Targetable domains for the design of peptide-dendrimer inhibitors of SARS-CoV-2.

We have recently witnessed that considerable progresses have been made in the rapid detection and appropriate treatments of COVID-19, but still this virus remains one of the main targets of world research. Based on the knowledge of the complex mechanism of viral infection we designed peptide-dendrimer inhibitors of SARS-CoV-2with the aim to block cell infection through interfering with the host-pathogen interactions. We used two different strategies: i) the first one aims at hindering the virus anchorage to the human cell; ii) the second -strategy points to interfere with the mechanism of virus-cell membrane fusion. We propose the use of different nanosized carriers, formed by several carbosilane dendritic wedges to deliver two different peptides designed to inhibit host interaction or virus entry. The antiviral activity of the peptide-dendrimers, as well as of free peptides and free dendrimers was evaluated through the use of SARS-CoV-2 pseudotyped lentivirus. The results obtained show that peptides designed to block host-pathogen interaction represent a valuable strategy for viral inhibition.

Identification of an ultra-rare Alu insertion in the CFTR gene: Pitfalls and challenges in genetic test interpretation.

Cystic fibrosis (CF) is a life-limiting genetic disorder characterized by defective chloride ion transport due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Early detection through newborn screening programs significantly improves outcomes for individuals with CF by enabling timely intervention. Here, we report the identification of an Alu element insertion within the exon 15 of CFTR gene, initially overlooked in standard next-generation sequencing analyses. However, using traditional molecular techniques, based on polymerase chain reaction and Sanger sequencing, allowed the identification of the Alu element and the reporting of a correct diagnosis. Our analysis, based on bioinformatics tools and molecular techniques, revealed that the Alu element insertion severely affects the gene expression, splicing patterns, and structure of CFTR protein. In conclusion, this study emphasizes the importance of how the integration of human expertise and modern technologies represents a pivotal step forward in genomic medicine, ensuring the delivery of precision healthcare to individuals affected by genetic diseases.

In silico analysis and theratyping of an ultra-rare CFTR genotype (W57G/A234D) in primary human rectal and nasal epithelial cells.

Mutation targeted therapy in cystic fibrosis (CF) is still not eligible for all CF subjects, especially for cases carrying rare variants such as the CFTR genotype W57G/A234D (c.169T>G/c.701C>A). We performed in silico analysis of the effects of these variants on protein stability, which we functionally characterized using colonoids and reprogrammed nasal epithelial cells. The effect of mutations on cystic fibrosis transmembrane conductance regulator (CFTR) protein was analyzed by western blotting, forskolin-induced swelling (FIS), and Ussing chamber analysis. We detected a residual CFTR function that increases following treatment with the CFTR modulators VX661±VX445±VX770, correlates among models, and is associated with increased CFTR protein levels following treatment with CFTR correctors. In vivo treatment with VX770 reduced sweat chloride concentration to non-CF levels, increased the number of CFTR-dependent sweat droplets, and induced a 6% absolute increase in predicted FEV1% after 27 weeks of treatment indicating the relevance of theratyping with patient-derived cells in CF.

L1077P CFTR pathogenic variant function rescue by Elexacaftor-Tezacaftor-Ivacaftor in cystic fibrosis patient-derived air-liquid interface (ALI) cultures and organoids: in vitro guided personalized therapy of non-F508del patients.

Cystic fibrosis (CF) is caused by defects of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR-modulating drugs may overcome specific defects, such as the case of Trikafta, which is a clinically approved triple combination of Elexacaftor, Tezacaftor and Ivacaftor (ETI) that exhibited a strong ability to rescue the function of the most frequent F508del pathogenic variant even in genotypes with the mutated allele in single copy. Nevertheless, most rare genotypes lacking the F508del allele are still not eligible for targeted therapies. Via the innovative approach of using nasal conditionally reprogrammed cell (CRC) cell-based models that mimic patient disease in vitro, which are obtainable from each patient due to the 100% efficiency of the cell culture establishment, we theratyped orphan CFTR mutation L1077P. Protein studies, Forskolin-induced organoid swelling, and Ussing chamber assays congruently proved the L1077P variant function rescue by ETI. Notably, this rescue takes place even in the context of a single-copy L1077P allele, which appears to enhance its expression. Thus, the possibility of single-allele treatment also arises for rare genotypes, with an allele-specific modulation as part of the mechanism. Of note, besides providing indication of drug efficacy with respect to specific CFTR pathogenic variants or genotypes, this approach allows the evaluation of the response of single-patient cells within their genetic background. In this view, our studies support in vitro guided personalized CF therapies also for rare patients who are nearly excluded from clinical trials.

