Clinical and functional efficacy of elexacaftor/tezacaftor/ivacaftor in people with cystic fibrosis carrying the N1303K mutation.

BACKGROUND: Elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA) significantly improves health outcomes in people with cystic fibrosis (pwCF) carrying one or two F508del mutations. According to in vitro assays performed in FRT cells, 178 additional mutations respond to ELX/TEZ/IVA. The N1303K mutation is not included in this list of mutations. Recent in vitro data suggested that ELX/TEZ/IVA increases N1303K-CFTR activity. Based on the in vitro response, eight patients commenced treatment with ELX/TEZ/IVA. METHODS: Two homozygotes; and six compound heterozygotes N1303K/nonsense or frameshift mutation pwCF were treated off label with ELX/TEZ/IVA. Clinical data before and 8 weeks after starting treatment were prospectively collected. The response to ELX/TEZ/IVA was assessed in intestinal organoids derived from 5 study patients and an additional patient carrying N1303K that is not receiving treatment. RESULTS: Compared to the values before commencing treatment, mean forced expiratory volume in 1 second increased by 18.4 percentage points and 26.5% relative to baseline, mean BMI increased by 0.79 Kg/m2, and mean lung clearance index decreased by 3.6 points and 22.2%. There was no significant change in sweat chloride. Nasal potential difference normalized in four patients and remained abnormal in three. Results in 3D intestinal organoids and 2D nasal epithelial cultures showed a response in CFTR channel activity. CONCLUSIONS: This report supports the previously reported in vitro data, performed in human nasal and bronchial epithelial cells and intestinal organoids, that pwCF who carry the N1303K mutation have a significant clinical benefit by ELX/TEZ/IVA treatment.

Correlation of Electrophysiological and Fluorescence-Based Measurements of Modulator Efficacy in Nasal Epithelial Cultures Derived from People with Cystic Fibrosis.

It has been suggested that in vitro studies of the rescue effect of CFTR modulator drugs in nasal epithelial cultures derived from people with cystic fibrosis have the potential to predict clinical responses to the same drugs. Hence, there is an interest in evaluating different methods for measuring in vitro modulator responses in patient-derived nasal cultures. Commonly, the functional response to CFTR modulator combinations in these cultures is assessed by bioelectric measurements, using the Ussing chamber. While this method is highly informative, it is time-consuming. A fluorescence-based, multi-transwell method for assaying regulated apical chloride conductance (Fl-ACC) promises to provide a complementary approach to theratyping in patient-derived nasal cultures. In the present work, we compared Ussing chamber measurements and fluorescence-based measurements of CFTR-mediated apical conductance in matching, fully differentiated nasal cultures derived from CF patients, homozygous for F508del (n = 31) or W1282X (n = 3), or heterozygous for Class III mutations G551D or G178R (n = 5). These cultures were obtained through a bioresource called the Cystic Fibrosis Canada-Sick Kids Program in Individual CF Therapy (CFIT). We found that the Fl-ACC method was effective in detecting positive responses to interventions for all genotypes. There was a correlation between patient-specific drug responses measured in cultures harbouring F508del, as measured using the Ussing chamber technique and the fluorescence-based assay (Fl-ACC). Finally, the fluorescence-based assay has the potential for greater sensitivity for detecting responses to pharmacological rescue strategies targeting W1282X.

Validating organoid-derived human intestinal monolayers for personalized therapy in cystic fibrosis.

Highly effective drugs modulating the defective protein encoded by the CFTR gene have revolutionized cystic fibrosis (CF) therapy. Preclinical drug-testing on human nasal epithelial (HNE) cell cultures and 3-dimensional human intestinal organoids (3D HIO) are used to address patient-specific variation in drug response and to optimize individual treatment for people with CF. This study is the first to report comparable CFTR functional responses to CFTR modulator treatment among patients with different classes of CFTR gene variants using the three methods of 2D HIO, 3D HIO, and HNE. Furthermore, 2D HIO showed good correlation to clinical outcome markers. A larger measurable CFTR functional range and access to the apical membrane were identified as advantages of 2D HIO over HNE and 3D HIO, respectively. Our study thus expands the utility of 2D intestinal monolayers as a preclinical drug testing tool for CF.

