Figure 1.The CFRD prediction model accurately (±95% CI) predicts CFRD diagnosis in the subsequent 1-10 years in an independent validation set of 524 individuals with CF.Strata are the lowest 25%ile (red), middle 50%ile (green), and highest 25%ile (blue) of predicted CFRD risk.Conclusions: Implementation of a CFRD risk-prediction model could enable individualized screening recommendations for PwCF, potentially decreasing the burden of annual screening via OGTT in individuals at low risk of CFRD without delaying diagnosis.We are expanding the model to analyze pancreatic sufficiency and CFTR modulator use.Future directions include prospective validation of the model with an external data set.

Methods: Primary hNECs as described above were electroporated with ABE8e/sgRNA and allowed to differentiate for 21 days on air-liquid interface culture before being dissociated and prepared for single-cell library preparation performed using 10X Genomics v3.1 3 Library Preparation chemistry with a dual indexing system.Sequencing was performed at the Johns Hopkins Genomic Resources Core Facility via paired-end sequencing 2 × 100 cycles using a NovaSeq 6000 Illumina sequencing machine.Analysis of scRNA-seq results was performed using 10X Genomics Cell Ranger 3.1.0followed by the Seurat package (version 4.1) in R created by the Satija Lab.Subsetting was determined based on comparisons of the number of genes expressed, unique molecular identifier count, and percentage of reads mapped to mitochondrial RNA.Log normalization with a scale factor of 10,000 was used.Results: Analysis of scRNA-seq of 8,791 cells across two samples of primary hNECs (unedited Control, edited 4L) compared CFTR-positive cells and expression levels.Although these cells are from an individual harboring missense variant G480S that produces stable CFTR RNA, an increase in RNA expression can be attributed to the edited 3120+1 allele that generates stable full-length wt RNA.There was a consistent difference in CFTR+ cells between unedited and edited cells across all defined cell type clusters, most noticeably in ciliated/deuterosomal (500% more in edited cells) and cycling basal cells (175% more) (Figure 1A).Cells expressing CFTR increase from 2.5% of total cells in unedited samples to 3.96% in edited samples, and normalized read count per sample increased from 0.049 to 0.081.Editing increased the percentage of basal and cycling basal cells expressing CFTR, indicating that correction was achieved in airway progenitor cells (Figure 1B). Figure 1.(A) Percentage increase in CFTR+ cells between unedited and edited samples.(B) Comparison of percentage of CFTR+ cells in cell type clusters between unedited and edited samples.Conclusions: Base editing of primary hNECs carrying the 3120+1G >A variant show levels of CFTR function after editing equivalent to more than 50% of wt, exceeding the established clinically significant threshold of approximately 10%.We demonstrate that editing is occurring in progenitor cells after delivery of RNA encoding CRISPR along with guide RNAs by electroporation to undifferentiated primary hNECs.In ongoing work, we are repeating single-cell analysis and investigating whether delivery of this technology using nanoparticles corrects splicing of the 3120+1G >A variant in progenitor airway cells as a prelude to in vivo delivery.

reversed by ivacaftor treatment.Additionally, ivacaftor treatment partially corrects airway inflammation and reduces concentrations of the airway gel-forming mucins Muc5b and Muc5ac in these rats.We used the hG551D rat model to understand how ivacaftor treatment alters the course of chronic lung infections with bacteria such as Pseudomonas aeruginosa.Methods: Six-month-old hG551D rats were treated with ivacaftor or methylcellulose vehicle before or after intratracheal inoculation with 10 6 CFUs of a P. aeruginosa mucoid clinical isolate PAM 57-15 in agarose-laden beads.Rats were euthanized 3 days after infection to study acute infection and 28 days after infection to study chronic infection.Lung tissue and bronchoalveolar lavage fluid (BALF) were collected at harvest.CFU enumeration from the lung homogenate was assessed to determine bacterial lung burden.Immune cells in the BALF were counted with differentiation.Mucins and cytokines were measured in the BALF using modified dot blot and enzyme-linked immunosorbent assay.Mucociliary transport was analyzed via micro-optical coherence tomography (OCT) imaging.Results: Rats treated with ivacaftor in the acute phase of infection had lower bacterial burden, a smaller percentage of neutrophils in the BALF, and less Muc5b and Muc5ac content in the BALF than the methylcellulose vehicle group.In this infection scheme, P. aeruginosa colonies recovered from the vehicle group were all mucoid, whereas 50% of the P. aeruginosa colonies recovered from rats treated with ivacaftor had reverted to a nonmucoid phenotype.In the second infection scheme, rats treated with ivacaftor before infection also had lower bacterial burden in the chronic phase than the vehicle group but no difference in neutrophil percentage or mucin content in the BALF.In the final scheme, hG551D rats that initiated modulator therapy after infection had established chronicity had no difference in bacterial burden, inflammation, or mucin from the vehicle group.Rats in the chronic infection experiment were also analyzed via μOCT, which found that mucociliary transport and mucus viscosity were no different in the rats treated with ivacaftor and the vehicle group.Conclusions: These data indicate that reductions in P. aeruginosa burden after initiation of highly effective modulator therapies such as ivacaftor depend on the phase of infection, with the most promising results acquired before the infection becomes chronic.These data also show that the beneficial effect of ivacaftor on mucociliary clearance is abrogated during chronic infection, suggesting that, to eradicate infection completely, additional therapeutic options will be needed.