Theratyping of the Rare CFTR Genotype A559T in Rectal Organoids and Nasal Cells Reveals a Relevant Response to Elexacaftor (VX-445) and Tezacaftor (VX-661) Combination.

Despite the promising results of new CFTR targeting drugs designed for the recovery of F508del- and class III variants activity, none of them have been approved for individuals with selected rare mutations, because uncharacterized CFTR variants lack information associated with the ability of these compounds in recovering their molecular defects. Here we used both rectal organoids (colonoids) and primary nasal brushed cells (hNEC) derived from a CF patient homozygous for A559T (c.1675G>A) variant to evaluate the responsiveness of this pathogenic variant to available CFTR targeted drugs that include VX-770, VX-809, VX-661 and VX-661 combined with VX-445. A559T is a rare mutation, found in African-Americans people with CF (PwCF) with only 85 patients registered in the CFTR2 database. At present, there is no treatment approved by FDA (U.S. Food and Drug Administration) for this genotype. Short-circuit current (Isc) measurements indicate that A559T-CFTR presents a minimal function. The acute addition of VX-770 following CFTR activation by forskolin had no significant increment of baseline level of anion transport in both colonoids and nasal cells. However, the combined treatment, VX-661-VX-445, significantly increases the chloride secretion in A559T-colonoids monolayers and hNEC, reaching approximately 10% of WT-CFTR function. These results were confirmed by forskolin-induced swelling assay and by western blotting in rectal organoids. Overall, our data show a relevant response to VX-661-VX-445 in rectal organoids and hNEC with CFTR genotype A559T/A559T. This could provide a strong rationale for treating patients carrying this variant with VX-661-VX-445-VX-770 combination.

The Immune Response to SARS-CoV-2 Vaccine in a Cohort of Family Pediatricians from Southern Italy.

In Italy, from January 2021, the Ministry of Health indicated a vaccination plan against COVID for frail patients and physicians based on a three-dose scheme. However, conflicting results have been reported on which biomarkers permit immunization assessment. We used several laboratory approaches (i.e., antibodies serum levels, flow cytometry analysis, and cytokines release by stimulated cells) to investigate the immune response in a cohort of 53 family pediatricians (FPs) at different times after the vaccine. We observed that the BNT162b2-mRNA vaccine induced a significant increase of specific antibodies after the third (booster) dose; however, the antibody titer was not predictive of the risk of developing the infection in the six months following the booster dose. The antigen stimulation of PBMC cells from subjects vaccinated with the third booster jab induced the increase of the activated T cells (i.e., CD4+ CD154+); the frequency of CD4+ CD154+ TNF-α+ cells, as well as the TNF-α secretion, was not modified, while we observed a trend of increase of IFN-γ secretion. Interestingly, the level of CD8+ IFN-γ+ (independently from antibody titer) was significantly increased after the third dose and predicts the risk of developing the infection in the six months following the booster jab. Such results may impact also other virus vaccinations.

Patient-derived cell models for personalized medicine approaches in cystic fibrosis.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel that perturb anion transport across the epithelia of the airways and other organs. To treat cystic fibrosis, strategies that target mutant CFTR have been developed such as correctors that rescue folding and enhance transfer of CFTR to the apical membrane, and potentiators that increase CFTR channel activity. While there has been tremendous progress in development and approval of CFTR therapeutics for the most common (F508del) and several other CFTR mutations, around 10-20% of people with cystic fibrosis have rare mutations that are still without an effective treatment. In the current decade, there was an impressive evolution of patient-derived cell models for precision medicine. In cystic fibrosis, these models have played a crucial role in characterizing the molecular defects in CFTR mutants and identifying compounds that target these defects. Cells from nasal, bronchial, and rectal epithelia are most suitable to evaluate treatments that target CFTR. In vitro assays using cultures grown at an air-liquid interface or as organoids and spheroids allow the diagnosis of the CFTR defect and assessment of potential treatment strategies. An overview of currently established cell culture models and assays for personalized medicine approaches in cystic fibrosis will be provided in this review. These models allow theratyping of rare CFTR mutations with available modulator compounds to predict clinical efficacy. Besides evaluation of individual personalized responses to CFTR therapeutics, patient-derived culture models are valuable for testing responses to developmental treatments such as novel RNA- and DNA-based therapies.

