Papers by Dmitry Kostyushev
Impaired therapeutic efficacy of bone marrow cells from post-myocardial infarction patients in the TIME and LateTIME clinical trials
PLOS ONE
International Journal of Molecular Sciences
CRISPR/Cas technologies have advanced dramatically in recent years. Many different systems with n... more CRISPR/Cas technologies have advanced dramatically in recent years. Many different systems with new properties have been characterized and a plethora of hybrid CRISPR/Cas systems able to modify the epigenome, regulate transcription, and correct mutations in DNA and RNA have been devised. However, practical application of CRISPR/Cas systems is severely limited by the lack of effective delivery tools. In this review, recent advances in developing vehicles for the delivery of CRISPR/Cas in the form of ribonucleoprotein complexes are outlined. Most importantly, we emphasize the use of extracellular vesicles (EVs) for CRISPR/Cas delivery and describe their unique properties: biocompatibility, safety, capacity for rational design, and ability to cross biological barriers. Available molecular tools that enable loading of desired protein and/or RNA cargo into the vesicles in a controllable manner and shape the surface of EVs for targeted delivery into specific tissues (e.g., using targeting...
International Journal of Molecular Sciences
Restriction of foreign DNA is a fundamental defense mechanism required for maintaining genomic st... more Restriction of foreign DNA is a fundamental defense mechanism required for maintaining genomic stability and proper function of mammalian cells. APOBEC cytidine deaminases are crucial effector molecules involved in clearing pathogenic DNA of viruses and other microorganisms and improperly localized self-DNA (DNA leakages). Mastering the expression of APOBEC provides the crucial means both for developing novel therapeutic approaches for combating infectious and non-infectious diseases and for numerous research purposes. In this study, we report successful application of a CRISPRa approach to effectively and specifically overexpress APOBEC3A and APOBEC3B deaminases and describe their effects on episomal and integrated foreign DNA. This method increased target gene transcription by >6–50-fold in HEK293T cells. Furthermore, CRISPRa-mediated activation of APOBEC3A/APOBEC3B suppressed episomal but not integrated foreign DNA. Episomal GC-rich DNA was rapidly destabilized and destroyed b...
Vaccines
Telomerase reverse transcriptase (TERT) is a classic tumor-associated antigen overexpressed in ma... more Telomerase reverse transcriptase (TERT) is a classic tumor-associated antigen overexpressed in majority of tumors. Several TERT-based cancer vaccines are currently in clinical trials, but immune correlates of their antitumor activity remain largely unknown. Here, we characterized fine specificity and lytic potential of immune response against rat TERT in mice. BALB/c mice were primed with plasmids encoding expression-optimized hemagglutinin-tagged or nontagged TERT or empty vector and boosted with same DNA mixed with plasmid encoding firefly luciferase (Luc DNA). Injections were followed by electroporation. Photon emission from booster sites was assessed by in vivo bioluminescent imaging. Two weeks post boost, mice were sacrificed and assessed for IFN-γ, interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-α) production by T-cells upon their stimulation with TERT peptides and for anti-TERT antibodies. All TERT DNA-immunized mice developed cellular and antibody response against...
Infectious diseases are a global health problem affecting billions of people. Developing rapid an... more Infectious diseases are a global health problem affecting billions of people. Developing rapid and sensitive diagnostic tools is key for successful patient management and curbing disease spread. Currently available diagnostics are very specific and sensitive but time-consuming and require expensive laboratory settings and well-trained personnel; thus, they are not available in resource-limited areas, for the purposes of large-scale screenings and in case of outbreaks and epidemics. Developing new, rapid, and affordable point-of-care diagnostic assays is urgently needed. This review focuses on CRISPR-based technologies and their perspectives to become platforms for point-of-care nucleic acid detection methods and as deployable diagnostic platforms that could help to identify and curb outbreaks and emerging epidemics. We describe the mechanisms and function of different classes and types of CRISPR-Cas systems, including pros and cons for developing molecular diagnostic tests and appli...
