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Project titleMolecular mechanisms of the oncolytic vaccinia virus therapeutic action on malignant brain tumors

Keywordsglioma; cell cultures; tumor models; oncolytic virus; full transcriptome analysis of gene expression; virotherapy; molecular mechanisms

Annotation
Malignant neoplasms of the brain and central nervous system are characterized by one of the highest mortality rates in the structure of oncological diseases in the Russian Federation and throughout the world. The currently available treatment regimens for brain tumors do not provide a significant improvement in the quality and increase in the life expectancy of patients. The development of effective therapeutic methods for this type of tumor using tumor models that preserve the molecular biological characteristics of the primary tumor (patient-derived cell cultures), and a detailed analysis of the mechanism of action of the proposed drugs are undoubtedly urgent tasks of modern biomedical research. The goal of this project is to create new methods of malignant brain tumor therapy based on the oncolytic vaccinia virus, taking into account a detailed analysis of the mechanisms of its action on the proliferation, invasion and metastasis of patient-derived glioma cell cultures in vitro and in vivo. For this purpose, the project will use the oncolytic vaccinia virus VV-GMCSF-Lact, developed by the authors of the project earlier. Previously, we found that VV-GMCSF-Lact suppresses the growth and metastasis of human breast tumors, and the available data show the potential for using this virus for the treatment of malignant brain tumors. In this project, new cellular models of malignant brain tumors will be obtained – patient-derived glioma cell cultures that preserve the genetic and molecular biological characteristics of primary tumors. The resulting patient-derived glioma cell cultures will be characterized in detail by modern molecular biological and cytological techniques. The tumorigenic and metastatic potential of new patient-derived glioma cell cultures in the in vivo system when transplanted into SCID mice will be determined. A detailed analysis of the response of glioma cells to therapy with oncolytic viruses in culture cells, as well as in tumors inoculated in SCID mice in vivo, will be carried out. For a large-scale data analysis of the biological processes of growth, invasion, metastasis and the response of cells of malignant brain tumors to virotherapy, the project will conduct a detailed analysis of changes in the transcriptome using the method of high-throughput RNA sequencing. A detailed bioinformatic analysis of a whole transcriptome data of patient-derived glioma cell cultures, as well as tumors and organs of SCID mice with metastases from human tumor xenograft, will reveal individual transcripts, gene groups, transcription factors, microRNAs and other genetic and molecular biological characteristics of tumor growth in the body. Differential expression analysis of glioma cells under conditions of therapy with the oncolytic vaccinia virus VV-GMCSF-Lact, in vitro and in vivo studies, will make it possible to establish key biological processes - signaling cascades, pro- and anti-apoptotic events that modulate the response of brain tumor cells for virotherapy. The new patient-derived glioma cell cultures obtained in the project, combined with the results of a whole transcriptome analysis of gliomas response to the action of the oncolytic vaccinia virus VV-GMCSF-Lact in vitro and in vivo, as well as data on the combined action of the oncolytic virus and chemotherapy drugs used in the clinic, will make it possible to propose and experimentally confirm novel diagnostic markers, create new approaches for the therapy of malignant brain tumors.

