Comprehensive Assessment of CFTR Modulators' Therapeutic Efficiency for N1303K Variant.

p.Asn1303Lys (N1303K) is a common missense variant of the CFTR gene, causing cystic fibrosis (CF). In this study, we initially evaluated the influence of CFTR modulators on the restoration of N1303K-CFTR function using intestinal organoids derived from four CF patients expressing the N1303K variant. The forskolin-induced swelling assay in organoids offered valuable insights about the beneficial effects of VX-770 + VX-661 + VX-445 (Elexacaftor + Tezacaftor + Ivacaftor, ETI) on N1303K-CFTR function restoration and about discouraging the prescription of VX-770 + VX-809 (Ivacaftor + Lumacaftor) or VX-770 + VX-661 (Ivacaftor + Tezacaftor) therapy for N1303K/class I patients. Then, a comprehensive assessment was conducted on an example of one patient with the N1303K/class I genotype to examine the ETI effect on the restoration of N1303K-CFTR function using in vitro the patient's intestinal organoids, ex vivo the intestinal current measurements (ICM) method and assessment of the clinical status before and after targeted therapy. All obtained results are consistent with each other and have proven the effectiveness of ETI for the N1303K variant. ETI produced a significant positive effect on forskolin-induced swelling in N1303K/class I organoids indicating functional improvement of the CFTR protein; ICM demonstrated that ETI therapy restored CFTR function in the intestinal epithelium after three months of treatment, and the patient improved his clinical status and lung function, increased his body mass index (BMI) and reduced the lung pathogenic flora diversity, surprisingly without improving the sweat test results.

Advances in the Study of Common and Rare CFTR Complex Alleles Using Intestinal Organoids.

Complex alleles (CAs) arise when two or more nucleotide variants are present on a single allele. CAs of the CFTR gene complicate the cystic fibrosis diagnosis process, classification of pathogenic variants, and determination of the clinical picture of the disease and increase the need for additional studies to determine their pathogenicity and modulatory effect in response to targeted therapy. For several different populations around the world, characteristic CAs of the CFTR gene have been discovered, although in general the prevalence and pathogenicity of CAs have not been sufficiently studied. This review presents examples of using intestinal organoid models for assessments of the two most common and two rare CFTR CAs in individuals with cystic fibrosis in Russia.

Clinical and Genetic Characteristics of a Patient with Cystic Fibrosis with a Complex Allele [E217G;G509D] and Functional Evaluation of the CFTR Channel.

The intricate nature of complex alleles presents challenges in the classification of CFTR gene mutations, encompassing potential disease-causing, neutral, or treatment-modulating effects. Notably, the complex allele [E217G;G509D] remains absent from international databases, with its pathogenicity yet to be established. Assessing the functionality of apical membrane ion channels in intestinal epithelium employed the intestinal current measurements (ICM) method, using rectal biopsy material. The effectivity of CFTR-targeted therapy was evaluated using a model of intestinal organoids of a patient harboring the genotype F508del/[E217G;G509D]. ICM analysis revealed diminished chloride channel function. Remarkably, [E217G;G509D] presence within intestinal organoids correlated with heightened residual CFTR function. Employing CFTR modulators facilitated the restoration of the functional CFTR protein. This multifaceted study intertwines genetic investigations, functional analyses, and therapeutic interventions, shedding light on the intricate interplay of complex alleles within CFTR mutations. The results highlight the potential of targeted CFTR modulators to restore functional integrity, offering promise for advancing precision treatments in cystic fibrosis management.

Natural Guanine Derivatives Exert PARP-Inhibitory and Cytoprotective Effects in a Model of Cardiomyocyte Damage under Oxidative Stress.

