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Grant Projects (ongoing in the year 2022)
View ongoing grants in the year: 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, 2022, 2023, 2024, 2025, 2026, 2027, 2028.
Circular RNAs and their relation to RNA splicing in the pathogenesis of myelodysplastic syndromes
20-19162S [2020 – 2022]
Ing. Michaela Dostálová Merkerová, Ph.D.
Doc. Ing. Jiří Kléma, Ph.D., FEL ČVUT Praha
Mutations in RNA splicing factors represent the most common molecular alterations in myelodysplastic syndromes (MDS). Despite some advances in the understanding of their roles in MDS pathogenesis, the impact of mutated splicing factors on circular RNAs (circRNAs) has not been studied. CircRNAs constitute a class of RNAs with a covalently closed continuous loop that has recently been discovered to be widespread and abundant. Because most circRNAs are produced by backsplicing, we hypothesize that the aberrant splicing seen in MDS also affects circRNA formation. With respect to the various regulatory functions of circRNAs, their deregulation likely contributes to MDS pathogenesis. Moreover, altered levels of particular circRNAs might serve as new molecular biomarkers for the prognosis and therapeutic response in MDS management. Moreover, circRNAs might form a new class of molecular targets for MDS treatment. In this project, we will use next generation sequencing and perform integrative data analyses and functional studies to identify and characterize circRNA roles in MDS pathogenesis.
Study of molecular pathogenesis of bone marrow failure and identification of clinically relevant biomarkers in hypoplastic myelodysplastic syndrome and acquired aplastic anemia
AZV NU21-03-00565 [2021 – 2024]
Hypoplastic myelodysplastic syndrome (hMDS) and acquired aplastic anemia (AA) are severe hematopoietic disorders whose clinicopathological features overlap that makes diagnosis complicated. In both disorders, bone marrow (BM) failure occurs due to damage to hematopoietic cells by cytotoxic T-lymphocytes. However, the molecular mechanisms involved in this process are still unclear. In this context, a comprehensive genome analysis in hMDS and AA will be performed in the proposed project to characterize the molecular basis of BM failure and to define clinically relevant biomarkers for easier differential diagnosis, prediction of disease progression, and evaluation of the effect of immunosuppressive therapy on pathological clone evolution. Whole-exome sequencing will be used for characterization of genomic landscape, the transcriptome will be analyzed by RNASeq at the level of CD34+ BM cells and CD3+ lymphocytes, and mRNA and lnRNA expression profiles will be determined. Due to the etiology of the diseases, we will focus on the factors mediating the immune response and the T-cell population that will be studied in detail by flow cytometry. Knowledge of the molecular background of hMDS/AA will provide us an understanding of the disease development and will also enable to define new biomarkers which may strengthen the personalized approach to patients.
Pre-clinical validation of cGMP production of CAR T-cells for solid tumorstherapy
AZV 19-08-00147 [2019 – 2022]
Doc. RNDr. Irena Krontorád Koutná, Ph..D., FNUSA Brno
MUDr. Pavel Otáhal, Ph.D., Mgr. Pavel Šimara, Ph.D., MU Brno
Chimeric antigen receptor (CAR) T-cell is a cutting edge technology for targeted cell therapy of oncologic diseases. Promising clinical results were reported for hematological malignancies, but the results in solid tumors are not that encouranging yet. Here we propose to validate protocols for the production of CAR T-cells against solid tumor antigens under cGMP rules. We will focus mainly on target antigens GD2, PSMA, and PSCA. Standard operation protocols and analytical certificates will be presented to the State Institute for Drug Control for their approval. The consortium of three prominent research facilities will participate on this project: (i) International Clinical Research Center of St. Anne's University Hospital Brno (FNUSA-ICRC), (ii) Centre for Biomedical Image Analysis at Masaryk University Brno (MU-CBIA), and (iii) Institute of Hematology and Blood Transfusion in Prague (UHKT). Our main aim is to establish production of CAR T-cells for anti-solid tumor therapy which can be translated into clinical applications.
