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Project titleDevelopment of a novel nanoparticle formulation for mRNA based therapeutic vaccination

KeywordsHIV, mRNA, vaccine, non-viral delivery, polymeric microcapsules, immune response, antigen, antibody, prodrugs, dendritic cells

Annotation
The project is aimed at solving the most important tasks of medical immunology and bioengineering - the creation of an effective means of delivering mRNA vaccines for therapeutic vaccination for HIV infection based on hybrid microcapsules. Despite the great advances in antiretroviral therapy (ART), the HIV1 pandemic remains one of the main challenges to the global health care system. AIDS ranks 6th on the list of causes of death, taking more than 1 million lives annually. One promising new approach designed to improve the ART therapy is therapeutic vaccination of HIV-infected people, aimed at restoring the patient’s specific T-cell response to HIV and reducing the HIV reservoir in the infected organism. Among a number of tested therapeutic vaccination strategies, it is mRNA vaccines that seem to be the most promising approach. The peroral route of therapeutic mRNA vaccination, planned for research in the proposed project, is very promising and new, because gut-associated lymphoid tissue (GALT) is one of the most important organism reservoirs of HIV. With any vaccine administration strategy, a key factor in its final effectiveness is the choice of mRNA delivery vehicle to target antigen-presenting cells. In the proposed project, we plan to use polyelectrolyte nano and microcapsules. The unique advantages of the mRNA encapsulation technology are the large capacity of encapsulation, low toxicity, biodegradability, a high level of capture of microcapsules by antigen-presenting cells, effective release of mRNA from endosomes and the possibility of additional chemical modification of the surface of the capsules. The prospect of using polyelectrolyte microcapsules for therapeutic mRNA vaccination in HIV infection (with RNA sequences of model antigens, tumor antigens and HIV antigens) in this project will be evaluated in numerous in vitro and in vivo immunological tests, including dendritic cells of different origin, normal and transgenic mice, and at the final stage of the work of macaques as the most relevant animal model of HIV infection. In general, using the above approaches, we plan to achieve significant progress in solving the extremely urgent and significant problem of creating an effective therapeutic mRNA - a vaccine for HIV infection.

Expected results
The main result of this project should be the solution of the most important task of creating an effective means of delivering mRNA sequences of HIV antigens in vivo for therapeutic vaccination. This task is of fundamental and practical medical importance in the fight against the HIV pandemic. It is the limitations associated with the low efficiency of the delivery of functional mRNA to target antigen-presenting cells that are the main obstacle to the successful introduction of therapeutic mRNA vaccines into the medical practice of treating HIV infection. It is planned to develop an effective procedure for the synthesis of polyelectrolyte microcapsules coated with a silicate shell using the sol-gel technology, and their modification for peroral administration. During the project, a comprehensive characterization of the preparation being developed (physicochemical properties, stability, cytotoxicity, cell uptake, expression of the target antigen, biodistribution in vivo with different routes of administration) will be carried out, as well as a large set of modern immunological tests to assess the immunogenicity of the encapsulated mRNA vaccines. For the most complete assessment of the immunogenicity of a candidate vaccine, various in vitro and in vivo models will be used (dendritic cells originating from human, mice, macaques, normal and transgenic mice, primates) and a number of immunological analysis methods - ELISPOT, flow cytometry, immunohistochemistry, fluorescent microscopy, in vivo imaging, tetramer analysis. In general, these studies will reliably and fully assess the potential for practical use of the encapsulated mRNA vaccine under study.

Annotation of the results obtained in 2022
In 2022, studies were conducted on the immunogenicity of mRNAs encapsulated in non-viral delivery systems being developed during the project. The experiments were carried out on model objects: immunocompetent human cells and laboratory animals. Data were obtained on three types of carriers for non-viral delivery of genetic material. Based on the data, it was found that of all candidate carriers, nanoparticles based on cationic lipids currently demonstrate the highest transfection activity, despite the low immunogenicity and toxicity of all delivery systems developed under the project. The research results obtained in the third year of the project were published in two high-ranking journals (Q1, Q2): Applied Materials Today (Postovalova et al., 2022) IF = 8.66, ACS Applied Biomaterials (Yakubova et al., 2022) IF= 3.25. Oral and poster presentations were also presented at the XVI International Symposium “Hematopoietic stem cell transplantation. Gene and Cell Therapy”, St. Petersburg September 15-17, 2022. These works helped in the implementation of the overall project, making it possible to evaluate various variants of carriers for the delivery of mRNA. Also, in the course of the research, it was possible to carry out work in vivo with the study of mRNA delivery systems in laboratory animals using imaging systems and fluorescent markers. The final experiments were carried out jointly with Belgian partners on the analysis of the immune response of capsules in the lymph nodes of laboratory animals showed that antigen expression is observed in the lymph nodes of animals. According to these data, the final joint publication with the title of “Immune response of polymer based nano- and microcarriers: in vitro and in vivo studies” will be submitted in the journal of ACS Applied Materials and Interfaces, which will be published after the end of the project next year. Thus, the work planned during the reporting period for execution by the research team of the project on the basis of SPbPU was completed in full.

