The development of effective strategies for the treatment of bone defects is based on gene therapy methods aimed at regulating the differentiation of osteoprogenitor cells. One approach is the development of knockdown systems of inhibitory genes of osteogenic cell differentiation using siRNA molecules. In this work, we developed approaches to induce osteogenic differentiation of mesenchymal stem cells (MSCs) by knockdown of GSK3β using siRNAs in cultures of MSCs derived from human adipose tissue (AD-MSCs). For this purpose, we performed a comparative evaluation of the efficacy of lipoplexes and polyplexes formed with one of the 4 siRNA molecules and 5 commercial transfection agents most commonly used in laboratory practice. The most effective transfection agent appeared to be PEI, which demonstrated high cytocompatibility in free form and as part of polyplexes even when maximum concentrations were used. Using the polyplexes formed by siRNA molecule designed for the first time and PEI, we developed a highly efficient GSK3β gene knockdown system, which showed its effectiveness in cultures of AD-MSCs. As a result, we demonstrated the osteoinductive properties of GSK3β siRNA molecules in these cultures. The results obtained can be applied in the development of gene therapy strategies based on siRNA molecules in human bone tissue diseases.

Introduction. The development of gene-activated osteoplastic materials opens up new opportunities for effective treatment of patients with bone deficiency. Materials and methods. The study was carried out using multipotent mesenchymal stromal cells (MSCs) isolated from adipose tissue of rats. We obtained matrices based on polylactide acid granules (PLA granules), type I collagen, and fibrin that were impregnated with adenovirus with the BMP2 gene (Ad-BMP2) and ibuprofen. Cell adhesion on the matrices was assessed with scanning electron microscopy. The release kinetics of adenoviruses and ibuprofen was studied with spectrophotometry and UV spectrophotometry, respectively. We used real-time PCR to evaluate the efficiency of osteogenic differentiation of cells with the expression of genes of osteogenic markers; enzyme-linked immunosorbent assay to assess the production of proteins; and alizarin red staining to analyze the activity of alkaline phosphatase and the mineralization of the MSCs extracellular matrix. Results. The addition of ibuprofen during transduction of MSCs with adenoviruses with the BMP2 gene not only contributed to the effective delivery of the target gene and production of the BMP-2 protein, but also ensured more pronounced osteogenic differentiation of cells. The inclusion of ibuprofen in matrices based on PLA granules, type I collagen, and fibrin made it possible to achieve long-term release of the drug from the material. The developed matrices impregnated with Ad-BMP2 and ibuprofen are characterized by high adhesive properties. Viruses released from the matrices effectively transduced cells and induced osteogenic differentiation of MSCs. Conclusion. The developed matrices containing adenovirus with the BMP2 gene and ibuprofen have osteogenic properties in vitro and a high potential use in the treatment of bone defects. Keywords: adenoviral vectors, ibuprofen, bone morphogenetic protein, polylactide, collagen, fibrin, osteogenic differentiation

The use of 3D-printed gene-activated bone grafts represents a highly promising approach in the fields of dentistry and orthopedics. Bioresorbable poly-lactic-co-glycolic acid (PLGA) scaffolds, infused with adenoviral constructs that carry osteoinductive factor genes, may provide an effective alternative to existing bone grafts for the reconstruction of extensive bone defects. This study aims to develop and investigate the properties of 3D scaffolds composed of PLGA and adenoviral constructs carrying the BMP2 gene (Ad-BMP2), both in vitro and in vivo. The elastic modulus of the disk-shaped PLGA scaffolds created using a specialized 3D printer was determined by compressive testing in both axial and radial directions. In vitro cytocompatibility was assessed using adipose-derived stem cells (ADSCs). The ability of Ad-BMP2 to transduce cells was evaluated. The osteoinductive and biocompatible properties of the scaffolds were also assessed in vivo. The Young’s modulus of the 3D-printed PLGA scaffolds exhibited comparable values in both axial and radial compression directions, measuring 3.4 ± 0.7 MPa for axial and 3.17 ± 1.4 MPa for radial compression. The scaffolds promoted cell adhesion and had no cytotoxic effect on ADSCs. Ad-BMP2 successfully transduced the cells and induced osteogenic differentiation in vitro. In vivo studies demonstrated that the 3D-printed PLGA scaffolds had osteoinductive properties, promoting bone formation within the scaffold filaments as well as at the center of a critical calvarial bone defect.

