Cystic Fibrosis Conductance Regulator Research Articles

Highly Effective Modulator Therapy: Implications for the Microbial Landscape in Cystic Fibrosis

Cystic fibrosis (CF) is an autosomal recessive multisystem disorder caused by mutations in the cystic fibrosis conductance regulator (CFTR) anion channel. In the lungs specifically, CFTR mutations lead to changes in mucus viscosity and defective mucociliary clearance. Moreover, people with CF (pwCF) mount an insufficient immune response to invading pathogens, which predisposes individuals to chronic airway disease associated with chronic inflammation, colonization, and recurrent infections by mainly opportunistic pathogens. These chronic infections in the CF lung are typically polymicrobial and frequently harbour multidrug-resistant pathogens, making both treatment and eradication very challenging. During the last decade, the development of highly effective CFTR modulator therapy (HEMT) has led to a breakthrough in treatment options for pwCF. While the majority of pwCF now live longer and have fewer CF exacerbations, colonisation with common respiratory pathogens persists, thereby contributing to chronic inflammation and infection. Interestingly, there are limited reports examining the lung microbiome in the post-modulator era. Since ETI treatment is still quite novel and has only been used for about five years by now, this review will be one of the first discussing the current literature on the effect of ETI on CF pathogens. In addition, we will identify unanswered questions that remain from the effect of HEMT on the CF microbiome.

A nonnatural peptide targeting the A-kinase anchoring function of PI3Kγ for therapeutic cAMP modulation in pulmonary cells

A-kinase anchoring proteins (AKAPs) are key orchestrators of cyclic AMP (cAMP) signaling that act by recruiting protein kinase A (PKA) in proximity of its substrates and regulators to specific subcellular compartments. Modulation of AKAPs function offers the opportunity to achieve compartment-restricted modulation of the cAMP/PKA axis, paving the way to new targeted treatments. For instance, blocking the AKAP activity of PI3Kγ improves lung function by inducing cAMP-mediated bronchorelaxation, ion transport and anti-inflammatory responses. Here, we report the generation of a non-natural peptide, DRI-Pep #20, optimized to disrupt the AKAP function of PI3Kγ. DRI-Pep #20 mimicked the native interaction between the N-terminal domain of PI3Kγ and PKA, demonstrating nanomolar affinity for PKA, high resistance to protease degradation and high permeability to the pulmonary mucus barrier. DRI-Pep #20 triggered cAMP elevation both in vivo in the airway tract of mice upon intratracheal administration, and in vitro in bronchial epithelial cells of cystic fibrosis (CF) patients. In CF cells, DRI-Pep #20 rescued the defective function of the cAMP-operated channel cystic fibrosis conductance regulator (CFTR), by boosting the efficacy of approved CFTR modulators. Overall, this study unveils DRI-Pep #20 as a potent PI3Kγ/PKA disruptor for achieving therapeutic cAMP elevation in chronic respiratory disorders.

Phosphatidylserine liposomes induce a phagosome acidification-dependent and ROS-mediated intracellular killing of Mycobacterium abscessus in human macrophages.

Mycobacterium abscessus (Mab) is an opportunistic nontuberculous mycobacterium responsible of difficult-to-treat pulmonary infections in vulnerable patients, such as those suffering from Cystic Fibrosis (CF), where it represents a major cause of morbidity and mortality. Additionally, due to the intrinsic extensive antimicrobial resistance spectrum displayed by this species and the side effects reported for some available antibiotics, the therapeutic management of such infections remains extremely difficult. In the present study, we show that phosphatidylserine liposomes (PS-L) enhance intracellular mycobacterial killing of Mab infected human macrophages with functional or pharmacologically inhibited cystic fibrosis conductance regulator (CFTR), by a mechanism involving phagosome acidification and reactive oxygen species (ROS) production. Additionally, PS-L significantly reduce proinflammatory response of Mab infected macrophages in terms of NF-kB activation and TNF-α production, irrespective of CFTR inhibition. Altogether, these results represent the proof of concept for a possible future development of PS-L as a therapeutic strategy against difficult-to-treat Mab infection.

