Method of packaging a protein, an RNA or a protein-RNA complex into a particle

Abstract

The invention relates to the field of medicine, and more particularly to bioengineering, genetic engineering, gene editing, molecular medicine, nanotechnology, biotechnology, nanoengineering and protein engineering. The invention can be used for packaging Cas proteins and guide RNA (guide RNA, single-guide RNA, crRNA, tracrRNA), separately or together in the form of ribonucleoprotein complexes, and for packaging a Cas protein and a guide RNA of any class, type, form, origin and modification, as well as for packaging any proteins and RNA of any structure and sequence.

Description

[0001] A method of packaging a protein, RNA, or protein-RNA complex into a particle

[0002] Field of technology

[0003] The invention relates to the field of medicine, namely bioengineering, genetic engineering, gene editing, molecular medicine, nanotechnology, biotechnology, nanoengineering and protein engineering. The invention can be used for packaging Cas proteins and guide RNA guides (guide RNA, single-guide RNA, crRNA, tracrRNA) separately or together in the form of ribonucleoprotein complexes, for packaging Cas protein and guide RNA of any class, type, kind, origin and modification, as well as for packaging any proteins and RNA of any structure and sequence.

[0004] State of the art

[0005] CRISPR / Cas systems are tools for controlling and modifying DNA or RNA that allow for genetic (changes in the nucleotide sequence of nucleic acids), epigenetic (introduction or removal of chemical changes in histone and non-histone proteins associated with DNA, or directly in DNA nucleotides), epitranscriptomic (introduction or removal of chemical modifications in RNA) and protein modifications, which are actively used to develop new methods for treating human diseases (hereditary, infectious, oncological, genetic, metabolic, etc.), creating new and improved strains of microorganisms, plants, animals, creating animal models, cells, tissues and human organs, etc. [Brezgin, S., Kostyusheva, A., Kostyushev, D., & Chulanov, V. (2019). Dead Cas Systems: Types, Principles, and Applications. International journal of molecular sciences, 20(23), 6041. https: / / doi.org / 10.3390 / ijms20236041]

[0006] There are thousands of variants of CRISPR / Cas systems and their analogs in eukaryotic and prokaryotic organisms [Makarova, KS, et al. "Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants" / / Nature Reviews Microbiology 18.2 (2020): 67-83]. There are two main classes of CRISPR / Cas (1 and 2). Class 1 includes 3 types of systems (I-III). For type I, the signature protein is the Cas3 protein, the effector complex consists of a complex of various proteins that have activity against single-stranded DNA, while the proteins can exhibit collateral activity (the ability to cut or modify nucleic acids in the environment). Type III includes systems that can target both DNA and RNA, while the effector complex consists of a complex of proteins, of which the CaslO protein is the signature. For type IV, the signature protein is Csfl, which is part of a multisubunit complex.In class 2, the main types of systems II, V and VI are distinguished, the signature proteins are Cas9, Casl2, Casl3 and Cas 14, which are active against double-stranded DNA, single-stranded DNA, and may or may not exhibit collateral activity [Kostyusheva, A., Brezgin, S., Babin, Y, Vasilyeva, I., Glebe, D., Kostyushev, D., & Chulanov, V. (2022). CRISPR-Cas systems for diagnosing infectious diseases. Methods (San Diego, Calif.), 203, 431-446. https: / / doi.Org / 10.1016 / j.ymeth.2021.04.007]. At the same time, every year dozens of new CRISPR / Cas systems are described with various characteristics, which can differ significantly in properties, composition, principles of targeting (recognition of the target site and action on the target site), enzymatic activity and size.

[0007] In addition, using the methods of directed evolution, rational engineering, in silico design methods, machine learning technologies, and site-directed mutagenesis technologies, it is possible to introduce various modifications, add additional components, remove components, or modify both Cas proteins (or components of complexes with Cas proteins or Cas-like proteins) and guide RNA guides (sgRNA, gRNA, crRNA, tracrRNA, or their variants) [Lee M. (2023). Deep learning in CRISPR-Cas systems: a review of recent studies. Frontiers in bioengineering and biotechnology, 11, 1226182. https: / / doi.org / 10.3389 / fbioe.2023.1226182].Cas proteins can also be used as split proteins or in combination with affinity interaction systems, due to dimerization or dissociation domains, degradation domains upon introduction or removal of a stimulus, which allows their functioning to be regulated by switching them on or off, or by a combined action [Wright, A. V, Sternberg, S. H., Taylor, D. W., Staahl, B. T., Bardales, J. A., Kornfeld, J. E., & Doudna, J. A. (2015). Rational design of a split-Cas9 enzyme complex. Proceedings of the National Academy of Sciences of the United States of America, 112(10), 2984–2989. https: / / doi.org / 10.1073 / pnas.1501698112jf Aschenbrenner, S., Kallenberger, S. M., Hoffmann, M. D., Huck, A., Eils, R., & Niopek, D. (2020). Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 target specificity. Science advances, 6(6), eaay0187. https: / / doi.org / 10.1126 / sciadv.aay0187].Modifications to components of CRISPR / Cas systems can significantly alter their properties and adapt them to perform new functions, such as editing nucleotide bases, changing DNA and RNA sequences, introducing epigenetic modifications, epitranscriptomic modifications, visualizing DNA or RNA sequences, editing proteins, changing the three-dimensional structure of chromatin, attracting proteins and RNA with enzymatic activity to specified loci of DNA, RNA, proteins or components of cells, tissues, organs and organ systems.

[0008] CRISPR / Cas systems can be used to introduce certain modifications into the body's biomolecules to treat hereditary diseases, metabolic disorders, correct genetic mutations, start or block genes, modulate gene activity, change the three-dimensional localization of chromatin, change the composition and properties of DNA, RNA, proteins and their complexes. Their use in humans and animals is possible for the treatment of infectious diseases: stopping viral replication, blocking viral infections, eliminating viruses in acute and chronic viral infections.They can also be used to make beneficial changes in the body to improve the properties of cells, tissues, organs and / or organ systems, eliminate negative changes (e.g., age-related) or improve the original characteristics of humans and animals, including in the fields of medicine, cosmetology, sports and high-performance sports, mental abilities, in the creation of new species of animals and producer cells, as well as in the modification of animal and human cells, the treatment of cancer, correcting tumor resistance genes to drugs, overcoming the resistance of microorganisms to antibacterial drugs, increasing the sensitivity of tumors and healthy tissues to the action of drugs, improving the absorption of drugs, nutrients, high-molecular compounds, low-molecular compounds, nanocarriers and their combinations [Mir, A., Edraki, A., Lee, J., & Sontheimer, E. J. (2018).Туре П-С CRISPR-Cas9 biology, mechanism, and application. ACS chemical biology, 13(2), 357-365].

[0009] Selected approaches to correct genetic mutations in genes such as PCSK9, BCL11A, HBG1 / HBG2 for the treatment of sickle cell anemia and beta-thalassemia, ATTR for the treatment of transthyretin amyloidosis, as well as for modifying T cells and treating human immunodeficiency virus-1 infection are already in early stages of clinical trials [H. Frangoul, D. Altshuler, MD Cappellini, Y.-S. Chen, J. Domm, BK Eustace, J. Foell, J. de la Fuente, S. Grupp, R. Handgretinger, CRISPR-Cas9 gene editing for sickle cell disease and P-thalassemia, N. Engl. J. Med. 384 (2021) 252-260] [A. Sharma, J. -J. Boelens, M. Cancio, J. S. Hankins, P. Bhad, M. Azizy, A. Lewandowski, X. Zhao, S. Chitnis, R. Peddinti, CRISPR-Cas9 editing of the HBG1 and HBG2 promoters to treat sickle cell disease, N. Engl. J. Med. 389 (2023) 820-832.] [J. D. Gillmore, E. Gane, J. Taubel, J. Kao, M. Fontana, M. L. Maitland, J. Seitzer, D. O'Connell, K. R. Walsh, K.Wood, CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis, N. Engl. J. Med. (2021). ][ L. Xu, J. Wang, Y. Liu, L. Xie, B. Su, D. Mou, L. Wang, T. Liu, X. Wang, B. Zhang, CRISPR-edited stem cells in a patient with HIV and acute lymphocytic leukemia, N. Engl. J. Med. 381 (2019) 1240-1247. ][ D. Kostyushev, A. Kostyusheva, S. Brezgin, N. Ponomareva, NF Zakirova, A. Egorshina, D. V Yanvarev, E. Bayurova, A. Sudina, I. Goptar, Depleting hepatitis B virus relaxed circular DNA is necessary for resolution of infection by CRISPR-Cas9, Mol. Ther. Acids. 31 (2023) 482-493.]. Thousands of approaches have also been created to treat diseases and other disorders in human health, as well as to create new species and breeds of animals.

