GAPDH controls extracellular vesicle biogenesis and enhances the therapeutic potential of EV mediated siRNA delivery to the brain.

Extracellular vesicles (EVs) are biological nanoparticles with important roles in intercellular communication, and potential as drug delivery vehicles. Here we demonstrate a role for the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in EV assembly and secretion. We observe high levels of GAPDH binding to the outer surface of EVs via a phosphatidylserine binding motif (G58), which promotes extensive EV clustering. Further studies in a Drosophila EV biogenesis model reveal that GAPDH is required for the normal generation of intraluminal vesicles in endosomal compartments, and promotes vesicle clustering. Fusion of the GAPDH-derived G58 peptide to dsRNA-binding motifs enables highly efficient loading of small interfering RNA (siRNA) onto the EV surface. Such vesicles efficiently deliver siRNA to multiple anatomical regions of the brain in a Huntington's disease mouse model after systemic injection, resulting in silencing of the huntingtin gene in different regions of the brain

Measurements of CFTR-Mediated Cl- Secretion in Human Rectal Biopsies Constitute a Robust Biomarker for Cystic Fibrosis Diagnosis and Prognosis

BACKGROUND: Cystic Fibrosis (CF) is caused by ∼1,900 mutations in the CF transmembrane conductance regulator (CFTR) gene encoding for a cAMP-regulated chloride (Cl(-)) channel expressed in several epithelia. Clinical features are dominated by respiratory symptoms, but there is variable organ involvement thus causing diagnostic dilemmas, especially for non-classic cases. METHODOLOGY/PRINCIPAL FINDINGS: To further establish measurement of CFTR function as a sensitive and robust biomarker for diagnosis and prognosis of CF, we herein assessed cholinergic and cAMP-CFTR-mediated Cl(-) secretion in 524 freshly excised rectal biopsies from 118 individuals, including patients with confirmed CF clinical diagnosis (n=51), individuals with clinical CF suspicion (n=49) and age-matched non-CF controls (n=18). Conclusive measurements were obtained for 96% of cases. Patients with "Classic CF", presenting earlier onset of symptoms, pancreatic insufficiency, severe lung disease and low Shwachman-Kulczycki scores were found to lack CFTR-mediated Cl(-) secretion (<5%). Individuals with milder CF disease presented residual CFTR-mediated Cl(-) secretion (10-57%) and non-CF controls show CFTR-mediated Cl(-) secretion ≥ 30-35% and data evidenced good correlations with various clinical parameters. Finally, comparison of these values with those in "CF suspicion" individuals allowed to confirm CF in 16/49 individuals (33%) and exclude it in 28/49 (57%). Statistical discriminant analyses showed that colonic measurements of CFTR-mediated Cl(-) secretion are the best discriminator among Classic/Non-Classic CF and non-CF groups. CONCLUSIONS/SIGNIFICANCE: Determination of CFTR-mediated Cl(-) secretion in rectal biopsies is demonstrated here to be a sensitive, reproducible and robust predictive biomarker for the diagnosis and prognosis of CF. The method also has very high potential for (pre-)clinical trials of CFTR-modulator therapies.This work was supported by grants TargetScreen2 (EU/FP6/LSH/2005/037365), PIC/IC/83103/2007; PTDC/MAT/118335/2010; PEstOE/BIA/UI4046/2011 (to BioFIG) and PEstOE/MAT/UI0006/2011 (to CEAUL) from FCT (Portugal); and FAPESP (SPRF, Brazil), CNPq (40.8924/2006/3, Brazil) and Mukoviszidose e.V. S02/10 (Germany). MS and IU are recipients of SFRH/BD/35936/2007 and SFRH/BD/69180/2010 PhD fellowships (FCT, Portugal), respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Analysis of CFTR mutants in epithelial cells/tissues and testing of CFTR-repairing therapies