Blood plasma individual variability and pathophysiology and their influence on the interactions with synthetic antifouling surfaces
GA ČR 20-10845S [2020 – 2022]
Prof. Ing. Tomáš Riedel, Ph.D., Ústav makromolekulární chemie AV ČR
Ing. Pavel Májek, Ph.D.
Early biomarker detection improves treatment outcomes, survival and quality of life. In particular optical biosensors are promising technology for early diagnostics: minimally invasive procedures (plasma, saliva), rapid and sensitive detection, low sample consumption. The main pitfall of optical biosensors is the inability to differentiate a specific signal from an interfering signal caused by adsorption of particularly proteins during the contact of artificial surfaces with biological media (fouling). Fouling can result in complement or coagulation initiation, etc. It has prevented the advance of biosensors into clinical applications. In principle, fouling is influenced by individual biological variability and pathophysiology – with a few exceptions, these factors and their influence are unknown although they play a key role in clinical applicability. The aim of this project is to identify these factors and their effects, and to design sample pre-treatments to minimize or eliminate these factors.
Advanced Immunomonitoring and Immunotherapy of Hematological and Hemato-oncological Patients
OP VVV CZ.02.1.01/0.0/0.0/16_025/0007428 [2018 – 2022]
The development of diagnostic methods for determination of immunosuppressive escape mechanisms of leukemic cells, that should serve for the selection of a appropriate personalized immunotherapy of hemato- oncological patients, Development of procedures for the expansion of T-lymphocytes capable of recognizing antigens of leukemic cells in AML patients, Implementation of methods for monitoring the immune system reconstitution of patients after HSCT for identification of patients with insufficient immune system reconstitution. Identification of markers predicting the longterm in vivo persistence of transferred multi-virus specific T-lymphocytes and their tracking in treated patients, the construction of a new generation of CAR T-lymphocytes.
Multifunctional nanostructures for plasmonic sensing of biomolecules related to onco-hematological diseases
GA ČR 22-27329S [2022 – 2024]
prof. Ing. Jiří Homola CSc., DSc., Ústav fotoniky a elektrotechniky AV ČR
Ing. Jiří Suttnar, CSc., Ing. Andrés de los Santos Pereira Ph.D., Ústav makromolekulární chemie AV ČR
This is a multidisciplinary project that aims to advance research in plasmonics, functional coatings and onco-hematological diseases with the goal of developing a new generation of optical biosensors that enable simultaneous and sensitive detection of microRNA and protein biomarkers of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). This goal will be achieved by the development of new multi-functional plasmonic nanostructures that support surface plasmons in VIS/NIR and UV spectral regions, and functionalization methods using UV-triggered chemical reactions, and their combination for site-specific receptor immobilization in plasmonic hotspot regions. The biosensor technology to be developed in this project will provide a new tool for the research of onco-hematological diseases, open up new opportunities for monitoring MDS/AML patients and pave the way to personalized therapies.
Role of acute myeloid leukemia-associated nucleophosmin mutations in the NPM-p53-Mdm2 regulatory network
GA ČR 22-03875S [2022 – 2024]
prof. RNDr. Petr Heřman CSc., MFF UK Praha
Mgr. Barbora Brodská, Ph.D.
Nucleophosmin (NPM) mutations characteristic for the acute myeloid leukemia (AML) cause aberrant cytoplasmic localization of NPM and many of its interaction partners, e.g. of tumor suppressor p53. The p53 level is regulated at several stages by mdm2, which is also known to interact with NPM. Impact of the mutation on the dynamic equilibria within the NPM-p53-mdm2 network, mutual protein interactions, and potential formation of the ternary complex in live cells will be examined by microscopic and immunochemical methods. We will focus on the role of NPM in the regulation of p53 activity with the possibility to increase sensitivity and response of cells with mutated NPM to cytostatics. We will evaluate impact of the NPM mutations on relations between specific phosphorylations and oligomerization of p53 and NPM. Experiments will be done with cell lines transfected with fluorescent proteins and verified on primary cells from AML patients. Results will contribute to understanding of the role of NPM mutation in leukemogenesis and will open new strategies for AML treatment.
