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The low knock-in efficiency, especially in primary human cells, limits the use of the genome editing technology for therapeutic purposes, rendering it important to develop approaches for increasing the knock-in levels. In this work, the efficiencies of several approaches were studied using a model of knock-in of a construct coding for the peptide HIV fusion inhibitor MT-C34 into the human CXCR4 locus in the CEM/R5 T cell line. First, donor DNA modification was evaluated as a means to improve the efficiency of plasmid transport into the nucleus. The donor plasmid was modified to include the simian virus 40 (SV40) DNA nuclear targeting sequence (DTS) or binding sites for the transcription factor NF-κB, whose effects on the knock-in levels have not been described. The modification was ineffective in the model of MT-C34 knock-in into the CXCR4 locus. A second approach consisted in modification of Cas9 nuclease by introducing two additional nuclear localization signals (NLSs) and increased the knock-in level by 30%. Finally, blocking DNA repair via the nonhomologous end joining (NHEJ) pathway with DNA-dependent protein kinase inhibitors caused a 1.8-fold increase in knock-in. A combination of the last two approaches caused an additive effect. Thus, increasing the number of NLSs in the Cas9 protein and inhibiting DNA repair via the NHEJ pathway significantly increased the level of knock-in of the HIV-1 fusion inhibitory peptide into the clinically relevant locus CXCR4. The finding can be used to develop effective gene therapy approaches for treating HIV infection. [ABSTRACT FROM AUTHOR]

To successfully apply the genome editing technology using the CRISPR/Cas9 system in the clinic, it is necessary to achieve a high efficiency of knock-in, which is insertion of a genetic construct into a given locus of the target cell genome. One of the approaches to increase the efficiency of knock-in is to modify donor DNA with the same Cas9 targeting sites (CTS) that are used to induce double-strand breaks (DSBs) in the cell genome (the double-cut donor method). Another approach is based on introducing truncated CTS (tCTS), including a PAM site and 16 proximal nucleotides, into the donor DNA. Presumably, tCTS sites do not induce cleavage of the donor plasmid, but can support its transport into the nucleus by Cas9. However, the exact mechanisms whereby these two donor DNA modifications increase the knock-in level are unknown. In this study, the modifications were tested for effect on the knock-in efficiency of the MTC34 genetic construct encoding the HIV-1 fusion inhibitory peptide MT-C34 into the CXCR4 locus of the CEM/R5 T-cell line. When full-length CTSs were introduced into the donor plasmid DNA, the knock-in level was doubled regardless of the CTS number or position relative to the donor sequence. Modifications with tCTSs did not affect the knock-in levels. In vitro, both CTS and tCTS were efficiently cleaved by Cas9. To understand the mechanism of action of these modifications in detail, it is necessary to evaluate their cleavage both in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Delivery of ribonucleoprotein complexes of Cas9 nuclease and guide RNA into target cells with virus-like particles (VLP) is one of the novel methods of genome editing and is suitable for gene therapy of human diseases in the future. The efficiency of genome editing with VLPs depends on the Cas9 packaging into VLPs, the process mediated by the viral Gag protein. To improve the packaging of Cas9 into NanoMEDIC VLPs, plasmid constructs for Cas9 and Gag expression were modified by adding the HIV Rev response element (RRE), which was expected to increase the nuclear export of RRE-containing transcripts into the cytosol via the Rev accessory protein, as described for a Vpr-Cas9-based VLP system. The Cas9 and Gag protein levels in cell lysates were found to increase upon cotransfection with either the Rev-expressing plasmid or the empty control plasmid. The effect was independent of the presence of RRE in the transcript. Moreover, AP21967-induced dimerization of FRB and FKBP12, but not plasmid modification with RRE and/or cotransfection with the Rev-expressing plasmid, was shown to play the major role in Cas9 packaging into NanoMEDIC VLPs. The data indicated that it is impractical to use the RRE–Rev module to enhance the packaging of Cas9 nuclease into VLPs. [ABSTRACT FROM AUTHOR]