tezacaftor-ivacaftor (ETI) may lend insight into the role of CFTR in CFLD and help determine which model best explains CFLD pathogenesis, which is a critical step toward developing therapies.Treatment with ETI is followed by a decrease in the fecal inflammatory measure calprotectin, but it is unknown what effects ETI has on the fecal microbiota and hepatic disease.The aim of this study was to characterize the fecal microbiota of PwCF and advanced CFLD (aCFLD) before and after ETI to compare the microbiomes in people with and without aCFLD and the effects of ETI.Methods: This is an ancillary analysis of stool samples from subjects in PROMISE, a prospective observational study of PwCF aged 12 and older (NCT04038047).Liver involvement was classified based on laboratory data, physical exam, liver histology, and imaging.Included subjects had (aCFLD) (non-cirrhotic PHT and cirrhosis with and without PHT) or were agematched PwCF without liver disease (CFnoLD).Stool samples were collected from participants within 30 days before (baseline) and 1 and 6 months after initiating ETI.Fecal microbiota were defined by shotgun metagenomic sequencing.Results: We analyzed 94 samples from 34 participants (11 aCFLD, 23 CFnoLD).At baseline, aCFLD had statistically significantly higher relative abundances of Streptococcus and Veillonella than CFnoLD, primarily driven by S. salivarius and V. parvula, and a trend toward lower relative abundance of Ruminococcus.Four of the 11 participants with aCFLD (36%) had an initially abnormal fecal calprotectin level nthat normalized after 6 months of ETI.This change correlated with a statistically significant decrease in S. salivarius and a trend toward decreasing V. parvula (Figure 1).Conclusions: In PwCF and aCFLD, we found statistically significant higher fecal relative abundances of S. salivarius and V. parvula at baseline than in CFnoLD.Fecal abundances of both species decreased after 6 months of ETI in aCFLD, correlating with normalization of fecal calprotectin.V. parvula is pathogenic, producing lipopolysaccharide, which upregulates inflammatory pathways.Levels of both species have been observed to be high in people with non-CF cirrhosis and inflammatory bowel disease.These results support the model linking dysbiosis to intestinal inflammation in CFLD, with demonstrable improvement in both after initiation of ETI.

Beta regression models were used to determine significant association between BV5:TBV ratio and age and between BV5:TBV ratio and ppFEV1.Natural cubic spline functions were used to determine points of inflection for ppFEV1 and age in association with BV5:TBV ratio.The optimum number of knots used in the spline function were derived from the adjusted R2 values.Results: We report the results of 79 subjects (median age 15.3 [IQR 5.3, 21.0], median ppFEV 1 87 [IQR 40, 121], 59.5% female, 97.5% white).BV5:TBV ratio decreased with decreasing ppFEV 1 ( p = 0.004) (Figure 1A) and increasing age ( p < 0.0001) (Figure 1B).The spline fit for the relationship between ppFEV 1 and BV5:TBV showed that the point of inflection is a ppFEV 1 of 95%.The spline fit for the relationship between BV5:TBV ratio and age showed a gradual decline since young age and point inflection, with a steep decline around the age of 17. Conclusions: The loss of small blood vessels in CF lung disease measured by the ratio of blood volume in vessels less that 5mm 2 to total lung blood volume begins to decline at young age and normal FEV1% predicted.Further investigation of the role of CFTR in angiogenesis and vasculogenesis may explain mechanisms involved in loss of lung function in CF lung disease.

