Key Proteins of Replication Stress Response and Cell Cycle Control as Cancer Therapy Targets.

Replication stress (RS) is a characteristic state of cancer cells as they tend to exchange precision of replication for fast proliferation and increased genomic instability. To overcome the consequences of improper replication control, malignant cells frequently inactivate parts of their DNA damage response (DDR) pathways (the ATM-CHK2-p53 pathway), while relying on other pathways which help to maintain replication fork stability (ATR-CHK1). This creates a dependency on the remaining DDR pathways, vulnerability to further destabilization of replication and synthetic lethality of DDR inhibitors with common oncogenic alterations such as mutations of TP53, RB1, ATM, amplifications of MYC, CCNE1 and others. The response to RS is normally limited by coordination of cell cycle, transcription and replication. Inhibition of WEE1 and PKMYT1 kinases, which prevent unscheduled mitosis entry, leads to fragility of under-replicated sites. Recent evidence also shows that inhibition of Cyclin-dependent kinases (CDKs), such as CDK4/6, CDK2, CDK8/19 and CDK12/13 can contribute to RS through disruption of DNA repair and replication control. Here, we review the main causes of RS in cancers as well as main therapeutic targets-ATR, CHK1, PARP and their inhibitors.

Genetically modified mice as a tool for the study of human diseases.

Modeling a human disease is an essential part of biomedical research. The recent advances in the field of molecular genetics made it possible to obtain genetically modified animals for the study of various diseases. Not only monogenic disorders but also chromosomal and multifactorial disorders can be mimicked in lab animals due to genetic modification. Even human infectious diseases can be studied in genetically modified animals. An animal model of a disease enables the tracking of its pathogenesis and, more importantly, to test new therapies. In the first part of this paper, we review the most common DNA modification technologies and provide key ideas on specific technology choices according to the task at hand. In the second part, we focus on the application of genetically modified mice in studying human diseases.

Anti-Cancer Potential of Transiently Transfected HER2-Specific Human Mixed CAR-T and NK Cell Populations in Experimental Models: Initial Studies on Fucosylated Chondroitin Sulfate Usage for Safer Treatment.

Human epidermal growth factor receptor 2 (HER2) is overexpressed in numerous cancer cell types. Therapeutic antibodies and chimeric antigen receptors (CARs) against HER2 were developed to treat human tumors. The major limitation of anti-HER2 CAR-T lymphocyte therapy is attributable to the low HER2 expression in a wide range of normal tissues. Thus, side effects are caused by CAR lymphocyte "on-target off-tumor" reactions. We aimed to develop safer HER2-targeting CAR-based therapy. CAR constructs against HER2 tumor-associated antigen (TAA) for transient expression were delivered into target T and natural killer (NK) cells by an effective and safe non-viral transfection method via nucleofection, excluding the risk of mutations associated with viral transduction. Different in vitro end-point and real-time assays of the CAR lymphocyte antitumor cytotoxicity and in vivo human HER2-positive tumor xenograft mice model proved potent cytotoxic activity of the generated CAR-T-NK cells. Our data suggest transient expression of anti-HER2 CARs in plasmid vectors by human lymphocytes as a safer treatment for HER2-positive human cancers. We also conducted preliminary investigations to elucidate if fucosylated chondroitin sulfate may be used as a possible agent to decrease excessive cytokine production without negative impact on the CAR lymphocyte antitumor effect.

Genetically Engineered Mice Unveil In Vivo Roles of the Mediator Complex.

The Mediator complex is a multi-subunit protein complex which plays a significant role in the regulation of eukaryotic gene transcription. It provides a platform for the interaction of transcriptional factors and RNA polymerase II, thus coupling external and internal stimuli with transcriptional programs. Molecular mechanisms underlying Mediator functioning are intensively studied, although most often using simple models such as tumor cell lines and yeast. Transgenic mouse models are required to study the role of Mediator components in physiological processes, disease, and development. As constitutive knockouts of most of the Mediator protein coding genes are embryonically lethal, conditional knockouts and corresponding activator strains are needed for these studies. Recently, they have become more easily available with the development of modern genetic engineering techniques. Here, we review existing mouse models for studying the Mediator, and data obtained in corresponding experiments.

Physiological and Functional Effects of Dominant Active TCRα Expression in Transgenic Mice.