International Journal of Molecular Sciences
The gene editing tool CRISPR-Cas has become the foundation for developing numerous molecular syst... more The gene editing tool CRISPR-Cas has become the foundation for developing numerous molecular systems used in research and, increasingly, in medical practice. In particular, Cas proteins devoid of nucleolytic activity (dead Cas proteins; dCas) can be used to deliver functional cargo to programmed sites in the genome. In this review, we describe current CRISPR systems used for developing different dCas-based molecular approaches and summarize their most significant applications. We conclude with comments on the state-of-art in the CRISPR field and future directions.
Oxidative Medicine and Cellular Longevity
HIV-induced immune suppression results in the high prevalence of HIV/AIDS-associated malignancies... more HIV-induced immune suppression results in the high prevalence of HIV/AIDS-associated malignancies including Kaposi sarcoma, non-Hodgkin lymphoma, and cervical cancer. HIV-infected people are also at an increased risk of “non-AIDS-defining” malignancies not directly linked to immune suppression but associated with viral infections. Their incidence is increasing despite successful antiretroviral therapy. The mechanism behind this phenomenon remains unclear. Here, we obtained daughter clones of murine mammary gland adenocarcinoma 4T1luc2 cells expressing consensus reverse transcriptase of HIV-1 subtype A FSU_A strain (RT_A) with and without primary mutations of drug resistance. In in vitro tests, mutations of resistance to nucleoside inhibitors K65R/M184V reduced the polymerase, and to nonnucleoside inhibitors K103N/G190S, the RNase H activities of RT_A. Expression of these RT_A variants in 4T1luc2 cells led to increased production of the reactive oxygen species (ROS), lipid peroxidati...
Microorganisms
Background: Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is the major cause... more Background: Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is the major cause of viral persistence in patients with chronic HBV infection. Understanding the mechanisms underlying stability and persistence of HBV cccDNA in hepatocytes is critical for developing novel therapeutics and managing chronic hepatitis B. In this study, we observed an unexpected increase in HBV cccDNA levels upon suppression of transcription by de novo DNA methyltransferase DNMT3A and uncovered additional mechanisms potentially involved in HBV cccDNA maintenance. Methods: HBV-expressing cell lines were transfected with a DNMT3A-expressing plasmid. Real-time PCR and HBsAg assays were used to assess the HBV replication rate. Cell cycling was analyzed by fluorescent cell sorting. CRISPR/Cas9 was utilized to abrogate expression of APOBEC3A and APOBEC3B. Alterations in the expression of target genes were measured by real-time PCR. Results: Similar to previous studies, HBV replication induced DN...
Viruses
Chronic hepatitis B virus infection (CHB) caused by the hepatitis B virus (HBV) is one of the mos... more Chronic hepatitis B virus infection (CHB) caused by the hepatitis B virus (HBV) is one of the most common viral infections in the world. Reactivation of HBV infection is a life-threatening condition observed in patients with CHB receiving chemotherapy or other medications. Although HBV reactivation is commonly attributed to immune suppression, other factors have long been suspected to play a role, including intracellular signaling activated in response to DNA damage. We investigated the effects of DNA-damaging factors (doxorubicin and hydrogen peroxide) on HBV reactivation/replication and the consequent DNA-damage response. Dose-dependent activation of HBV replication was observed in response to doxorubicin and hydrogen peroxide which was associated with a marked elevation in the mRNA levels of ataxia-telangiectasia mutated (ATM) and ATM- and RAD3-related (ATR) kinases. Downregulation of ATM or ATR expression by shRNAs substantially reduced the levels of HBV RNAs and DNA. In contras...
Implantation of bone marrow-derived cells (BMCs) into mouse hearts post-myocardial infarction (MI... more Implantation of bone marrow-derived cells (BMCs) into mouse hearts post-myocardial infarction (MI) limits cardiac functional decline. However, clinical trials of post-MI BMC therapy have yielded conflicting results. While most laboratory experiments use healthy BMC donor mice, clinical trials use post-MI autologous BMCs. Post-MI mouse BMCs are therapeutically impaired, due to inflammatory changes in BMC composition. Thus, therapeutic efficacy of the BMCs progressively worsens after MI but recovers as donor inflammatory response resolves. The availability of post-MI patient BM mononuclear cells (MNCs) from the TIME and LateTIME clinical trials enabled us to test if human post-MI MNCs undergo a similar period of impaired efficacy. We hypothesized that MNCs from TIME trial patients would be less therapeutic than healthy human donor MNCs when implanted into post-MI mouse hearts, and that therapeutic properties would be restored in MNCs from LateTIME trial patients. Post-MI SCID mice rec...