The anion exchanger slc26a3 regulates colonic mucus expansion during steady state and in response to prostaglandin E2, while Cftr regulates de novo mucus release in response to carbamylcholine

SummaryThe intestinal epithelium is covered by mucus that protects the tissue from the luminal content. Studies have shown that anion secretion via the cystic fibrosis conductance regulator (Cftr) regulates mucus formation in the small intestine. However, mechanisms regulating mucus formation in the colon are less understood. The aim of this study was to explore the role of anion transport in the regulation of mucus formation during steady state and in response to carbamylcholine (CCh) and prostaglandin E2 (PGE2). The broad-spectrum anion transport inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS), CftrdF508 (CF) mice, and the slc26a3 inhibitor SLC26A3-IN-2 were used to inhibit anion transport. In the distal colon, steady-state mucus expansion was reduced by SLC26A3-IN-2 and normal in CF mice. PGE2 stimulated mucus expansion without de novo mucus release in wild type (WT) and CF colon via slc26a3 sensitive mechanisms, while CCh induced de novo mucus secretion in WT but not in CF colon. However, when added simultaneously, CCh and PGE2 stimulated de novo mucus secretion in the CF colon via DIDS-sensitive pathways. A similar response was observed in CF ileum that responded to CCh and PGE2 with DIDS-sensitive de novo mucus secretion. In conclusion, this study suggests that slc26a3 regulates colonic mucus expansion, while Cftr regulates CCh-induced de novo mucus secretion from ileal and distal colon crypts. Furthermore, these findings demonstrate that in the absence of a functional Cftr channel, parallel stimulation with CCh and PGE2 activates additional anion transport processes that help release mucus from intestinal goblet cells.

Influence of highly effective modulator therapy on the sputum proteome in cystic fibrosis

BackgroundThere have been dramatic clinical improvements in people with cystic fibrosis (PwCF) commenced on the cystic fibrosis conductance regulator (CFTR) modulator elexacaftor/tezacaftor/ivacaftor (ETI). Sputum proteomics is a powerful research technique capable of identifying important airway disease mechanisms. Using this technique, we evaluated how ETI changes the sputum proteome in PwCF. MethodsSputum samples from 21 CF subjects pre- and post- ETI, 6 CF controls ineligible for ETI, and 15 healthy controls were analysed by liquid chromatography mass spectrometry. ResultsPost-ETI, mean FEV1 % increased by 13.7 % (SD 7.9). Principal component and hierarchical clustering analysis revealed that the post-ETI proteome shifted to an intermediate state that was distinct from pre-ETI and healthy controls, even for those achieving normal lung function. Functional analysis showed incomplete resolution of neutrophilic inflammation. The CF control sputum proteome did not alter. At the protein-level many more proteins increased in abundance than decreased following ETI therapy (80 vs 30; adjusted p value <0.05), including many that have anti-inflammatory properties. Of those proteins that reduced in abundance many were pro-inflammatory neutrophil-derived proteins. Several important respiratory proteases were unchanged. ConclusionsSputum proteomics can provide insights into CF lung disease mechanisms and how they are modified by therapeutic intervention, in this case ETI. This study identifies imbalances in pro- and anti- inflammatory proteins in sputum that partially resolve with ETI even in those achieving normal spirometry values. This post-ETI intermediate state could contribute to ongoing airway damage and therefore its relevance to clinical outcomes needs to be established.

Spectrum of Cystic Fibrosis Conductance Regulator Gene Mutations Reported in Pakistani Descent Cystic Fibrosis Patients

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. This study aims to determine the genotypic and phenotypic spectrum of the CFTR gene mutations reported in the literature for Pakistani-origin CF patients. Databases were searched for such studies from 1947-2019 for sample size, method of diagnosis, and CFTR gene mutations. The authors identified 12 studies reporting 33 CFTR gene mutations, both intronic as well as exonic in Pakistani origin patients. The most widely tested mutation was D508 with a frequency of 17%-60%. No hotspot zone was identified and not all reported mutations were causing disease. There is a need to identify common mutations in the Pakistani population to develop population-specific CFTR mutations panel. This will enable the researchers to perform phenotype-genotype correlation studies to improve the CF detection rate. Key Words: Cystic fibrosis, Pakistan, Mutations, CFTR.