Expected results
Brain tumors are one of the most pressing problems of modern neuro-oncology. Today the average survival rate of patients diagnosed with glioblastoma is no more than 15 months. At the same time, understanding the mechanisms of drugs action, as well as the mechanisms that provide tumor resistance to therapy, is crucial in the development of new drugs and diagnostic tools. In addition, an important aspect of research is the relevance of cell and tumor models used to assess the potential of the developed diagnostic and therapeutic approaches. Tumor models obtained on the basis of patient-derived cell cultures, reflecting as much as possible genetic, molecular-biological and morphological characteristics of tumor cells, are currently the most relevant for assessing the antitumor effect of both those already used in the clinic and newly created drugs. Drugs based on oncolytic viruses are currently one of the most promising anticancer agents for the treatment of malignant neoplasms, including brain tumors. Preclinical trials of the oncolytic virus VV-GMCSF-Lact have already demonstrated its safety. The data obtained in the course of solving the problems of this project will fully assess the prospects for effective treatment of human glioblastoma using VV-GMCSF-Lact and draw a conclusion about the relevance of further preclinical and clinical trials. The goal of this project is to create novel therapeutic methods for malignant brain tumors based on the oncolytic vaccinia virus, taking into account a detailed analysis of the mechanisms of its action on the proliferation, invasion and metastasis of patient-derived glioma cell cultures in vitro and in vivo. When executing the project, the following results will be obtained: 1. Primary glioma cell cultures from samples of tumors of various degrees of malignancy, as well as patient-derived cell cultures, containing neurospheres, to create a glioma tumor model with high tumorigenic and metastatic potential, will be obtained. 2. The resulting patient-derived cell cultures, containing neurospheres, will be characterized by the main tumor stem cell markers (CD44, CD15, SOX-2, c-Myc), as well as by other neuronal and embryonic stem cell markers. 3. Data will be obtained on the effect of the oncolytic virus VV-GMCSF-Lact on the proliferation, viability and activation of cell death of human gliomas of various degrees of malignancy (immortalized and patient-derived cells) in vitro. 4. Sets of experimental whole-transcriptome NGS data - arrays of RNA sequences of cells of immortalized and patient-derived glioma cell cultures will be obtained. Bioinformatic analysis of experimental data sets of high throughput RNA sequencing, in combination with meta-analysis of previously published data from international projects GENCODE, The Cancer Genome Atlas (TCGA), etc., will allow describing the contribution of individual transcripts, changes in RNA expression and alternative splicing variants, and genomic rearrangements, mutations associated with the development of malignant brain tumors. 5. Differentially expressed mRNA, lncRNA and microRNA, as well as other small and long non-coding RNAs - potential diagnostic markers of malignant brain tumors will be identified and described. 6. The optimal combinations of chemotherapeutic drugs used in the clinic (temozolomide, procarbazine, lomustine, vincristine) with the oncolytic vaccinia virus VV-GMCSF-Lact will be determined, causing the maximum suppression of growth and metastasis and inducing death of glioma cells of various degrees of malignancy in vitro. 7. Experimental NGS data on changes in RNA expression of glioma cells under cytotoxic effect of VV-GMCSF-Lact will be obtained. Bioinformatic analysis of NGS data will be carried out, which will allow describing the molecular processes of suppressing the viability of cells sensitive to virotherapy and, separately, the processes of developing cell resistance to virotherapy. 8. Based on patient-derived glioma cell cultures a tumor model of glioblastoma with high tumorigenic and metastatic potential for obtaining a transplantable tumor in SCID mice will be developed. 9. Data on the antitumor efficacy of VV-GMCSF-Lact against glioblastoma xenografts obtained by subcutaneous transplantation of cells of immortalized and patient-derived glioblastoma lines with high tumorigenic and metastatic potential will be obtained. 10. Experimental NGS data will be obtained on differential gene expression in human glioma xenografts and in organs of mice (with and without metastases) of the experimental (VV-GMCSF-Lact treated) and control (saline injected) groups. The obtained NGS data arrays will make it possible to establish the molecular mechanisms of interaction of tumor cells with stromal cells, to identify groups of genes that promote invasion and metastasis of tumors, genes for tumor response to therapy with the oncolytic virus VV-GMCSF-Lact, as well as indicator and diagnostic changes in the transcriptome of cells, organs and tissues in response to the growth and metastasis of glioma. 11. The description of the fundamental molecular processes of gliomas growth, metastasis and the mechanisms of gliomas response to therapy will be proposed. 12. Using the experimental and theoretical results of the project, schemes for the treatment of human glioblastoma with the viral drug VV-GMCSF-Lact in monotherapy mode and in combination with chemotherapy drugs used in the clinic will be proposed. Thus, the expected results of the project are fully consistent with the world class of research, are of high scientific and social significance. If the research is successfully completed, an innovative drug for effective therapy of glioblastoma will appear on the market, which will improve the quality of life and increase the survival rate of patients with this diagnosis who have a poor prognosis when using standard treatment methods. Thus, a contribution will be made to the implementation of one of the tasks of the Action Plan of the Ministry of Health of the Russian Federation for the period 2019 - 2024 (approved by the Ministry of Health of the Russian Federation on January 28, 2019) to combat cancer.