Uptake of the culturally appropriate ASQ-TRAK developmental screening tool in the Australian Aboriginal and Torres Strait Islander context.

BACKGROUND: Recently in Australia, access to culturally safe developmental practices for Aboriginal and Torres Strait Islander families has been enhanced by the availability of a culturally appropriate developmental screening tool, the Ages and Stages Questionnaire - Talking about Raising Aboriginal Kids (ASQ-TRAK). This paper aims (i) to describe the uptake of the ASQ-TRAK developmental screening tool in Aboriginal Community Controlled Organizations and mainstream services in Australia and (ii) to explore the extent to which organizations using the ASQ-TRAK have engaged training for staff. METHODS: A retrospective review of ASQ-TRAK sales and training records from January 2015 to May 2020 to determine the ASQ-TRAK distribution by jurisdiction and service type and the number of services that have engaged training. RESULTS: Five hundred ASQ-TRAK kits have been distributed across 77 agencies. Of those, 100 kits (20%) have been purchased by Aboriginal Community Controlled Organizations. Most have been distributed in the Northern Territory (NT) (178, 36%), Western Australia (165, 33%) and South Australia (64, 13%). Of the 15 ASQ-TRAK training workshops, nine have been in the NT. Of the 196 practitioners trained, 25 were identified as facilitators for their organization. CONCLUSION: Despite substantive research translation across Australia, with evidence of its acceptability in different contexts, most Aboriginal Community Controlled Organizations have not yet accessed the ASQ-TRAK, and most organizations have not participated in training. There is an imperative to progress knowledge translation to improve quality and accessibility of culturally appropriate developmental care. Adequately resourced ASQ-TRAK implementation support is needed to ensure sustainable implementation at scale.

Genetic evidence supports the development of SLC26A9 targeting therapies for the treatment of lung disease.

Over 400 variants in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) are CF-causing. CFTR modulators target variants to improve lung function, but marked variability in response exists and current therapies do not address all CF-causing variants highlighting unmet needs. Alternative epithelial ion channel/transporters such as SLC26A9 could compensate for CFTR dysfunction, providing therapeutic targets that may benefit all individuals with CF. We investigate the relationship between rs7512462, a marker of SLC26A9 activity, and lung function pre- and post-treatment with CFTR modulators in Canadian and US CF cohorts, in the general population, and in those with chronic obstructive pulmonary disease (COPD). Rs7512462 CC genotype is associated with greater lung function in CF individuals with minimal function variants (for which there are currently no approved therapies; p = 0.008); and for gating (p = 0.033) and p.Phe508del/ p.Phe508del (p = 0.006) genotypes upon treatment with CFTR modulators. In parallel, human nasal epithelia with CC and p.Phe508del/p.Phe508del after Ussing chamber analysis of a combination of approved and experimental modulator treatments show greater CFTR function (p = 0.0022). Beyond CF, rs7512462 is associated with peak expiratory flow in a meta-analysis of the UK Biobank and Spirometa Consortium (p = 2.74 × 10-44) and provides p = 0.0891 in an analysis of COPD case-control status in the UK Biobank defined by spirometry. These findings support SLC26A9 as a therapeutic target to improve lung function for all people with CF and in individuals with other obstructive lung diseases.

Emerging preclinical modulators developed for F508del-CFTR have the potential to be effective for ORKAMBI resistant processing mutants.

BACKGROUND: F508del is prototypical of Class 2 CFTR mutations associated with protein misprocessing and reduced function. Corrector compounds like lumacaftor partially rescue the processing defect of F508del-CFTR whereas potentiators like ivacaftor, enhance its channel activity once trafficked to the cell surface. We asked if emerging modulators developed for F508del-CFTR can rescue Class 2 mutations previously shown to be poorly responsive to lumacaftor and ivacaftor. METHODS: Rescue of mutant CFTRs by the correctors: AC1, AC2-1 or AC2-2 and the potentiator, AP2, was studied in HEK-293 cells and in primary human nasal epithelial (HNE) cultures, using a membrane potential assay and Ussing chamber, respectively. RESULTS: In HEK-293 cells, we found that a particular combination of corrector molecules (AC1 plus AC2-1) and a potentiator (AP2) was effective in rescuing both the misprocessing and reduced function of M1101K and G85E respectively. These findings were recapitulated in patient-derived nasal cultures, although another corrector combination, AC1 plus AC2-2 also improved misprocessing in these primary tissues. Interestingly, while this corrector combination only led to a modest increase in the abundance of mature N1303K-CFTR it did enable its functional expression in the presence of the potentiator, AP2, in part, because the nominal corrector, AC2-2 also exhibits potentiator activity. CONCLUSIONS: Strategic combinations of novel modulators can potentially rescue Class 2 mutants thought to be relatively unresponsive to lumacaftor and ivacaftor.