The restoration of bone defects resulting from tooth loss, periodontal disease, severe trauma, tumour resection and congenital malformations is a crucial task in dentistry and maxillofacial surgery. Growth factor- and gene-activated bone graft substitutes can be used instead of traditional materials to solve these problems. New materials will overcome the low efficacy and difficulties associated with the use of traditional bone substitutes in complex situations. One of the most well-studied active components for bone graft substitutes is bone morphogenetic protein-2 (BMP-2), which has strong osteoinductive properties. The aim of this review was to examine the use of BMP-2 protein and gene therapy for bone regeneration in the oral and maxillofacial region and to discuss its future use.

About 13% of all variants causing cystic fibrosis are splice variants of the CFTR gene; at the moment, such variants are not well studied.The aim was to investigate the effect of the splicing variant 2789+5G>A on the phenotypic manifestations of cystic fibrosis and the effectiveness of targeted therapy in an intestinal organoids model in the patients with genotypes 2789+5G>A/L732X and 2789+5G>A/CFTRdele2,3.Methods. The medical history data, intestinal current measurement (ICM), and forskolin-induced swelling assay in the intestinal organoids were used.Results. The clinical features of the course of CF in 2 patients, carriers of the 2789+5G>A variant in a heterozygous state with genetic variants of the CFTR gene of classes I and VII, are presented. Clinical picture: the disease is of moderate severity; the pancreas is sufficient. The ICM method demonstrated the preservation of the chloride channel function. Assays on the cultured organoids obtained from the intestinal tissue showed moderate residual functional activity of the chloride channels. In addition, the 2789+5G>A variant is sensitive to all tested CFTR modulators.Conclusions. The splicing variant 2789+5G>A is characterized by moderate residual functional activity of the CFTR channel and is sensitive to all targeted treatments. However, the most effective CFTR modulator for this genetic variant is tezacaftor.

Complex alleles of the CFTR gene complicate the diagnosis of cystic fibrosis (CF), the classification of its pathogenic variants, affect the clinical picture of the disease and can affect the efficiency of targeted drugs. The total frequency of complex allele [L467F;F508del] in the Russian population of patients with CF is 0.74%, and in patients with the F508del/F508del genotype, its frequency reaches 8%. This article presents multi-faceted study of the complex allele [L467F;F508del] in a cohort of patients with genotypes [L467F;F508del]/class I (c.3532_3535dup, c.1766+2T>C, W1310X, 712-1G>T), and data for a unique patient with the genotype [L467F;F508del]/[L467F;F508del]. Using the intestinal current measurement method, it was demonstrated the absence of CFTR function for [L467F;F508del]/class I and [L467F;F508del]/[L467F;F508del] genotypes. In intestinal organoids, it was shown that [L467F;F508del] in combination with class I variants and in the homozygotes abolishes the efficacy of both two-component (ivacaftor+lumacaftor; ivacaftor+tezacaftor) and three-component (ivacaftor+tezacaftor+elexacaftor) targeted drugs. When prescribing ivacaftor+tezacaftor+elexacaftor to three patients, they did not have a clinical effect after 6–12 months.

A comparative study of gelation processes in aqueous solutions of sodium alginate initiated by their chemical crosslinking with divalent ions Ca2+ or Ba2+ was carried out. 3D cryoprinted hydrogel scaffolds of certain architectonics were formed from the studied compositions containing various concentrations of crosslinking agents. It is shown that the increase in the crosslinking agent concentration from 2 to 10 wt. % leads to the improvement in the strength characteristics of alginate structures. At the same time, scaffolds crosslinked with 10 % aqueous solutions of both CaCl2 and BaCl2 retain their structural stability during 21 days of incubation in a culture medium at a temperature of 37 °C. During in vitro experiments on cultures of mesenchymal stem cells derived from rat adipose tissue, samples crosslinked with Ba2+ ions were found to demonstrate a moderate cytotoxic effect and showed their inability to maintain cell adhesion, unlike non-toxic scaffolds crosslinked in CaCl2 aqueous solutions. In vivo analysis of experimental samples on an intramuscular implantation model for male Wistar rats also confirmed moderate cytotoxicity of alginate scaffolds formed using Ba2+ ions as crosslinking agents. The results obtained allow us to assert that of all the studied by us materials, sodium alginate crosslinked with a 10% CaCl2 aqueous solution can be considered the most promising for various biomedical applications.