Background: CF is a progressive genetic disease leading to reduction in function or complete loss of the CFTR chloride channel that is normally located on the apical plasma membrane of specialized respiratory epithelial cells.Treatments combining CFTR modulators and a potentiator has been very successful, but a significant fraction of people with CF are not amenable to currently approved treatments.To address this unmet need, ReCode Therapeutics is advancing an mRNA-based treatment to rescue CFTR function using its proprietary lipid nanoparticle (LNP) platform to deliver optimized CFTR mRNA as an inhaled aerosol.Methods: Sequence-optimized CFTR (hemagglutinin tagged) mRNA containing modified nucleotides was used in patient-derived primary human bronchial epithelial cells (hBECs).Different CF genotypes (R553X/W1282X, W1282X/W1282X, F508del/F508del, G542X/F508del) and non-CF hBECs were grown at an air-liquid interface (ALI) and treated with aerosolized mRNA LNPs using commercially available mesh nebulizers and exposure systems.Read-outs for the nebulization experiments included transepithelial resistance (TEER) and forskolin-induced chloride conductance measured by multi-transepithelial current clamp (MTECC24), protein levels (Western blot), cytotoxicity (lactate dehydrogenase), and cell tropism (immunofluorescence). Results: We evaluated the impact of cell tropism and mucus accumulation on rescue of CFTR function in hBECs derived from people with different genotypes.Forskolin-induced Cl -currents showed significant rescue after a single dose of aerosolized SORT LNPs.Analysis of protein expression by Western blot indicated successful delivery of mRNA using optimized LNPs and expression of CFTR protein in all donor cultured cells.Data suggest that CFTR is being translated, folded, and glycosylated, whereas immunofluorescence results showed that the newly translated hemagglutinin-tagged protein trafficked to the apical membrane in pulmonary ionocytes and secretory cells.ReCode's leading formulation to treat primary ciliary dyskinesia, which was developed for delivery of DNAI1 mRNA to ciliated cells for rescue ciliary activity, promoted inefficient CFTR functional rescue.Mucus accumulation was studied by subjecting hBECs to stringent dithiothreitol washes (3 mM or 10 mM).Chloride flux results showed a modest effect of sterile mucus in transfection efficiency after LNP exposure.Further studies are being conducted to explore the connection between LNP-specific cell tropism patterns and ability to restore CFTR-dependent Cl -currents.We are also investigating the impact of mucus on LNP survivability and cell transfection in patient-derived mucus and in vivo models.Conclusions: Our results demonstrate the capability of proprietary ReCode SORT LNPs to deliver LNP-formulated CFTR mRNA as an aerosol and increase CFTR function in well-differentiated CF hBEC cultures, including cells from people with mutations unresponsive to current modulator therapies.ReCode's leading formulations to treat CF efficiently transfect pulmonary ionocytes and secretory cells in vitro.These cell-based observations support further development and may provide future treatment options for a significant fraction of people with CF who do not benefit from current CFTR modulator therapy.

acid tolerance genes-a potential by-product of decreased acidity due to improved CFTR function in the gut.Conclusions: This study represents the first comprehensive analysis of the intestinal microbiome in children with CF receiving ETI.