Clinical outcomes of a large cohort of individuals with the F508del/5T;TG12 CFTR genotype

BackgroundIn recent years, patients with cystic fibrosis (CF) conductance regulator (CFTR) variant poly(T) sequences have been increasingly reported with a wide spectrum of clinical severity. We describe the long-term clinical outcomes and progression to a CF diagnosis over time in a large Italian cohort of patients carrying the CFTR F508del/5T;TG12 genotype. MethodsA retrospective analysis of subjects from 10 CF centres in Italy with the F508del/5T;TG12 genotype was performed. Demographic, clinical, microbiological, and biochemical data, as well as information about the follow-ups and complications of the enroled patients, were collected. ResultsA total of 129 subjects (54 females; median age: 15.0 years, range: 0–58 years; 59 older than 18 years) were included. In terms of initial diagnoses, 30 were CF (23.3%), 41 were CFTR-related disorder (CFTR-RD) (31.7%), and 58 were CF transmembrane conductance regulator-related metabolic syndrome/cystic fibrosis screen positive, inconclusive diagnosis (CRMS/CFSPID) (45.0%). After a median follow-up of 6.7 years (range 0.2–25 years), 15 patients progressed to CF, bringing the total number of CF diagnoses to 45/129 (34.9%). Most of these patients had mild lung diseases with pancreatic sufficiency and a low prevalence of CF-related complications. ConclusionsAt the end of the study, 34.9% of subjects with the CFTR F508del/5T;TG12 genotype were diagnosed with CF. We suggest including patients with the F508del/5T;TG12 genotype in long-term follow-ups.

POSA271 A Comparative Analysis of Pricing and Reimbursement of Cystic Fibrosis Medicines in Europe

Cystic fibrosis conductance regulator (CFTR) gene modulators, ivacaftor, lumacaftor/ivacaftor and tezacaftor/ivacaftor, have substantially improved patients’ lives yet significantly burden healthcare budgets. This analysis aims to compare pricing and reimbursement of aforementioned cystic fibrosis medicines, developed by Vertex Pharmaceuticals, Inc., across European countries. Clinical trial registries, national databases, health technology assessment reports and grey literature of Belgium, the Netherlands, Ireland, the United Kingdom, France, Germany, Denmark, Sweden, Switzerland, Austria, Spain and Poland were consulted. Publicly available efficacy results, prices, reimbursement statuses, economic evaluations, budget impacts and managed entry agreements of CFTR modulators were reported. All interventions improved lung clearance and expiratory volume. Lowest prices were captured in Switzerland at €426 per DDD, €307 per DDD and €186 per ADD for Kalydeco®, Orkambi® and Symkevi®, respectively. Spain had the highest prices for Kalydeco® and Symkevi® at €851 per DDD and €337 per ADD, whereas Orkambi® was most expensive in Poland at €983 per DDD. In all countries, treatments were deemed not to be cost-effective. The annual budget impact of these CFTR gene modulators varied between countries and depended on factors such as local product prices, size of target population, scope of costs and discounting. However, all modulators were fully reimbursed in majority of countries except for France, Germany, Sweden and Poland that decided on partial or no reimbursement. Managed entry agreements were confidential but commonly adopted to address financial uncertainties. Discrepancies in reimbursement decisions and prices for Kalydeco®, Orkambi® and Symkevi® across European countries were detected. More transparency and increased country collaboration might stimulate uniform patient access to these cystic fibrosis medicines.

An Innovative Protocol for Metaproteomic Analyses of Microbial Pathogens in Cystic Fibrosis Sputum.

