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Vol 14, No 4 (2019)

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Open Access Open Access
Restricted Access Access granted
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Gene therapy approaches to the duchenne muscular dystrophy theatment

Zaynitdinova M.I., Smirnikhina S.A., Lavrov A.V., Eremin I.I., Pulin A.A.


Duchenne muscular dystrophy (DMD) is a common genetic disease which develops as a result of a mutation in the gene encoding dystrophin. In this review, the main experimental therapeutic approaches based on gene therapy are described. Independence of the type of mutation in the DMD gene is an advantage of the viral delivery of micro- and minidystrophin in muscle cells, but this method provides only a temporary effect. The specificity of the mutation also does not matter with an increase in the level of utrophin, however, this protein cannot fully replace dystrophin. The drugs which promote reading through the stop codon have low efficiency and are suitable for only 10-15% of patients with DMD. The most promising approach for the treatment of DMD is the exon skipping, which will suit 90% of patients. It can be implemented by antisense oligonucleotides or using the CRISPR/Cas9 genome editing system. CRISPR/Cas9-mediated exon skipping is thought to be the most promising approach, because it allows to make the necessary changes in the genome with great efficiency after single application.
Genes & Cells. 2019;14(4):6-18
pages 6-18 views

Mutation lanscape of acute myeloid leukemia in elderly patients

Vinogradov A.V., Rezaykin A.V., Sazonov S.V., Sergeev A.G., Kapitonova M.Y.


The molecular genetic landscape of acute myeloid leukemia (AML) have specific features in elderly patients, and these features correlates with hematopoietic progenitor cells senescence. Aim: to estimate the frequency of mutations in DNMT3A, FLT3, KIT, NPM1, NRAS, TP53 and WT1 genes in AML patients in elderly. Bone marrow and peripheral blood samples obtained from 54 AML patients aged over 60 years old. Distribution of the patients according to FAB-classification was as follows: AML M0 - 2, M1 - 6, M2 - 27, М3 - 2, M4 - 11, M5 - 1, M6 - 3, acute myelofibrosis - 1, blastic plasmacytoid dendritic cell neoplasm - 1. Detection of mutations in DNMT3A, FLT3, KIT, NPM1, NRAS, TP53 and WT1 genes performed by automatic direct sequencing technique. In the study group were more common patients with unfavorable (33.3%, n=18) and unspecified (42.6%, n=23) cytogenetics. The average frequency of functionally significant mutations in all investigated genes among the treated AML patients was 38.9% (n=21), including 3 cases (27.3%) with normal karyotype, 11 cases (61.1%) with unfavorable cytogenetics, 7 cases (30.4%) with unspecified karyotype. Average frequency of mutations in TP53 gene exons 4-11 was 20.0%, FLT3 gene exons 12-15 and 19-21 18.4%, DNMT3A exons 18-26 - 9.1%, NRAS gene exons 1-4 - 7.7%, KIT gene exons 7-12 and 16-19 - 5.9%, NPM1 gene exons 9-12 - 5.4% (n=2), DNMT3A - 9.1% (n=1). Multiple point mutations in investigated genes were detected in 11.1% AML specimens (n=6, usually FLT3 gene mutations, including FLT3 ITD in 4 cases). Additional gene mutations detection using direct sequencing allowed to clarify the prognostic stratification of AML from groups of unspecified and intermediate prognosis in 35.9% (n=10). In all cases, they were associated with an unfavorable prognosis. Thus, using of cytogenetic and additional molecular genetic research, a favorable prognosis of overall survival was established in 2 cases (3.7%), intermediate - in 9 cases (16.7%), unfavorable - in 27 cases (50.0%), and unspecified - in 16 (29.6%).
Genes & Cells. 2019;14(4):19-24
pages 19-24 views

Effect of pCMV-VEGF165 plasmides on the reparation of full-layer skin wound in experiment

Shestakova V.G., Banin V.V., Deev R.V., Bazhenov D.V.