Background: An increasing pregnancy rate in women with cystic fibrosis (WwCF) means increasing numbers of women who breastfeed.Pregnancy and lactation reduce bone mineral density (BMD) [1] and increase metabolic demand, associations of clinical significance for WwCF [2].We describe available prenatal characteristics and outcomes regarding breastfeeding (BF), BMD, and nutrition from a large adult CF center that provides a dedicated CF reproductive and maternal health monthly clinic.Methods: This was a single-center retrospective review of electronic medical records of WwCF who gave birth between September 2020 and February 2023.Cases were categorized as BF (exclusively or in combination) at 6 to 8 weeks or not breastfeeding (NBF).WwCF who gave birth before October 2022 were included in a sub-analysis of BF 6 months postpartum and NBF.Results were analyzed using paired t-tests.Results: Twenty-three of 37 cases (62%) were BF at 6 to 8 weeks.WwCF who were BF were older (mean 27.9 ± 6 s vs. 32.3 ± 4, p = 0.01).Fewer WwCF who were BF were on CFTR modulators (BF n = 16 (69.6%)vs. NBF n = 14 (100%)).Although not statistically significant, preconception mean ppFEV 1 was lower in NBF WwCF at 6 to 8 weeks than in those who were BF (67.3 ± 21.3% vs. 79.3 ± 22.6%, p = 0.07).There was no significant decrease in ppFEV 1 in either group at 6 to 8 weeks (BF median 1.0 [IQR -9.0-4.0] vs. NBF 1.5 [IQR -5.0-5.0],p = 0.57).Prenatally, 11 (31%) WwCF were osteopenic, and two (6%) were osteoporotic (two had no previous bone scan).Of these 13 with low BMD, seven (54%) were BF at 6 to 8 weeks.Only two women had postpartum DEXA scans; one was osteopenic before pregnancy, and although she was still osteopenic, her BMD increased by 10%.The other had normal BMD (T score = -0.2);neither was BF at 6 to 8 weeks.Mean preconception BMI was similar (BF 21.6 ± 2.8 kg/m 2 vs. NBF 22.6 ± 3.1 kg/m 2 , p = 0.34).At 6 to 8 weeks, there was no significant difference in change in mean BMI between BF and NBF women (BF 1.11 ± 1.72 kg/m 2 vs. NBF 0.33 ± 2.28 kg/m 2 , p = 0.41).Of 32 women included in the 6-month postpartum subanalysis, 12 (37.5%)were BF.There was no difference in median change in ppFEV 1 from 6 to 8 weeks to 6 months in BF or NBF women (BF 1% [-2%, 3%], NBF -0.5% [-2%, 4%], p = 0.86).Median decrease in BMI from 6 to 8 weeks to 6 months was 1.1 kg/ m 2 (IQR -1.3, -0.6 kg/m 2 ) in those who continued to breast feed and 0.1 kg/m 2 [IQR -0.5, 0.3 k/m 2 ( p = 0.05) in those who did not, although both groups had a mean BMI of 22 kg/m 2 at 6 months.Conclusions: In this single-center study, BF had no detrimental effect on spirometry in women continuing to breast fee for at least 6 months, but it affected nutritional status.With relatively high BF rates (vs.49.3% in England overall at 6 to 8 weeks [3]), CF teams should be cognizant of this risk factor for nutritional decline and lower BMD in WwCF, particularly those with low BMI or BMD before pregnancy.

Background: More than 20% of people with cystic fibrosis (CF) carry at least one copy of a Class I mutation in the CF transmembrane conductance regulator (CFTR) gene.Class I CFTR mutations (also known as X-mutations) result in nonsense and canonical splice-site mutations, leading to complete absence of CFTR protein.Major Class I mutations include G542X and W1282X.People with a homozygous Class I mutation do not benefit from elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA), the primary CF drug that has gained Food and Drug Administration's approval for treating patients with one or two copies of the most prevalent CFTR-F508del (dF) mutation or any other CFTR modulator drugs on the market such as lumacaftor/ ivacaftor.Recently we reported that inhibitors of the sodium-dependent glucose cotransporter 1/2 (SGLT1/2), sotagliflozin and phloridzin, promoted swelling of dF/dF and dF/G551D human proximal lung organoids (HLOs) derived from induced pluripotent stem cells (iPSCs) in forskolinstimulated organoid swelling assay.In addition to its SGLT1/2 inhibiting capacity, phloridzin is also known as an inhibitor of the Na+/K+-ATPase.Methods: We derived HLOs from Class I CF iPSCs with the G542X/G542X or W1282X/W1282X mutation.We evaluated the effects of ELX/TEZ/IVA, sotagliflozin, phloridzin, or amiloride (an epithelial sodium channel inhibitor) on these Class I CF HLOs in the forskolin-stimulated swelling assay.Results: Only phloridzin promoted swelling of G542X/G542X and W1282X/W1282X HLOs, to a similar extent achieved by the CFTR modulators lumacaftor/ivacaftor on dF/dF HLOs, whereas none of the other compounds (ELX/TEZ/IVA, sotagliflozin, amiloride) had any effects.Conclusions: Our data demonstrate a promoting effect of SGLT1/2 inhibitor phloridzin on Class I CF HLOs.The differential results by sotagliflozin and phloridzin suggest a potential contribution of Na+/K+-ATPase on the forskolin-promoted swelling of Class I CF HLOs.Follow-up studies are needed to delineate the roles of SGLT1/2 and Na+/K+-ATPase in Class I CF mutations.

neutrophil migration and LTB 4 production were restored.PA14DleuB displayed normal levels of motility and adhesion but showed less chemotaxis toward neutrophils than WT PA14.Conclusions: These findings suggest that leuB plays an important role in the neutrophil response to P. aeruginosa infection in the airway, perhaps as a component of a signal peptide that induces neutrophil chemotaxis.Future research is necessary to further elucidate the role of leuB in driving airway inflammation in CF and to identify targets for therapeutics to mitigate airway damage.