Tese de doutoramento, Bioquímica (Genética Molecular), Universidade de Lisboa, Faculdade de Ciências, 2015Cystic fibrosis (CF), the most common life-shortening genetic disorder among Caucasians, is caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, an anion (chloride/ bicarbonate) channel that is expressed at the apical membrane of epithelial cells to control salt and water transport. Clinically, CF is characterized by multiple manifestations in different organs, but the disease is dominated by the respiratory symptoms, the main cause of morbidity and mortality. The very thick mucus, a hallmark of CF lead to inefficient mucociliary clearance (MCC) and recurrent chronic bacterial infections, mostly by Pseudomonas aeruginosa, and to chronic exacerbated inflammation contributing to progressive loss of respiratory function and ultimately to death. Other CF classical symptoms include elevated sweat electrolytes, exocrine pancreatic insufficiency (85%), intestinal obstruction (meconium ileus in 15-20% of CF newborns and/or distal intestinal obstruction syndrome at a later age) and male infertility (95%). To date almost 2,000 CFTR gene mutations (most disease-causing) were reported, but one single mutation - F508del that impairs CFTR plasma membrane (PM) traffic – occurs in ~85% of CF patients. Although there is some degree of correlation between the CFTR genotype and disease severity, there is also a huge variation in the severity of clinical phenotypes, namely in lung disease. CFTR protein is an atypical ABC transporter (ABCC7) that functions as an apical membrane channel in epithelial cells and causes CF when defective. Unique to CFTR is its regulatory domain (RD) which includes a highly conformationally dynamic region - the regulatory extension (RE). The first nucleotide-binding domain (NBD1), where the most common CF-causing mutation (F508del) is located, contains the regulatory insertion (RI) also equally dynamic. Because CFTR is a negative regulator of the epithelial Na+ channel (ENaC), besides impaired cAMP-dependent chloride (Cl-)/ bicarbonate secretion, enhanced sodium (Na+) absorption by ENaC also occurs in CF airways. Novel therapies modulating the basic defect caused by F508del and other CFTR mutations are emerging. Indeed, high-throughput screens (HTS) have identified several novel small molecules with potential to treat the basic defect in CF and some have approved for clinical use. These include the potentiator VX-770/ivacaftor for patients with G551D and other mutations affecting channel gating and the VX-809/lumacaftor corrector (that partially rescues the trafficking defect of F508del-CFTR) in combination with VX-770 for F508del-homozygous patients However, the latter combination drug only showed 3% improvement in lung function and the respective mechanism of action (MoA) is not established, a step that is considered essential to identify more efficacious drugs. Despite these advances, not all CFTR mutations can be pharmacologically rescued (e.g., frameshift mutations or large deletions). For these, major hopes are on the ‘bypass’ therapeutic strategies which aim to correct the ionic imbalance in CF by downregulating ENaC hyperactivity and/or stimulating non-CFTR Cl- channels, such as the calcium (Ca2+)-, activated Cl- channels (CaCCs), to compensate for the absence of functional CFTR. The first objective (Chapter I) of this doctoral work was to assess the expression and function of the CFTR mutants in epithelial cells and tissues from the CF patients to determine its respective molecular/cellular defect and to evaluate the effect of potential therapeutic compounds in correcting the CF basic defect. One of these compounds is INO-4995 which is shown here to activate the endogenous CaCCs-TMEM16A in both CF and non-CF primary human bronchial epithelial cells. Within this objective, we also established a new methodology to culture human nasal epithelial cells from the nasal brushings in order to perform electrophysiological measurements in this novel cellular model, namely to assess patients’ responses to therapies in their own cells in a personalized pharmacotherapeutic approach. The second objective (Chapter II) of the present work, aimed to study the mechanisms that regulate processing and function of CFTR protein (both wt- and F508del) upon removal of two dynamic regions – the regulatory extension (RE) and the regulatory insertion (RI), which are absent in the other ABC transporters. Our results show that the deletion of the RE region leads to a dramatic stabilization of the immature form of the mutant protein. Moreover, our data demonstrate that VX-809 significantly rescued ΔRI-F508del-CFTR processing. More strikingly, its activity was only restored to normal CFTR levels when RE was also absent. Altogether, these data suggest that the two dynamic regions negatively impact the rescuing of F508del-CFTR by VX-809, but while RI deletion is essential to achieve full processing, only by RE removal is maximal channel function reached. The third and final objective (Chapter III) of this work, aimed to functionally characterize 270 genes, localized at two loci identified in a genomewide association and linkage study (GWAS) as significantly associated with severity of lung disease in CF, by finding their possible role in CFTR traffic or ENaC activity. To this end, we used cell-based microscopy assays and a loss-of function approach, (siRNA reverse transfection) to assess the respective effects by high-content automatic microscopy. We thus identified 66 genes regulating the traffic of wt-CFTR, 49 regulators of F508del-CFTR traffic and 41 regulators of ENaC function. Further, in order to clarify the MoA by which the hit siRNAs rescue F508del-CFTR, the 127 most significant genes were selected for the secondary screen using similar double-tagged constructs of F508del-CFTR but containing revertant mutations, i.e. second-site mutations which revert the F508del-CFTR traffic defect. Some of the hits identified within this study are potential drug targets that enhance CFTR traffic or decrease ENaC function in the context of CF lung disease. Further hit characterization is expected to generate a valuable resource for the community regarding both insight into those pathways and the establishment of novel non-CFTR therapeutic targets for CF. Altogether, results included in the present thesis bring new insights into our understanding of expression, traffic, function and regulation of the normal and mutant CFTR protein both in heterologous cellular systems and in primary human airway cell cultures. The relevance of developing the “by-pass therapies” aiming at overcoming the ionic defects caused in epithelia by the loss of CFTR via ENaC and CaCCs is also a major point of the current work, such as understanding the mechanism of action of the novel therapeutical compounds approved for a use in CF patients, such as VX-770 and VX-809. The characterization of genetically identified modifier genes by functional approaches (siRNA screens), constitutes an original approach linking genetics to functional genomics towards a global understanding of disease pathways.Fundação para a Ciência e a Tecnologia (FCT), SFRH/BD/69180/201