The study of the regeneration of a full-layer skin wound under conditions of stimulated angiogenesis was carried out by paravulnare injection of a solution containing the plasmid pCMV-VEGF165. The inflammatory reaction in the experimental group of rats was more intense and already on day 7, the leukocyte shaft in the control was 1.3 times wider, and on day 14 in the experimental group, this indicator was 2.2 times less than the control figures. The indicators characterizing the development of granulation tissue on day 7, in the second group, 1.3 prevail over the control values, after 14 days, due to the maturation of young connective tissue - 1.1 times. The greatest differences in the comparison groups were found in relation to the degree of epithelialization of the defect. The epithelium thickness at the border is 1.4 times the control, the epithelial layer thickness is 1.7 times, and the length is 1.5 times. Compared with the control values, the number of vessels in the experimental group on day 7 were 2.3 times higher, on day 14 - 1.8 times and on day 21 - 1.7 times. The healing of the full-thickness wound of the skin in the experimental group proceeded more intensively compared with the control, and epithelization was completed 2 days earlier. In addition, stimulation of angiogenesis led to the formation of an organ-specific regenerate practically indistinguishable from intact skin.
Genes & Cells. 2019;14(4):25-28
pages 25-28 views

A comparative analysis of cultivation techniques of epithelial stem cells of cornea and creation of biomedical cell product (biocomposite) on the basis biocompatible matrix and stem cells

Kvacheva Z.B., Vasilevich I.B., Chekina A.Y., Marchenko L.N., Dzhumova M.F., Pinchuk C.V., Fedulov A.S., Volotovski I.D.


In recent years the autologous stem cells of front cornea epithelium (CSC) are increasingly used in the treatment of degenerative diseases of this transparent part of the outer shell of the eye. Limb is considered as a specialized storage for these cells in eye. A number of techniques for isolation of stem cells from limb epithelium and their cultivation in vitro were developed. Goal of the work: To analyze the possibilities of two methods of cultivation of epithelial stem cells front epithelium of cornea (CSC), explants and suspension ones by criteria of efficiency of proliferation, time consumption and constancy of morphology and function parameters of cell cultures. To study of biocompatibility of CSC with commercial gel preparations for its application as an ingredient of biocomposite to use in clinical practice. The primary multilayer CSC cultures were obtained by two methods (explant and suspension) from biopsies of intact parts of cornea limb of 10 patients at the age from 39 to 73 years suffering different types of ceratopathies. Under comparative analysis of CSC cultures their viability, proliferation potential and immunophenotype were investigated. It was shown that explant method is more effective as compared with suspension one as it allows to gain the significant amount of cells with epithelium like morphology. The cell population was presented primarily by epithelium CSC: 95% of cells synthesized K19+ protein. CSC proliferation index of cells isolated by explant method was by 3.5-4 times higher, the time of doubling of population at cultivation in DMEM/F12 medium with 10 ng/ml of EGF was 48 h. Using explant method from one limb biopsy (1 х2 mm) a biomass of CSC in amount of 4 ± 1,2 mln during 3-4 weeks (1-2 passages) was obtained. It was show under investigation for biocompatibility of cells with a number of commercial gel preparations that preparation Provisk is the best on this indicator and this allows to recommend it as a carrier for cells when biomedical cell product are produced.
Genes & Cells. 2019;14(4):29-34
pages 29-34 views

Comparison of the effectiveness of available sources of autologous colony-forming endothelial cells

Matveeva V.G., Antonova L.V., Velikanova E.A., Sardin E.S., Barbarash O.L.


Endothelial Colony-forming cells (ECFCs) are valuable material for tissue vascular engineering and cell therapy of ischemic tissues. Difficult isolation is the main problem for using of ECFCs. ECFCs isolation from peripheral blood and adipose tissue has been previously described. In the presented research we compared effectiveness of peripheral blood, subcutaneous and epicardial adipose tissue for the ECFCs isolation without cell sorting. ECFCs was isolated from peripheral blood, subcutaneous and epicardial adipose tissue obtained from coronary heart disease patients (males, n=8) undergoing elective coronary artery bypass surgery. The stromal-vascular fraction of subcutaneous (SVF-ST) and epicardial (SVF-ET) adipose tissue as well as the mononuclear blood fraction (MNF) were cultivated in the complied EGM-2 medium. Cell cultures phenotyping was performed by flow cytometry and confocal microscopy. Their angiogenic (Matrigel) and proliferative activity (xCELLigence analyzer) in vitro were studied. ECFCs were isolated from MNF in 50% of cases, from SVF-ST in 12.5% and SVF-ET in 37.5%. The proliferative activity of ECFCs isolated from adipose tissue was low while cultures from MNF showed high and medium activity in 75% of cases. Pure ECFCs (more 99%) were obtained from MNF to third passage without cell sorting. Cultures from adipose tissue were contaminated by mesenchymal-stromal cells (MSCs) and contained ECFCs and MSCs. Thus, peripheral blood is the most effective source of autologous ECFCs compared with adipose tissue for this isolation method. However, adipose tissue is a suitable source of MSC and mixed cultures of MSC and endothelial cells.
Genes & Cells. 2019;14(4):35-45
pages 35-45 views