[0010] CRISPR / Cas systems for the above-mentioned purposes can be used in the form of coding DNA (usually coding plasmids or viral vectors), mRNA and RNA, as well as complexes of the Cas protein with a guide RNA guide, or in other variants where one of the above-mentioned types of use appears [Kostyushev, Dmitry, et al. "Gene editing by extracellular vesicles." International Journal of Molecular Sciences 21.19 (2020): 7362.].Delivery of CRISPR / Cas coding elements is possible through the use of viral vectors (integrating, non-integrating, or viruses that replicate both with the integration of genetic material into the cell genome and in episomal form), such as adeno-associated viral vectors (AAV) of various serotypes, from different species of organisms and with different genetic modifications, adenoviruses (AdV), herpesviruses (e.g., HSV-1), lentiviruses, retroviruses, as well as with the help of coding DNA sequences, vectors, episomes, minicircles, etc. [Asmamaw Mengstie M. Viral vectors for the in vivo delivery of crispr components: advances and challenges / / Frontiers in Bioengineering and Biotechnology. - 2022. - V. 10. - C. 895713.] Delivery in the form of coding mRNA and / or RNA is usually carried out using non-viral methods such as LNPs, liposomes, polymeric compounds, polyplexes, etc. [Miller, Jason B., et al."Non-viral CRISPR / Cas gene editing in vitro and in vivo enabled by synthetic nanoparticle co-delivery of Cas9 mRNA and sgRNA." Angewandte Chemie 129.4 (2017): 1079–1083.] It is known that expression of proteins and, in particular, CRISPR / Cas systems with coding DNA is accompanied by difficult-to-control expression, which is fraught with the induction of side reactions, including off-target cleavage [Fu, Yanfang, et al. "High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells." Nature biotechnology 31.9 (2013): 822–826.]. Delivery in the form of mRNA / RNA is safer, but has a shorter expression duration, requires synchronization of protein synthesis with mRNA in the presence of a guide RNA, while mRNA / RNA themselves are unstable in cells, require chemical stabilization, which significantly increases the cost of their production process, and also increases the toxicity of the approach [Yin, Hao, et al. "Structure-guided chemical modification of guide RNA enables potent non-viral in vivo genome editing." Nature biotechnology 35.12 (2017): 1179-1187.]. It is known that ribonucleoprotein complexes (complexes of the Cas protein with the guide RNA, or RNP) have the highest activity, the highest speed of action, their use is associated with the lowest probability of off-target effects on non-target molecules, while RNP complexes are short-lived and, as a rule, the duration of their existence inside cells is no more than 24 hours, but can be increased or decreased artificially [Chen, Sean, et al. "Highly efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes." Journal of Biological Chemistry 291.28 (2016): 14457-14467.]. In addition, delivery in the form of RNPs does not require the synthesis of a Cas protein foreign to humans by the cell's translational apparatus, which significantly reduces the likelihood of T-cell immune clearance of the cells into which the RNPs were delivered [Chew, Wei Leong. "Immunity to CRISPR Cas9 and Casl2a therapeutics." Wiley Interdisciplinary Reviews: Systems Biology and Medicine 10.1 (2018): el408.]. Thus, delivery as RNPs is the most preferred option for using CRISPR / Cas.

[0011] However, to date, there are no effective methods for packaging CRISPR / Cas RNP complexes into micro- and nanoparticles. Micro- and nanoparticles are carriers that can consist of materials of organic (natural, synthetic or semi-synthetic), inorganic origin, their derivatives or combinations. The most well-known include liposomes, micro- and nanoparticles based on cationic lipids, amphiphilic peptides, DNA nanoclaws, gold nanoparticles, graphene-based nanoparticles using CPP (cell-penetrating peptides), as well as biological nanoparticles (exosomes, virus-like particles - VLPs) or biomimetic nanoparticles, as well as hybrid nanoparticles [Kostyushev, Dmitry, et al. "Gene editing by extracellular vesicles." International Journal of Molecular Sciences 21.19 (2020): 7362.].Packaging of RNPs into such types of nanoparticles is possible due to rational design of nanoparticles, modification of RNPs using chemical (treatment with surface-active compounds, etc.), physical (electroporation, freezing, sonication, etc., the principle is the formation of pores in the membranes of nanoparticles) or genetic (in the case of biological nanoparticles) methods. The highest efficiency of genetic editing was demonstrated by VLPs, into which it was possible to load up to 100 RNP molecules, while pseudotyping of VLPs with vesicular stomatitis virus glycoprotein (VSV-G) ensured high efficiency of transduction of human and mammalian cells [Banskota, Samagya, et al. "Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins." Cell 185.2 (2022): 250-265.].However, VLPs are immunogenic nanocarriers that induce an immunological response and are effectively captured and destroyed by macrophages of the reticuloendothelial system (RES), which reduces the efficiency of delivery even with the first administration of the drug before the formation of an adaptive immune response [Mohsen, Mona O., et al. "Interaction of viral capsid-derived virus-like particles (VLPs) with the innate immune system." Vaccines 6.3 (2018): 37.]. The result of the response to immunogenic virus particles are severe immune-mediated adverse reactions (hepatotoxicity, neurotoxicity, nephrotoxicity, etc.) up to the death of patients (cases of death of patients who received high and low doses of adeno-associated gene therapy, adenovirus-based gene therapy, etc., from systemic or local immune and inflammatory reactions) [Lek, Angela, et al. "Death after High-Dose rAAV9 Gene Therapy in a Patient with Duchenne's Muscular Dystrophy." New England Journal of Medicine 389.13 (2023): 1203-1210.]. At the same time, the disadvantages of other nanocarriers include high immunogenicity, molecular, cellular and tissue toxicity, the lack of universal methods for packaging any variants of CRISPR / Cas RNP, and, often, the inability to simultaneously package the Cas protein and the guide RNA. Indeed, it was previously demonstrated that Cas proteins can be packaged into secreted exosomes or extracellular vesicles: packaging can occur stochastically (randomly) or due to special inducers (treatment with light of a certain wavelength, a chemical compound) [Chen, Ran, et al. "Friend or foe? Evidence indicates endogenous exosomes can deliver functional gRNA and Cas9 protein." Small 15.38 (2019): 1902686.].In this case, co-packaging of the guide RNA occurs either randomly or requires the creation of complex genetic constructs based on ribozymes, components of bacterial hairpin recognition complexes, or similar technologies [Gee, Peter, et al. "Extracellular nanovesicles for packaging of CRISPR-Cas9 protein and sgRNA to induce therapeutic exon skipping." Nature communications 11.1 (2020): 1334.].