Full Rescue of F508del-CFTR Processing and Function by CFTR Modulators Can Be Achieved by Removal of Two Regulatory Regions

Cystic Fibrosis (CF) is caused by mutations in the CF Transmembrane conductance Regulator (CFTR), the only ATP-binding cassette (ABC) transporter functioning as a channel. Unique to CFTR is a regulatory domain which includes a highly conformationally dynamic region&mdash;the regulatory extension (RE). The first nucleotide-binding domain of CFTR contains another dynamic region&mdash;regulatory insertion (RI). Removal of RI rescues the trafficking defect of CFTR with F508del, the most common CF-causing mutation. Here we aimed to assess the impact of RE removal (with/without RI or genetic revertants) on F508del-CFTR trafficking and how CFTR modulator drugs VX-809/lumacaftor and VX-770/ivacaftor rescue these variants. We generated cell lines expressing &Delta;RE and &Delta;RI CFTR (with/without genetic revertants) and assessed CFTR expression, stability, plasma membrane levels, and channel activity. Our data demonstrated that &Delta;RI significantly enhanced rescue of F508del-CFTR by VX-809. While the presence of the RI seems to be precluding full rescue of F508del-CFTR processing by VX-809, this region appears essential to rescue its function by VX-770, suggesting some contradictory role in rescue of F508del-CFTR by these two modulators. This negative impact of RI removal on VX-770-stimulated currents on F508del-CFTR can be compensated by deletion of the RE which also leads to the stabilization of this mutant. Despite both regions being conformationally dynamic, RI precludes F508del-CFTR processing while RE affects mostly its stability and channel opening

Measurements of Functional Responses in Human Primary Lung Cells as a Basis for Personalized Therapy for Cystic Fibrosis

Background: The best investigational drug to treat cystic fibrosis (CF) patients with the most common CF-causing mutation (F508del) is VX-809 (lumacaftor) which recently succeeded in Phase III clinical trial in combination with ivacaftor. This corrector rescues F508del-CFTR from its abnormal intracellular localization to the cell surface, a traffic defect shared by all Class II CFTR mutants. Our goal here is to test the efficacy of lumacaftor in other Class II mutants in primary human bronchial epithelial (HBE) cells derived from CF patients. Methods: The effect of lumacaftor was investigated in primary HBE cells from non-CF and CF patients with F508del/F508del, A561E/A561E, N1303K/G542X, F508del/G542X and F508del/Y1092X genotypes by measurements of Forskolin plus Genistein-inducible equivalent short-circuit current (Ieq-SC-Fsk + Gen) in perfused open-circuit Ussing chambers. Efficacy of corrector C18 was also assessed on A561E/A561E and F508del/F508del cells. Results: Our data indicate that A561E (when present in both alleles) responds positively to lumacaftor treatment at equivalent efficacy of F508del in primary HBE cells. Similarly, lumacaftor has a positive impact on Y1092X, but not on N1303K. Our data also show that cells with only one copy of F508del-CFTR respond less to VX-809. Moreover, there is great variability in lumacaftor responses among F508del-homozygous cells from different donors. Compound C18 failed to rescue A561E-CFTR but not in F508del-CFTR, thus plausibly it has a different mechanism of action distinct from lumacaftor. Conclusions: CF patients with A561E (and likely also those with Y1029X) can potentially benefit from lumacaftor. Moreover, the methodology used here exemplifies how ex vivo approaches may apply personalized therapies to CF and possibly other respiratory diseases