Inhibitors of human poly(ADP-ribose) polymerase (PARP) are considered as promising agents for treatment of cardiovascular, neurological, and other diseases accompanied by inflammation and oxidative stress. Previously, the ability of natural compounds 7-methylguanine (7mGua) and 8-hydroxy-7-methylguanine (8h7mGua) to suppress activity of the recombinant PARP protein was demonstrated. In the present work, we have investigated the possibility of PARP-inhibitory and cytoprotective action of 7mGua and 8h7mGua against the rat cardiomyoblast cultures (undifferentiated and differentiated H9c2). It was found that 7mGua and 8h7mGua rapidly penetrate into the cells and effectively suppress the H2O2-stimulated PARP activation (IC50 = 270 and 55 µM, respectively). The pronounced cytoprotective effects of 7mGua and 8h7mGua were shown in a cellular model of oxidative stress, and effectiveness of 8h7mGua exceeded the classic PARP inhibitor 3-aminobenzamide. The obtained data indicate promise for the development of PARP inhibitors based on guanine derivatives and their testing using the models of ischemia-reperfusion tissue damage.

Airway and Lung Organoids from Human-Induced Pluripotent Stem Cells Can Be Used to Assess CFTR Conductance.

Airway and lung organoids derived from human-induced pluripotent stem cells (hiPSCs) are current models for personalized drug screening, cell-cell interaction studies, and lung disease research. We analyzed the existing differentiation protocols and identified the optimal conditions for obtaining organoids. In this article, we describe a step-by-step protocol for differentiating hiPSCs into airway and lung organoids. We obtained airway and lung organoids from a healthy donor and from five donors with cystic fibrosis. Analysis of the cellular composition of airway and lung organoids showed that airway organoids contain proximal lung epithelial cells, while lung organoids contain both proximal and distal lung epithelial cells. Forskolin-induced swelling of organoids derived from a healthy donor showed that lung organoids, as well as airway organoids, contain functional epithelial cells and swell after 24 h exposure to forskolin, which makes it a suitable model for analyzing the cystic fibrosis transmembrane conductance regulator (CFTR) channel conductance in vitro. Thus, our results demonstrate the feasibility of generating and characterizing airway and lung organoids from hiPSCs, which can be used for a variety of future applications.

Clinical and Functional Characteristics of the E92K CFTR Gene Variant in the Russian and Turkish Population of People with Cystic Fibrosis.

The pathogenic variant E92K (c.274G > A) of the CFTR gene is rare in America and Europe, but it is common for people with cystic fibrosis from Russia and Turkey. We studied the effect of the E92K genetic variant on the CFTR function. The function of the CFTR channel was studied using the intestinal current measurements (ICM) method. The effects of CFTR modulators on the restoration of the CFTR function were studied in the model of intestinal organoids. To assess the effect of E92K on pre-mRNA splicing, the RT-PCR products obtained from patients' intestinal organoid cultures were analyzed. Patients with the genetic variant E92K are characterized by an older age of diagnosis compared to homozygotes F508del and a high frequency of pancreatic sufficiency. The results of the sweat test and the ICM method showed partial preservation of the function of the CFTR channel. Functional analysis of CFTR gene expression revealed a weak effect of the E92K variant on mRNA-CFTR splicing. Lumacaftor (VX-809) has been shown to restore CFTR function in an intestinal organoid model, which allows us to consider the E92K variant as a promising target for therapy with CFTR correctors.

The Effect of Complex Alleles of the CFTR Gene on the Clinical Manifestations of Cystic Fibrosis and the Effectiveness of Targeted Therapy.

The authors of this article analyzed the available literature with the results of studying the prevalence of complex alleles of the CFTR gene among patients with cystic fibrosis, and their pathogenicity and influence on targeted therapy with CFTR modulators. Cystic fibrosis (CF) is a multisystemic autosomal recessive disease caused by a defect in the expression of the CFTR protein, and more than 2000 genetic variants are known. Clinically significant variants are divided into seven classes. Information about the frequency of complex alleles appears in a number of registers, along with the traditional presentation of data on genetic variants. Complex alleles (those with the presence of more than two nucleotide variants on one allele) can complicate the diagnosis of the disease, and change the clinical manifestations of cystic fibrosis and the response to treatment, since each variant in the complex allele can contribute to the functional activity of the CFTR protein, changing it both in terms of increasing and decreasing function. The role of complex alleles is often underestimated, and their frequency has not been studied. At the moment, characteristic frequently encountered complex alleles have been found for several populations of patients with cystic fibrosis, but the prevalence and pathogenicity of newly detected complex alleles require additional research. In this review, more than 35 complex alleles of the CFTR gene from existing research studies were analyzed, and an analysis of their influence on the manifestations of the disease and the effectiveness of CFTR modulators was also described.