Genomic characterization of adult B-precursor acute lymphoblastic leukemia for prediction of treatment response to targeted therapy
AZV NU22-03-00210 [2022 – 2025]
doc. MUDr. Mgr. Cyril Šálek, Ph.D.
Prof. MUDr. Tomáš Stopka, Ph.D., 1. LF UK Praha, Priv.-Doz. Dr. rer. nat. habil. Radislav Sedláček, ÚMG AV ČR Praha
Subtypes of B-precursor acute lymphoblastic leukemias (BCP-ALL) with distinct genomic or gene expression profile have been recently revealed using high-throughput sequencing technology, including BCR-ABL1-like ALL, ALL with DUX4, ZNF384, MEF2D and NUTM1 rearrangements, ALL with PAX5 alterations or IKZFplus ALL. They are mostly associated with poor response to standard chemotherapy and impaired progression-free survival. This can be overcome with targeted pharmaceuticals including inhibitors of distinct kinases, monoclonal antibodies (including bispecific agents and immunoconjugates), and chimeric antigen receptor T-cells (CAR-T). These drugs have shown unprecedent therapeutic efficacy in relapsed/refractory cohorts. Czech Leukemia Study Group is running three academic trials with targeted agents in frontline settings: 1) Blina-CELL Study with anti-CD19/anti-CD3 bispecific monoclonal antibody in the induction phase for BCR-ABL1-negative ALL); 2) EWALL-INO Study with anti-CD22 immunoconjugate in the induction phase for BCR-ABL1-negative ALL; and 3) Pona-CELL Study combining third generation tyrosine kinase inhibitor ponatinib with reduced-intensity chemotherapy in BCR-ABL1-positive ALL. These trials are aiming to accelerate treatment response and induce negativity of minimal residual disease (MRD) at early phases of the therapy. The aim of proposed project is to perform detailed genomic characterization and search for specific genomic patterns in subjects enrolled to above mentioned academic studies and to compare their genomic profile with the treatment response. Prospective cohort treated with targeted agents will be compared to a consecutive retrospective cohort treated with standard chemotherapy. The goal is to identify specific genomic signatures that confer a high risk of treatment failure on standard chemotherapy protocols and to prove that high-risk genomic features can be overcome with modern targeted agents if administered early in the front-line therapy.
Gene engineered killer T cells for the therapy of acute myeloid leukemia
AZV NU22-05-00374 [2022 – 2025]
MUDr. Pavel Otáhal, Ph.D.
Prof. MUDr. Tomáš Stopka, Ph.D., 1. LF UK Praha, Priv.-Doz. Dr. rer. nat. habil. Radislav Sedláček, ÚMG AV ČR Praha
The aim of the project is to develop new applied methods of cell therapy of AML based on genetically modified autologous T lymphocytes. This procedure is based on a genetic modification by a recombinant T-cell receptor (TCR), which redirects the specificity of T lymphocytes to the selected antigen, and their subsequent in vitro expansion. One of the targets studied in this way is Wilms Tumor Antigen 1 (WT1), which is expressed by AML cells and is at the same time effectively recognized by the immune system and has therefore been used as a target in a number of clinical trials. This identical TCR reacting with the RMFNAPYL antigen antigen WT1 in the HLA A2 complex was prepared and tested at the same time as a number of other TCR control TCR reacting with other relevant antigens such as NY-ESO-1. The main goal of the project will be to develop a GMP-certified method of preparing WT1-specific TCR-T cells using the non-viral gene transduction method using piggyBac transposon, in a similar way as CD19-specific CAR-T cells are currently being prepared in UHKT. The function of prepared cells will be studied using in vitro and in vivo using mouse models. The anticancer effect will be monitored by in-vivo bio-luminescent methods using standard cell lines of acute myeloid leukaemia and primary AML cells. In addition, humanized mouse models will be introduced to allow a more detailed study of a number of immunological aspects of the treatment of acute myeloid leukemia via TCR-T cells. In the next objective of the project, we will prepare new types of TCR vectors that will allow to target additional epitopes from antigens WT1, or PRAME and we will develop new types of TCR vectors with enhanced activity via co-expression of cytokines such as IL-15 and IL-21. The applied output of the project will be the GMP production of TCR-T lymphocytes specific to the WT1 antigen, ands the submission of an application for a clinical trial (Phase I) in patients with AML.
