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Rapid progress in gene editing based on clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) has revolutionized functional genomic studies and genetic disease correction. While numerous gene editing applications have been easily adapted by experimental science, the clinical utility of CRISPR/Cas remains very limited due to difficulty in delivery to primary cells and possible off-target effects. The use of CRISPR in the form of a ribonucleoprotein (RNP) complex substantially reduces the time of DNA exposure to the effector nuclease and minimizes its off-target activity. The traditional electroporation and lipofection methods lack the cell-type specificity of RNP delivery, can be toxic for cells, and are less efficient when compared to nanoparticle transporters. This review focuses on CRISPR/Cas RNP packaging and delivery using retro/lentiviral particles and exosomes. First, we briefly describe the natural stages of viral and exosomal particle formation, release and entry into the target cells. This helps us understand the mechanisms of CRISPR/Cas RNP packaging and uncoating utilized by the current delivery systems, which we discuss afterward. Much attention is given to the exosomes released during viral particle production that can be passively loaded with RNPs as well as the mechanisms necessary for particle fusion, RNP release, and transportation inside the target cells. Collectively, together with specific packaging mechanisms, all these factors can substantially influence the editing efficiency of the system. Finally, we discuss ways to improve CRISPR/Cas RNP delivery using extracellular nanoparticles.

Lymphocyte phosphatase-associated phosphoprotein (LPAP) is a transmembrane protein tightly associated with the phosphatase CD45, which regulates antigen specific lymphocyte activation. Although LPAP is positioned in close proximity to key signaling molecules, its function remains unknown. In this study, we investigated signaling pathways involved in LPAP phosphorylation. Using phosphospecific antibodies generated in our laboratory, we analyzed changes in LPAP phosphorylation in response to various stimuli. Cross-linking with antibodies against TCR or BCR, as well as ionophores and Thapsigargin, resulted in rapid dephosphorylation at Ser172 and Ser99 followed by phosphorylation at Ser163. A panel of inhibitors allowed us to show that PMA and TCR cross-linkage engages the MEK-ERK pathway to drive phosphorylation of LPAP at Ser163. The ERK1/2 kinase was the most distal element in the cascade, which when inhibited prevented changes in LPAP phosphorylation. Supporting this, we found that ERK1 is capable of phosphorylating LPAP at Ser163 in vitro. Although the functional role of this event is yet to be revealed, we provide evidence for a new ERK1/2 target in lymphocytes, namely LPAP, representing a potential regulatory mechanism in the signaling cascade.

The COVID-19 pandemic caused by SARS-CoV-2 has posed a global threat to human lives and economics. One of the best ways to determine protection against the infection is to quantify the neutralizing activity of serum antibodies. Multiple assays have been developed to validate SARS-CoV-2 neutralization, most of them utilized lentiviral or vesicular stomatitis virus-based particles pseudotyped with the spike (S) protein, making them safe and acceptable to work with in many labs. However, these systems are only capable of measuring infection with purified particles. This study has developed a pseudoviral assay with replication-dependent reporter vectors that can accurately quantify the level of infection directly from the virus producing cell to the permissive target cell. Comparative analysis of cell-free and cell-to-cell infection revealed that the neutralizing activity of convalescent sera was more than tenfold lower in cell cocultures than in the cell-free mode of infection. As the pseudoviral system could not properly model the mechanisms of SARS-CoV-2 transmission, similar experiments were performed with replication-competent coronavirus, which detected nearly complete SARS-CoV-2 cell-to-cell infection resistance to neutralization by convalescent sera. These findings suggest that the cell-to-cell mode of SARS-CoV-2 transmission, for which the mechanisms are largely unknown, could be of great importance for treatment and prevention of COVID-19.

The COVID-19 pandemic caused by SARS-CoV-2 has posed a global threat to human lives and economics. One of the best ways to determine protection against the infection is to quantify the neutralizing activity of serum antibodies. Multiple assays have been developed to validate SARS-CoV-2 neutralization; most of them utilized lentiviral or vesicular stomatitis virus-based particles pseudotyped with the spike (S) protein, making them safe and acceptable to work with in many labs. However, these systems are only capable of measuring infection with purified particles. This study has developed a pseudoviral assay with replication-dependent reporter vectors that can accurately quantify the level of infection directly from the virus producing cell to the permissive target cell. Comparative analysis of cell-free and cell-to-cell infection revealed that the neutralizing activity of convalescent sera was more than tenfold lower in cell cocultures than in the cell-free mode of infection. As the pseudoviral system could not properly model the mechanisms of SARS-CoV-2 transmission, similar experiments were performed with replication-competent coronavirus, which detected nearly complete SARS-CoV-2 cell-to-cell infection resistance to neutralization by convalescent sera. Based on available studies, this is the first attempt to quantitatively measure SARS-CoV-2 cell-to-cell infection, for which the mechanisms are largely unknown. The findings suggest that this route of SARS-CoV-2 transmission could be of great importance for treatment and prevention of COVID-19.ImportanceImmune surveillance of viral or bacterial infections is largely mediated by neutralizing antibodies. Antibodies against the SARS-CoV-2 spike protein are produced after vaccination or infection, but their titers only partly reflect the degree of protection against infection. To identify protective antibodies, a neutralization test with replicating viruses or pseudoviruses (PVs) is required. This study developed lentiviral-based PV neutralization assays that, unlike similar systems reported earlier, enable quantitative measurement of SARS-CoV-2 neutralization in cell cocultures. Using both PVs and replication-competent virus, it was demonstrated that SARS-CoV-2 cell-to-cell infection is considerably more resistant to serum neutralization than infection with purified viral particles. The tests are easy to set up in many labs, and are believed to be more informative for monitoring SARS-CoV-2 collective immunity or entry inhibitor screening.