Rescue of multiple class II CFTR mutations by elexacaftor+tezacaftor+ivacaftor mediated in part by the dual activities of elexacaftor as both corrector and potentiator.

Positive results in pre-clinical studies of the triple combination of elexacaftor, tezacaftor and ivacaftor, performed in airway epithelial cell cultures obtained from patients harbouring the class II cystic fibrosis transmembrane conductance regulator (CFTR) mutation F508del-CFTR, translated to impressive clinical outcomes for subjects carrying this mutation in clinical trials and approval of Trikafta.Encouraged by this correlation, we were prompted to evaluate the effect of the elexacaftor, tezacaftor and ivacaftor triple combination on primary nasal epithelial cultures obtained from individuals with rare class II CF-causing mutations (G85E, M1101K and N1303K) for which Trikafta is not approved.Cultures from individuals homozygous for M1101K responded better than cultures harbouring G85E and N1303K after treatment with the triple combination with respect to improvement in regulated channel function and protein processing. A similar genotype-specific effect of the triple combination was observed when the different mutations were expressed in HEK293 cells, supporting the hypothesis that these modulators may act directly on the mutant proteins. Detailed studies in nasal cultures and HEK293 cells showed that the corrector, elexacaftor, exhibited dual activity as both corrector and potentiator, and suggested that the potentiator activity contributes to its pharmacological activity.These pre-clinical studies using nasal epithelial cultures identified mutation genotypes for which elexacaftor, tezacaftor and ivacaftor may produce clinical responses that are comparable to, or inferior to, those observed for F508del-CFTR.

Preclinical Studies of a Rare CF-Causing Mutation in the Second Nucleotide Binding Domain (c.3700A>G) Show Robust Functional Rescue in Primary Nasal Cultures by Novel CFTR Modulators.

The combination therapies ORKAMBITM and TRIKAFTATM are approved for people who have the F508del mutation on at least one allele. In this study we examine the effects of potentiator and corrector combinations on the rare mutation c.3700A>G. This mutation produces a cryptic splice site that deletes six amino acids in NBD2 (I1234-R1239del). Like F508del it causes protein misprocessing and reduced chloride channel function. We show that a novel cystic fibrosis transmembrane conductance regulator CFTR modulator triple combination (AC1, corrector, AC2-2, co-potentiator and AP2, potentiator), rescued I1234-R1239del-CFTR activity to WT-CFTR level in HEK293 cells. Moreover, we show that although the response to ORKAMBI was modest in nasal epithelial cells from two individuals homozygous for I1234-R1239del-CFTR, a substantial functional rescue was achieved with the novel triple combination. Interestingly, while both the novel CFTR triple combination and TRIKAFTATM treatment showed functional rescue in gene-edited I1234-R1239del-CFTR-expressing HBE cells and in nasal cells from two CF patients heterozygous for I1234-R1239del/W1282X, nasal cells homozygous for I1234-R1239del-CFTR showed no significant response to the TRIKAFTATM combination. These data suggest a potential benefit of CFTR modulators on the functional rescue of I1234-R1239del -CFTR, which arises from the rare CF-causing mutation c.3700A>G, and highlight that patient tissues are crucial to our full understanding of functional rescue in rare CFTR mutations.