Three-dimensional scaffolds were made from a solution of poly(lactide-co-glycolide) mixed with tetraglycol using antisolvent 3D printing. The elastic properties and the structure of the obtained matrices were studied. MTT-test and staining with PKH-26, Calcein-AM, DAPI with subsequent fluorescence microscopy were used to study biological properties. The three-dimensional scaffolds had good mechanical properties. Young’s modulus value was 18±2 MPa, tensile strength was 0.43±0.05 MPa. The relative survival rate of cells after the first day was 99.58±2.28%, on the 14th day – 98.14±2.22%. The structure of the scaffold promoted cell adhesion and spreading on its surface. The poly(lactide-co-glycolide) matrices produced by antisolvent printing have high porosity, biocompatibility and good mechanical properties. It is allowed to use them in the future as a basis for personalized constructions for the replacement of extensive bone defects.

BACKGROUND: When new, most often rare CFTR gene variants, are detected in patients with cystic fibrosis, intestinal organoids are used, which allows for the assessment of the residual functional activity of the CFTR channel and the effect of targeted drugs to determine the possibility of further pathogenetic therapy. Clinical trials of the effectiveness of the targeted drugs in patients with rare CFTR variants are expensive, and patient groups are extremely small. A forskolin-induced swelling assay on a patient’s intestinal organoids allows for a personalized approach when studying rare or even single CFTR variants. AIM: To examine the effect of the CFTR potentiator ivacaftor and the combination of ivacaftor with the CFTR correctors lumacaftor, tezacaftor, and elexacaftor on the restoration of CFTR channel functions on a culture of intestinal organoids obtained from a patient with two rare CFTR variants c.264_268delATATT and c.3139+1GC. MATERIALS AND METHODS: To assess the activity of the CFTR channel, the forskolin-induced swelling assay on organoids and intestinal current measurements on rectal biopsy samples method were used. The clinical picture of a patient with the c.264_268delATATT/c.3139+1GC genotype was described. RESULTS: In vitro, the genetic variants c.264_268delATATT and c.3139+1GC lead to a complete loss of the functional CFTR protein, whereas CFTR modulators do not positively affect and do not lead to the restoration of CFTR function, in contrast from F508del/F508del control. The disease severity in a child is consistent with the results of functional tests. CONCLUSION: Both CFTR variants are classified as “severe” and cause a complete loss of chloride channel activity. No effective CFTR modulators were found for the c.264_268delATATT and c.3139+1GC variants; thus, targeted therapy cannot be recommended to the patient.

Human-induced airway basal cells (hiBCs) derived from human-induced pluripotent stem cells (hiPSCs) offer a promising cell model for studying lung diseases, regenerative medicine, and developing new gene therapy methods. We analyzed existing differentiation protocols and proposed our own protocol for obtaining hiBCs, which involves step-by-step differentiation of hiPSCs into definitive endoderm, anterior foregut endoderm, NKX2.1+ lung progenitors, and cultivation on basal cell medium with subsequent cell sorting using the surface marker CD271 (NGFR). We derived hiBCs from two healthy cell lines and three cell lines with cystic fibrosis (CF). The obtained hiBCs, expressing basal cell markers (NGFR, KRT5, and TP63), could differentiate into lung organoids (LOs). We demonstrated that LOs derived from hiBCs can assess cystic fibrosis transmembrane conductance regulator (CFTR) channel function using the forskolin-induced swelling (FIS) assay. We also carried out non-viral (electroporation) and viral (recombinant adeno-associated virus (rAAV)) serotypes 6 and 9 and recombinant adenovirus (rAdV) serotype 5 transgene delivery to hiBCs and showed that rAAV serotype 6 is most effective against hiBCs, potentially applicable for gene therapy research.