Annotation of the results obtained in 2023
Virotherapy is currently one of the most promising technologies for the treatment of malignant neoplasms. However, despite the obvious advantages of this approach, there are a number of limitations, such as the ability of the tumor to evade the immune response even with its additional induction due to the spread of viral particles; the viscosity of the extracellular matrix, which affects the spread of viral particles from cell to cell; production of virus-neutralizing antibodies that reduce the effectiveness of virotherapy with repeated injections of the virus, etc. These limitations can be minimized by using viral drugs in combination with other therapeutic agents, in particular with chemotherapeutic drugs. The use of oncolytic viruses in combination with chemotherapy can enhance the antitumor effect due to the influence of the components on various signaling pathways and cell systems, for example, the DNA repair system, which is a key factor in the formation of resistance of tumor cells to chemotherapy [Kanai et.al., 2012]. At the same time, one of the most informative tools is a whole-transcriptome analysis of changes in gene expression in cells, tumors and tissues, which, in combination with modern bioinformatics approaches, makes it possible to describe not only changes in the levels of individual RNAs, but also to identify group characteristics, including key tumor, tissue and cellular processes. Thus, to develop the most effective treatment regimens, it is necessary to comprehensively study the antitumor effect of the drug, both in monotherapy and in combination with other antitumor agents, as well as investigation the signaling pathways and individual molecules involved in modulating the response to the therapy in order to form a list of additional targets, the impact on which will enhance the antitumor effect of the combination used. As a result of the third stage of the project (2023), the following work was carried out and results were obtained: 1. The antitumor efficacy of the recombinant vaccinia virus VV-GMCSF-Lact was assessed against tumors obtained by subcutaneous transplantation of immortalized glioblastoma cells and patient-derived cultures of human gliomas with high tumorogenic potential into immunodeficient mice. VV-GMCSF-Lact has been shown to have antitumor efficacy against human glioblastoma in vivo. Intratumoral administration of the virus completely eliminated 30% of U343 MG tumors and 10% of MG4ns tumors (personalized culture of human glioblastoma) https://doi.org/10.3390/life11101084. 2. Using a whole-transcriptome analysis of RNA from human glioblastoma xenografts transplanted into SCID mice, it was established that infection with the oncolytic virus VV-GMCSF-Lact causes the following changes: - In transplanted human glioma cells, transcription factors of the IRF, NFY, ESR families are activated and the activity of the transcription factor SOX2 is suppressed, which leads to activation of gene expression: interferon response; response to hypoxia; focal adhesion; membranes of the endocytic and phagocytic pathways, as well as suppression of the formation of the spindle and cell cycle. - In the stroma of transplanted tumors, the expression of genes controlled by transcription factors of the IRF, NFKB, STAT families is activated, and the expression of genes controlled by the factors SUZ12, MYOD1, CEBPA and MEF2C is suppressed. In this case, the interferon response genes are activated, the processes of response to hypoxia are suppressed, glycolysis is suppressed, as well as the processes of epithelial-mesenchymal transition. The transcriptome of the VV-GMCSF-Lact virus in infected tumors is represented by RNA products of the EL3, BR15R, BR8, etc. genes, which encode modulators of the immune response, as well as RNA of the recombinant CSN3 gene, encoding the proapoptotic peptide, lactaptin. 