[0012] Biological nanoparticles are a general term for nanocarriers created on the basis of or using components of biological origin (biomimetics) [Parodi, Alessandro, et al. "Synthetic nanoparticles functionalized with biomimetic leukocyte membranes possess cell-like functions." Nature nanotechnology 8.1 (2013): 61-68.]. The best known biological nanoparticles are exosomes (extracellular vesicles, or EVs) 50-150 nm in size, secreted by all types of human cells. Secreted biological nanoparticles also include various microvesicles (100-1000 nm in size), oncosomes (1000-10,000 nm in size), and apoptotic bodies (50-5000 nm in size) [Kostyushev, Dmitry, et al. "Gene editing by extracellular vesicles." International Journal of Molecular Sciences 21.19 (2020): 7362.]. In addition, there are technologies for producing exosome-mimetic nanovesicles (EMNV) [Brezgin, Sergey, et al."Hydroxychloroquine Enhances Cytotoxic Properties of Extracellular Vesicles and Extracellular Vesicle-Mimetic Nanovesicles Loaded with Chemotherapeutics." Pharmaceutics 15.2 (2023): 534.], which are obtained by one of the technologies associated with cell extrusion through membranes with different pore diameters, as well as nanoghosts (NG), which are nanoparticles created from components or fragments of biological membranes of human cells obtained by hypotonic lysis, sonication, purification using gradient centrifugation and subsequent extrusion [Oieni, Jacopo, et al. "Nano-ghosts: Novel biomimetic nano-vesicles for the delivery of antisense oligonucleotides." Journal of Controlled Release 333 (2021): 28-40.]. In addition, it is possible to create hybrid nanoparticles and biomimetics based on inorganic and organic components, as well as components of biological nanoparticles.The key characteristics of biological nanoparticles include high biocompatibility; the ability to overcome biological barriers; safety; lack of immunogenicity; the ability to program the properties of biological nanoparticles using chemical, physical and biological (genetic) methods, high loading capacity, and reduced clearance by RES macrophages.

[0013] Thus, the disadvantages of the above-mentioned known technical solutions are that they:

[0014] (1) do not provide efficient packaging of Cas proteins; (2) do not provide efficient co-packaging of guide RNAs; (3) are not universal and require optimization for a specific class, type, kind and variant of the CRISPR / Cas system; (4) do not provide the ability to package chemically modified guide RNAs or toxic guide RNAs, the expression of which in producer cells is impossible, worsens the characteristics of the nanoparticles or reduces or completely disrupts the characteristics of the resulting product in the form of nanoparticles and / or CRISPR / Cas complexes; (5) use immunogenic, toxic and non-biocompatible nanocarriers; (6) are technically complex and expensive to manufacture. The WW-Ndfipl system is known from the prior art [Sterzenbach, U.; Putz, U.; Low, L.-H.; Silke, J.; Tan, S.-S.; Howitt, J. Engineered Exosomes as Vehicles for Biologically Active Proteins. Mol. Ther.2017, 25, 1269-1278], which ensures the packaging of proteins into extracellular vesicles (EV) due to the WW-Ndfipl interaction, where the WW sequence can be located on any proteins. However, the disadvantages of this invention include the possibility of using only WW for packaging, the toxic effect of Ndfipl, which is necessary for packaging the target protein, as well as the low efficiency of packaging of target proteins by EVs using this technology (the StCas9-Nedd4 variant was used as a control in this work, where Nedd4 is the WW-Ndfipl interaction factor).

[0015] Methods for packaging proteins, and in particular Cas proteins, into EVs using chemical dimerization-dissociation systems are known [Gee, P, Lung, MSY, Okuzaki, Y. et al. Extracellular nanovesicles for packaging of CRISPR-Cas9 protein and sgRNA to induce therapeutic exon skipping. Nat Commun 11, 1334 (2020), doi 10.1038 / s41467-020-14957-y] based on the FKBP12-FRB dimerization system, where dimerization is carried out by the chemical agent rapamycin.

[0016] The disadvantages of this system are: (1) The need to use rapamycin, an immunosuppressant that has a toxic effect on EV-producing cells. As a result, when using rapamycin, EV-producing cells may die, and by-products associated with cell death may be packaged into EVs. In addition, rapamycin and cell death products may be transferred to target cells, which reduces the safety profile and may lead to severe side effects;

[0017] (2) The possibility of using this system only for EVs; (3) the impossibility of simultaneous co-packaging of guide RNAs. In this system, a complex system of packaging and release of guide RNAs is used for packaging of guide RNAs, based on flanking of the guide RNA with hepatitis D virus ribozyme sequences, as well as an encapsidation signal. The dependence of the release of guide RNAs on ribozyme sequences makes the process of self-excision of ribozyme regions in EVs, and not only in the cell cytoplasm, random. The presence of a complex construct also increases the number of coding plasmids required for transfection to 4-5.

[0018] A method for packaging proteins into EV EXPLORs is known based on the dimerization domains of CIBN-CRY2 under irradiation with blue spectrum light [Yim, Nambin, et al. "Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein-protein interaction module." Nature communications 7.1 (2016): 12277.]. The disadvantages of this method are: (1) the need to use blue spectrum light, which is toxic to cells; (2) the possibility of using the method only for packaging proteins into EVs; (3) the lack of the possibility of co-packaging RNA.

[0019] A method for packaging RNA into EVs is known based on the CIBN-CRY2 dimerization system by creating constructs encoding one of the dimerization domains, which is attached to the constitutive EV protein, and the other to the MCP domain [Hung, Michelle E., and Joshua N. Leonard. "A platform for actively loading cargo RNA to elucidate limiting steps in EV-mediated delivery." Journal of extracellular vesicles 5.1 (2016): 31027]. The RNA for packaging was modified with an MS2 hairpin. Thus, when a stimulus was applied (blue light, with a wavelength of 450-488 nm), it ensured the dimerization of MCP with the second dimerization domain, and the MS2 hairpin interacted with MCP, thereby ensuring the loading of the M82-modified RNA into EVs. The disadvantages of the system are:

[0020] (1) The need to use blue spectrum light, but this light is toxic to producer cells, causes the formation of reactive oxygen species, cell death, which can lead to co-packaging of toxic compounds in EVs and cause side reactions in target cells.

[0021] (2) This system can only be used for EV.

[0022] (3) The possibility of using this system only for packaging RNA, no information on the possibility of co-packaging with the protein of interest.

[0023] (4) The possibility of using CRISPR / Cas systems or analogs for packaging guide RNAs has not been demonstrated; it has only been shown for short interfering RNAs.

[0024] (5) Potential lack of dissociation between MS2 and MCP upon entry into the target cell, making release of the packaged RNA difficult or impossible.

[0025] A method for non-reversible packaging of RNA into vesicles, microvesicles, and EVs is known using the TAMEL platform [Hung M.E., Leonard J.N. A platform for actively loading cargo RNA to elucidate limiting steps in EV-mediated delivery. / / J Extracell Vesicles. 2016 May 13;5:31027, doi: 10.3402 / jev.v5.31027], based on the interaction of high-affinity RNA hairpin groups with affinity domains linked to the CD63 or Lamp2b protein. The disadvantage of this method is the irreversible (i.e. without dissociation and release of active RNA into the vesicle cavity) interaction of RNA with the CD63 protein, which cancels the possibility of manifestation of the functional activity of RNA, as well as the lack of the possibility of using other types of particles - exosome-mimetic nanovesicles (EMNV), nanoghosts (NG) and hybrids.

[0026] Based on the set of essential features, the method indicated in the article by Oieni, Jacopo, et al. / / Journal of Controlled Release 333 (2021): 28-40 is the closest to the proposed invention and was chosen as a prototype.

[0027] At the same time, the disadvantages of the methods in the prototype of the present invention (and in other analogues) are that: (1) they do not provide the possibility of packaging RNA into NG;

[0028] (2) they do not provide the possibility of packaging chemically and genetically modified RNA, hybrid and chimeric molecules, such as RNA-DNA;

[0029] (3) they do not provide the possibility of packaging RNA into micro- and nanoparticles enriched with the protein of interest with or without the formation of ribonucleoprotein complexes;

[0030] (4) they do not provide the ability to control the size of the resulting NG and hybrid micro- and nanoparticles using physical methods (extrusion, homogenization, sonication), which is necessary for various types of delivery, such as systemic delivery;

[0031] (5) they do not provide the possibility of using the developed technologies for the purposes of genetic editing, the purposes of vaccination and therapeutic vaccines, the purposes of gene therapy, genetic engineering, the delivery of therapeutic and prophylactic RNA sequences and their modified analogues, the delivery of therapeutic and prophylactic proteins, their complexes, protein and RNA complexes, their medical, biomedical, veterinary use, the creation of reagents and methods for the delivery of proteins, RNA and their complexes into human and animal cells;

[0032] (6) the packaging of the contents into the obtained particles is carried out by the electroporation method, which leads to damage and aggregation of the particles and / or their contents.

