Funding: European Regional Development Fund (ERDF) “On Implementation of Activity 1.1.1.2 “Post-doctoral Research Aid” of the Specific Aid Objective 1.1.1 “To increase the research and innovative capacity of scientific institutions of Latvia and the ability to attract external financing, investing in human resources and infrastructure” of the Operational Programme “Growth and Employment”
Project Title: „Engineering of extracellular vesicles to transfer tumor suppressor miRNA to cancer cells”
Project No.: 1.1.1.2/VIAA/1/16/203
Period: 36 month (1st September 2017 – 31st August 2020)
Project costs: 133 806,00 EUR
Project implementer: Dr.biol. A. Ābols
The aim of this study is to engineer donor cells to produce EVs that contain therapeutically relevant miRNAs and target ER+ breast and thyroid cancer cells thus providing a proof-of-principle that engineered EVs can be exploited for targeted drug delivery in cancer.
This project is expected to deliver novel engineered EVs with a surface marker allowing to conjugate them with different cancer specific antibodies and enclosed tumour suppressor miRNAs in order to treat different cancer types. These EVs will serve as the basis for the development of new therapies for different types of cancer.
Information published: 01.09.2017.
Progress of the project
1 September 2017 – 30 November 2017
The latest literature was studied and detailed plan of the project execution was developed during these months. The plan was presented to the scientific consultant. Also, the topic of the project was explained to the general public in non – scientific language during “Zinātnieku nakts” event. The modification of the stem cells was started by selecting the best gene candidates and modification protocols from literature and schematic overview of the modified genes were developed.
Information published: 30.11.2017.
Progress of the project
1 December 2017 – 28 February 2018
During this period there was created cell cultures stocks used in this project and tested for mycoplasma. In order to create hybrid proteins primers were designed and some fragments were already amplified, trimmed and purified for further manipulations. Also, there was prepared and presented lecture about project aims and topics in scientific forum “Ko nozīmē būt zinātniekam” and organised several excursions and demonstrations for students interested in this field during these months. Preparation for the next years “Zinātnieku nakts” event was started.
Information published: 28.02.2018.
Progress of the project
1 March 2018 – 31 May 2018
During this period there was finished cell cultures stocks used in this project and created three lentivirus vectors with different hybrid protein genes. They where retransformed, amplified, purified and prepared for packaging in order to modify mesenchymal stem cells. Also, there was prepared and presented lecture about project aims and topics for student corporations and project achievements where discussed with project scientific consultant as planed in project description.
Information published: 31.05.2018.
Progress of the project
1 June 2018 – 31 August 2018
HEK293T cells where prepared for packaging of lentivirus plasmids during this period. Cells where grown till certain density and numbers in specialised medium, followed by transfection of plasmids, cultivation for virus particle production, virus particle extraction from media, quantification, confirmation, that virus particles contain plasmid of interest. Samples where aliquoted and frozen for further applications.
Also during this period project topic and aims with first results were presented in student council of Latvian Biomedical research and study centre organised seminar: “Discoveries of cancer research in the last years”. Project achievements where discussed with project scientific consultant as planed in project description.
Information published: 31.08.2018.
Progress of the project
1 September, 2018 – 31 November, 2018
Mesenchimal stem cells used in project where infected with various concentrations of previously produced lentivirus particles in order to find the most optimal virus to cell ratio. Several additional experiments where performed to increase the efficiency of infection due to the poor infection results. After infection cells are continuously grown in media with previously selected puromicyn concentration, that will help to select the cell clones that contain gene of interest. Also, during this period project topics where explained in popular scientific language to public in “Zinātnieku nakts 2018” and project progress was discussed with project scientific consultant according to project description.
Information published 30.11.2018.
Progress of the project
1 December, 2018 – 28 February, 2019
After second infection attempt with freshly made viruses cells where cultivated into selective media for a few weeks. Infection was successful based on the control virus and ZsGreen protein expression into target cells. Infected cells where amplified, counted and frozen into aliquots for future experiments. Few aliquots where grown in huge amounts into adherent and suspension cultures for vesicle isolation. Cells where collected to isolate DNA and RNA to test hybrid gene cloning and expression on mRNA level by PCR method. Vesicles where quantified by BSA test and size was determined by NTA analysis. Result of experiments where discussed with scientific consultant of the project according to project plan. Also few secondary school students were introduced into daily laboratory work and theme of the project that visited BMC on “shadow day”.