Cystic Fibrosis Patients with F508del/Minimal Function Genotype: Laboratory and Nutritional Evaluations after One Year of Elexacaftor/Tezacaftor/Ivacaftor Treatment.

The last ten years have been characterized by an enormous step forward in the therapy and management of patients with Cystic Fibrosis (CF), thanks to the development and combination of Cystic Fibrosis Transmembrane Receptor (CFTR) correctors and potentiators. Specifically, the last approved triple combination elexacaftor/tezacaftor/ivacaftor has been demonstrated to improve lung function in CF patients with both homozygous Phe508del and Phe508del/minimal function genotypes. Here we have assessed the effect of elexacaftor/tezacaftor/ivacaftor in patients carrying the Phe508del/minimal function genotype (n = 20) after one year of treatments on liver function and nutrient absorption with a focus on lipid metabolism. We show that weight, BMI, and albumin significantly increase, suggesting a positive impact of the treatment on nutrient absorption. Furthermore, cholesterol levels as a biomarker of lipid metabolism increased significantly after one year of treatment. Most importantly, we suggest that these results were not dependent on the diet composition, possibly indicating that the drug improves the hepatic synthesis and secretion of proteins and cholesterol.

Effectiveness of Elexacaftor/Tezacaftor/Ivacaftor Therapy in Three Subjects with the Cystic Fibrosis Genotype Phe508del/Unknown and Advanced Lung Disease.

We evaluated the effectiveness and safety of elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA) in three subjects carrying the Phe508del/unknown CFTR genotype. An ex vivo analysis on nasal epithelial cells (NEC) indicated a significant improvement of CFTR gating activity after the treatment. Three patients were enrolled in an ELX/TEZ/IVA managed-access program, including subjects with the highest percent predicted Forced Expiratory Volume in the 1st second (ppFEV1) < 40 in the preceding 3 months. Data were collected at baseline and after 8, 12 and 24 weeks of follow-up during treatment. All patients showed a considerable decrease of sweat chloride (i.e., meanly about 60 mmol/L as compared to baseline), relevant improvement of ppFEV1 (i.e., >8) and six-minute walk test, and an increase in body mass index after the first 8 weeks of treatment. No pulmonary exacerbations occurred during the 24 weeks of treatment and all domains of the CF Questionnaire-Revised improved. No safety concerns related to the treatment occurred. This study demonstrates the benefit from the ELX/TEZ/IVA treatment in patients with CF with the Phe508del and one unidentified CFTR variant. The preliminary ex vivo analysis of the drug response on NEC helps to predict the in vivo therapeutic endpoints.

Elexacaftor-Tezacaftor-Ivacaftor Therapy for Cystic Fibrosis Patients with The F508del/Unknown Genotype.

The new CFTR modulator combination, elexacaftor/tezacaftor/ivacaftor (Trikafta) was approved by the FDA in October 2019 for treatment of Cystic Fibrosis in patients 6 years of age or older who have at least one F508del mutation in one allele and a minimal-function or another F508del mutation in the other allele. However, there is a group of patients, in addition to those with rare mutations, in which despite the presence of a F508del in one allele, it was not possible to identify any mutation in the other allele. To date, these patients are excluded from treatment with Trikafta in Italy, where the CF patients carrying F508del/unknown represent about 1.3% (71 patients) of the overall Italian CF patients. In this paper we show that the Trikafta treatment of nasal epithelial cells, derived from F508del/Unknown patients, results in a significant rescue of CFTR activity. Based on our findings, we think that the F508del/Unknown patients considered in this study could obtain clinical benefits from Trikafta treatment, and we strongly suggest their eligibility for this type of treatment. This study, adding further evidence in the literature, once again confirms the validity of functional studies on nasal cells in the cystic fibrosis theratyping and personalized medicine.

Ex vivo model predicted in vivo efficacy of CFTR modulator therapy in a child with rare genotype.

BACKGROUND: New drugs that target the basic defect in cystic fibrosis (CF) patients may now be used in a large number of patients carrying responsive mutations. Nevertheless, further research is needed to extend the benefit of these treatments to patients with rare mutations that are still uncharacterized in vitro and that are not included in clinical trials. For this purpose, ex vivo models are necessary to preliminary assessing the effect of CFTR modulators in these cases. METHOD: We report the clinical effectiveness of lumacaftor/ivacaftor therapy prescribed to a CF child with a rare genetic profile (p.Phe508del/p.Gly970Asp) after testing the drug on nasal epithelial cells. We observed a significant drop of the sweat chloride value, as of the lung clearance index. A longer follow-up period is needed to define the effects of therapy on pancreatic status, although an increase of the fecal elastase values was found. CONCLUSION: Drug response obtained on nasal epithelial cells correlates with changes in vivo therapeutic endpoints and can be a predictor of clinical efficacy of novel drugs especially in pediatric patients.