The CFTR Mutation c.3453G > C (D1152H) Confers an Anion Selectivity Defect in Primary Airway Tissue that Can Be Rescued by Ivacaftor.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene variant, c.3453G > C (D1152H), is associated with mild Cystic Fibrosis (CF) disease, though there is considerable clinical variability ranging from no detectable symptoms to lung disease with early acquisition of Pseudomonas aeruginosa. The approval extension of ivacaftor, the first CFTR modulator drug approved, to include D1152H was based on a positive drug response of defective CFTR-D1152H chloride channel function when expressed in FRT cells. Functional analyses of primary human nasal epithelial cells (HNE) from an individual homozygous for D1152H now revealed that while CFTR-D1152H demonstrated normal, wild-type level chloride conductance, its bicarbonate-selective conductance was impaired. Treatment with ivacaftor increased this bicarbonate-selective conductance. Extensive genetic, protein and functional analysis of the nasal cells of this D1152H/D1152H patient revealed a 90% reduction of CFTR transcripts due to the homozygous presence of the 5T polymorphism in the poly-T tract forming a complex allele with D1152H. Thus, we confirm previous observation in patient-derived tissue that 10% normal CFTR transcripts confer normal, wild-type level chloride channel activity. Together, this study highlights the benefit of patient-derived tissues to study the functional expression and pharmacological modulation of CF-causing mutations, in order to understand pathogenesis and therapeutic responses.

A helper-dependent adenoviral vector rescues CFTR to wild-type functional levels in cystic fibrosis epithelial cells harbouring class I mutations.

Cystic fibrosis (CF) is a genetic disorder affecting multiple organs, including the pancreas, hepatobiliary system and reproductive organs; however, lung disease is responsible for the majority of morbidity and mortality. Management of CF involves CF transmembrane conductance regulator (CFTR) modulator agents including corrector drugs to augment cellular trafficking of mutant CFTR as well as potentiators that open defective CFTR channels. These therapies are poised to help most individuals with CF, with the notable exception of individuals with class I mutations where full-length CFTR protein is not produced. For these mutations, gene replacement has been suggested as a potential solution.In this work, we used a helper-dependent adenoviral vector (HD-CFTR) to express CFTR in nasal epithelial cell cultures derived from CF subjects with class I CFTR mutations.CFTR function was significantly restored in CF cells by HD-CFTR and reached healthy control functional levels as detected by Ussing chamber and membrane potential (FLIPR) assay. A dose-response relationship was observed between the amount of vector used and subsequent functional outcomes; small amounts of HD-CFTR were sufficient to correct CFTR function. At higher doses, HD-CFTR did not increase CFTR function in healthy control cells above baseline values. This latter observation allowed us to use this vector to benchmark in vitro efficacy testing of CFTR-modulator drugs.In summary, we demonstrate the potential for HD-CFTR to inform in vitro testing and to restore CFTR function to healthy control levels in airway cells with class I or CFTR nonsense mutations.

Functional rescue of c.3846G>A (W1282X) in patient-derived nasal cultures achieved by inhibition of nonsense mediated decay and protein modulators with complementary mechanisms of action.

BACKGROUND: The nonsense mutation, c.3846G>A (aka: W1282X-CFTR) leads to a truncated transcript that is susceptible to nonsense-mediated decay (NMD) and produces a shorter protein that is unstable and lacks normal channel activity in patient-derived tissues. However, if overexpressed in a heterologous expression system, the truncated mutant protein has been shown to mediate CFTR channel function following the addition of potentiators. In this study, we asked if a quadruple combination of small molecules that together inhibit nonsense mediated decay, stabilize both halves of the mutant protein and potentiate CFTR channel activity could rescue the functional expression of W1282X-CFTR in patient derived nasal cultures. METHODS: We identified the CFTR domains stabilized by corrector compounds supplied from AbbVie using a fragment based, biochemical approach. Rescue of the channel function of W1282X.-CFTR protein by NMD inhibition and small molecule protein modulators was studied using a bronchial cell line engineered to express W1282X and in primary nasal epithelial cultures derived from four patients homozygous for this mutation. RESULTS: We confirmed previous studies showing that inhibition of NMD using the inhibitor: SMG1i, led to an increased abundance of the shorter transcript in a bronchial cell line. Interestingly, on top of SMG1i, treatment with a combination of two new correctors developed by Galapagos/AbbVie (AC1 and AC2-2, separately targeting either the first or second half of CFTR and promoting assembly, significantly increased the potentiated channel activity by the mutant in the bronchial epithelial cell line and in patient-derived nasal epithelial cultures. The average rescue effect in primary cultures was approximately 50% of the regulated chloride conductance measured in non-CF cultures. CONCLUSIONS: These studies provide the first in-vitro evidence in patient derived airway cultures that the functional defects incurred by W1282X, has the potential to be effectively repaired pharmacologically.