3. Generalization of the project results made it possible to formulate a cumulative scheme for modulating the activity of transcription factors and biological processes, as well as the connection of these processes with the sensitivity of cells to the cytotoxic effect of VV-GMCSF-Lact and, separately, with processes in glioblastoma metastases. Individual transcripts, the level of which changes during VV-GMCSF-Lact infection, were identified, and group characteristics of sets of transcripts, such as transcription factors, signaling cascades, biological processes, etc., were collected and analyzed for the entire data set. The data obtained indicate that the activity of transport processes, including transport involving the endoplasmic reticulum and the Golgi apparatus, reduces the cytotoxic effect of VV-GMCSF-Lact. At the same time, the activity of nuclear processing of Pol II transcripts and mRNA splicing increases the sensitivity of cells to the cytotoxic effect of the virus. Importantly, the expression of nuclear Pol II transcript processing and mRNA splicing genes is increased in human tumor metastasis cells transplanted into SCID mice. https://www.mdpi.com/2073-4409/12/22/2616. 4. A study of the antitumor efficacy of the oncolytic virus VV-GMCSF-Lact in monotherapy and in combination with temozolomide on a model of human glioblastoma xenografts was conducted. It was shown that VV-GMCSF-Lact, both in monotherapy and in combination with TMZ, has high antitumor efficacy against glioblastoma. No significant differences in the antitumor efficacy of VV-GMCSF-Lact in monotherapy and various combinations of the viral drug with TMZ were detected. TGI indices for the “VV-GMCSF-Lact”, “VV-GMCSF-Lact+TMZ” and “TMZ+VV-GMCSF-Lact” groups were 93%, 93% and 96%, respectively. However, according to histological analysis of tumors in experimental animals, it has been shown that the use of a combination of drugs (“VV-GMCSF-Lact+TMZ” and “TMZ+VV-GMCSF-Lact”) leads to more significant destruction of tumor tissue. Thus, VV-GMCSF-Lact in combination with temozolomide was shown to be effective against human glioblastoma in vivo. A treatment regimen has been proposed, which includes treatment of the resection field with an oncolytic virus and a subsequent course of temozolomide no earlier than 7 days after the use of VV-GMCSF-Lact. The results of the project were presented at the following conferences: 1. IV All-Russian scientific and practical conference “Development of physical and chemical biology and biotechnology at the present stage”, Irkutsk, October 2023, Kuligina E.V. “Pharmacological efficacy of the oncolytic virus VV-GMCSF-Lact against tumor models of gliomas in vitro and in vivo,” Oral presentation. 2. VIII All-Russian Conference on Molecular Oncology with international participation, December 20-22, 2023, Moscow, Vasilyeva N.S. "Oncolytic virus VV-GMCSF-Lact as a means of therapy for human gliomas", oral presentation 3. VIII All-Russian Conference on Molecular Oncology with international participation, December 20-22, 2023, Moscow, Ageenko A.B. "Efficacy of therapy for human glioblastoma with a combination of oncolytic virus VV-GMCSF-Lact and temozolomide", poster presentation All work planned for the third stage of the project has been completed in full. The results obtained correspond to the project objectives for 2023. References 1. Kanai R. и др. Oncolytic Virus-Mediated Manipulation of DNA Damage Responses: Synergy With Chemotherapy in Killing Glioblastoma Stem Cells // JNCI: Journal of the National Cancer Institute. 2012. V. 104. № 1. P. 42–55.