Background/Objectives: The replenishment of bone deficiency remains a challenging task in clinical practice. The use of gene-activated matrices (GAMs) impregnated with genetic constructs may be an innovative approach to solving this problem. The aim of this work is to develop collagen-based matrices with the addition of platelet-rich plasma, carrying polyplexes with the BMP2 gene, to study their biocompatibility and osteogenic potential in vitro and in vivo. Methods: The cytocompatibility of the materials during incubation with adipose-derived stem cells (ADSCs) was studied using the MTT test and fluorescent microscopy. Biocompatibility was assessed during intramuscular implantation, followed by histological analysis. Osteogenic differentiation was determined by the expressions of Alpl and Bglap using real-time PCR and extracellular matrix (ECM) mineralization by alizarin red staining. The efficiency of bone regeneration was studied using micro-CT and analysis of histological sections stained according to Masson. Results: After the incubation of ADSCs with GAS, significant increases in the expressions of the Alpl and Bglap genes by 3 ± 0.1 and 9.9 ± 0.6 times, relative to the control, as well as mineralization of the ECM, were observed. The volume of newly formed bone was 37.2 ± 6.2% after implantation of GAS, 20.9 ± 1.2%—non-activated Col/PRP, and 2.6 ± 1.5% in an empty defect. Conclusions: The use of Col/PRP-based matrices is an effective method for delivering of the osteoinductor gene to the site of bone tissue damage. The highest degree of healing was observed after the implantation of Col/PRP-TF/pBMP2 into the critical size defect compared to the other groups.

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.

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.

Изучение редких вариантов в гене CFTR является актуальным в эпоху таргетной терапии CFTR-модуляторами. В данном исследовании изучена клиническая картина муковисцидоза, а также функция CFTR-канала у гомозиготного носителя редкого патогенного варианта c.1329_1350del (p.Asp443GlufsX19), относящегося к I классу нарушений в гене CFTR. Получена полная корреляция результатов функциональных тестов с фенотипической картиной муковисцидоза и потовым тестом. Метод определения разности кишечных потенциалов продемонстрировал отсутствие функциональной активности хлорного CFTR канала в ректальном биоптате; при стимулировании форсколином органоиды пациента не ответили набуханием, что свидетельствует о полном нарушении образования функционального белка CFTR. Воздействие на кишечные органоиды всеми зарегистрированными для терапии таргетными препаратами не привело к их набуханию, в отличие от контрольной культуры, гомозиготной по F508del, таким образом, хлорный канал пациента с генотипом p.Asp443GlufsX19/p.Asp443GlufsX19 оказался нечувствителен ни к одному CFTR-модулятору. The study of rare variants in the CFTR gene is relevant in the era of targeted therapy with CFTR modulators. In this study, we studied the clinical picture of cystic fibrosis, as well as the function of the CFTR channel in a homozygous carrier of a rare pathogenic variant c.1329_1350del (p.Asp443GlufsX19), belonging to class I disorders in the CFTR gene. A complete correlation of the results of functional tests with the phenotypic pattern of CF and data of a sweat test was obtained. The method for determining the difference in intestinal potentials demonstrated the absence of functional activity of the chloride CFTR channel in the rectal biopsy, and when stimulated with forskolin, the patient’s organoids did not swell, which indicates a complete violation of the functional CFTR protein production. Exposure to intestinal organoids by all registered for therapy targeted drugs did not lead to their swelling, unlike the control culture, homozygous for F508del. The chloride channel of a patient with genotype p.Asp443GlufsX19/p.Asp443GlufsX19 turned out to be insensitive to any CFTR modulator.

An important factor influencing the severity of cystic fibrosis (CF) and the effectiveness of treatment is the presence of complex alleles in the CFTR gene ≥ 2 variants in one allele. The influence of such alleles on the manifestations of CF has not been sufficiently studied.The aim of this study was to investigate the effect of the complex allele [L467F;F508del] on the phenotypic manifestations of CF and the efficacy of targeted therapy in an intestinal organoid (IO) model in a patient with the [L467F;F508del]/W1310X genotype.Methods. Methods for determining the difference in intestinal potentials (IDP), the KO method, and the forskolin test are presented using the medical history of a patient with the [L467F;F508del]/ W1310X genotype as an example.Results. The patient was diagnosed with progressive disease with obvious deterioration of pulmonary function. The ORKP method showed the absence of chlorine channel function. An assay with a KO culture from intestinal tissue showed a complete loss of functional activity of the chloride channel. In addition, the complex allele [L467F;F508del] is not sensitive to the effect of all tested CFTR modulators.Conclusion. The complex allele [L467F;F508del] causes a complete loss of functional CFTR protein and is not sensitive to the effect of any of the registered targeted drugs.