[0033] Thus, the objective of the present invention is to create a new method for packaging protein, RNA and their complexes into micro- and nanoparticles.

[0034] Technical results:

[0035] - increasing the efficiency of packaging of proteins, RNA and their complexes;

[0036] - expansion of the spectrum of molecules subject to packaging into micro- and nanoparticles;

[0037] - expansion of the arsenal of methods for packaging protein, RNA and their complexes into a particle. The stated problem is solved and the technical result is achieved by creating a method for packaging a protein, RNA or a protein-RNA complex into a particle, which includes the following stages: a. the original cells are lysed to obtain a cell lysate; b. the cell membranes are sedimented from the cell lysate obtained in stage (a), followed by washing to obtain a suspension of cell membranes; c. RNA is chemically synthesized or obtained using in vitro transcription, then the RNA is mixed with the protein of interest, the mixture is incubated until a ribonucleoprotein complex is formed; d. the cell membrane suspension obtained in stage (b) is mixed with the complex collected in stage (c), then the resulting mixture is extruded by pressing through at least one extruder membrane to form spherical particles of the required size; e.The particles obtained in step (d) are purified from unpacked components using chromatography.

[0038] According to preferred embodiments, the said technical result is also achieved by the fact that:

[0039] - the particles obtained according to the mentioned method are microparticles;

[0040] - the particles obtained according to the mentioned method are nanoparticles;

[0041] - the particles obtained according to the mentioned method are nanoparticles (NG);

[0042] - the cells are HEK293, HT1080, HeLa, PER.C6, CHO cells, mesenchymal stem cells (MSCs), induced pluripotent (iPSCs), totipotent, multipotent cells of various origins, NIH-3T3, BHK-21, bEnd.3, COS-7, HB54, HB55, HCA2, HMEC, HeLa and other cell lines, including those of tumor origin, as well as cultures of primary cells and tissues of various origins and morphologies; - HEK293 cells are HEK293 cells of any modifications selected from the group consisting of HEK293, HEK293T, HEK293F, HEK293FT, HEK Expi293F;

[0043] - mesenchymal stem cells (MSCs) are MSCs of various origins, selected from a group including placental, adipose, bone marrow, MSCs from umbilical cord blood, amniotic fluid, peripheral blood, synovial fluid, dental pulp, endometrium, skin, muscle tissue, salivary glands;

[0044] - in step (e), the particles were purified from unpacked complexes using gel exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, ultracentrifugation affinity chromatography, density gradient ultracentrifugation, ultrafiltration, tangential filtration, precipitation, asymmetric flow fractionation, or a combination of these methods or other methods;

[0045] - the protein is a protein or peptide of any amino acid sequence, including chemically modified or stabilized proteins containing chemically modified amino acids or amino acid derivatives, recombinant proteins, genetically modified proteins, enzymes, antigenic proteins or fragments thereof, Cas proteins of various classes, types, species, origins and modifications, Cas proteins from various species of organisms, Cas-like proteins, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), meganucleases, human proteins, animal proteins, proteins of viruses, bacteria, archaea, bacteria, chimeric proteins;

[0046] - the protein complex is a complex of a recombinant protein, a genetically modified protein, an enzymatic complex, an antigen fragment, a complex of a protein with RNA or DNA, a Cas protein, a Cas-like protein, a zinc finger nuclease, a nuclease based on an effector similar to a transcription activator (TALENs), meganucleases, human proteins, animal proteins, proteins of viruses, bacteria, archaea, bacteria, chimeric proteins, proteins associated with or used together with low molecular weight compounds, polymers, adjuvants, materials of organic or inorganic origin;

[0047] - RNA is mRNA, long non-coding RNA, RNA of CRISPR-Cas systems, hybrid RNA consisting of various elements, optionally containing ribozyme sequences, aptamers, ribosome entry sites, encapsidation signals, translation initiation and termination signals, sequences that change RNA stability, RNA localization in cells, RNA translation, RNA splicing, wherein chemically modified RNA optionally contains pseudouridine, or is modified by sugar or nucleoside residues, or contains cap structures at the 5'-end, a poly A sequence or modified polyA sequences in combination with other nucleotides at the 3'-end, or contains fluorescent labels, sites of interaction with proteins, is a hybrid of RNA with DNA, contains additional hairpins for dimerization, dissociation, interaction, destruction, stabilization of proteins, or additional effector elements;

[0048] - CRISPR-Cas system RNAs are short RNAs (crRNAs), tracrRNAs, or single guide RNAs (sgRNAs);

[0049] - modifications of Cas proteins are complete Cas nucleases or proteins based on catalytically inactive dead-Cas (dCas), containing inactivating mutations in the nuclease domains, or Cas nicases (nCas), containing a mutation in only one domain;

[0050] - Cas, dCas, nCas proteins are wild-type proteins, or improved or modified variants of Cas, dCas, nCas proteins obtained by random or directed evolution, mutagenesis with or without optimization of protein-coding codons, and optionally have additional elements in the coding construct;

[0051] - the Cas, dCas or nCas proteins are Cas9, Casl2a / b or similar proteins, or improved variants selected from Cas9-HF, eSpCas9, HypaCas9, xCas9, SpRY / SpG, Fokl-fused dCas9; - the Cas, dCas or nCas are a single protein or consist of several components that assemble into a single protein in target cells, producer cells or within particles;

[0052] - Cas, dCas, nCas proteins contain additional domains, including domains for transcription activation, domains for transcription repression, domains for editing DNA or RNA bases, reverse transcriptase domains of Prime Editing systems and their modifications and / or improved variants obtained by directed evolution or mutagenesis, RNA demethylase or RNA methyltransferase domains, DNA visualization systems;

[0053] - domains for transcription activation are the domains VP48, VP64, VP160, VP192, p65, p65-HSF1, p300, VPR, TET1, as well as their modifications according to the principles of Scaffold, Casilio, SAM, TREE, SunTag or similar principles;

[0054] - domains for transcription suppression are KRAB, EZH2, KRAB-MeCP2, DNMT3A, DNMT3A-3L, LSD1 domains, or their modifications or combinations thereof;

[0055] - domains for editing DNA or RNA bases are the rAPOBEC1, ABOVECA, ASh, TadA, ADAR2 domains, as well as their orthologs, their homologs and / or their modified variants obtained by mutagenesis or directed evolution;

[0056] - the domains of RNA demethylases or RNA methyltransferases are the domains of METTL3, METTL14, METTL16, FTO, ALKHB5, or their combinations or their modified variants obtained by mutagenesis or directed evolution;

[0057] - DNA visualization systems are selected from the group including CRISPR-FISHer, CRISPR-SIRIUS, CRISPRainbow modified CRISPR systems with fluorescent protein domains attached and / or recruited to a Cas protein and / or guide RNA;

[0058] - RNA is chemically modified at nucleotide residues, RNA nucleotides or sugar residues, including 2'-O-methyl derivatives (M), 2'-O-methyl-3'-phosphorothioate derivatives (MS), or 2'-O-methyl-3'-thiophosphonoacetate (MSP), pseudouridine *P, N1-methylpseudouridine (mlT), Nl-methylpseudouridine (m5C), 5-hydroxymethylcytosine (5hmC), 5-methiouridine (m5U), 2-thiouridine (s2U), aminomethylene-containing nucleotides (BNANC[N-Me]), locked nucleic acid (LNA), 2'-O-methyl-3'-phosphonoacetate (MP), ethyl group (cEt), fluoro group (F), phosphorothioate (PS), tetraethylene glycol (TEG), phosphonoacetate (PACE) or their combinations;

[0059] - RNA contains cap structures at the 5' end, a poly A sequence or modified polyA sequences in combination with other nucleotides at the 3' end, or contains fluorescent labels, sites of interaction with proteins, or is a hybrid of RNA with DNA, or contains additional hairpins for dimerization, dissociation, interaction, destruction, stabilization of proteins and other molecules, as well as for attracting additional effector elements;

[0060] - packaging of proteins, RNA or their complexes is carried out at the stage of obtaining the mentioned particles or after isolation of the mentioned particles;