Hallmarks of cystic fibrosis (CF) are increased viscosity of mucus and impaired mucociliary clearance within the airways due to mutations of the cystic fibrosis conductance regulator gene. This facilitates the colonization of the lung by microbial pathogens and the concomitant establishment of chronic infections leading to tissue damage, reduced lung function, and decreased life expectancy. Although the interplay between key CF pathogens plays a major role during disease progression, the pathophysiology of the microbial community in CF lungs remains poorly understood. Particular challenges in the analysis of the microbial population present in CF sputum is (I) the inhomogeneous, viscous, and slimy consistence of CF sputum, and (II) the high number of human proteins masking comparably low abundant microbial proteins. To address these challenges, we used 21 CF sputum samples to develop a reliable, reproducible and widely applicable protocol for sputum processing, microbial enrichment, cell disruption, protein extraction and subsequent metaproteomic analyses. As a proof of concept, we selected three sputum samples for detailed metaproteome analyses and complemented and validated metaproteome data by 16S sequencing, metabolomic as well as microscopic analyses. Applying our protocol, the number of bacterial proteins/protein groups increased from 199-425 to 392-868 in enriched samples compared to nonenriched controls. These early microbial metaproteome data suggest that the arginine deiminase pathway and multiple proteases and peptidases identified from various bacterial genera could so far be underappreciated in their contribution to the CF pathophysiology. By providing a standardized and effective protocol for sputum processing and microbial enrichment, our study represents an important basis for future studies investigating the physiology of microbial pathogens in CF in vivo – an important prerequisite for the development of novel antimicrobial therapies to combat chronic recurrent airway infection in CF.

Dysregulation of ion transport in the lung epithelium infected with SARS-CoV-2.

Dysregulation of ion transport in the lung epithelium infected with SARS-CoV-2.

Cell engineering method using fluorogenic oligonucleotide signaling probes and flow cytometry.

ObjectiveChromovert® Technology is presented as a new cell engineering technology to detect and purify living cells based on gene expression.MethodsThe technology utilizes fluorogenic oligonucleotide signaling probes and flow cytometry to detect and isolate individual living cells expressing one or more transfected or endogenously-expressed genes.ResultsResults for production of cell lines expressing a diversity of ion channel and membrane proteins are presented, including heteromultimeric epithelial sodium channel (αβγ-ENaC), sodium voltage-gated ion channel 1.7 (NaV1.7-αβ1β2), four unique γ-aminobutyric acid A (GABAA) receptor ion channel subunit combinations α1β3γ2s, α2β3γ2s, α3β3γ2s and α5β3γ2s, cystic fibrosis conductance regulator (CFTR), CFTR-Δ508 and two G-protein coupled receptors (GPCRs) without reliance on leader sequences and/or chaperones. In addition, three novel plasmid-encoded sequences used to introduce 3′ untranslated RNA sequence tags in mRNA expression products and differentially-detectable fluorogenic probes directed to each are described. The tags and corresponding fluorogenic signaling probes streamline the process by enabling the multiplexed detection and isolation of cells expressing one or more genes without the need for gene-specific probes.ConclusionsChromovert technology is provided as a research tool for use to enrich and isolate cells engineered to express one or more desired genes.

Therapeutic peptides for the treatment of cystic fibrosis: Challenges and perspectives.

Cystic fibrosis (CF) is the most common amongst rare genetic diseases, affecting more than 70.000 people worldwide. CF is characterized by a dysfunctional chloride channel, termed cystic fibrosis conductance regulator (CFTR), which leads to the production of a thick and viscous mucus layer that clogs the lungs of CF patients and traps pathogens, leading to chronic infections and inflammation and, ultimately, lung damage. In recent years, the use of peptides for the treatment of respiratory diseases, including CF, has gained growing interest. Therapeutic peptides for CF include antimicrobial peptides, inhibitors of proteases, and modulators of ion channels, among others. Peptides display unique features that make them appealing candidates for clinical translation, like specificity of action, high efficacy, and low toxicity. Nevertheless, the intrinsic properties of peptides, together with the need of delivering these compounds locally, e.g. by inhalation, raise a number of concerns in the development of peptide therapeutics for CF lung disease. In this review, we discuss the challenges related to the use of peptides for the treatment of CF lung disease through inhalation, which include retention within mucus, proteolysis, immunogenicity and aggregation. Strategies for overcoming major shortcomings of peptide therapeutics will be presented, together with recent developments in peptide design and optimization, including computational analysis and high-throughput screening.