A T cell receptor (TCR) consists of α- and ß-chains. Accumulating evidence suggests that some TCRs possess chain centricity, i.e., either of the hemi-chains can dominate in antigen recognition and dictate the TCR's specificity. The introduction of TCRα/ß into naive lymphocytes generates antigen-specific T cells that are ready to perform their functions. Transgenesis of the dominant active TCRα creates transgenic animals with improved anti-tumor immune control, and adoptive immunotherapy with TCRα-transduced T cells provides resistance to infections. However, the potential detrimental effects of the dominant hemi-chain TCR's expression in transgenic animals have not been well investigated. Here, we analyzed, in detail, the functional status of the immune system of recently generated 1D1a transgenic mice expressing the dominant active TCRα specific to the H2-Kb molecule. In their age dynamics, neither autoimmunity due to the random pairing of transgenic TCRα with endogenous TCRß variants nor significant disturbances in systemic homeostasis were detected in these mice. Although the specific immune response was considerably enhanced in 1D1a mice, responses to third-party alloantigens were not compromised, indicating that the expression of dominant active TCRα did not limit immune reactivity in transgenic mice. Our data suggest that TCRα transgene expression could delay thymic involution and maintain TCRß repertoire diversity in old transgenic mice. The detected changes in the systemic homeostasis in 1D1a transgenic mice, which are minor and primarily transient, may indicate variations in the ontogeny of wild-type and transgenic mouse lines.

Limitations of Tamoxifen Application for In Vivo Genome Editing Using Cre/ERT2 System.

Inducible Cre-dependent systems are frequently used to produce both conditional knockouts and transgenic mice with regulated expression of the gene of interest. Induction can be achieved by doxycycline-dependent transcription of the wild type gene or OH-tamoxifen-dependent nuclear translocation of the chimeric Cre/ERT2 protein. However, both of these activation strategies have some limitations. We analyzed the efficiency of knockout in different tissues and found out that it correlates with the concentration of the hydroxytamoxifen and endoxifen-the active metabolites of tamoxifen-measured by LC-MS in these tissues. We also describe two cases of Cdk8floxed/floxed/Rosa-Cre-ERT2 mice tamoxifen-induced knockout limitations. In the first case, the standard scheme of tamoxifen administration does not lead to complete knockout formation in the brain or in the uterus. Tamoxifen metabolite measurements in multiple tissues were performed and it has been shown that low recombinase activity in the brain is due to the low levels of tamoxifen active metabolites. Increase of tamoxifen dosage (1.5 fold) and duration of activation (from 5 to 7 days) allowed us to significantly improve the knockout rate in the brain, but not in the uterus. In the second case, knockout induction during embryonic development was impossible due to the negative effect of tamoxifen on gestation. Although DNA editing in the embryos was achieved in some cases, the treatment led to different complications of the pregnancy in wild-type female mice. We propose to use doxycycline-induced Cre systems in such models.

AAV infection of bovine embryos: Novel, simple and effective tool for genome editing.

Adeno-associated viruses (AAV) are widely used in the field of genetically modified organism production. In this work, transduction of bovine embryos by AAV was selected as a potential approach to perform genetic modifications: we have used recombinant AAV to produce GFP-positive bovine embryos. Five different AAV serotypes were used to evaluate their ability to deliver genetic material into the bovine embryos. AAV9 serotype demonstrated minimal effectiveness (38,10%) as the genetic material transfer tool. Four other serotypes of AAVs (AAV1, AAV2, AAV6 and AAV-DJ) showed very close transduction efficiency (52,94-58,33%). CD209 is a C-type lectin receptor which is presented on the surface of macrophages and dendritic cells. CD209 recognizes a broad range of pathogens in a rather nonspecific manner. Production of CD209 knock-out is relevant for better understanding of infection mechanisms. Potentially, production of such knock-out may enable animals to become resistant to various infections. We have analyzed DNA samples from 22 blastocysts obtained after in vitro culture of zygotes subjected to recombinant AAV action. We have detected that 3 of 22 analyzed blastocysts contained mosaic CD209 frameshifts. Therefore, we have demonstrated proof of principle that application of AAV as a genome editing tool is an effective method for obtaining genetically modified cattle embryos.

Gene Transcription as a Therapeutic Target in Leukemia.