Subject. Currently, about 530 types of microorganisms that live in supragingival and subgingival plaque have been discovered. To periodontal pathogens today carry a little more than ten types. The main role in the occurrence of periodontal disease is played by microorganisms included in the “red complex” of BANA+: Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia. With the simultaneous identification of these types can be judged on the high risk of developing periodontal disease. Objective ― calculating the frequency of occurrence of various types of dentition defects of patients with varying severity of periodontal disease, establishing the connection between the severity of periodontal disease and the number of periodontal pathogenic microflora. Methodology. A clinical examination of a patients group which consists of 32 women and 10 men in the age from 38 to 66 years was conducted. Anamnesis was collected using a survey and a special questionnaire. External examination and examination of the oral cavity allowed to find the clinical conditions and anatomical features that leads to periodontal disease’s development. Results. Based on the data of clinical and radiological examination, the patients were divided into three groups according to the severity of periodontal disease. During the clinical examination, we calculated the frequency of occurrence of various types of dentition defects in patients with varying degrees of severity of periodontal disease. Findings. It has been found that the course of chronic generalized periodontitis depends on the number of types of aggressive forms of periodontal pathogens.

Lymphocyte phosphatase-associated phosphoprotein (LPAP) is a small transmembrane protein that is found in lymphocytes and is tightly associated with the phosphatase CD45. The function of LPAP is still unknown. Studies of the LPAP interactome may reveal new details of how C45 and lymphocyte signaling in general are regulated. LPAP binding partners were sought using coimmunoprecipitation coupled with mass spectrometry, stabilization of protein complexes with chemical crosslinkers, and Blue Native electrophoresis. In addition to CD45, several proteins were identified as LPAP partners, including CD71, CD98, cytoskeletal proteins, the amino acid transporter SLC1A4, and the cell signaling component HS1. It was confirmed that more than 70% of LPAP molecules were bound with CD45 in a 1 : 1 complex. The effect of CD45 on LPAP was studied in CEM and Jurkat cells with a CD45 knockout. The LPAP levels in the cells were 10% of the level in wild-type cells. In the absence of CD45, LPAP phosphorylation at Ser-153 and Ser-163 was not affected, whereas phosphorylation at Ser-99 and Ser-172 decreased significantly. Based on the results, CD45 was assumed to play a role in regulating LPAP expression and phosphorylation status.

Lymphocyte phosphatase-associated phosphoprotein (LPAP) is a molecular partner of CD45 phosphatase that plays a key role in the regulation of antigen-specific activation of lymphocytes. The functions of LPAP still remain unknown. We believe that studying LPAP phosphorylation pathways could shed light on its functions. In this work, we studied the phosphorylation of LPAP ectopically expressed in non-lymphoid cells in order to determine the effect of LPAP interaction partners on its phosphorylation. We found that phosphorylation at Ser153 and Ser163 in non-hematopoietic HEK293 cells was conserved, while phosphorylation at Ser99 and Ser172 was almost absent. The pattern of LPAP phosphorylation in K562 erythroid and U937 myeloid cells expressing endogenous CD45 protein was similar to that observed in T and B lymphocytes. We demonstrated for the first time that LPAP is a substrate for protein kinase CK2 that phosphorylates it at Ser153, presumably ensuring LPAP resistance to degradation.