Publications

1. Dmitriy V. Semenov, Natalia S. Vasileva, Maya A. Dymova, Sergey V. Mishinov, Yulya I. Savinovskaya, Alisa B. Ageenko, Anton S. Dome, Nikita D. Zinchenko, Grigory A. Stepanov, Galina V. Kochneva, Vladimir A. Richter, and Elena V. Kuligina Transcriptome Changes in Glioma Cells upon Infection with the Oncolytic Virus VV-GMCSF-Lact MDPI, Cells 2023, 12(22), 2616 (year - 2023) https://doi.org/10.3390/cells12222616

Annotation of the results obtained in 2021
The currently available treatments for brain tumors do not significantly improve the quality and survival of patients. The development of effective methods of therapy for this type of tumor using tumor models that maximally preserve the molecular biological characteristics of the primary tumor (personalized cell cultures), as well as a detailed analysis of the mechanism of action of the proposed drugs are undoubtedly urgent tasks of modern biomedical research. During the project implementation, a collection of cell cultures obtained from patient tumor samples (patient-derived cultures) was created. For today the collection created includes 10 cultures of human glioma cells obtained from the samples of primary tumors of various grades of malignancy (6 cultures of glioblastoma of 4th grade of malignancy, 3 cultures of astrocytoma of 3rd grade of malignancy and 1 culture of oligodendroglioma of 3 grade of malignancy). Tumor samples and data of samples’ histological analysis were provided by Novosibirsk research institute of traumatology and orthopedics n.a. Ya.L. Tsivyan (Novosibirsk, Russia) with the patients’ informed consent. It was found that five personalized cultures are able to form neurospheres under cultivation in the absence of serum and with the addition of growth factors and nutritional supplements. The obtained cultures can be the basis for the development of a relevant tumor models for assessing the potential of antitumor agents in vitro and in vivo. Personalized cultures (both adherent and containing neurospheres), as well as immortalized U87 MG and U343 MG cultures were characterized by the present of cancer stem cell (CSC) markers: CD133, CD44, CD15, CD171, SOX2 and MYC, also the present of receptors epidermal growth factor (EGFR) and platelet-derived growth factor (PDGFRA). The representation of CD133, CD44, CD15, and CD171 and EGFR and PDGFRA receptors on cells was assessed by flow cytometry using appropriate antibodies. The level of SOX2 and MYC markers in the cells of the studied cultures was assessed by RT-PCR and Western blot analysis. It was shown that the levels of studied markers in the cell cultures under investigation vary significantly and do not depend on the primary tumor grade of malignancy. The study of the cytotoxic activity of the oncolytic virus – the recombinant vaccinia virus VV-GMCSF-Lact against the cells of the obtained cultures showed that VV-GMCSF-Lact effectively reduces the cell viability of all studied cultures. Astrocytoma BR2.20 and AS2 cells (grade 3) were the most sensitive to the virus action, while astrocytoma BR5.21 cells and glioblastoma BR4.21 (grade 4) cells were the most resistant. The sensitivity to the virus also differed in the pairs "adherent culture - culture containing neurospheres" of the respective cultures. It was found cell cultures which are more sensitive to the VV-GMCSF-Lact action are characterized by a larger population of cells carrying CSC markers, in particular, CD133 + / CD44 + cells. According to the data obtained, the sensitivity of cells to the oncotoxic VV-GMCSF-Lact action does not directly depend on the grade of tumor malignancy. Analysis of changes in the proliferative potential and migration activity of cells of the obtained patient-derived cultures of human glioma under the influence of VV-GMCSF-Lact showed that VV-GMCSF-Lact effectively suppresses cell proliferation of all studied glioma cultures, and also affects negatively their ability to migrate. The study of apoptotic processes in human glioma cells under the influence of the oncolytic virus VV-GMCSF-Lact showed that both necrotic and apoptotic pathways of cell death are realized in the cells of the studied patient-derived cultures. The cell death of immortalized glioblastoma cells under the virus action occurs predominantly by the necrosis pathway. Highly efficient RNA sequencing (Illumina 1500) of patient-derived culture cells (both adherent and containing neurospheres) of human brain tumors was carried out. Bioinformatic analysis of NGS data was evaluated, which allows a detailed assessment of the transcriptomes of these glioma cells: - to identify indicator transcripts, the level of which is significantly different in comparison with a large set of transcriptomes of human cells; - to identify diagnostic transcripts of glioblastoma, the level of which is increased or decreased in comparison with cell cultures - the nearest non-malignant transcriptome homologues; - to identify SNPs that were not previously associated with gliomas; - to describe the changes in alternative pre-mRNA / lncRNA splicing variants compared to transcripts of non-malignant brain cells; - to identify and catalog chromosomal translocations characteristic of the studied tumor cultures. Meta-analysis of the data from The Cancer Genome Atlas (TCGA) project allowed compiling a set of transcripts, the level of which in human brain tumor samples can be used as a diagnostic criterion for classifying tumors into glioblastoma and low-grade glioma; to identify a number of transcripts, the level of which positively or negatively correlates with the overall survival of patients. Thus, the tasks of the first stage of the project are fully completed: - a collection of patient-derived cultures of human glioma cells, including those containing neurospheres, was created; - the obtained glioma cultures were characterized by the present of cancer stem cell (CSC) markers and epidermal growth factor (EGFR) and platelet-derived growth factor (PDGFRA) receptors; - the effect of the VV-GMCSF-Lact oncolytic virus on the viability, proliferation and migration of immortalized and patient-derived glioma cell cultures was estimated; - a study of apoptotic processes in human glioma cells under the influence of the oncolytic virus VV-GMCSF-Lact was carried out; - a detailed study of transcriptomes of personalized cultures of human gliomas obtained in the project was carried out.; - identified potential diagnostic transcripts for the detection of glioma and differential diagnosis of glioma. Including: by levels of transcript sets; by single nucleotide polymorphisms; changes in splicing patterns, chromosomal translocation; - systems for RT-PCR analysis of mRNA and regulatory lncRNA levels in glioma have been developed, which can be used to study the oncolytic virus VV-GMCSF-Lact action at the next stages of the project; - the obtained data of NGS analysis can be used to identify the key factors of the pro-neural-mesenchymal transition of malignant brain tumors.