[0061] - the complex of protein and RNA is the CRISPR / Cas ribonucleoprotein;

[0062] - human and mammalian cells are used as source cells, including genetically and / or chemically modified cells, cell lines, transiently transfected cell lines and other cell components or cell lines; the extrusion step is carried out using a manual extruder, an automated extruder of cell mass or a suspension of cells or cell membranes or particles in a solvent;

[0063] - from 0.5 ml to more than 1000 l of cell mass or cell suspension or cell membranes or particles are subjected to extrusion;

[0064] - the solvent affects the production of nanoparticles, and / or changes the properties of the membranes of cells or particles, in particular their fluidity, and / or affects the cellular viability or integrity of the particles;

[0065] - extrusion is carried out under the pressure of inert gases or by mechanical extrusion, or by centrifugal extrusion or vacuum filtration;

[0066] - at the extrusion stage, ultrasonic treatment is additionally carried out; - membranes made of polycarbonate, polyethylene terephthalate, polypropylene, regenerated cellulose or cellulose ethers, polytetrafluoroethylene, polyvinylidene fluoride, nitrocellulose, nylon, and polyethersulfone are used for extrusion;

[0067] - the membrane for extrusion is standard or track;

[0068] - the extrusion membrane has a hydrophilic or hydrophobic coating;

[0069] - membranes for extrusion have pore diameters selected from the range from 10 to 0.01 µm;

[0070] - membranes for extrusion have pore diameters selected from the group consisting of 10 μm, 8 μm, 5 μm, 2 μm, 1;2 μm, 0.8 μm, 0.45 μm, 0.4 μm, 0.22 μm, 0.2 μm, 0.1 μm, 0.05 μm, 0.03 μm, 0.01 μm;

[0071] - extrusion is carried out using drainage discs; cells are treated with high-molecular and / or low-molecular compounds that are capable of influencing the production of nanoparticles, expression of proteins and / or RNA;

[0072] - cell membranes, purified from cytoplasmic components, intracellular components and cell nuclei, are optionally fragmented by ultrasound after hypotonic lysis of cells, or fragmentation is carried out chemically or physically, and is used for extrusion in a liquid containing ready proteins, protein-RNA complexes or CRISPR / Cas ribonucleoprotein;

[0073] - membranes are modified to change their charge or affinity for proteins, protein-RNA complexes, or CRISPR / Cas ribonucleoprotein, for more efficient packaging;

[0074] - proteins, protein-RNA complexes or CRISPR / Cas RNPs are also modified to change the charge, size and / or affinity to membranes for more efficient packaging, or multifactorial modifications of membranes and packed cargo are introduced to ensure more efficient packaging into nanoparticles. The stated problem is also solved, and the technical result is achieved by creating a particle obtained by the above-mentioned method.

[0075] The stated task is also solved, and the technical result is achieved by creating a pharmaceutical composition for genetic editing, for genome modification, for correction of genetic mutations, for modification of the epigenome, for modification of the epitranscriptome, for editing RNA, for DNA destruction, for RNA destruction, for modification of nucleotide bases, for modification of nucleotides, for deamination of DNA or RNA, for integration of DNA or RNA sequences into the human genome or mitochondria, for DNA visualization, for studying the spatial structure of chromatin, for changing RNA metabolism, for protein modification, for protein editing, for control of biological processes, for control of physiological processes, for the purposes of vaccine prophylaxis, for creation of therapeutic vaccines based on RNA, proteins, peptides, their mixtures or combinations, for creation of medicinal products, for creation of diagnostic products, for cosmetic products,for use in regenerative medicine, for the correction or introduction of mutations, including drug resistance mutations, in reconstructive medicine, for the treatment of oncological diseases, for the treatment of tumor metastases, for the treatment of autoimmune diseases, for the treatment of hereditary diseases, for the treatment of metabolic diseases, for the treatment of dysbacteriosis, for the impact on the microbial, for the treatment or prevention of mental illnesses, psychogenic disorders, psychosomatic disorders, neuroses, addictions, mood disorders, schizophrenia and related diseases, psychoses, eating disorders, for the treatment and prevention of physical, mechanical, chemical, biological and psychogenic diseases and disorders, for theranostics, for diagnostic purposes, for the treatment and prevention of disorders of the structure and function of tissues, organs and organ systems, in the form of probiotics, prebiotics, synbiotics, for in vitro or ex vivo modification of cell lines,primary cultures or individual cells, including animal embryos, for obtaining, modifying or differentiating stem cells, for obtaining genetically modified animals, for obtaining animal cell lines, and / or for use in veterinary medicine, containing an effective amount of said particles and at least one pharmaceutically acceptable excipient. The stated problem is also solved and the technical result is achieved by using said particle for genetic editing, for genome modification, for correcting genetic mutations, for modifying the epigenome, for modifying the epitranscriptome, for editing RNA, for destroying DNA, for destroying RNA, for modifying nucleotide bases, for modifying nucleotides, for deaminating DNA or RNA, for integrating DNA or RNA sequences into the human genome or mitochondria, for visualizing DNA, for studying the spatial structure of chromatin, for studying RNA metabolism,for protein modification, for protein editing, for the control of biological processes, for the control of physiological processes, for the purposes of vaccine prophylaxis, for the creation of therapeutic vaccines based on RNA, proteins, peptides, mixtures or combinations thereof, for the creation of medicinal products, for the creation of diagnostic products, for cosmetic products, for use in regenerative medicine, for the correction of drug resistance mutations, for reconstructive medicine, for the treatment of oncological diseases, for the treatment of autoimmune diseases, for the treatment of hereditary diseases, for the treatment of metabolic diseases, for in vitro or ex vivo modification of cell lines, primary cultures or individual cells, including animal embryos, to obtain genetically modified animals, to obtain animal cell lines, and / or for use in veterinary medicine.

[0076] According to preferred embodiments, the said technical result is also achieved by the fact that:

[0077] - the mentioned particles are administered by systemic administration into the body and / or by local administration and / or by inhalation and / or subcutaneously and / or directly into organs and tissues in a certain dosage form, and / or by oral administration, by injection into a vein, into a muscle, by administration into the space surrounding the spinal cord, under the skin, by placement under the tongue, between the gums and cheek, in the form of intravenous infusion, intramuscular injection, catheter, enema, rectal administration, inhalation, instillation, insufflation, intravaginally, intranasally, by injection, irrigation, parenteral administration, oral administration, using a powder inhaler, transbuccally, using a tracheal tube, using vascular access; - systemic administration into the body is intravenous, intra-arterial, bolus, intraperitoneal, subarachnoid, epidural or intrasternal administration, or administration into the sinuses, into the cavities of the body, or administration subcutaneously, intramuscularly, or administration into specific organs and tissues;

[0078] - local administration into the body is application to the surface of the skin and mucous membranes, the surface of organs, or washing of organs;