Significance of T cell response to vaccination against SARS-CoV2 for leukemic patients with weakend immune system
AZV NU22-E-138 [2022 – 2023]
The project is aimed on choosing optimal methods for measurement of total and SARS-CoV-2 specific immunity through quantification of specific effectors of adaptive immunity of haemato-oncologic patients treated with cellular therapy, that will be suitable for optimizing of vaccination against COVID-19 and for evaluation of its immunogenicity. The state of immune system of patients will be characterized and the impact of its deterioration on immune response to vaccination will be assessed. Immune responses to vaccination of recipients of various types of cellular therapy and of healthy individuals will be compared. Impact of vaccination on the course of therapy of underlying disease will be determined. The goal of the project includes proposing a recommendation of a suitable method for examination of specific anti-SARS-CoV-2 cellular immunity, acceptable for clinical praxis.
Spectrum of NGS detected somatic mutations and their association with prognosis and outcome of adolescent and young adult patients with Ph-positive leukemias
AZV NU21-07-00225 [2021 – 2024]
doc. Mgr. Kateřina Machová Poláková, Ph.D.
Prof. MUDr. Jan Trka,, Ph.D., Fakultní nemocnice v Motole, Praha
Adolescent and young adult (AYA) patients with Ph+ leukemias comprise a unique subgroup of patients with hematologic malignancies. The disease demands the careful monitoring and good compliance. Up to now, except for few clinical trials, the specific aspects and biology of Ph+ leukemias of AYA patients have not been studied in detail. Currently, most patients with chronic myeloid leukemia in chronic phase (CML-CP) have a normal life expectancy. Before tyrosine kinase inhibitors (TKIs) introduction into the clinical practice, the higher age was a negative prognostic factor. In the current era of TKIs it seems that younger patients with CML-CP at age 15-39, defined by National Comprehensive Cancer Network Guidelines as AYAs, have worse prognosis and response to TKIs. In recent years, somatic mutations in hematologic malignancies are of importance and have been highly reported. However, very little is known about the genomic landscape of somatic mutations outside the oncogene BCR-ABL1 in Ph positive leukemias - CML and Ph positive acute lymphoblastic leukemia (Ph+ALL) - of AYAs and their relation to the resistance to TKI treatment and relapses. We expect that the complex NGS-based screening of somatic mutations proposed in this project will allow us to determine whether the clonal hematopoiesis represents a worse prognostic factor for AYA patients with Ph+ leukemias. We anticipate that the similar spectrum of mutations can be identified in CML and Ph+ALL AYA patients, but with different patterns of clonal evolution in association with the different treatment protocols. This project may identify high risk AYA CML patients, who may profit from more intensive treatment protocol, similar to protocol of the Ph+ALL.