Comparative impact analysis of neuronal and glial progenitors conditioned medium on cerebellar neurons under glutamate exitotoxicity

Salikhova D.I., Leonov G.E., Bukharova T.B., Kornienko Z.V., Bulatenko N.V., Efremova A.S., Makhnach O.V., Makarov A.V., Elchaninov A.V., Fathudinov T.H., Goldshtein D.V.


One of the main causes of cell death in neurodegenerative diseases is excitotoxicity. Today the potential directions of treatment neurodegenerative diseases are including cell therapy, the purpose of which is to replace lost nerve tissue with donor cells. Transplanted cells along with replaced lost tissues have a paracrine effect, which requires careful study. The aim of this work was to study the effect of conditioned media, obtaining from neuronal and glial progenitor cells, on a primary culture of cerebellar neurons in a model of glutamate excitotoxicity. The cell viability, expression of marker genes for apoptosis and neuritogenesis, and the number of necrotic and apoptotic cells were determined in the culture of cerebellar neurons. The composition of the studied conditioned media was analyzed for the content of neurotrophins. A comparative analysis was revealed differences in the secretion of neurotrophins between the obtained cultures: the amount of brain-derived neurotrophic factor, nerve growth factor, ciliary neurotrophic factor and glial neurotrophic factor was higher in the secretion of glial progenitors. It was shown that the addition of conditioned media from neuronal cells does not significantly affect the viability of cerebellar neurons, whereas preincubation with media from glial progenitors has a neuroprotective effect by increasing the viability of cerebellar neurons, and during long-term cultivation promotes the growth of neurites by increasing the expression level of MAP2 and GAP43 genes.
Genes & Cells. 2019;14(4):46-53
pages 46-53 views

The effect of TiO2 nanotubes layered on surfaces of titanium to be used in implantology on the proliferative and secretory activity of fibroblasts

Fadeyev F.A., Khrunyk Y.Y., Belikov S.V., Lugovets D.V., Gubaeva O.V., Leontyev S.L., Sazonov S.V., Popov A.A.


Nowadays titanium and its alloys are the most widely used metallic materials in medicine. In comparison with other metals, titanium has several advantages including biocompatibility, good mechanic properties and corrosion resistance. This research was focused on the studies of proliferative and secretory characteristics of human fibroblasts, cultured on nanotube-layered titanium surfaces as well as the levels of collagen and non-collagenous proteins deposition. Experiments were performed with 2 fibroblast lines isolated from skin samples of 2 donors. Fibroblasts were grown on titanium disks with untreated and anodized surfaces and on the tissue culture treated plastic. Cells were fixed after 3, 5, 7 and 9 days of cultivation. At each time point six samples were analyzed for each surface type. Cell density was estimated by counting cell nuclei, stained with DAPI. IL-6, IL-8/CXCL8 and pro-collagen I concentrations were measured by ELISA, the quantity of collagen and non-collagenic proteins on surfaces was calculated by measuring the level of absorption of Sirius Red and Fast Green dyes, respectively. The results of experiments indicate that the modification of titanium surface with nanotubes does not trigger the formation of fibrous capsule during osseointegration. However, elevated levels of secreted chemokine IL-8/CXCL8, which attracts neutrophils, were observed on anodized samples thus implying possible increased inflammatory response. To get more insights on the role of nanotubes in osseointegration further research is needed.
Genes & Cells. 2019;14(4):54-60
pages 54-60 views

Pathological changes of myocardium in the model of chronic renal insufficiency at application of low-protein diet

Shved N.V., Baykov V.V.