Impact of Physical Activity on Cognitive Functions: A New Field for Research and Management of Cystic Fibrosis.

Cystic Fibrosis (CF) is a genetic disease inherited by an autosomal recessive mechanism and characterized by a progressive and severe multi-organ failure. Mutations in Cystic Fibrosis Conductance Regulator (CFTR) protein cause duct obstructions from dense mucus secretions and chronic inflammation related to organ damage. The progression of the disease is characterized by a decline of lung function associated with metabolic disorders and malnutrition, musculoskeletal disorders and thoracic deformities, leading to a progressive decrement of the individual’s quality of life. The World Health Organization (WHO) qualifies Physical Activity (PA) as a structured activity produced by skeletal muscles’ movements that requires energy consumption. In the last decade, the number of studies on PA increased considerably, including those investigating the effects of exercise on cognitive and brain health and mental performance. PA is recommended in CF management guidelines, since it improves clinic outcomes, such as peripheral neuropathy, oxygen uptake peak, bone health, glycemic control and respiratory functions. Several studies regarding the positive effects of exercise in patients with Cystic Fibrosis were carried out, but the link between the effects of exercise and cognitive and brain health in CF remains unclear. Animal models showed that exercise might improve learning and memory through structural changes of brain architecture, and such a causal relationship can also be described in humans. Indeed, both morphological and environmental factors seem to be involved in exercise-induced neural plasticity. An increase of gray matter volume in specific areas is detectable as a consequence of regular training in humans. Neurobiological processes associated with brain function improvements include biochemical modifications, such as neuromodulator or neurohormone release, brain-derived neurotrophic factor (BDNF) production and synaptic activity changes. From a functional point of view, PA also seems to be an environmental factor enhancing cognitive abilities, such as executive functions, memory and processing speed. This review describes the current state of research regarding the impacts of physical activity and exercise on cognitive functions, introducing a possible novel field of research for optimizing the management of Cystic Fibrosis.

Anti-Infectives Restore ORKAMBI® Rescue of F508del-CFTR Function in Human Bronchial Epithelial Cells Infected with Clinical Strains of P. aeruginosa.

Chronic infection and inflammation are the primary causes of declining lung function in Cystic Fibrosis (CF) patients. ORKAMBI® (Lumacaftor-Ivacaftor) is an approved combination therapy for Cystic Fibrosis (CF) patients bearing the most common mutation, F508del, in the cystic fibrosis conductance regulator (CFTR) protein. It has been previously shown that ORKAMBI®-mediated rescue of CFTR is reduced by a pre-existing Pseudomonas aeruginosa infection. Here, we show that the infection of F508del-CFTR human bronchial epithelial (HBE) cells with lab strain and four different clinical strains of P. aeruginosa, isolated from the lung sputum of CF patients, decreases CFTR function in a strain-specific manner by 48 to 88%. The treatment of infected cells with antibiotic tobramycin or cationic antimicrobial peptide 6K-F17 was found to decrease clinical strain bacterial growth on HBE cells and restore ORKAMBI®-mediated rescue of F508del-CFTR function. Further, 6K-F17 was found to downregulate the expression of pro-inflammatory cytokines, interleukin (IL)-8, IL-6, and tumor necrosis factor-α in infected HBE cells. The results provide strong evidence for a combination therapy approach involving CFTR modulators and anti-infectives (i.e., tobramycin and/or 6K-F17) to improve their overall efficacy in CF patients.

Modulation of Hepatic MRP3/ABCC3 by Xenobiotics and Pathophysiological Conditions: Role in Drug Pharmacokinetics.