Gene editing using the CRISPR/Cas9 system provides new opportunities to treat human diseases. Approaches aimed at increasing the efficiency of genome editing are therefore important to develop. To increase the level of editing of the CXCR4 locus, which is a target for gene therapy of HIV infection, the Cas9 protein was modified by introducing additional NLS signals and ribonucleoprotein complexes of Cas9 and guide RNA were stabilized with poly-L-glutamic acid. The approach allowed a 1.8-fold increase in the level of CXCR4 knockout in the CEM/R5 T cell line and a 2-fold increase in the level of knock-in of the HIV-1 fusion peptide inhibitor MT-C34 in primary CD4+ T lymphocytes. [ABSTRACT FROM AUTHOR]

Tn antigen is a tumor-associated antigen that appears on cancer cells as a result of aberrant O-glycosylation. The most studied form of Tn antigen is found in mucins, in particular, in mucin 1 (MUC1). Antibodies against this form of Tn antigen are used to diagnose tumors, as well as to generate T-killers with a chimeric receptor. Some carcinomas do not carry MUC1 and antibodies of a different specificity are required to detect Tn antigen on these cells. In our work, we searched for anti-Tn antibodies without preliminary assumptions about the proteins that may be carriers of the Tn antigen. For this purpose, we obtained several pairs of isogenic cell lines with the wild type and knockout of the Cosmc gene, which is essential for correct protein O-glycosylation. Using the created lines as immunogens, we generated a monoclonal antibody AKC3, which reacted with the Cosmc-deficient A549 lung adenocarcinoma cells and did not bind to the wild-type cells. Using mass spectrometry, as well as co-immunoprecipitation, it was shown that the AKC3 antibody recognized the Tn antigen in the context of CD44 protein – a protein important for tumor growth. The AKC3 antibody can be used for tumor diagnosis, and to generate T cells with a chimeric receptor for treatment of tumors that do not express mucins. [ABSTRACT FROM AUTHOR]

Currently, more than 37 million individuals worldwide are infected with the human immunodeficiency virus (HIV). Antiretroviral therapy may control the viral infection but is incapable of eradicating it. It is important to understand how cells respond to HIV-1 infection and what cellular factors are involved in this process to develop novel classes of antiviral drugs. This review summarizes the current understanding of the HIV restriction mechanism. We discuss the ambiguous role of HIV restriction factors in viral infection and counteraction mediated by HIV-1 accessory proteins. [ABSTRACT FROM AUTHOR]

Lymphocyte phosphatase-associated phosphoprotein (LPAP) is a transmembrane protein with unknown function. The available data on its close association with phosphatase CD45 and its phosphorylation depending on cell activation suggest that LPAP can play a significant role in the antigenic stimulation of lymphocytes. We have localized three antigenic epitopes of the LPAP molecule that can be detected using monoclonal antibodies prepared earlier. Experiments on reactions of antibodies with point mutants and shortened forms of the LPAP protein revealed regions of the amino acid sequence that correspond to the epitopes recognized by the antibodies. [ABSTRACT FROM AUTHOR]

This study was undertaken to compare the concentrations of pro- and anti-angiogenic growth factors, nitric oxide (NO) stable metabolites in maternal serum and embryonic left ventricular (LV) isovolumic relaxation time (IRT, ms) during the first trimester in two groups of women: with pregnancy conceived by assisted reproductive technologies (ART,n = 39) and normally conceived (control group,n = 68) pregnancy. The concentration of vasoconstrictor endothelin 1 was 45.5 times more in ART than in control group. On the contrary, the concentrations of NO stable metabolites in ART were 1.9 times less than in control women. The assessment of angiogenic suppressors in ART women demonstrates the decrease in s-endoglin concentration was 1.6 times and in soluble receptor to vascular endothelial growth factor concentration was 2.0 times in comparison with control group. There was a significant increase in LV IRT in ART embryos in comparison to control ones. These data suggest significant changes in pro- anti-angiogenic factors balance and increase in vascular impedance in ART-conceived embryos. [ABSTRACT FROM PUBLISHER]

The experiments show that it is possible in principle to use new methods of calcining clays into grogs in equipment of the vortex type. In spite of the very short firing time, the clay substance undergoes extensive mullite formation, although the process of sintering cannot be considered as fully completed. The grog obtained can be used for making foamed lightweight fireclay refractories. This grog will find use for making heat-resistant concretes and mortars and also for refractory bricks. The experimental work on the calcination of refractory clays in a vortex furnace should be continued to explain the technical and economic effectiveness of firing and to produce coarse grogs and also data for planning an experimental-industrial plant. [ABSTRACT FROM AUTHOR]