Scientific Reports
of cccDNA by non-homologous end-joining (NHeJ). NHeJ has been suggested to enhance anti-HBV activ... more of cccDNA by non-homologous end-joining (NHeJ). NHeJ has been suggested to enhance anti-HBV activity of CRISPR/Cas9 and increase cccDNA mutation. In this study, we assessed anti-HBV activity of CRISPR/Cas9 and cccDNA repair outcomes in an altered NHEJ/HR environment. NU7026, a strong inhibitor of NHEJ, prevented CRISPR/Cas9-mediated degradation of cccDNA and resulted in frequent on-target deletions. We conclude that CRISPR/Cas9 is a highly effective tool to degrade cccDNA and first demonstrate that inhibiting NHEJ impairs cccDNA degradation.
Genes
Covalently closed circular DNA (cccDNA) is a persistent form of the HBV genome that maintains HBV... more Covalently closed circular DNA (cccDNA) is a persistent form of the HBV genome that maintains HBV chronicity. Decades of extensive research resulted in the two therapeutic options currently available: nucleot(s)ide analogs and interferon (IFN) therapy. A plethora of reliable markers to monitor HBV patients has been established, including the recently discovered encapsidated pregenomic RNA in serum, which can be used to determine treatment end-points and to predict the susceptibility of patients to IFN. Additionally, HBV RNA splice variants and cccDNA and its epigenetic modifications are associated with the clinical course and risks of hepatocellular carcinoma (HCC) and liver fibrosis. However, new antivirals, including CRISPR/Cas9, APOBEC-mediated degradation of cccDNA, and T-cell therapies aim at completely eliminating HBV, and it is clear that the diagnostic arsenal for defining the long-awaited sterilizing cure is missing. In this review, we discuss the currently available tools for detecting and measuring HBV RNAs and cccDNA, as well as the state-of-the-art in clinical implications of these markers, and debate needs and goals within the context of the sterilizing cure that is soon to come.
Orthologous CRISPR/Cas9 systems for specific and efficient degradation of covalently closed circular DNA of hepatitis B virus
Cellular and Molecular Life Sciences
Molecular therapy : the journal of the American Society of Gene Therapy, Jan 5, 2018
Treatment of myocardial infarction (MI) with bone marrow cells (BMCs) improves post-MI cardiac fu... more Treatment of myocardial infarction (MI) with bone marrow cells (BMCs) improves post-MI cardiac function in rodents. However, clinical trials of BMC therapy have been less effective. While most rodent experiments use young healthy donors, patients undergoing autologous cell therapy are older and post-MI. We previously demonstrated that BMCs from aged and post-MI donor mice are therapeutically impaired, and that donor MI induces inflammatory changes in BMC composition including reduced levels of B lymphocytes. Here, we hypothesized that B cell alterations in bone marrow account for the reduced therapeutic potential of post-MI and aged donor BMCs. Injection of BMCs from increasingly aged donor mice resulted in progressively poorer cardiac function and larger infarct size. Flow cytometry revealed fewer B cells in aged donor bone marrow. Therapeutic efficacy of young healthy donor BMCs was reduced by depletion of B cells. Implantation of intact or lysed B cells improved cardiac function,...
Journal of the American Heart Association, Jan 6, 2016
Circulating angiogenic cells (CACs) are peripheral blood cells whose functional capacity inversel... more Circulating angiogenic cells (CACs) are peripheral blood cells whose functional capacity inversely correlates with cardiovascular risk and that have therapeutic benefits in animal models of cardiovascular disease. However, donor age and disease state influence the efficacy of autologous cell therapy. We sought to determine whether age or coronary artery disease (CAD) impairs the therapeutic potential of CACs for myocardial infarction (MI) and whether the use of ex vivo gene therapy to overexpress endothelial nitric oxide (NO) synthase (eNOS) overcomes these defects. We recruited 40 volunteers varying by sex, age (< or ≥45 years), and CAD and subjected their CACs to well-established functional tests. Age and CAD were associated with reduced CAC intrinsic migration (but not specific response to vascular endothelial growth factor, adherence of CACs to endothelial tubes, eNOS mRNA and protein levels, and NO production. To determine how CAC function influences therapeutic potential, w...