[0079] - the dosage form is selected from the group including an aerosol for inhalation, an aerosol for topical use, an aerosol for external use, an aerosol for application to the oral mucosa, a nasal aerosol, a sublingual aerosol, a transdermal aerosol, an ear aerosol, a vaginal gel, an eye gel, an injection gel, a gel for topical use, a gel for application to the gums, a gel for external use, a gel for subcutaneous administration, a gel for the preparation of a suspension for oral administration, a gel for oral administration, a gel for application to the oral mucosa, a dental gel, an intestinal gel, a nasal gel, a periodontal gel, a rectal gel, a dental gel, a transdermal gel, a urethral gel, an ear gel, an endocervical gel, granules, granules for preparation, drops, solutions, suspensions, granules for resorption, enteric-coated granules, enteric-coated granules with prolonged release, film-coated granules,cut-pressed granules, granules with modified release, granules with prolonged release, effervescent granules, dispersion for infusion, dispersion for injection, dispersion for intravenous administration, dispersion for intradermal administration, liquid for inhalation, liquid for external use, liquid for oral administration, implant, intravitreal implant, eye drops, eye drops with prolonged release, drops for inhalation, drops for topical use, drops for application to the oral mucosa, drops for oral administration, dental drops, nasal drops, sublingual drops, ear drops, capsules, vaginal capsules, intrauterine capsules, chewable capsules, enteric-coated capsules, enteric-coated capsules with prolonged release, sublingual capsules, rectal capsules, capsules with modified release, capsules with powder for inhalation, prolonged-release capsules, concentrate for preparation,dispersion, solution, suspension, emulsion, vaginal cream, eye cream, cream for topical use, cream for external use, cream for application to the oral mucosa, nasal cream, rectal cream, ear cream, vaginal liniment, liniment for topical use, liniment for external use, periodontal liniment, endocervical liniment, lyophilisate for the preparation of, dispersion, drops, concentrate, solution, spray, suspension, emulsion, vaginal ointment, eye ointment, inhalation ointment, ointment for topical use, ointment for application to the oral mucosa, ointment for external use, nasal ointment, rectal ointment, ear ointment, inhalation oil, oil for topical use, oil for external use, oil for internal use, tincture, inhalation tincture, tincture for topical applications, tincture for external use, dental sticks, periodontal sticks, nasal sticks, urethral sticks,ear sticks, paste for application to gums, paste for external use, paste for preparation of suspension for internal use, paste for internal use, paste for application to oral mucosa, medicinal dental paste, vaginal foam, intrauterine foam, foam for external use, rectal foam, patch, patch for oral mucosa, transdermal patch, eye films, films for sticking to gums, buccal films, films dispersible in the oral cavity, periodontal films, sublingual films, dosed inhalation powder, powder for external use, powder for preparation of gel, dispersion, drops, solution, paste, syrup, spray, suspension, powder for internal use, nasal powder, periodontal powder, ear powder, effervescent powder, vaginal solution, intrauterine solution, solution for intra-arterial administration, solution for intraperitoneal administration, solution for intravenous administration,solution for intraocular administration, solution for intradermal administration, solution for intracoronary administration, solution for intramuscular administration, solution for intracavitary administration, solution for intravesical administration, solution for intra-articular administration, solution for gastrointestinal administration, solution for hemodialysis, solution for hemodiafiltration, solution for hemofiltration, solution for inhalation, solution for intra-amniotic administration, solution for intra-lymphatic administration, solution for infusion, solution for injection, solution for local use, solution for cutaneous scarification application, solution for application to the gums, solution for external use, solution for periarticular administration, solution for gastric irrigation, solution for bladder irrigation, solution for parabulbar administration, solution for peritoneal dialysis, solution for subcutaneous administration, solution for rinsing, solution for oral administration, solution for prick test,eye wash solution, nasal wash solution, oral wash solution, ear canal wash solution, oral mucosa solution, subconjunctival solution, extra-amniotic solution, endo-sinusial solution, endo-tracheal solution, dental solution, rectal solution, transdermal solution, syrup, vaginal therapeutic system, intrauterine therapeutic system, topical spray, external spray, oral mucosa spray, nasal spray, sublingual metered spray, transdermal spray, ear spray, vaginal suppositories, rectal suppositories, vaginal suspension, intradermal suspension, intramuscular suspension, intra-articular suspension, gastrointestinal suspension, injection suspension,injection suspension with prolonged release, suspension for implantation, suspension for inhalation, suspension for topical use, suspension for cutaneous scarification application, suspension for external use, suspension for periarticular administration, suspension for subcutaneous administration, suspension for oral administration, suspension for oral mucosa, suspension for endo-sinusial administration, suspension for endotracheal administration, dental suspension, rectal suspension, tablets, vaginal tablets, vaginal effervescent tablets, intrauterine tablets, orodispersible tablets, dispersible tablets, tablets for implantation, tablets for inhalation, tablets for the preparation of drops, solution, suspension, lozenges, chewable tablets, buccal tablets, buccal mucoadhesive tablets, enteric-coated tablets, enteric-coated tablets with prolonged release, lyophilisate tablets, sublingual tablets,film-coated tablets, film-coated tablets, soluble tablets, modified-release tablets, prolonged-release tablets, effervescent tablets, medicated vaginal tampons, medicated inhalation tampons, medicated ear tampons, vaginal emulsion, intrauterine emulsion, emulsion for intravenous administration, emulsion for intramuscular administration, emulsion for gastrointestinal administration, emulsion for inhalation, emulsion for infusion, emulsion for injection, emulsion for topical use, emulsion for external use, emulsion for oral administration, emulsion for rinsing the ear canal, dental emulsion, rectal emulsion, medicated sponge, dragee, medicated pencil, medicated nail polish, medicated lozenges, medicated pastilles, medicated plates, tiles, medicated absorbable wipes, medicated shampoo, elixir, pills,

[0080] Brief description of the drawings

[0081] The invention is illustrated by the following drawings.

[0082] Fig. 1 shows general schemes of protein and RNA packaging into NG nanoparticles. Human or animal cells are lysed using hypotonic solution(s), ultrasonic or other type of homogenization; the lysed cells are used to obtain purified cell membranes by centrifugation and a series of washes. The purified cell membranes are mixed with recombinant proteins, RNA or assembled ribonucleoprotein complexes, and the resulting mass is passed through serial extrusion. The result is a mixture of NG loaded with protein, RNA or ribonucleoprotein complexes, and unpackaged proteins, RNA or ribonucleoprotein complexes. Alternatively, NG can be obtained by closure of membrane fragments into spherical particles with packed contents after a series of freeze-thaw cycles, ultrasonic treatment, microfluidics and other methods. Purification of loaded NG is carried out using chromatography (gel filtration, affinity, anion exchange, etc.)., tangential filtration, ultrafiltration, etc.), the particles are concentrated, and a finished NG preparation loaded with proteins, RNA or ribonucleoprotein complexes of interest is obtained.

[0083] Fig. 2 shows the characterization of biological nanoparticles - nanoghosts (NG). (A) Cryoelectron images, (B) z-potential, (C) size distribution of nanoparticles loaded with Cas / RNA-guide complexes using one of the developed technologies. All the obtained nanoparticles have a similar size with a peak in the region of 100 nm. Error bars correspond to standard deviations. Fig. 3 shows the results of the analysis of Cas protein packaging in biological nanoparticles using the developed technologies in calculating the number of Cas protein copies per nanoparticle. Neg cntrl NP is the background value of the Cas protein signal in nanoparticles without Cas protein. StCas9 RNP NG is the packaging of StCas9 protein in NG nanoparticles. Error bars correspond to standard deviations.

[0084] Fig. 4 shows the results of RNA packaging using the developed loading technology. The data are presented in RNA copies per nanoparticle, cntrl - negative control without RNA; EV - stochastic packaging in EV; NG - packaging in NG; Error bars correspond to standard deviations.

[0085] Preferred embodiments of the invention

[0086] The inventors have created a method for packaging proteins, RNA and protein and RNA complexes in NG. General schemes for packaging proteins, RNA and their complexes are shown in Fig. 1. Micro- and nanoparticles may or may not contain additional components, including pH-sensitive linkers and elements, endosomal exit inducers, smart materials, light-sensitive and enzyme-sensitive and responsive elements, ribozymes loaded or linked to RNA guides, with biological nanoparticles, or with proteins.