High-Content siRNA Screen Reveals Global ENaC Regulators and Potential Cystic Fibrosis Therapy Targets

Dysfunction of ENaC, the epithelial sodium channel that regulates salt and water reabsorption in epithelia, causes several human diseases, including cystic fibrosis (CF). To develop a global understanding of molecular regulators of ENaC traffic/function and to identify of candidate CF drug targets, we performed a large-scale screen combining high-content live-cell microscopy and siRNAs in human airway epithelial cells. Screening over 6,000 genes identified over 1,500 candidates, evenly divided between channel inhibitors and activators. Genes in the phosphatidylinositol pathway were enriched on the primary candidate list, and these, along with other ENaC activators, were examined further with secondary siRNA validation. Subsequent detailed investigation revealed ciliary neurotrophic factor receptor (CNTFR) as an ENaC modulator and showed that inhibition of (diacylglycerol kinase, iota) DGKι, a protein involved in PiP2 metabolism, downgrades ENaC activity, leading to normalization of both Na+ and fluid absorption in CF airways to non-CF levels in primary human lung cells from CF patients. [Display omitted] •High-content siRNA screens reveal cystic-fibrosis-linked sodium channel modulators•Phosphatidylinositol pathway genes are enriched in the primary screen data set•Ciliary neurotrophic factor regulator (CNFTR) implicated in channel control•DGKι inhibitor normalizes Na+ and fluid absorption in human CF primary airway cells Screening of live human cells reveals both inhibitors and activators of the epithelial Na+ channel, including an extensive network of phosphatidylinositol pathway genes and DGKι, which can be inhibited to revert ion flow and fluid absorption in cells from CF patient airways

Correlations between clinical outcomes and CFTR-mediated I_sc in rectal biopsies.

<p>NOTE: Pearson coefficients (r) and p-values showing statistical correlations (<i>p</i><0.05) are highlighted (n = 95).</p

Results from Ussing chamber measurements in rectal biopsies from 113 individuals.

<p>Original recordings of the effects of cholinergic (CCH, 100 µM) and cAMP-dependent (IBMX, 100 µM and Fsk, 2 µM, basolateral) activation on transepithelial voltage in rectal tissues from (<b>A</b>) a healthy control presenting CCH lumen-negative responses; (<b>B</b>) a Non-Classic CF patient showing biphasic responses, thus presenting residual CFTR function; and (<b>C</b>) a Classic CF patient with no detectable Cl⁻ secretion, presenting only lumen-positive responses. All the experiments were performed in the presence of Amiloride (Amil, 20 µM, luminal) and Indomethacin (Indo, 10 µM, basolateral). (<b>D</b>) Summary of activated short-circuit currents (ΔI_sc) for basal CCH (I_sc-CCH), IBMX/Fsk (I_sc-IBMX/Fsk) and CCH following IBMX/Fsk application (I_{sc-CCH(IBMX/Fsk)}) in rectal biopsies from controls (lumen-negative, filled circles, n = 18), Non-Classic CF (biphasic, filled diamonds, n = 5), Classic CF (lumen positive, filled triangles, n = 46) and CF suspicious patients (open squares, n = 44) showing lumen-negative, biphasic and lumen-positive responses. Data represent the mean of the mean measurements on 2–5 rectal biopsies per individual. Black solid line shows mean ΔI_sc values for each group represented in µA/cm²: Controls (I_sc-CCH =  −109.28±14.47; I_sc-IBMX/Fsk =  −55.38±11.12; I_{sc-CCH(IBMX/Fsk)} =  −162.07±19.64); Non-Classic CF (I_sc-CCH = 47.20±17.86; I_sc-IBMX/Fsk =  −16.27±2.83; I_{sc-CCH(IBMX/Fsk)} =  −21.64±8.01); Classic CF (I_sc-CCH = 30.13±2.66; I_sc-IBMX/Fsk = 11.30±0.78; I_{sc-CCH(IBMX/Fsk)} = 27.17±2.22); and CF suspicious with similar ΔI_sc distribution.</p