Assisting PNA transport through cystic fibrosis human airway epithelia with biodegradable hybrid lipid-polymer nanoparticles.

Cystic fibrosis (CF) is characterized by an airway obstruction caused by a thick mucus due to a malfunctioning Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein. The sticky mucus restricts drugs in reaching target cells limiting the efficiency of treatments. The development of new approaches to enhance drug delivery to the lungs represents CF treatment's main challenge. In this work, we report the production and characterization of hybrid core-shell nanoparticles (hNPs) comprising a PLGA core and a dipalmitoylphosphatidylcholine (DPPC) shell engineered for inhalation. We loaded hNPs with a 7-mer peptide nucleic acid (PNA) previously considered for its ability to modulate the post-transcriptional regulation of the CFTR gene. We also investigated the in vitro release kinetics of hNPs and their efficacy in PNA delivery across the human epithelial airway barrier using an ex vivo model based on human primary nasal epithelial cells (HNEC) from CF patients. Confocal analyses and hNPs transport assay demonstrated the ability of hNPs to overcome the mucus barrier and release their PNA cargo within the cytoplasm, where it can exert its biological function.

Impaired cholesterol metabolism in the mouse model of cystic fibrosis. A preliminary study.

This study aims to investigate cholesterol metabolism in a mouse model with cystic fibrosis (CF) by the comparison of affected homozygous versus wild type (WT) mice. In particular, we evaluated the effects of a diet enriched with cholesterol in both mice groups in comparison with the normal diet. To this purpose, beyond serum and liver cholesterol, we analyzed serum phytosterols as indirect markers of intestinal absorption of cholesterol, liver lathosterol as indirect marker of de novo cholesterol synthesis, liver cholestanol (a catabolite of bile salts synthesis) and the liver mRNA levels of LDL receptor (LDLR), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoAR), acyl CoA:cholesterol acyl transferase 2 (ACAT2), cytochrome P450 7A1 (CYP7A1) and tumor necrosis factor alpha (TNFα). CF mice showed lower intestinal absorption and higher liver synthesis of cholesterol than WT mice. In WT mice, the cholesterol supplementation inhibits the synthesis of liver cholesterol and enhances its catabolism, while in CF mice we did not observe a reduction of LDLR and HMG-CoAR expression (probably due to an altered feed-back), causing an increase of intracellular cholesterol. In addition, we observed a further increase (5-fold) in TNFα mRNA levels. This preliminary study suggests that in CF mice there is a vicious circle in which the altered synthesis/secretion of bile salts may reduce the digestion/absorption of cholesterol. As a result, the liver increases the biosynthesis of cholesterol that accumulates in the cells, triggering inflammation and further compromising the metabolism of bile salts.

Lung Microbiome in Cystic Fibrosis.

The defective mucociliary clearance due to CFTR malfunctioning causes predisposition to the colonization of pathogens responsible for the recurrent inflammation and rapid deterioration of lung function in patients with cystic fibrosis (CF). This has also a profound effect on the lung microbiome composition, causing a progressive reduction in its diversity, which has become a common characteristic of patients affected by CF. Although we know that the lung microbiome plays an essential role in maintaining lung physiology, our comprehension of how the microbial components interact with each other and the lung, as well as how these interactions change during the disease's course, is still at an early stage. Many challenges exist and many questions still to be answered, but there is no doubt that manipulation of the lung microbiome could help to develop better therapies for people affected by CF.

TAS2R38 is a novel modifier gene in patients with cystic fibrosis.