[0087] In preferred embodiments of the invention, the proteins may be any proteins, protein compounds or their derivatives of any amino acid sequence, including chemically modified or stabilized proteins, their complexes, compounds or derivatives containing or not containing chemically modified amino acids or amino acid derivatives, genetically modified proteins or their complexes, protein complexes with RNA or DNA, including Cas proteins of any classes, types, species and modifications, Cas proteins from various types of organisms, Cas-like proteins, zinc-finger nucleases, transcription activator-like effector nucleases (TALENs), meganucleases, human proteins, animal proteins, proteins of viruses, bacteria, archaea.Proteins can be packaged in a free form, in the form of complexes or in a bound form together with low-molecular compounds, polymers, adjuvants, materials of organic or inorganic origin. Any sequences of ribonucleic acids, their derivatives, ribonucleotides, RNA components, including RNA guides of CRISPR / Cas systems or similar systems, their derivatives due to genetic modification, addition or removal of elements of RNA guides, hybrid RNAs associated with various elements that can include sequences of ribozymes, aptamers, ribosome landing sites, encapsidation signals, translation initiation and termination signals, signals for improving or changing translation, sequences that change RNA stability, RNA localization in cells, RNA splicing can act as RNA.RNA can be chemically modified, for example, contain pseudouridine, can be modified by sugar or nucleoside residues, contain cap structures at the 5' end, poly A sequence or modified poly A sequences in combination with other nucleotides at the 3' end, contain fluorescent labels, sites of interaction with proteins, be a hybrid of RNA with DNA, contain additional hairpins for dimerization, dissociation, interaction, destruction, stabilization of proteins and other molecules, attraction of additional effector elements. RNA can be RNA for translation and protein production or for regulation of gene transcription, regulation of translation, regulation of the epigenetic state of the human genome, the genome of transformed and tumor tissues, animals or pathogenic microorganisms. Packaging of proteins, RNA or their complexes in the case of NG occurs from human or animal cells.Animal or human cells may be primary cells, transplanted, transformed or tumor cells, genetically modified cells or cells modified using genetic (transfection, transduction, fusion with other cells) or non-genetic methods (treatment with low- or high-molecular compounds, metabolic effects, thermal effects, atmospheres with different gas contents, contact with the surface of cells).

[0088] In preferred embodiments of the invention, the cells for producing NG may be mesenchymal stromal / stem cells, derivatives of mesenchymal stromal / stem cells, induced pluripotent cells or embryonic stem cells, adult, terminally or non-terminally differentiated cells of humans or animals, such as cells of the immune system (peripheral mononuclear cells, leukocytes, macrophages, monocytes, natural killers, etc.), cells of skeletal or smooth muscle, cardiac tissue, epithelium, brain, etc., or cells derived from any germ layers of humans or animals.

[0089] Protein, RNA molecules or their complexes may contain additional elements that can provide or enhance interaction with membranes of biological nanoparticles to enhance packaging efficiency. Protein, RNA or their complexes are packaged according to the general scheme shown in Fig. 1. through filters with a pore diameter of <200 nm (for systemic administration) or >30 nm but <5000 nm (for local use or administration directly into tissues, organs and cavities of the body). The next step involves purification of the nanoparticles using chromatography with preliminary and / or subsequent concentration by TFF or chromatography, filtration through cut-off pores (ultrafiltration, UF), sedimentation or precipitation, or without additional concentration.

[0090] In the case of NG, human or animal cells (whether or not expressing proteins, RNA, or ribonucleoprotein complexes) are lysed using hypotonic solutions, ultrasonic or mechanical homogenization, followed by sedimentation of the cell membranes and their washing. The purified cell membranes are mixed with protein, RNA, or their complexes, and the resulting mixture is passed through serial extrusion.

[0091] Extrusion can be performed using a manual, semi-industrial or industrial extruder with different pressure applied to the mass of cell membranes or biological particles for extrusion. Extrusion can be performed manually, by pressure of inert gases or gas mixtures or mechanical pressure (for example, centrifugal force during centrifugation or using presses). The material of the membranes for extrusion can be different, it can be hydrophilic or hydrophobic, such as polycarbonate, polyethylene terephthalate, polyimide, etc. As a result, the cargo of interest is packed in NG. Purification from unpackaged cargo is performed using chromatography or other methods, including TFF, UF, gradient centrifugation, AF4 or other methods with preliminary and / or subsequent concentration by TFF, filtration through cut-off pores (UF), precipitation or sedimentation or without additional concentration.In this case, in order to improve the quality of nanoparticles, the efficiency of packaging, improve purification and improve the quality of the final product, additional stages, elements or components can be introduced into the declared general scheme, which can include instrumental methods, analytical methods or additives to increase the stability of nanoparticles, increase the stability and increase the efficiency of cargo packaging in the form of inhibitors of RNases, DNases, proteases, in the form of factors increasing the elasticity and fluidity of cell membranes, etc., as well as the possible use of lyophilization or spray drying, etc.

[0092] A method for packaging proteins, RNA or protein-RNA complexes, in particular ribonucleoprotein (RNP) CRISPR / Cas complexes or their analogues, has been created, which ensures packaging of RNA, proteins or ready-made RNP CRISPR / Cas complexes into NG micro- and nanoparticles. The present method consists in packaging a cargo consisting of any proteins, RNA or their complexes regardless of their class, type, kind, origin, variant and modification, and can also package additional compounds, factors, molecules or their derivatives, including low-molecular compounds, high-molecular compounds, polymers, biomolecules and their derivatives. RNA can be obtained using chemical synthesis, in vitro transcription, may or may not contain additional structural or non-structural elements, chemical or physical modifications, DNA sequences.NG are purified components of biological membranes of humans or animals that form micellar bilayer spherical structures with a size of 20 nm to 50 μm, which are formed as a result of hypotonic lysis, ultrasonic treatment and subsequent extrusion, or using other physical or chemical methods, and can be obtained from individual cell lines, mixtures of cell lines or cell populations, including genetically or chemically modified cells, components of cell membranes. NG may contain additional inorganic, organic, high-molecular or low-molecular components.Technologically, the method consists of packing any cargo of any size, charge and properties into purified biological membranes by forming them into spherical capsules of any size in the range from 20 nm to 50 μm, while in the process of extrusion (or other physical action) the cell membranes purified from cytoplasm components, intracellular components and cell nuclei are placed in a mixture with the cargo required for loading. As a result of extrusion, capsules loaded with cargo with different particle sizes are formed. Changing the pore diameter of membrane filters (or other physical barriers with a pore size from 20 nm to 5 μm) allows you to regulate the size of the resulting micro- and / or nanoparticles with loaded cargo.The cell membranes can be purified and fragmented by combinations of hypotonic cell lysis or without lysis, ultrasonic or mechanical fragmentation, or the disintegration of the cell membranes can occur using other chemical or physical methods, with the mandatory component of the technology being the extrusion of the cell membranes and the packed cargo or the spontaneous or induced closure of the membrane fragments into spherical particles around the packed cargo. The cell membranes can be modified genetically, chemically or physically, with a change in the membrane size, component composition of the membranes, membrane charge, modifications of membrane components, while in the mixture of the packed cargo it is possible to introduce changes in the composition, charge and size, as well as modifications of the cargo, including proteins, RNA or their complexes, which can affect the efficiency of nanoparticle formation, membrane fluidity, and the efficiency of cargo packaging.In addition, additional proteins can be included in NG to improve the properties of the carrier, for example, GALA peptides to improve endosomal release, viral or bacterial components, factors increasing import into the nucleus or localization in the cytoplasm or cytoplasmic organelles, anchoring in the membrane, etc. The properties, packaging efficiency and amount of the resulting NG and the packed cargo can also be influenced by the mode of obtaining nanoparticles, including the temperature regime, methods and approaches for extrusion or ultrasonic treatment, the pore sizes of the membranes during extrusion, the procedures for isolating the membranes used to obtain the nanoparticles, the concentration and properties of the packed cargo, as well as other additives and supplements.

[0093] Preferred embodiments of the invention

[0094] A method for producing a particle by packaging proteins, RNA and protein and RNA complexes, in particular CRISPR / Cas RNP, consisting of using NG. Packaging of proteins, RNA or their complexes can be carried out at the stage of obtaining the above nanoparticles or after isolating the said particles. Protein molecules, RNA or their complexes can contain additional elements that can provide or enhance interaction with membranes of biological nanoparticles to enhance the packaging efficiency or improve the biological properties of the resulting nanoparticles, for example, enhancing endosomal release in target cells.

[0095] Any proteins and peptides of any amino acid sequence may act as proteins, including chemically modified or stabilized proteins containing chemically modified amino acids or amino acid derivatives, or protein complexes, recombinant proteins, genetically modified proteins or their complexes, enzymes or enzymatic complexes, antigen fragments, as well as protein complexes with RNA or DNA, including Cas proteins of various classes, types, species, origins and modifications, Cas proteins from various species of organisms, Cas-like proteins, zinc-finger nucleases, transcription activator-like effector nucleases (TALENs), meganucleases, human proteins, proteins of animal origin, proteins of viruses, bacteria, archaea, bacteria, chimeric proteins. Proteins associated with or used together with low molecular weight compounds, polymers, adjuvants, materials of organic or inorganic origin.