Publications

1. Natalia Vasileva, Alisa Ageenko, Maria Dmitrieva, Anna Nushtaeva, Sergey Mishinov, Galina Kochneva, Vladimir Richter and Elena Kuligina Double Recombinant Vaccinia Virus: a Candidate Drug against Human Glioblastoma Life, Т. 11, выпуск 10 (year - 2021) https://doi.org/10.3390/life11101084

2. Maya A. Dymova, Elena V. Kuligina and Vladimir A. Richter Molecular Mechanisms of Drug Resistance in Glioblastoma Int. J. Mol. Sci., Т. 22, выпуск 12, С.6385 (year - 2021) https://doi.org/10.3390/ijms22126385

3. Dome A.S., Stepanov G.A., Semenov D.V., Kuligina E.V., Richter V.A. Направленная регуляция экспрессии длинных некодирующих РНК и их роль в патогенезе глиобластомы III ОБЪЕДИНЕННЫЙ НАУЧНЫЙ ФОРУМ ФИЗИОЛОГОВ, БИОХИМИКОВ И МОЛЕКУЛЯРНЫХ БИОЛОГОВ, НАУЧНЫЕ ТРУДЫ, том 2, том 2, стр. 34 (year - 2021)

Annotation of the results obtained in 2022
The current mainstay of treatment for glioblastoma is a combination of maximal surgical resection, radiotherapy, and chemotherapy. Tumor resection reduces mass effect and provides tissue for subsequent histological analysis and molecular characterization [1]. However, recurrence occurs even with maximal surgical resection because glioma cells are able to migrate to distant locations in the brain and form extracranial metastases [2, 3]. The standard adjuvant chemotherapy for glioblastoma is temozolomide, which was approved for clinical use in 2005 [4]. Another type of chemotherapy to treat glioblastoma is also used - the PCV regimen. PCV is a combination of the alkylating agents procarbazine and lomustine (CCNU) and the microtubule inhibitor vincristine [5]. The implementation of the second phase of the project (2022) has carried out the following work and achieved results: 1. The sensitivity to the action of chemotherapeutic agents (temozolamide, lomustine, procarbazine, and vincristine) was assessed for the cells of immortalized human glioblastoma U87 MG and U343 MG lines and personalized BR2.20 and BR5.21 glioblastoma cultures obtained at the previous stage of the project. It was shown that the studied cultures have different sensitivity to chemotherapy drugs. U343 MG cells are the most sensitive to lomustine, vincristine and temozolomide. The most resistant to temozolomide are U87 MG cells, to vincristine and lomustine - BR5.21 cells. Cells of all the studied cultures are resistant to procarbazine. 2. The effect of VV-GMCSF-Lact in combination with temozolomide was analyzed for the cells of immortalized lines U87 MG and U343 MG and personalized cultures BR2.20 and BR5.21 with different sensitivity to VV-GMCSF-Lact. The sensitivity of the cells to the virus decreases in the line BR2.20>U 343 MG>U87 MG>BR5.21. 2.1. It was shown that the cytotoxic activity of the "VV-GMCSF-Lact - temozolomide" combination to the cells of the BR5.21 culture, the most resistant to VV-GMCSF-Lact, is higher than the oncotoxic activity of the virus in the monotherapy. The cells of the other cultures were resistant to the combination "VV-GMCSF-Lact - temozolomide" when temozolomide was added 24 hours after the virus. However, when temozolomide was added 60 hours after VV-GMCSF-Lact, a synergistic or additive effects of the drugs used were observed for all cell cultures, except U87 MG. Based on the data obtained, it can be concluded the vaccinia virus is able to inhibit the action of temozolomide. 2.2. When evaluating the antitumor efficacy of the combination "temozolomide - VV-GMCSF-Lact", when adding the virus 24 hours after temozolomide, such combination of drugs provides synergistic or additive effect for cells of the most cultures studied. However, to the BR2.20 cells, the most sensitive to VV-GMCSF-Lact, the use of the "temozolomide - VV-GMCSF-Lact" combination leads to the antagonistic effect of the drugs. Based on the data obtained, a combination therapy scheme was proposed: the use of temozolomide into the therapeutics should occur not earlier than 8–9 days after treatment of the resection field by VV-GMCSF-Lact, which will minimize the risk of inhibition of the temozolomide action by a viral drug. 3. To identify the genetic features, biological processes, and fundamental characteristics of human brain tumor cells under the VV-GMCSF-Lact therapy, a full-transcriptome analysis of RNA of cells of immortalized and personalized cultures of brain tumors before and after incubation with the virus was