concentrations of cognate tRNAs. CONCLUSIONS: Our results highlight ways in which the presence of synonymous SNVs may alter local kinetics of mRNA translation; and thus, impact protein biogenesis and function. This effect is likely to influence results from mechansistic analysis and/or drug screeining efforts, and establishes importance of cereful model system selection based on genetic variation profile.

Ribosomal Stalk Protein Silencing Partially Corrects the ΔF508-CFTR Functional Expression Defect.

The most common cystic fibrosis (CF) causing mutation, deletion of phenylalanine 508 (ΔF508 or Phe508del), results in functional expression defect of the CF transmembrane conductance regulator (CFTR) at the apical plasma membrane (PM) of secretory epithelia, which is attributed to the degradation of the misfolded channel at the endoplasmic reticulum (ER). Deletion of phenylalanine 670 (ΔF670) in the yeast oligomycin resistance 1 gene (YOR1, an ABC transporter) of Saccharomyces cerevisiae phenocopies the ΔF508-CFTR folding and trafficking defects. Genome-wide phenotypic (phenomic) analysis of the Yor1-ΔF670 biogenesis identified several modifier genes of mRNA processing and translation, which conferred oligomycin resistance to yeast. Silencing of orthologues of these candidate genes enhanced the ΔF508-CFTR functional expression at the apical PM in human CF bronchial epithelia. Although knockdown of RPL12, a component of the ribosomal stalk, attenuated the translational elongation rate, it increased the folding efficiency as well as the conformational stability of the ΔF508-CFTR, manifesting in 3-fold augmented PM density and function of the mutant. Combination of RPL12 knockdown with the corrector drug, VX-809 (lumacaftor) restored the mutant function to ~50% of the wild-type channel in primary CFTRΔF508/ΔF508 human bronchial epithelia. These results and the observation that silencing of other ribosomal stalk proteins partially rescue the loss-of-function phenotype of ΔF508-CFTR suggest that the ribosomal stalk modulates the folding efficiency of the mutant and is a potential therapeutic target for correction of the ΔF508-CFTR folding defect.

Channel Gating Regulation by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) First Cytosolic Loop.

In this study, we present data indicating a robust and specific domain interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) first cytosolic loop (CL1) and nucleotide binding domain 1 (NBD1) that allows ion transport to proceed in a regulated fashion. We used co-precipitation and ELISA to establish the molecular contact and showed that binding kinetics were not altered by the common clinical mutation F508del. Both intrinsic ATPase activity and CFTR channel gating were inhibited severely by CL1 peptide, suggesting that NBD1/CL1 binding is a crucial requirement for ATP hydrolysis and channel function. In addition to cystic fibrosis, CFTR dysregulation has been implicated in the pathogenesis of prevalent diseases such as chronic obstructive pulmonary disease, acquired rhinosinusitis, pancreatitis, and lethal secretory diarrhea (e.g. cholera). On the basis of clinical relevance of the CFTR as a therapeutic target, a cell-free drug screen was established to identify modulators of NBD1/CL1 channel activity independent of F508del CFTR and pharmacologic rescue. Our findings support a targetable mechanism of CFTR regulation in which conformational changes in the NBDs cause reorientation of transmembrane domains via interactions with CL1 and result in channel gating.

Trafficking and function of the cystic fibrosis transmembrane conductance regulator: a complex network of posttranslational modifications.

Posttranslational modifications add diversity to protein function. Throughout its life cycle, the cystic fibrosis transmembrane conductance regulator (CFTR) undergoes numerous covalent posttranslational modifications (PTMs), including glycosylation, ubiquitination, sumoylation, phosphorylation, and palmitoylation. These modifications regulate key steps during protein biogenesis, such as protein folding, trafficking, stability, function, and association with protein partners and therefore may serve as targets for therapeutic manipulation. More generally, an improved understanding of molecular mechanisms that underlie CFTR PTMs may suggest novel treatment strategies for CF and perhaps other protein conformational diseases. This review provides a comprehensive summary of co- and posttranslational CFTR modifications and their significance with regard to protein biogenesis.

Combination therapy with cystic fibrosis transmembrane conductance regulator modulators augment the airway functional microanatomy.

Recently approved therapies that modulate CFTR function have shown significant clinical benefit, but recent investigations regarding their molecular mechanism when used in combination have not been consistent with clinical results. We employed micro-optical coherence tomography as a novel means to assess the mechanism of action of CFTR modulators, focusing on the effects on mucociliary clearance. Primary human airway monolayers from patients with a G551D mutation responded to ivacaftor treatment with increased ion transport, airway surface liquid depth, ciliary beat frequency, and mucociliary transport rate, in addition to decreased effective viscosity of the mucus layer, a unique mechanism established by our findings. These endpoints are consistent with the benefit observed in G551D patients treated with ivacaftor, and identify a novel mechanism involving mucus viscosity. In monolayers derived from F508del patients, the situation is more complicated, compounded by disparate effects on CFTR expression and function. However, by combining ion transport measurements with functional imaging, we establish a crucial link between in vitro data and clinical benefit, a finding not explained by ion transport studies alone. We establish that F508del cells exhibit increased mucociliary transport and decreased mucus effective viscosity, but only when ivacaftor is added to the regimen. We further show that improvement in the functional microanatomy in vitro corresponds with lung function benefit observed in the clinical trials, whereas ion transport in vitro corresponds to changes in sweat chloride. Functional imaging reveals insights into clinical efficacy and CFTR biology that significantly impact our understanding of novel therapies.

PRE-CLINICAL AND CLINICAL VALIDATION OF AN ANTI-CANCER MODALITY THAT ABLATES REFRACTORY, LOW GROWTH FRACTION TUMORS.

Intratumoral expression of the E. coli purine nucleoside phosphorylase (PNP) gene was originally described by our laboratories as a means to inhibit growth of solid tumors in vivo. The approach generates purine bases that disrupt DNA, RNA, and protein synthesis, a unique mechanism when compared with all approved or experimental cancer therapeutics. Use of PNP has been validated by numerous laboratories worldwide against human tumor xenografts (lung, liver, pancreas, bladder, glioma, and prostate, among others). Data from a recently completed phase 1 clinical trial has indicated substantial anti-cancer activity in human subjects with no serious toxicities.

A functional anatomic defect of the cystic fibrosis airway.

RATIONALE: The mechanisms underlying cystic fibrosis (CF) lung disease pathogenesis are unknown. OBJECTIVES: To establish mechanisms linking anion transport with the functional microanatomy, we evaluated normal and CF piglet trachea as well as adult swine trachea in the presence of selective anion inhibitors. METHODS: We investigated airway functional microanatomy using microoptical coherence tomography, a new imaging modality that concurrently quantifies multiple functional parameters of airway epithelium in a colocalized fashion. MEASUREMENTS AND MAIN RESULTS: Tracheal explants from wild-type swine demonstrated a direct link between periciliary liquid (PCL) hydration and mucociliary transport (MCT) rates, a relationship frequently invoked but never experimentally confirmed. However, in CF airways this relationship was completely disrupted, with greater PCL depths associated with slowest transport rates. This disrupted relationship was recapitulated by selectively inhibiting bicarbonate transport in vitro and ex vivo. CF mucus exhibited increased viscosity in situ due to the absence of bicarbonate transport, explaining defective MCT that occurs even in the presence of adequate PCL hydration. CONCLUSIONS: An inherent defect in CF airway surface liquid contributes to delayed MCT beyond that caused by airway dehydration alone and identifies a fundamental mechanism underlying the pathogenesis of CF lung disease in the absence of antecedent infection or inflammation.