Blood malignancies often arise from undifferentiated hematopoietic stem cells or partially differentiated stem-like cells. A tight balance of multipotency and differentiation, cell division, and quiescence underlying normal hematopoiesis requires a special program governed by the transcriptional machinery. Acquisition of drug resistance by tumor cells also involves reprogramming of their transcriptional landscape. Limiting tumor cell plasticity by disabling reprogramming of the gene transcription is a promising strategy for improvement of treatment outcomes. Herein, we review the molecular mechanisms of action of transcription-targeted drugs in hematological malignancies (largely in leukemia) with particular respect to the results of clinical trials.

Novel transgenic mice with Cre-dependent co-expression of GFP and human ACE2: a safe tool for study of COVID-19 pathogenesis.

The current coronavirus disease (COVID-19) pandemic remains one of the most serious public health problems. Increasing evidence shows that infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes a very complex and multifaceted disease that requires detailed study. Nevertheless, experimental research on COVID-19 remains challenging due to the lack of appropriate animal models. Herein, we report novel humanized mice with Cre-dependent expression of hACE2, the main entry receptor of SARS-CoV-2. These mice carry hACE2 and GFP transgenes floxed by the STOP cassette, allowing them to be used as breeders for the creation of animals with tissue-specific coexpression of hACE2 and GFP. Moreover, inducible expression of hACE2 makes this line biosafe, whereas coexpression with GFP simplifies the detection of transgene-expressing cells. In our study, we tested our line by crossing with Ubi-Cre mice, characterized by tamoxifen-dependent ubiquitous activation of Cre recombinase. After tamoxifen administration, the copy number of the STOP cassette was decreased, and the offspring expressed hACE2 and GFP, confirming the efficiency of our system. We believe that our model can be a useful tool for studying COVID-19 pathogenesis because the selective expression of hACE2 can shed light on the roles of different tissues in SARS-CoV-2-associated complications. Obviously, it can also be used for preclinical trials of antiviral drugs and new vaccines.

Adoptive Immunotherapy Based on Chain-Centric TCRs in Treatment of Infectious Diseases.

Complications after vaccination, lack of vaccines against certain infections, and the emergence of antibiotic-resistant microorganisms point to the need for alternative ways of protection and treatment of infectious diseases. Here, we proposed a therapeutic approach to control salmonellosis based on adoptive cell therapy. We showed that the T cell receptor (TCR) repertoire of salmonella-specific memory cells contains 20% of TCR variants with the dominant-active α-chain. Transduction of intact T lymphocytes with the dominant salmonella-specific TCRα led to their enhanced in vitro proliferation in response to salmonella. Adoptive transfer of transduced T cells resulted in a significant decrease in bacterial loads in mice infected with salmonella before or after the adoptive transfer. We demonstrated that adoptive immunotherapy based on T cells, transduced with dominant-specific TCRα could be successfully applied for treatment and prevention of infectious diseases and represent a useful addition to vaccination and existing therapeutic strategies.

Persistence of plasmid-mediated expression of transgenes in human mesenchymal stem cells depends primarily on CpG levels of both vector and transgene.

BACKGROUND: Gene therapy and cell modification for clinical applications using plasmid vectors are considered to be a safe and promising strategy. One of the major problems with plasmid vector-based constructs is a rapid decline of transgene expression in cells in vitro and in vivo. An important role of CpG motifs or bacterial vector backbone in expression silencing has been suggested. METHODS: To address the effects of CpG motifs on transgene expression maintenance in stem cells in vitro, we constructed a novel pMBR2 plasmid vector containing 13 CpG motifs only. pMBR2 constructs with CpG-free and CpG-replete firefly luciferase inserts were introduced into cultured human adipose-derived mesenchymal stem (MSCs) by electroporation, and luciferase expression levels were monitored for 3 weeks. RESULTS: The pMBR2 vector with CpG-free luciferase insert demonstrated the highest persistence of expression, whereas the wild-type luciferase insert containing 97 CpG motifs demonstrated lower expression maintenance in the same vector. In comparison, the same inserts in the CpG-replete pCDNA3 vector demonstrated significantly lower expression levels and only a minimal persistence of expression. ß-galactosidase and enhanced green fluorescent protein genes inserted into pMBR2 vector also demonstrated higher expression levels and better maintenance compared to the same genes in pCDNA3 vector. CONCLUSIONS: The persistence of plasmid vector expression in human MSCs is determined primarily by CpG content of both vector and transgene. The data obtained in the present study indicate that the pMBR2 vector with a minimized number of CpG motifs is appropriate for extended plasmid-mediated expression of transgenes in MSCs and possibly other types of stem cells.