ORKAMBI-Mediated Rescue of Mucociliary Clearance in Cystic Fibrosis Primary Respiratory Cultures Is Enhanced by Arginine Uptake, Arginase Inhibition, and Promotion of Nitric Oxide Signaling to the Cystic Fibrosis Transmembrane Conductance Regulator Channel.

ORKAMBI, a combination of the corrector, lumacaftor, and the potentiator, ivacaftor, partially rescues the defective processing and anion channel activity conferred by the major cystic fibrosis-causing mutation, F508del, in in vitro studies. Clinically, the improvement in lung function after ORKAMBI treatment is modest and variable, prompting the search for complementary interventions. As our previous work identified a positive effect of arginine-dependent nitric oxide signaling on residual F508del-Cftr function in murine intestinal epithelium, we were prompted to determine whether strategies aimed at increasing arginine would enhance F508del-cystic fibrosis transmembrane conductance regulator (CFTR) channel activity in patient-derived airway epithelia. Now, we show that the addition of arginine together with inhibition of intracellular arginase activity increased cytosolic nitric oxide and enhanced the rescue effect of ORKAMBI on F508del-CFTR-mediated chloride conductance at the cell surface of patient-derived bronchial and nasal epithelial cultures. Interestingly, arginine addition plus arginase inhibition also enhanced ORKAMBI-mediated increases in ciliary beat frequency and mucociliary movement, two in vitro CF phenotypes that are downstream of the channel defect. This work suggests that strategies to manipulate the arginine-nitric oxide pathway in combination with CFTR modulators may lead to improved clinical outcomes. SIGNIFICANCE STATEMENT: These proof-of-concept studies highlight the potential to boost the response to cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulators, lumacaftor and ivacaftor, in patient-derived airway tissues expressing the major CF-causing mutant, F508del-CFTR, by enhancing other regulatory pathways. In this case, we observed enhancement of pharmacologically rescued F508del-CFTR by arginine-dependent, nitric oxide signaling through inhibition of endogenous arginase activity.

The CF Canada-Sick Kids Program in individual CF therapy: A resource for the advancement of personalized medicine in CF.

BACKGROUND: Therapies targeting certain CFTR mutants have been approved, yet variations in clinical response highlight the need for in-vitro and genetic tools that predict patient-specific clinical outcomes. Toward this goal, the CF Canada-Sick Kids Program in Individual CF Therapy (CFIT) is generating a "first of its kind", comprehensive resource containing patient-specific cell cultures and data from 100 CF individuals that will enable modeling of therapeutic responses. METHODS: The CFIT program is generating: 1) nasal cells from drug naïve patients suitable for culture and the study of drug responses in vitro, 2) matched gene expression data obtained by sequencing the RNA from the primary nasal tissue, 3) whole genome sequencing of blood derived DNA from each of the 100 participants, 4) induced pluripotent stem cells (iPSCs) generated from each participant's blood sample, 5) CRISPR-edited isogenic control iPSC lines and 6) prospective clinical data from patients treated with CF modulators. RESULTS: To date, we have recruited 57 of 100 individuals to CFIT, most of whom are homozygous for F508del (to assess in-vitro: in-vivo correlations with respect to ORKAMBI response) or heterozygous for F508del and a minimal function mutation. In addition, several donors are homozygous for rare nonsense and missense mutations. Nasal epithelial cell cultures and matched iPSC lines are available for many of these donors. CONCLUSIONS: This accessible resource will enable development of tools that predict individual outcomes to current and emerging modulators targeting F508del-CFTR and facilitate therapy discovery for rare CF causing mutations.

Antimicrobial prescription in patients dying from chronic obstructive pulmonary disease.