carried out. The time intervals of incubation of tumor cells with oncolytic virus (12 and 24 hours) and the multiplicity of viral infection (1 PFU per cell) were determined based on literature data and data of preliminary experiments. Arrays of experimental NGS data were obtained to describe changes in gene expression in cells of human brain tumors under conditions of VV-GMCSF-Lact infection. A unique, annotated base of reference sequences of the human genome (hg38) with the insertion of the VV-GMCSF-Lact genome was created, which allows for bioinformatic analysis of the levels of human cells RNA and, separately, the levels of RNA encoded by the virus genome, in infected cells. Data arrays were collected containing information on the contribution of individual transcripts (including viral ones) in the total set of cells RNAs of 4 personalized and 2 immortalized cultures of brain tumors and control non-malignant brain cells at different stages of VV-GMCSF-Lact infection. A detailed description of changes in the levels of viral transcripts during infection has been created, and viral genes whose activation is associated with the oncolytic effect of VV-GMCSF-Lact on human brain malignant tumor cells have been identified. A detailed description of changes in the levels of cellular transcripts has been created, groups of genes that are activated or suppressed during infection have been identified and described. In general, the results of bioinformatics analysis of the full transcriptome study data made it possible to create a detailed description of the processes of response to infection, to identify individual factors and their groups that determine the sensitivity of glioma/glioblastoma cells to the oncolytic effect of the VV-GMCSF-Lact virus. 4. Xenograft tumor models of glioblastoma have been developed using cells of personalized human glioblastoma cultures obtained at the first stage of the project. Xenograft models were obtained by transplantation of BR3.20 cells cultured under standard conditions and GK2.22ns cells (glioblastoma cell culture obtained from a patient's tumor sample in 2022) cultured under conditions of neurosphere formation into SCID mice. Cells of BR1.20ns, BR2.20ns, BR3.20ns, and BR5.21ns cultures were unable to initiate tumor formation. It is possible that the use of NSG mice, which lack an NK-cell response, for the development of tumor models will allow obtaining a wider panel of xenografts, however, the NSG line is currently not available in the SPF vivarium of the Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences. As part of the second stage of the project, 4 articles were published in peer-reviewed journals, 2 of them in journals of the first quartile (Q1) The results of the project were presented at the International Congress "Biotechnology: state and development prospects", October 31 - November 1, 2022, Moscow. Symposium: "Treatment of human oncological diseases with natural and recombinant viruses". Based on the results of the project, an article was published in "Science in Siberia" https://www.sbras.info/articles/nauka-dlya-obschestva/dannye-ob-izmenenii-transkriptoma-pomogut-razrabotat-metody-dlya and Interfax Russia https://www.interfax-russia.ru/view/virus-protiv-raka All works planned for the second stage of the project have been fulfielded. The results obtained are in line with the project objectives for 2022 References: 1. Omuro A, DeAngelis LM. Glioblastoma and other malignant gliomas: a clinical review. JAMA, 2013, Nov 6;310(17):1842-50. doi: 10.1001/jama.2013.280319. 2. A. Armento, J. Ehlers, S. Schötterl, U. Naumann, S. De Vleeschouwer. Molecular Mechanisms of Glioma Cell Motility. In: Glioblastoma [Internet]. Brisbane (AU): Codon Publications; 2017 Sep 27. Chapter 5. doi: 10.15586/codon.glioblastoma.2017.ch5 3. C. J Liu, G. A Shamsan, T. Akkin, D. J Odde. Glioma Cell Migration Dynamics in Brain Tissue Assessed by Multimodal Optical Imaging. Biophys J., 2019, Oct 1;117(7):1179-1188. doi: 10.1016/j.bpj.2019.08.010. 4. R. Stupp, W. P Mason, M. J van den Bent. Radiotherapy plus Concomitant and Adjuvant Temozolomide for Glioblastoma). N Engl J Med. 2005 Mar 10;352(10):987-96. doi: 10.1056/NEJMoa043330. 5. Solimando DA Jr, Waddell JA. Procarbazine, Lomustine, and Vincristine (PCV) Regimen for Central Nervous System Tumors, Hosp Pharm. 2017 Feb;52(2):98-104. doi: 10.1310/hpj5202-98.