Personalized Selection of a CFTR Modulator for a Patient with a Complex Allele [L467F;F508del].

The presence of complex alleles in the CFTR gene can lead to difficulties in diagnosing cystic fibrosis and cause resistance to therapy with CFTR modulators. Tezacaftor/ivacaftor therapy for 8 months in a patient with the initially established F508del/F508del genotype did not lead to an improvement in her condition-there was no change in spirometry and an increase in the patient's weight, while there was only a slight decrease in NaCl values, measured by a sweat test. The intestinal current measurements of the patient's rectal biopsy showed no positive dynamics in the rescue of CFTR function while taking tezacaftor/ivacaftor. The assumption that the patient had an additional mutation in the cis position was confirmed by sequencing the CFTR gene, and the complex allele [L467F;F508del] was identified. Based on the rescue of CFTR function by elexacaftor/tezacaftor/ivacaftor obtained using forskolin-induced swelling on intestinal organoids, the patient was prescribed therapy with this targeted drug. The use of elexacaftor/tezacaftor/ivacaftor for 7 months resulted in a significant improvement in the patient's clinical condition.

Evaluation of the Complex p.[Leu467Phe;Phe508del] CFTR Allele in the Intestinal Organoids Model: Implications for Therapy.

In the cohort of Russian patients with cystic fibrosis, the p.[Leu467Phe;Phe508del] complex allele (legacy name [L467F;F508del]) of the CFTR gene is understudied. In this research, we present the results of frequency evaluation of the [L467F;F508del] complex allele in the Russian Federation among patients with a F508del/F508del genotype, its effect on the clinical course of cystic fibrosis, the intestinal epithelium ionic channel function, and the effectiveness of target therapy. The frequency of the [L467F;F508del] complex allele among patients with homozygous F508del was determined with multiplex ligase-dependent probe amplification followed by polymerase chain reaction and fragment analysis. The function of ionic channels, including the residual CFTR function, and the effectiveness of CFTR modulators was analyzed using intestinal current measurements on rectal biopsy samples and the forskolin-induced swelling assay on organoids. The results showed that the F508del/[L467F;F508del] genotype is present in 8.2% of all Russian patients with F508del in a homozygous state. The clinical course of the disease in patients with the F508del/[L467F;F508del] genotype is severe and does not vary from the course in the cohort with homozygous F508del, although the CFTR channel function is significantly lower. For patients with the F508del/[L467F;F508del] genotype, we can recommend targeted therapy using a combined ivacaftor + tezacaftor + elexacaftor medication.

Health Characteristics of Patients with Cystic Fibrosis whose Genotype Includes a Variant of the Nucleotide Sequence c.3140-16T>A and Functional Analysis of this Variant.