BACKGROUND: Despite rising antimicrobial resistance, treatment guidelines for chronic obstructive pulmonary disease (COPD) exacerbations are frequently ignored. Patients with terminal conditions are often prescribed antimicrobials despite the goal of care to reduce burdensome treatments. The appropriate use of antimicrobials in patients who die from an exacerbation of COPD is unknown. AIM: To review antimicrobial prescription during the final admission in patients who died from an acute exacerbation of COPD. METHODS: A retrospective medical record audit was performed for 475 patients who died over 12 years (2004-2015). Patients were analysed within three groups: Group 1 - pneumonia on chest radiograph, Group 2 - infective exacerbation of COPD +/- raised inflammatory markers (white cell count, C-reactive protein) and Group 3 - non-infective exacerbation of COPD. RESULTS: A total of 221 patients died from COPD. The median age was 80 years, and 136 (60%) were male. Median respiratory function: forced expiratory volume in 1 s 0.8 L (41.0%), forced vital capacity 2.0 L (74.0%) and diffusing capacity for carbon monoxide 8 (40.5%). A total of 109 (49.3%) patients used home oxygen and 156 (70.6%) were ex-smokers. Of the cohort, 90.5% received antimicrobials. In Groups 1, 2 and 3, 68 (94.4%), 108 (92.3%) and 24 (75.0%) patients received antimicrobials respectively. Guideline-concordant therapy was administered to 31.7% of patients (Group 1: 79.2%, Group 2: 4.3%, Group 3: 25.0%), 60.2% of patients received ceftriaxone and 44.8% received azithromycin. The median duration of therapy was 4 days and 27.1% received antimicrobials at the time of death. CONCLUSION: Antimicrobials are overprescribed, and non-guideline antimicrobials are overused in patients who die from COPD. Further education of medical staff, regular medication reviews and the use of disease severity scores or clinical pathways may improve antimicrobial stewardship.

Transducing Airway Basal Cells with a Helper-Dependent Adenoviral Vector for Lung Gene Therapy.

A major challenge in developing gene-based therapies for airway diseases such as cystic fibrosis (CF) is sustaining therapeutic levels of transgene expression over time. This is largely due to airway epithelial cell turnover and the host immunogenicity to gene delivery vectors. Modern gene editing tools and delivery vehicles hold great potential for overcoming this challenge. There is currently not much known about how to deliver genes into airway stem cells, of which basal cells are the major type in human airways. In this study, helper-dependent adenoviral (HD-Ad) vectors were delivered to mouse and pig airways via intranasal delivery, and direct bronchoscopic instillation, respectively. Vector transduction was assessed by immunostaining of lung tissue sections, which revealed that airway basal cells of mice and pigs can be targeted in vivo. In addition, efficient transduction of primary human airway basal cells was verified with an HD-Ad vector expressing green fluorescent protein. Furthermore, we successfully delivered the human CFTR gene to airway basal cells from CF patients, and demonstrated restoration of CFTR channel activity following cell differentiation in air-liquid interface culture. Our results provide a strong rationale for utilizing HD-Ad vectors to target airway basal cells for permanent gene correction of genetic airway diseases.

The effectiveness of bariatric surgery on long term psychosocial quality of life - A systematic review.

BACKGROUND: Little is known about the psychological effects of bariatric surgery. This review aimed to assess long term effectiveness of bariatric surgery on psychosocial quality of life (QoL) of obese adults compared to non-surgical interventions. METHODS: A systematic review of the literature was conducted, six electronic databases, plus other relevant sources were searched from inception to January 2014. The main outcome sought was a QoL measure with a minimum 12 months follow-up. Three reviewers screened records, extracted data and independently read through full articles for eligibility and quality using standardised forms. Findings were analysed using narrative synthesis. RESULTS: Of 4383 identified references included in the review, 11 studies showed bariatric surgery to be effective long term for overall QoL than non-surgical treatments with specifically modest benefits psychosocially. Significant improvements in psychosocial QoL alongside improved physical QoL were observed after 2 year follow-up post-surgery compared to non-surgical interventions. However improvements in psychosocial QoL after surgery at 10 year follow up were minimal when compared to non-surgical interventions. When compared to untreated control groups, long term psychosocial QoL did not improve after bariatric surgery despite major improvements in physical QoL, significant weight loss and co-morbidities. CONCLUSIONS: Results on long term psychosocial QoL remain uncertain with some suggestion of psychological disorder persisting. This highlights need for psychological intervention post-surgery and further research to provide more data on long-term psychosocial QoL following bariatric surgery.