Publications

1. Maya A. Dymova, Natalia S. Vasileva, Elena V. Kuligina, Yulya I. Savinovskaya, Nikita D. Zinchenko, Alisa B. Ageenko, Sergey V. Mishinov, Grigory A. Stepanov, Vladimir A. Richter and Dmitry V. Semenov MicroRNA and mRNA Expression Changes in Glioblastoma Cells Cultivated under Conditions of Neurosphere Formation Current Issues in Molecular Biology, том 44, номер 11, стр. 5294-5311 (year - 2022) https://doi.org/10.3390/cimb44110360

2. N. S. Vasileva, A. B. Ageenko, O. A. Chinak, V. A. Richter, and E. V. Kuligina РОЛЬ ИНГИБИТОРА АПОПТОЗА XIAP В УСТОЙЧИВОСТИ КЛЕТОК ГЛИОБЛАСТОМЫ К ДЕЙСТВИЮ РЕКОМБИНАНТНОГО ВИРУСА ОСПОВАКЦИНЫ VV-GMCSF-Lact Биотехнология, том 38, № 5, с. 73–79 (year - 2022) https://doi.org/10.56304/S0234275822050155

3. Natalia S. Vasileva , Elena V. Kuligina , Maya A. Dymova , Yulya I. Savinovskaya, Nikita D. Zinchenko, Alisa B. Ageenko, Sergey V. Mishinov, Anton S. Dome, Grigory A. Stepanov, Vladimir A. Richter and Dmitry V. Semenov Transcriptome Changes in Glioma Cells Cultivated under Conditions of Neurosphere Formation Cells, том 11, номер 19, стр. 2-24 (year - 2022) https://doi.org/10.3390/cells11193106

4. Anton Dome, Maya Dymova *, Vladimir Richter and Grigory Stepanov Post‐Transcriptional Modifications of RNA as Regulators of Apoptosis in Glioblastoma Int J Mol Sci, Том 23, Выпуск 16, стр.2 - 20 (year - 2022) https://doi.org/10.3390/ijms23169272

5. - Данные об изменении транскриптомапомогут разработать методы для лечения глиобластомы Наука в Сибири, 24 ноября 2022 г. (year - )

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