Liver transporters play an important role in the pharmacokinetics and disposition of pharmaceuticals, environmental contaminants, and endogenous compounds. Among them, the family of ATP-Binding Cassette (ABC) transporters is the most important due to its role in the transport of endo- and xenobiotics. The ABCC sub-family is the largest one, consisting of 13 members that include the cystic fibrosis conductance regulator (CFTR/ABCC7); the sulfonylurea receptors (SUR1/ABCC8 and SUR2/ABCC9) and the multidrug resistanceassociated proteins (MRPs). The MRP-related proteins can collectively confer resistance to natural, synthetic drugs and their conjugated metabolites, including platinum-containing compounds, folate anti-metabolites, nucleoside and nucleotide analogs, among others. MRPs can be also catalogued into "long" (MRP1/ABCC1, -2/C2, -3/C3, -6/C6, and -7/C10) and "short" (MRP4/C4, -5/C5, -8/C11, -9/C12, and -10/C13) categories. While MRP2/ABCC2 is expressed in the canalicular pole of hepatocytes, all others are located in the basolateral membrane. In this review, we summarize information from studies examining the changes in expression and regulation of the basolateral hepatic transporter MPR3/ABCC3 by xenobiotics and during various pathophysiological conditions. We also focus, primarily, on the consequences of such changes in the pharmacokinetic, pharmacodynamic and/or toxicity of different drugs of clinical use transported by MRP3.

Restoration of F508-del Function by Transcomplementation: The Partners Meet in the Endoplasmic Reticulum.

Because of the small size of adeno-associated virus, AAV, the cystic fibrosis conductance regulator, CFTR, cDNA is too large to fit within AAV and must be truncated. We report here on two truncated versions of CFTR, which, when inserted into AAV1 and used to infect airway cells, rescue F508-del CFTR via transcomplementation. The purpose of this study is to shed light on where in the cell transcomplementation occurs and how it results in close association between the endogenous F508-del and truncated CFTR. We treated CF airway cells (CFBE41o-) with AAV2/1 (AAV2 inverted terminal repeats/AAV1 capsid) containing truncated forms of CFTR, ∆264 and ∆27-264 CFTR, who can restore the function of F508-del by transcomplementation. We addressed the aims of the study using a combination of confocal microscopy and short circuit currents measurements. For the latter, CF bronchial epithelial cells (CFBE) were grown on permeable supports. We show that both F508del and the truncation mutants colocalize in the ER and that both the rescued F508-del and the transcomplementing mutants reach the plasma membrane together. There was significant fluorescence resonance energy transfer (FRET) between F508-del and the transcomplementing mutants within the endoplasmic reticulum (ER), suggesting that transcomplementation occurs through a bimolecular interaction. We found that transcomplementation could increase the Isc in CFBE41o- cells stably expressing additional wt-CFTR or F508-del and in parental CFBE41o- cells expressing endogenous levels of F508-del. We conclude that the functional rescue of F508-del by transcomplementation occurs via a bimolecular interaction that most likely begins in the ER and continues at the plasma membrane. These results come at an opportune time for developing a gene therapy for CF and offer new treatment options for a wide range of CF patients.

Mechanisms of endothelial cell dysfunction in cystic fibrosis

Although cystic fibrosis (CF) patients exhibit signs of endothelial perturbation, the functions of the cystic fibrosis conductance regulator (CFTR) in vascular endothelial cells (EC) are poorly defined. We sought to uncover biological activities of endothelial CFTR, relevant for vascular homeostasis and inflammation. We examined cells from human umbilical cords (HUVEC) and pulmonary artery isolated from non-cystic fibrosis (PAEC) and CF human lungs (CF-PAEC), under static conditions or physiological shear. CFTR activity, clearly detected in HUVEC and PAEC, was markedly reduced in CF-PAEC. CFTR blockade increased endothelial permeability to macromolecules and reduced trans‑endothelial electrical resistance (TEER). Consistent with this, CF-PAEC displayed lower TEER compared to PAEC. Under shear, CFTR blockade reduced VE-cadherin and p120 catenin membrane expression and triggered the formation of paxillin- and vinculin-enriched membrane blebs that evolved in shrinking of the cell body and disruption of cell-cell contacts. These changes were accompanied by enhanced release of microvesicles, which displayed reduced capability to stimulate proliferation in recipient EC. CFTR blockade also suppressed insulin-induced NO generation by EC, likely by inhibiting eNOS and AKT phosphorylation, whereas it enhanced IL-8 release. Remarkably, phosphodiesterase inhibitors in combination with a β2 adrenergic receptor agonist corrected functional and morphological changes triggered by CFTR dysfunction in EC. Our results uncover regulatory functions of CFTR in EC, suggesting a physiological role of CFTR in the maintenance EC homeostasis and its involvement in pathogenetic aspects of CF. Moreover, our findings open avenues for novel pharmacology to control endothelial dysfunction and its consequences in CF.