The clinical manifestation of cystic fibrosis (CF) is heterogeneous also in patients with the same cystic fibrosis transmembrane regulator (CFTR) genotype and in affected sibling pairs. Other genes, inherited independently of CFTR, may modulate the clinical manifestation and complications of patients with CF, including the severity of chronic sinonasal disease and the occurrence of chronic Pseudomonas aeruginosa colonization. The T2R38 gene encodes a taste receptor and recently its functionality was related to the occurrence of sinonasal diseases and upper respiratory infections. We assessed the T2R38 genotype in 210 patients with CF and in 95 controls, relating the genotype to the severity of sinonasal disease and to the occurrence of P. aeruginosa pulmonary colonization. The frequency of the PAV allele i.e., the allele associated with the high functionality of the T2R38 protein, was significantly lower in i) CF patients with nasal polyposis requiring surgery, especially in patients who developed the complication before 14 years of age; and ii) in CF patients with chronic pulmonary colonization by P. aeruginosa, especially in patients who were colonized before 14 years of age, than in control subjects. These data suggest a role for T2R38 as a novel modifier gene of sinonasal disease severity and of pulmonary P. aeruginosa colonization in patients with CF.

Two CFTR mutations within codon 970 differently impact on the chloride channel functionality.

Pharmacological rescue of mutant cystic fibrosis transmembrane conductance regulator (CFTR) in cystic fibrosis (CF) depends on the specific defect caused by different mutation classes. We asked whether a patient with the rare p.Gly970Asp (c.2909G>A) mutation could benefit from CFTR pharmacotherapy since a similar missense mutant p.Gly970Arg (c.2908G>C) was previously found to be sensitive to potentiators in vitro but not in vivo. By complementary DNA transfection, we found that both mutations are associated with defective CFTR function amenable to pharmacological treatment. However, analysis of messenger RNA (mRNA) from patient's cells revealed that c.2908G>C impairs RNA splicing whereas c.2909G>A does not perturb splicing and leads to the expected p.Gly970Asp mutation. In agreement with these results, nasal epithelial cells from the p.Gly970Asp patient showed significant improvement of CFTR function upon pharmacological treatment. Our results underline the importance of controlling the effect of CF mutation at the mRNA level to determine if the pharmacotherapy of CFTR basic defect is appropriate.

High-throughput screening identifies FAU protein as a regulator of mutant cystic fibrosis transmembrane conductance regulator channel.

In cystic fibrosis, deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel causes misfolding and premature degradation. One possible approach to reducing the detrimental health effects of cystic fibrosis could be the identification of proteins whose suppression rescues F508del-CFTR function in bronchial epithelial cells. However, searches for these potential targets have not yet been conducted, particularly in a relevant airway background using a functional readout. To identify proteins associated with F508del-CFTR processing, we used a high-throughput functional assay to screen an siRNA library targeting 6,650 different cellular proteins. We identified 37 proteins whose silencing significantly rescued F508del-CFTR activity, as indicated by enhanced anion transport through the plasma membrane. These proteins included FAU, UBE2I, UBA52, MLLT6, UBA2, CHD4, PLXNA1, and TRIM24, among others. We focused our attention on FAU, a poorly characterized protein with unknown function. FAU knockdown increased the plasma membrane targeting and function of F508del-CFTR, but not of wild-type CFTR. Investigation into the mechanism of action revealed a preferential physical interaction of FAU with mutant CFTR, leading to its degradation. FAU and other proteins identified in our screening may offer a therapeutically relevant panel of drug targets to correct basic defects in F508del-CFTR processing.

Peptide Nucleic Acids as miRNA Target Protectors for the Treatment of Cystic Fibrosis.

Cystic Fibrosis (CF) is one of the most common life shortening conditions in Caucasians. CF is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene which result in reduced or altered CFTR functionality. Several microRNAs (miRNAs) downregulate the expression of CFTR, thus causing or exacerbating the symptoms of CF. In this context, the design of anti-miRNA agents represents a valid functional tool, but its translation to the clinic might lead to unpredictable side effects because of the interference with the expression of other genes regulated by the same miRNAs. Herein, for the first time, is proposed the use of peptide nucleic acids (PNAs) to protect specific sequences in the 3'UTR (untranslated region) of the CFTR messenger RNA (mRNA) by action of miRNAs. Two PNAs (7 and 13 bases long) carrying the tetrapeptide Gly-SerP-SerP-Gly at their C-end, fully complementary to the 3'UTR sequence recognized by miR-509-3p, have been synthesized and the structural features of target PNA/RNA heteroduplexes have been investigated by spectroscopic and molecular dynamics studies. The co-transfection of the pLuc-CFTR-3´UTR vector with different combinations of PNAs, miR-509-3p, and controls in A549 cells demonstrated the ability of the longer PNA to rescue the luciferase activity by up to 70% of the control, thus supporting the use of suitable PNAs to counteract the reduction in the CFTR expression.