Myocardial damage in patients with chronic renal failure histologically manifests as hypertrophy of cardiomyocytes, intramyocardial artery walls, development of diffuse sclerosis and a change in the number of capillaries. One of the components in the complex treatment of this category of patients is a low-protein diet, however, to date there is no data on the effect of a low-protein diet on structural changes in the myocardium. The aim of the work was to assess the effect of low protein diet on myocardial structural changes in the experimental model of chronic renal failure. To reproduce the experiment of chronic renal failure, a standardized 5/6 nephrectomy model was used in wistar rats. To determine the effect of a low-protein diet on structural changes in the myocardium, two types of diet were used - a standard one and a low-protein diet (Ketosteril). The animals were sacrificed 4 months after nephrectomy. For a comparative assessment of the effect of low-protein diet on structural changes in the myocardium, histological methods of investigation and morphometry were used. The use of low-protein diet was accompanied by a less pronounced violation of biochemical blood parameters (creatinine, urea, calcium, phosphorus), as well as maintaining normal blood pressure, myocardial mass and left ventricular wall thickness. Histologically, this was manifested by a decrease in the severity of dystrophic changes in cardiomyocytes, the degree of their hypertrophy, a decrease in the area of nuclei, and a decrease in the nuclear-cytoplasmic ratio. Indices of perivascular and diffuse sclerosis were lower compared with the control group. Also, when using low-protein diet in animals with a chronic renal failure model, a decrease in the total cross-sectional area of capillaries was noted with an increase in their number in comparison with animals of the control group. Thus, the use of a low-protein diet in an experimental model of chronic renal failure has a cardioprotective effect by reducing the severity of uremia and stabilizing biochemical parameters.
Genes & Cells. 2019;14(4):61-65
pages 61-65 views

CAR-dependent anti-metastatic activity of modified NK cell line YT

Koval O.A., Subrakova V.G., Nushtaeva A.A., Belovezhets T.N., Troitskaya O.A., Ermakov M.S., Varlamov M.E., Chikaev A.N., Kuligina E.V., Kulemzin S.V., Gorchakov A.A., Taranin A.V., Richter V.A.


Adoptive T- and NK-cells transfer with chimeric antigenic receptors (CAR) is considered as a promising anticancer strategy. Chimeric antigenic receptors are artificial molecules that provide activation of the cells carrying them when they contact with a specific antigen to induce cell death. Unlike T cells, NK cells have no T-cell receptors, which prevent "graft-versus-host” disease under allograft transplantation. The aim of this work was to study antimetastatic activity of a double-modified human NK cell line YT - Cyto-CAR-YT-Lact cells carrying CAR to the PSMA protein and expressing antiapoptotic protein lactaptin. The BrCCh4e-134 breast carcinoma line with high PSMA expression was constructed by lentiviral transduction. The YT double modified NK cell line, Cyto-CAR-YT-Lact, expressing functional anti-PSMA CAR and carrying a deletion of the Shp-2 gene encoding the Shp-2 protein, a negative regulator of NK cell activation, was used as effector cells. The cytotoxic activity of Cyto-CAR-YT-Lact cells was tested on PSMA-positive BrCCh4e-134 cells and on parental BrCCh4e cell in vitro in real-time mode. The antimetastasis activity of Cyto-CAR-YT-Lact cells was analyzed in SCID and NOD/SCID mice with spontaneously metastases and intravenously transplanted PSMA-positive BrCCh4e-134 cells. Cyto-CAR-YT-Lact cells efficiently reduced the viability of PSMA-positive tumor cells and moderately PSMA-negative target cells in vitro. As a tumor model, we used human breast cancer cells that overexpress PSMA as a transgene and are characterized by metastasis to the mediastinal lymph nodes being transplanted on mice. It was shown that a single intravenous administration of the Cyto-CAR-YT-Lact cells suppressed the development of metastases in the spontaneous metastasis model and when tumor cells were introduced into the bloodstream. The reaction of the primary tumor node to Cyto-CAR-YT-Lact therapy was not detected. Thus, the use of modified NK cells can be considered as a promising therapeutic approach for suppressing metastasis, but not for suppressing the growth of the primary tumor node.
Genes & Cells. 2019;14(4):66-71
pages 66-71 views

The effect of neoadjuvant chemotherapy on the level of bone marrow progenitor cells in the blood of patients with invasive breast carcinoma

Kaigorodova E.V., Perelmuter V.M., Orehov A.S., Fedulova N.V., Tarabanovskaya N.A., Simolina E.I., Savelieva O.E., Tashireva L.A., Cherdyntseva N.V.