Publications

1. Postovalova A.S., Karpov T.E., Akhmetova D.R., Rodimova S., Kuznetsova D. S., Antuganov D. O., Sysoev D. S., Muslimov A. R., Stanzhevsky A. A., Zyuzin M. V., Timin A.S. Preclinical studies of automated radiolabeled microcarriers for radiosynovectomy of inflammatory joint disease Elsevier, Applied Materials Today 29 (2022) 101571 (year - 2022) https://doi.org/10.1016/j.apmt.2022.101571

2. Talianov P. M., Yakubova A. A., Bukreeva A., Masharin M., Eliseev I. E., Zelenkov L., Muslimov A. R., Bukatin A.,Gordeeva A., Kudryavtseva V., Makarov S. V., Sukhorukov G. B., Timin A. S., and Zyuzin M. V. Incorporation of Perovskite Nanocrystals into Polymer Matrix for Enhanced Stability in Biological Media: In Vitro and In Vivo Studies American Chemical Society, ACS Appl. Bio Mater. 2022, 5, 5, 2411–2420 (year - 2022) https://doi.org/10.1021/acsabm.2c00295

Annotation of the results obtained in 2020
During the implementation of the project in 2020 the studies have been carried out to develop protocols for the synthesis of hybrid capsules for efficient encapsulation of biologically active compounds (plasmid DNA and messenger RNA of green fluorescent protein (GFP), followed by assessment of the biological activity of encapsulated substances in cell models in vitro. A comprehensive analysis of the structure and morphology of the obtained hybrid capsules has been performed using electron microscopy and confocal laser microscopy. The optimal conditions for the synthesis of hybrid capsules using the sol-gel technology were optimized. It was found that the maximum percentage (~ 95%) of the cells with internalized capsules was when the ratio of capsules to the cell equaled to 50:1. It should also be noted that the capsules do not show any cytotoxic effect at a concentration of more than 100 capsules per cell. Experimental data on the transfection capacity of hybrid capsules have been obtained using different cell types with capsules containing mRNA and pDNA encoding eGFP. Hybrid capsules are more efficient to deliver genetic materials compared to non-viral physical method such as electroporation. The results of the current studies were published in two high impact-factor journals such as “Journal of Materials Chemistry B” (Tarakanchikova et al., 2020) IF = 5.344 and “Biomaterials” (Mashel et al., 2020) IF = 10.317. Also the results were presented on the international conference Gratitude XIV Raisa Gorbacheva Memorial Meeting “Hematopoietic Stem Cell Transplantation. Gene and Cellular therapy ”.

Publications

1. T. V. Mashel, Y. V. Tarakanchikova, A. R. Muslimov, M. V. Zyuzin, A. S. Timin, K. V. Lepik, B. Fehse Overcoming the delivery problem for therapeutic genome editing: Current status and perspective of non-viral methods ELSEVIER, Biomaterials, - (year - 2020) https://doi.org/10.1016/j.biomaterials.2020.120282

2. Y. Tarakanchikova, A. Muslimov, I. Sergeev, K. Lepik, N. Yolshin, A. Goncharenko, K. Vasilyev, I. Eliseev, A. Bukatin, V. Sergeev, S. Pavlov, A. Popov, I. Meglinski, B. Afanasiev, B. Parakhonskiy, G. Sukhorukov and D. Gorin A highly efficient and safe gene delivery platform based on polyelectrolyte core–shell nanoparticles for hard-to-transfect clinically relevant cell types Journal of Materials Chemistry B, - (year - 2020) https://doi.org/10.1039/D0TB01359E

3. Y. V. Tarakanchikova, A. R. Muslimov, M. V. Zyuzin, I. Nazarenko, A. S. Timin, G. B. Sukhorukov, K. V. Lepik Layer-by-Layer-Assembled Capsule Size Affects the Efficiency of Packaging and Delivery of Different Genetic Cargo Wiley, - (year - 2021) https://doi.org/10.1002/ppsc.202000228

Annotation of the results obtained in 2021
During the implementation of the project in 2021, studies were carried out to assess the immunogenicity of encapsulated nucleic acids (plasmid DNA and messenger RNA of green fluorescent protein (GFP)) during their capture by human immunocompetent cells. A comprehensive assessment of immunological reactions was carried out using the methods of enzyme-linked immunosorbent assay (ELISA, flow cytometry) (by the SPbPU team of researchers together with Vrije Universiteit Brussel). Studies were carried out on the dynamics of the biodistribution of microcapsules inside the body of laboratory mice. According to the data obtained, microcapsules accumulate in the tissues of the lungs for 10-15 days. The absence of pathomorphological changes in tissues with the accumulation of carriers in them was confirmed. The results of the studies obtained in the second year of the project were published in «Materials Science and Engineering: C» (Tarakanchikova et al., 2021) IF = 7.328, «Expert Opinion on Drug Delivery» (Linnik et al., 2021) IF = 6.648 и «Particle & Particle Systems Characterization» (Tarakanchikova et al., 2021) IF = 3.310.

Publications

1. Tarakanchikova Y.V., Linnik D.S., Mashel T., Muslimov A.R., Pavlov S., Lepik K.V., Zyuzin M.V., Sukhorukov G.B., Timin A.S. Boosting transfection efficiency: A systematic study using layer-by-layer based gene delivery platform Materials Science and Engineering C, volume 126, P. 112161 (year - 2021) https://doi.org/10.1016/j.msec.2021.112161

2. Linnik D.S.,Tarakanchikova Y.V., Zyuzin M.V., Lepik K.V., Aerts J.L., Sukhorukov G., Timin A.S. Layer-by-Layer technique as a versatile tool for gene delivery applications Expert Opinion on Drug Delivery, volume 18 Issue8 Page1047-1065 (year - 2021) https://doi.org/10.1080/17425247.2021.1879790