Efficacy and safety of tyrosine kinase inhibitors’ discontinuation after two-step dose reduction in patients with chronic myeloid leukemia (A prospective multi-centre phase II clinical trial - HALF)
AZV NU22-03-00136 [2022 – 2025]
Prof. MUDr. Jiří Mayer, CSc., LF MU Brno
MUDr. Daniela Žáčková, Ph.D., FN Brno, doc. Mgr. Kateřina Machová Poláková, Ph.D., MUDr. Lukáš Stejskal, FN Ostrava, Prof. MUDr. Edgar Faber, CSc., FN Olomouc, MUDr. Michal Karas, Ph.D., RN Plzeň, MUDr. Petra Bělohlávková, Ph.D., FN Hradec Králové
Chronic myeloid leukemia (CML) patients’ treatment and prognosis improved significantly with tyrosine kinase inhibitors (TKI) introduction into clinical practice. However, long-lasting or even lifelong therapy can cause even serious adverse events and represents a substantial economic burden for health care systems. Treatment-free remission (TFR) has therefore become a new goal of CML therapy, since its feasibility and safety was confirmed by multiple clinical trials as well as clinical practice experience. However, usually abrupt therapy discontinuation has been successful only in about half of eligible patients and even if so, it can cause burdening symptoms known as a TKI withdrawal syndrome (TWS) in about 30% of patients. On top of that, sustainable deep molecular response as a main prerequisite for TKI discontinuation has been achieved in only 20-40% of patients. Thus, majority of CML patients need to be treated with lifelong therapy with all its drawbacks. Moreover, any robust clinical nor biological factor predictive for successful TFR has not been identified yet. We propose the project of multi-centre investigator-initiated phase II clinical trial (HALF) representing an original concept of the two-step TKI dose reduction (i.e. to half of standard dose during the first 6 months and to the same dose given alternatively during the next 6 months) before complete TKI withdrawal. We suppose that this approach will both induce higher rate of patients achieving successful TFR with less frequent/pronounce TWS and proof feasibility and safety of sequential TKI dose reduction as an alternative to the complete therapy interruption versus its lifelong administration. Besides our focus on efficacy and safety improvement, we plan to provide many analyses in order to identify both clinical and biological factors predictive for successful TFR. We also aim to evaluate and eventually reduce financial burden of the long-term therapy as well as improve patients’ quality of life
Role of transposable elements and PIWI-interacting RNAs in myelodysplastic syndrome and their potential clinical applications
AZV NU20-03-00412 [2020 – 2024]
Ing. Michaela Dostálová Merkerová, Ph.D.
Doc. Ing. Jiří Kléma, Ph.D., FEL ČVUT Praha
Myelodysplastic syndrome (MDS) is a malignant hematopoietic disorder characterized by aberrant differentiation of hematopoietic stem cells (HSCs). Genome instability is one of the key features of MDS HSCs and mobilization of transposable elements (TEs) is a known destabilizing factor of the genome integrity. PIWI-interacting RNAs (piRNAs) inhibit TE mobilization, functioning as central players in stem cell mechanisms to preserve genome integrity. Despite recent advances in the understanding of MDS pathogenesis, the role of TEs and piRNAs in development and progression of the disease has not been studied yet. We will analyze transcription of TEs and piRNAs by next generation sequencing and examine their activity in MDS. Besides the implication in the MDS pathogenesis, utilization of TEs and piRNAs as potential molecular markers of the disease progression will be tested. Moreover, possible effect of azacitidine treatment on TE and piRNA transcriptions will be addressed to prove their applicability as novel predictive markers for the drug responsiveness and relapse prevention.
Natural Killer cell based adoptive cellular immunotherapy of hemato-oncology disorders
AZV NU22-08-00287 [2022 – 2025]
The use of natural killer (NK) cells as a cellular immunotherapy has increased over the past decade, specifically in patients with hematologic malignancies. NK cell adoptive therapy is a promising cancer therapeutic approach, but there are significant challenges that are limiting its feasibility and clinical efficacy. Acute myeloid leukemia (AML) is a clinically and genetically heterogeneous disease with remaining unsatisfactory outcomes. In this project, we propose to develop methods for expansion and activation of NK cells from human peripheral blood mononuclear cells as well as clinical-scale method to produce NK cells for immunotherapy under Current Good Manufacturing Practices (cGMP) conditions. We plan to generate the data correlating the NK cells phenotype with the expansion and killing capacity allowing to stratify the NK cells donors in order to achieve reproducible and efficient product of cellular immunotherapy. In this study, we will use irradiated autologous peripheral blood mononuclear cells as cell-based feeder cells for large-scale expansion of highly purified cytotoxic NK cells. Along with those steps, we will investigate the possibilities of NK cell donor stratification based on the correlation of immunophenotypic markers and expansion and cytotoxic potential of NK cell product. The generated knowledge will be used for further improvements of the therapeutic strategy and more personalized donor selection. The ultimate goal of the project is to develop an immunotherapeutic protocol using adoptively transferred NK cells to achieve full remission of AML and myelodysplastic syndrome patients.