OBJECTIVE:To study the behavior of SHED cell culture on different types of materials for the regeneration of periodontal tissues with different porosity.MATERIAL AND METHODS:Porous collagen material Fibro-Gide (Geitstlich Pharma AG, Switzerland), designed to increase the volume of the gum and the barrier collagen membrane Bio-Gide (Geitstlich Pharma AG, Switzerland) were studied in vitro on SHED cultures. As a control sample, a Spongostan sponge made of gelatin (Johnson & Johnson Medical, UK) with the most pronounced porosity and wettability was used. Acute cytotoxicity was determined using a screening method for assessing the number of living cells in a sample (MTT test). SHED cells were sown on the materials to study the attachment of cells to materials and their migration inside the samples. Before seeding, the cells were stained with vital fluorescent dye PKH26 (red fluorescent cell linker kit, Sigma, Germany) for further visualization.RESULTS AND DISCUSSION:Using the MTT test it was shown that they do not have cytotoxic effects. At the same time by the 8th day of the experiment in the presence of Fibro-Gide and Bio-Gide the cells showed an increase in proliferative activity by 19% and 12%, respectively compared with the control group. The cells attached and spread out on the surface of the materials and migrated into the thickness of porous Fibro-Gide and Spongostan.CONCLUSION:The in vitro study showed that the most favorable material for SHED cell culture is the collagen material Fibro-Gide with sufficient porosity, elasticity and hydrophilicity. SHED cells attach to the collagen matrix and easily penetrate into the sample, filling the entire internal space, while the proliferative capacity of the cell culture increases.

Recombinant adeno-associated viruses (rAAVs) may be useful for the development of gene therapy for hereditary diseases. Patient-specific human induced pluripotent stem cells (hiPSCs) can be differentiated into a variety of cells which are difficult or impossible to obtain by biopsy. To date, few research on the efficiency of rAAV transduction of hiPSCs has been published, but the obtained data are very contradictory and do not answer the actual question: how effective are rAAVs for the delivery of transgenes into hiPSCs. In this work, we used rAAV serotypes 5, 6, and 9 carrying the GFP transgene. The transduction efficiency of rAAV2/9-GFP and rAAV2/6-GFP for the immortalized tracheal epithelial cell line derived from a patient with cystic fibrosis (CFTE29o-) was relatively high. At the same time, the efficiency of transduction of iPSCs from a healthy donor and a cystic fibrosis (CF) donor was extremely low. Thus, our results show that the efficiency of hiPSC transduction by rAAV serotypes 5, 6, and 9 is not suitable for the delivery of transgenes.

Objectives: The splicing variant 3272-16T>A (c.3140-16T>A) according to the Russian CF Patients Registry, occurs with a frequency of 0.33% (21 place in Russian CF Patients Registry). This genetic variant is not specified in the instructions for the modulators. The aim was to study the effect of CFTR modulators on the CFTR protein function in patients with CF having the genetic variant 3272-16T>A. Methods: The function of ion channels of the apical membrane of the intestinal epithelium was evaluated using the ICM method, using biopsy material of the patient's rectum. The effectiveness of CFTR modulators was evaluated on a model of intestinal organoids of a patient (boy, 7 years) with the genotype CFTRdele9/3272-16T>A. Results: The ICM method showed a reduced function of the chlorine channel. Stimulation of the organoid culture with forskolin at a concentration of 5 mM for 1 hour indicates the preservation of the functional activity of the CFTR channel: the size of intestinal organoids increases by 50% (AUC = 1400 ± 205.5). When combined with forskolin, ivacaftor/lumacaftor or ivacaftor/tezacaftor on organoids, the functional activity of the CFTR channel increases to a moderate extent (~70%, AUC values 2209 ± 518.5 and 2182.6 ± 124.5, respectively). When using elexacaftor/tezacaftor/ivacaftor, organoids increase by 2 times (AUC = 2697.3 ± 408.8). Thus, patients with CF who have a pathogenic variant 3272-16T>A in the CFTR genotype may be recommended therapy with elexacaftor/ tezacaftor/ ivacaftor. Conclusion: For patients with the genetic variant 3272-16T>A, a therapeutic effect can be expected from the use of the elexacaftor/tezacaftor/ivacaftor.