Stem cells and microenvironment: Integration of biochemical and mechanical factors
Biology Bulletin Reviews, 2014
ABSTRACT The role of the microenvironment in the regulation of the main stem cell functions is co... more ABSTRACT The role of the microenvironment in the regulation of the main stem cell functions is considered. Special attention is paid to the effects of mechanical interactions and mechanical properties of the substrate on self-renewal, maintenance of potency, and differentiation in stem cells in vivo and in vitro. Primary cilia, mechanosensitive channels, receptors coupled with G proteins, and the proteins of intercellular junctions can be mechanosensors. In turn, the major role in mechanotransduction belongs to integrins, a large family of extracellular matrix receptors. Integrins are part of focal adhesions. They form bridges to link the cell membrane to the cytoskeleton and nucleus. The study of the integration of the biochemical and mechanical factors of the microenvironment is essential for the introduction of stem cell technology into regenerative medicine.
Molecular and cellular targets as the basisof preclinical diagnosis of type 1 diabetes: problems and prospects
In autoimmune diabetes mellitus (IDDM), clinical manifestation is typically preceded by several y... more In autoimmune diabetes mellitus (IDDM), clinical manifestation is typically preceded by several years of latent autoimmune insulitis. The autoimmune process arises only in genetically predisposed people,and is usually provoked by an infectious pathogen. Dagnostics of IDDM in latent stage had been rare until recently, but now it has become possible to translate data collected by studies of human genome (genomics) and of protein profiles in healthy individuals and in conditions of pathology (proteomics) into clinical practice, creating biomarkers that allow diagnosing the disease at early preclinical stages, detecting genetic succeptability to IDDM, predicting prognosis and creating treatment programs for individual patients, accessing risk groups and giving scientifically based treatment and advice that would help to prevent the disease. In this review, we start by covering basic principles of selecting biomarkers of IDDM for clinical application. After that, we proceed to give infor...
Pharmacopreventive therapy of type 1 diabetes (t1d) from the positions of translational medicine: problems and prospects
Translational medicine is one of the critical components to overcome the impediments within the o... more Translational medicine is one of the critical components to overcome the impediments within the objectives of Predictive Preventive and Personalized Medicine. Identification of molecular mechanisms underlying subclinical stages of T1D pathogenesis is a key to detection of new biomarkers and development of drugs of new generation. To date, this is getting possible because of practical application of recent advances in translational medicine and translation of fundamentals in genomics, proteomics and metabolomics into clinical methods of preventive immunocorrection/immunomodulation and regeneration of beta-cells in Langerhans’ islands. In this paper, we describe novel immune and cell methods to prevent T1D, discuss the specifics in their application at subclinical stages and propose new approaches to modulate immune status in patients with T1D and regeneration of insulin-producing beta-cells by mesenchymal stem cells.
Ab-proteases and their potential applications in MS diagnostics and treatment
Mode of interaction between programmed exosomes (bionanosomes) with target cells: panoramic view on the role of a multitude of factors
Exosomes are nanoparticles of intercellular communication, cell derivatives of the body, they hav... more Exosomes are nanoparticles of intercellular communication, cell derivatives of the body, they have recently drawn an increasing attention, and found practical application and their own niche in different areas of medical and biological sciences, nanotechnology, teranostics and drug delivery. Composition of exosomes is variative, and differs depending on producer cells, as well as can be dissimilar even from the same cells if those are exposed to different conditions. Consequently, there is a unique opportunity to design exosomes which have similar structural, morphological and biochemical properties, but which trigger functional responses of different type, strength and longevity. Along with that, in some circumstances it is hard to reliably determine the response of target cells to exosomes. This is considered to be dependent upon the mechanism of exosome-cell interaction, mode of exosome internalization, rate of their penetration through cellular membranes and other factors that c...
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Papers by Dmitry Kostyushev