Previous studies suggest that short peptides from the heptad repeat 2 (HR2) domain of gp41 expressed on the cell surface are more potent inhibitors of HIV-1 entry than soluble analogs. However, their therapeutic potential has only been examined using lentiviral vectors. Here, we aimed to develop CRISPR/Cas9-based fusion inhibitory peptide knock-in (KI) technology for the generation and selection of HIV-1-resistant T cells. First, we cloned a series of HIV-1 fusion inhibitory peptides and embedded them in CD52, the shortest GPI-anchored protein, which efficiently delivers epitope tags to the cell surface and maintains a sufficient level of KI. Among the seven peptides tested, MT-C34, HP-23L, and 2P23 exhibited significant activity against both cell-free and cell-to-cell HIV-1 infection. Unlike membrane-bound peptides, the shed variant of MT-C34 provided insufficient protection against HIV-1 due to its low concentrations in the culture medium. Using Cas9 plasmids or ribonucleoprotein electroporation and cell sorting with antibodies raised against gp41 peptides, we generated CEM/R5 cells with biallelic KI of MT-C34 (embedded in CD52 for expression in lipid rafts) and 2P23 (N-terminally fused to CXCR4). In combination, these peptides provided a higher level of protection than individual KI. By extending homology arms and substituting PCR donor DNA with a plasmid containing signals for nuclear localization, we achieved KI of MT-C34 into CXCR4 loci and HIV-1 proviral DNA at levels of up to 35% in CEM/R5 cells and increased KI occurrence from undetectable to 4-5% in CD4 lymphocytes. Comparative analysis of lentiviral and HDR-based delivery strategies showed that KI led to the higher MT-C34 expression and stronger protection of primary CD4 lymphocytes from HIV-1 than lentiviral transduction, albeit the efficiency of KI needs further improvements in order to meet clinical requirements. Thus, the developed CRISPR/Cas9 platform offers a new opportunity for antiviral peptide delivery with a concomitant precise genetic modification of targeted locus that can be employed to strengthen cell protection against HIV.AUTHOR SUMMARYHIV is a human lentivirus that infects CD4-positive immune cells and, when left untreated, manifests in the fatal disease known as acquired immunodeficiency syndrome. Antiretroviral therapy (ART) is not leading to viral clearance, and HIV persists in the organism as a latent provirus. One way to control infection is to increase the population of HIV-resistant CD4 lymphocytes via entry molecule knockout or expression of different antiviral genes. Peptides from the heptad repeat (HR) domain of gp41 are potent inhibitors of HIV-1 fusion, especially when designed to express on the cell surface. Individual gp41 peptides encoded by therapeutic lentiviral vectors have been evaluated and some have entered clinical trials. However, a CRISPR/Cas9-based gp41 peptide delivery platform that operates through concomitant target gene modification has not yet been developed due to low knock-in (KI) rates in primary cells. Here, we systematically evaluated the antiviral activity of different HR2-peptides cloned into the shortest carrier molecule, CD52. The resulting small-size transgene constructs encoding selected peptides, in combination with improvements to enhance donor vector nuclear import, helped to overcome precise editing restrictions in CD4 lymphocytes. Using KI into CXCR4, we demonstrated different options for target gene modification, effectively protecting edited cells against HIV-1.

We studied argyrophilic proteins associated with nucleolar organizer regions (AgNOR) in non-small-cell cancer. We determined the area index (AI) and coefficient of variation (CV) of AgNOR. AI is associated with the key clinicomorphological parameters within the TNM system: T and N values, greatest tumor dimension up to 3 cm and more, disease stage, histogenesis, and tumor differentiation. CV is associated with T value, greatest tumor dimension up to 3 cm and more, histogenesis, and tumor differentiation. Survival of patients is longer in low AI or CV values versus high AI or CV values, longer in low AI and CV values (−AI/−CV type), shorter in high AI and CV values (+AI/+CV type), and intermediate in opposite AI and CV values (−AI/+CV and +AI/−CV types). Independent predictors in non-small-cell lung cancer include N value, greatest tumor dimension, histogenesis, and CV. Assessment of quantitative values and heterogeneity of AgNOR is important for differential diagnosis and prognosis of non-small-cell lung cancer.

Phosphatase CD45 regulates the activation of lymphocytes by controlling the level of receptor and signal molecule phosphorylation. However, it remains unknown which molecules mediate the phosphatase activity of CD45. A candidate for such a molecule is a small transmembrane adapter protein called lymphocyte phosphatase-associated phosphoprotein (LPAP). LPAP forms a supramolecular complex that consists of not only CD45 molecule but also CD4 and Lck kinase. The function of LPAP has not been defined clearly. In our study, we determined the pattern of LPAP expression in various cell types and characterized its proteoforms using new monoclonal antibodies generated against the intracellular portion of the protein. We show that LPAP is a pan-lymphocyte marker, and its expression in cells correlates with the expression of CD45. The majority of T, B and NK cells express high levels of LPAP, whereas monocytes, granulocytes, monocyte-derived dendritic cells, platelets and red blood cells are negative for LPAP. Using one- and two-dimensional protein gel electrophoresis, we demonstrate that LPAP has at least four sites of phosphorylation. The resting cells express at least six different LPAP phosphoforms representing mono-, di- and tri-phosphorylated LPAP. T and B cells differ in the distribution of the protein between phosphoforms. The activation of lymphocytes with PMA reduces the diversity of phosphorylated forms. Our experiments on Lck-deficient Jurkat cells show that Lck kinase is not involved in LPAP phosphorylation. Thus, LPAP is a dynamically phosphorylated protein, the function of which can be understood, when all phosphosites and kinases involved in its phosphorylation will be identified.