Information published 28.02.2019.
Progress of the project
1 March, 2019 – 31 May, 2019
Huge batch of modified EVs both from adherent and suspension cultures where produced and isolated from cell media in order to optimise WB method for hybrid protein expression and localisation confirmation. In parallel lentivirus vector was designed that contains three tumor-suppressor miRNA with EVs sorting sequence and potential company, that can produce such lentivirus vector were found. Result of experiments where discussed with scientific consultant of the project according to project plan. In this period several excursions for secondary school students where supervised in BMC with the aim to increase recognisability of the project between non scientific society. Also ZPD was defended that was developed within this project and was nominated with the 3. level degree in country.
Information published 31.05.2019.
Progress of the project
1 June, 2019 – 31 Augusts, 2019
Poster presentation was prepared and presented on conference: “THE FIRST ISEV & MRS JOINT EXTRACELLULAR VESICLE CONFERENCE FOCUSED ON CANCER”, Nashville, TN, USA, 2.08 – 4.082019. Also lentivirus vector containing tumor suppresor miRNA with EV sorting signal vector was redesigned to increase infection and cell growth success. Vector was ordered from company capable to designs such vector and new cell stocks where prepared for next infection experiments. Finally – plan of interactive game was designed for Science night in order to introduce society with project theme in a popular scientific language. Result of experiments where discussed with scientific consultant of the project according to project plan.
Information published 30.08.2019.
Progress of the project
1 September, 2019 – 30 November, 2019
Lentivirus vector containing tumor suppresor miRNA was amplified and purified, followed by virus construction and previously modified (with hybrid genes) mesenchimal stem cell modification. Additionally the same cells where infected with viruss conatining control vector without tumour supressor miRNA. Next – cancer cell lines where grown to test modified mesenchimal stem cell extracellular vesicles. Finally – supervision of interactive game during Science night in order to introduce society with project theme in a popular scientific language. Result of experiments where discussed with scientific consultant of the project according to project plan.
Information published 29.11.2019.
Progress of the project
1 December, 2019 – 29 February, 2020
Tumor suppresor miRNA-206 cloning and increased EV sorting into MSC was confirmed by RT-qPCR. Results where presented in the 78th international scientific conference of the University of Latvia, in the molecular biology session. Also cell lines containing hybrid gene and tumour suppressor miRNA where grown, isolated EVs and conjugated with antibodies against cancer surface markers in order to test their uptake and functionality. 8 secondary school students on national career day “shadow day” was supervised and introduced into project focus in popular scientific language. Result of experiments where discussed with scientific consultant of the project according to project plan.
Information published 28.02.2020.
Progress of the project
1 March, 2020 – 31 May, 2020
During this period, samples were prepared in the project for high-resolution flow cytometric analysis to determine what percentage of the modified vesicles contained the modified protein on the surface and whether it was able to functionally bind Ab. Stem cell modified extracellular vesicles were tested on breast cancer cell lines MCF7, MB-MDA-231 and thyroid cancer cell line 8305C. Decreased proliferation was observed in MCF7 cell lines and decreased expression of miRNA-206 target genes, whereas in MB-MDA231 and 8305C cells, no such effect was observed as expected. No differences were observed between modified EVs conjugated with or without antibodies to the target cell antigen EpCAM. In parallel, a publication manuscript on the use of stem cell vesicles in tumor therapy was prepared and submitted for publication and accepted. The scientific results of the project were discussed with the project’s scientific consultants in accordance with the project plan.
Information published 29.05.2020.
Progress of the project
1 June, 2020 – 31 August, 2020
During this period, modified extracellular vesicles of stem cells were tested on breast cancer cell lines MCF7 and MB-MDA-231. Reduced migration of MCF7 cells was observed, as well as reduced transcription of miRNA-206 target genes such as ESR1, VEGFA, CCND1 while ESR2 expression was not altered. This effect was not observed in MB-MDA231 cells as expected. At the same time, a poster on the use of stem cell vesicles in tumour therapy was prepared and presented for ISCT 2020 Paris Virtual. The scientific results of the project were discussed with the project’s scientific consultants in accordance with the project plan.
Information published 31.08.2020.