Cystic fibrosis (CF) is the most common monogenic autosomal recessive disease, associated with pathogenic variants in the CFTR gene. The splicing variant c.3140-16T>A (3272-16T>A) has been described previously and, according to the Russian CF Patients Registry, occurs with a frequency of 0.34%. The phenotypic features of CF patients with the c.3140-16T>A variant were compared with those of patients with the genotype F508del/F508del. Patients with the allele c.3140-16T>A had higher average age and age at diagnosis, and the allele was present in a greater proportion of adults. Patients carrying the c.3140-16T>A allele were characterised by better physical development indicators, both in adults and in children, had preserved pancreatic function, as well as the absence of a number of complications, and required pancreatic enzyme replacement therapy less often than patients with the F508del/F508del genotype. Sweat test values also were lower in patients with the c.3140-16T>A genotype. According to the results of clinical and laboratory studies, the phenotype of patients with the genetic variant c.3140-16T>A can be considered "mild". Functional CFTR protein activity in the presence of c.3140-16T>A was evaluated using intestinal current measurements (ICM) and the forskolin-induced swelling assay on organoids obtained from patients' rectal biopsies. c.3140-16T>A had high residual CFTR channel activity and was amenable to effective pharmacological correction with thea VX-770 potentiator. To evaluate the effect of the variant on CFTR pre-mRNA splicing we performed a minigene assay, as well as RT-PCR analysis of RNA isolated from the nasal epithelium and rectal biopsy of patients. We showed that the c.3140-16T>A variant creates a novel acceptor AG dinucleotide within CFTR intron 19, resulting in a 14-nucleotide extension of exon 20. This frameshift produces a premature termination codon and triggers mRNA degradation by the nonsense-mediated decay (NMD) mechanism. Moreover, we observed that the c.3140-16T>A allele could produce a residual amount of normally spliced transcript, thus explaining the patient's mild phenotype.

Genetically Encoded Fluorescent Sensor for Poly-ADP-Ribose.

Poly-(ADP-ribosyl)-ation (PARylation) is a reversible post-translational modification of proteins and DNA that plays an important role in various cellular processes such as DNA damage response, replication, transcription, and cell death. Here we designed a fully genetically encoded fluorescent sensor for poly-(ADP-ribose) (PAR) based on Förster resonance energy transfer (FRET). The WWE domain, which recognizes iso-ADP-ribose internal PAR-specific structural unit, was used as a PAR-targeting module. The sensor consisted of cyan Turquoise2 and yellow Venus fluorescent proteins, each in fusion with the WWE domain of RNF146 E3 ubiquitin ligase protein. This bipartite sensor named sPARroW (sensor for PAR relying on WWE) enabled monitoring of PAR accumulation and depletion in live mammalian cells in response to different stimuli, namely hydrogen peroxide treatment, UV irradiation and hyperthermia.

Disaccharide pyrimidine nucleosides and their derivatives: a novel group of cell-penetrating inhibitors of poly(ADP-ribose) polymerase 1.

Nearly 30 synthetic nucleosides were tested with human recombinant poly(ADP-ribose) polymerase 1 as potential inhibitors of this enzyme. The most active compounds were some disaccharide analogues of thymidine: 3'-O-ß-D-ribofuranosyl-5-iodo-dUrd (2d; IC50 = 45 µM), 3'-O-ß-D-ribofuranosyl-2'-deoxythymidine (2e; IC50 = 38 µM), and 3'-O-ß-D-ribofuranosyl-2'-deoxythymidine oxidized (4; IC50 = 25 µM). These compounds also reduced H2O2-induced synthesis of poly(ADP-ribose) in cultured human ovarian carcinoma (SKOV-3) cells in a dose-dependent manner. Furthermore, compounds 2d or 2e until a concentration of 1 mM did not affect growth of SKOV-3 cells, whereas dialdehyde compound 4, as well as thymidine, exhibited a significant cytotoxicity.

Isolation and characterization of high affinity aptamers against DNA polymerase iota.

Human DNA-polymerase iota (Pol ι) is an extremely error-prone enzyme and the fidelity depends on the sequence context of the template. Using the in vitro systematic evolution of ligands by exponential enrichment (SELEX) procedure, we obtained an oligoribonucleotide with a high affinity to human Pol ι, named aptamer IKL5. We determined its dissociation constant with homogenous preparation of Pol ι and predicted its putative secondary structure. The aptamer IKL5 specifically inhibits DNA-polymerase activity of the purified enzyme Pol ι, but did not inhibit the DNA-polymerase activities of human DNA polymerases beta and kappa. IKL5 suppressed the error-prone DNA-polymerase activity of Pol ι also in cellular extracts of the tumor cell line SKOV-3. The aptamer IKL5 is useful for studies of the biological role of Pol ι and as a potential drug to suppress the increase of the activity of this enzyme in malignant cells.