Small Molecule Induced Changes in the Trafficking of the Cystic Fibrosis Conductance Regulator

Small Molecule Induced Changes in the Trafficking of the Cystic Fibrosis Conductance Regulator

Chronic Pancreatitis in the 21st Century - Research Challenges and Opportunities: Summary of a National Institute of Diabetes and Digestive and Kidney Diseases Workshop.

A workshop was sponsored by the National Institute of Diabetes and Digestive and Kidney Diseases to focus on research gaps and opportunities in chronic pancreatitis (CP) and its sequelae. This conference marked the 20th year anniversary of the discovery of the cationic trypsinogen (PRSS1) gene mutation for hereditary pancreatitis. The event was held on July 27, 2016, and structured into 4 sessions: (1) pathophysiology, (2) exocrine complications, (3) endocrine complications, and (4) pain. The current state of knowledge was reviewed; many knowledge gaps and research needs were identified that require further investigation. Common themes included the need to design better tools to diagnose CP and its sequelae early and reliably, identify predisposing risk factors for disease progression, develop standardized protocols to distinguish type 3c diabetes mellitus from other types of diabetes, and design effective therapeutic strategies through novel cell culture technologies, animal models mimicking human disease, and pain management tools. Gene therapy and cystic fibrosis conductance regulator potentiators as possible treatments of CP were discussed. Importantly, the need for CP end points and intermediate targets for future drug trials was emphasized.

23. SiRNA and CRISPR/Cas9 Mediated Knockout of αENAC

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis conductance regulator (CFTR) gene, which encodes for a chloride channel. Loss of CFTR upregulates the epithelial sodium channel (ENaC) causing hyperabsorption of sodium, reducing the watery lining of the lung, leading to impaired mucociliary clearance and enabling inflammatory lung damage. Therefore, inhibition of ENaC has been proposed as a treatment in CF. In this study we are assessing the use of siRNA and CRISPR/Cas9 system as complementary methods of achieving disruption of ENaC expression in airway epithelial cells using receptor-targeted nanocomplexes. We sought to compare the efficiency and persistence of silencing, repeatability of delivery and its toxicity. We assessed silencing of the αENaC subunit by transfecting primary CFBE cells growing in Air-Liquid Interface cultures with siRNA-bearing nanocomplexes and achieved 30% silencing at the mRNA level. We then assessed their silencing efficiency in lungs of normal mice (C57BL/6) delivered by oropharyngeal instillation. Following a single dose, the siRNA formulations achieved ~30% knockdown of mouse αENaC. Following repeated delivery (3 doses at 48h intervals) we found out that 48h after the last administration ~50% silencing was achieved, with no adverse effects (as judged by body weight and histology), whilst following a single administration there was still ~30% silencing 7 days later. An algorithm was used to design 6 different guide RNA targets for αENaC. These were used to transfect cells along with Cas9 and showed varied levels of indel mutation rates in the αENaC gene by the T7 endonuclease I assay although two, T3 and T4, were optimal. In order to increase the amount of indels created we subjected the cells (CFTE and HBE) to sequential transfections of the same target at 48h intervals. This showed an accumulation of indels in cells targeted with T3 at a rate of 33.3% in HBE cells. This correlated with a 60% decrease in ENaC mRNA compared to controls and with 40% after a single dose to 65% decrease in protein levels after three doses. The siRNA-mediated silencing showed that we can repeatedly deliver the formulations with no adverse effects and that the effect of a single dose, although transient, can last for at least 1 week. CRISPR/Cas9 also mediated efficient gene disruption of αENaC, but with the advantage that this knockdown will be permanent in cells where the life span is quite long. Another encouraging finding was the accumulation of gene disruption with both systems when delivered by a non-immunogenic vector. This is very important as CF therapy would need repeated administrations. It is reasonable to anticipate that these systems offer new prospects to CF gene therapy.