The breast cancer occupies the first place in the structure of women cancer morbidity and mortality for many years. The treatment of this pathology includes two types of chemotherapy: neoadjuvant and adjuvant. Neoadjuvant chemotherapy (NACT) follows surgical treatment and makes it possible to assess the sensitivity of the tumor to the medication. The obtained data can be used to correct the subsequent adjuvant chemotherapy. However, there is a lot of evidence of the ability of NACT to increase the risk of progression of malignant tumors. The bone marrow progenitor cells are components of premetastatic niches. Objective: to assess the effect of neoadjuvant chemotherapy on the level of bone marrow progenitor cells in the blood of patients with breast cancer. In a prospective study were included 31 patients newly diagnosed with invasive breast cancer, of which 17 patients was performed neoadjuvant chemotherapy, 14 patients - without neoadjuvant chemotherapy. The method of multicolor flow cytometry was used to assess the dynamics of bone marrow progenitor cells (hematopoietic stem cells (HSC) and hematopoietic progenitor cells (HPC), endothelial progenitor cells (EPC), mesenchymal stem cells (MSC) in blood of patients with invasive carcinoma of a non-specific type during NACT. It has been shown that neoadjuvant chemotherapy leads to a statistically significant increase the number of endothelial cell precursor cells (EPC) in the blood of patients with invasive breast carcinoma (p = 0.036). The level of mesenchymal stem cells in the blood of patients with invasive breast carcinoma increases at the level of a statistical trend (p = 0.06) during neoadjuvant chemotherapy. Based on these data we can conclude that neoadjuvant chemotherapy enhances the recruitment of bone marrow cells involved in the formation of premetastatic niches.
Genes & Cells. 2019;14(4):72-76
pages 72-76 views

Clinical case of mandibular fibro-osseous lesions with giant multinuclear cells: molecular-genetic differential diagnostic analysis

Sviridov E.G., Deev R.V., Redko N.A., Drobyshev A.Y., Kadykova A.I.


The giant cell tumors of maxillofacial bones include several barely differentiable diseases with diverse prognoses. A case of a giant cell tumor in an 18-year-old man is described. Clinical, instrumental and laboratory examination did not allow to establish an accurate diagnosis. Molecular genetic test by New generation sequencing (NGS) of tumor tissues and blood identified mutation p. 1424 C> A; p.Pro475His in the SH3BP2 gene, previously described in patients with the so-called cherubism, that determined the tactic of treatment. Carrying out an oncogenetic study allows the differential diagnosis of such morphologically similar conditions with a giant cell component as a multiorgan form of fibrous dysplasia, reparative granulomas, Noonan syndrome, Sturge-Weber-Crabbe syndrome, juvenile ossifying fibroma.
Genes & Cells. 2019;14(4):77-81
pages 77-81 views

Legislative regulation and use of genetic information in the Russian Federation and abroad

Vulf M.A., Yurova K.A., Skuratovskaia D.A., Litvinova L.S.


In modern medicine, personalized approaches to the diagnosis, prevention and treatment of diseases are becoming increasingly important. The accumulated information on genetic mutations and their mapping led to the development of prognostic genetic tests. Created programs for clustering and analysis of the information received have served the development of pharmacogenetics and gene therapy. This has increased the effectiveness and safety of therapeutic approaches and increased average life expectancy. At the same time, the Russian Federation (RF) and the world community are actively discussing the protection of human rights, the inviolability of the person, storage, use of his personal data, including genetic information from unauthorized access. The purpose of this review was to generalize and detail information on legislative norms and legal regulation of the use of genetic information in the territory of the Russian Federation and in other countries of the world.
Genes & Cells. 2019;14(4):82-87
pages 82-87 views

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Genes & Cells. 2019;14(4):88-90
pages 88-90 views

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