[0096] Cas proteins are derived from bacteria or archaea or fungi or protozoa or algae.

[0097] In one embodiment of the invention, the modifications of Cas proteins may be complete Cas nucleases or proteins based on catalytically inactive dead-Cas (dCas) containing inactivating mutations in the nuclease domains, or Cas nickases (nCas) containing a mutation in only one domain.

[0098] In one embodiment of the invention, the Cas / dCas / nCas protein is a wild-type protein or an improved or modified variant of the Cas / dCas / nCas protein obtained by random or directed evolution, mutagenesis with / without optimization of protein-coding codons, and having additional elements in the coding construct. In one embodiment of the invention, the Cas, dCas or nCas protein is Cas9, Casl2a / b or similar proteins, or improved variants selected from Cas9-HF, eSpCas9, HypaCas9, xCas9, SpRY / SpG, Fokl-fused dCas9.

[0099] The bacteria used to produce Cas proteins are Streptococcus thermophilus, Streptococcus pyogenes, Staphilococcus aureus, Neisseria meningitidis, Francisella novicida, Acidaminococcus sp., Geobacillys stearothermophilus, Prevotella sp., Lachnaspiraceae bacterium, Campylobacter jejuni, Clostridium thermocellum, Streptococcus mutans, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Rhodococcus jostii, Vibrio cholerae, Bacteroides fragilis, Mycobacterium smegmatis, Thermus aquaticus.

[0100] Chlamydomonas reinhardtii, Phaeodactylum tricornutum, Nannochloropsis oceanica are used as algae.

[0101] Sulfolobus, Pyrococcus, and Methanococcus are used as archaea.

[0102] Tetrahymena thermophila and Trypanosoma brucei are used as protozoa.

[0103] The following fungi are used: Saccharomyces cerevisiae, Fusarium oxysporum, Candida albicans.

[0104] In one embodiment of the invention, Cas, dCas or nCas is a single protein or consists of several components that assemble into a single protein in target cells, producer cells or within particles.

[0105] In one embodiment of the invention, the Cas protein comprises additional domains, including domains for transcription activation (VP48, VP64, VP160, VP192, p65, p65-HSF1, VPR, TET1, as well as their modifications according to the Scaffold, Casilio, SAM, TREE, SunTag or similar principles), for transcription repression (KRAB, EZH2, KRAB-MeCP2, DNMT3A, DNMT3A-3L, LSD1, their modifications or combinations), for DNA or RNA base editing (rAPOBEC1, APOBECA3A, ASh, TadA, ADAR2, as well as their orthologs, homologs and modified variants obtained by mutagenesis or directed evolution), reverse transcriptase domains of Prime Editing systems and their modifications and / or improved variants obtained by directed evolution or mutagenesis, RNA demethylase or RNA methyltransferases (METTL3, METTL14, METTL16, FTO, ALKHB5, their combinations or modified variants obtained by mutagenesis or directed evolution), DNA visualization systems (CRISPR-FISHer, CRISPR-SIRIUS,modified CRISPR systems with fluorescent protein domains fused to and / or recruited to Cas protein and / or guide RNA) and other CRISPR-Cas-based systems.

[0106] In one embodiment of the invention, the RNA may be any RNA sequences, mRNA, long non-coding RNA, RNA of CRISPR-Cas systems of various types (crRNA, tracrRNA, sgRNA), oligo- and npe-gRNA, as well as hybrid RNA consisting of various elements that may contain ribozyme sequences, aptamers, ribosome entry sites, encapsidation signals, translation initiation and termination signals, sequences that alter RNA stability, RNA localization in cells, RNA translation, RNA splicing.

[0107] In one embodiment of the invention, the RNA may be chemically modified, such as at RNA nucleotide(s) residues or sugar residues, including 2'-O-methyl (M), 2'-O-methyl 3' phosphorothioate (MS) or 2'-O-methyl 3' thiopACE (MSP), pseudouridine *P, Nl-methylpseuduridine mlT, Nl-methylpseuduridine m5C, 5-hydroxymethylcytosine 5hmC, 5-methyluridine m5U, 2-thiouridine s2U, BNANC[N-Me], locked nucleic acids (LNA), 2'-O-methyl-3'-phosphonoacetate (MP), cEt, F, PS, tetraethylene glycol (TEG), phosphonoacetate (PACE), or combinations thereof, contain cap structures at the 5' end, a poly A sequence or modified polyA sequences in combination with other nucleotides (including non-canonical) at the 3' end, contain fluorescent labels, sites of interaction with proteins, be a hybrid of RNA with DNA, contain additional hairpins for dimerization, dissociation, interaction, destruction, stabilization of proteins and other molecules,attracting additional effector elements,

[0108] In one embodiment of the invention, the elements for improving the biological properties of the resulting nanoparticles, in particular, the endosomal release, include substances capable of enhancing the destabilization of the endosome and the release of the contents into the cytosol by various mechanisms, including pore formation, fusion with the endosomal membrane, the "proton sponge" mechanism, and destabilization of the endosomal membrane. The list of these substances includes, but is not limited to, cationic peptides (HA2, GALA, INF7, JTS1, ESCA, Mellitin and its analogs, LL37 and its analogs), anionic peptides (Tat, R8, EB1, Mellitin, KALA, SAP, H5WYG, ppTGl, LAH4), polycationic polymers and oligomers (spermine, spermidine), low molecular weight compounds (chloroquine, hydroxychloroquine, mefloquine, their analogs and derivatives).

[0109] A method for packaging a protein, RNA or a protein-RNA complex, in particular a CRISPR / Cas RNP, which ensures the packaging of ready-made CRISPR / Cas RNP complexes into NG nanoparticles. The method consists in packaging ready-made complexes consisting of a recombinant protein, in particular a Cas protein of any kind, class, type, origin, variant and modification, and a guide RNA obtained by chemical synthesis, in vitro transcription, containing or not containing additional structural or non-structural elements, chemical modifications, DNA sequences, into NG. NG are purified components of human and animal biological membranes that form micellar bilayer spherical structures with a size of 2 nm to 50 μm and can be obtained from individual cell lines, including genetically modified ones, from cell populations, components of cell membranes together or without additional inorganic, organic, high-molecular or low-molecular components.The packaging efficiency is determined by the quantity, concentration and properties of cell membranes, the procedure for isolating membranes used to obtain nanoparticles, the concentration and properties of the packed cargo, the protocol for forming NG from cell membranes (including the temperature regime for forming NG, the use of extrusion or ultrasonic treatment, the pore sizes of the membranes during extrusion, pre-treatment of the membranes and other factors), as well as additives and supplements that can affect the fluidity of biological membranes (e.g., DMSO, lipids with unsaturated fatty acids, surfactants, etc.), the quantity and quality of the obtained nanoparticles and the efficiency of cargo packaging.

[0110] Any human or mammalian cells can be used as producer cells, including genetically and / or chemically modified cells, cell lines, primary cell cultures, and transiently transfected cell lines.

[0111] In one embodiment of the invention, induced pluripotent stem cells are seeded in a rotary perfusion bioreactor, the cell mass is grown in a culture medium until 100% confluency is achieved. After confluency is achieved, the cells are washed with a phosphate-buffered saline solution and detached from the reactor substrate with a Versene solution. The cell mass is washed twice with a phosphate-buffered saline solution and resuspended in a hypotonic buffer TM solution (0.01 M Tris-HCl, 0.001 M MgCh) containing an inhibitor cocktail for proteases, with a density of up to 2x10 6cells / ml. The cell suspension is incubated at 0°C for 20 minutes and then sonicated. A 60% sucrose solution in TM buffer is added to the reactor containing the lysed cell suspension to a concentration of 0.25 M sucrose and centrifuged (6000g, 15 minutes). The supernatant is removed, a 0.25 M sucrose solution in TM buffer (pH 7.4) is added to the membrane tablet and mixed, the procedure of washing and removing the supernatant is repeated twice. A 0.25 M sucrose solution in TM buffer (pH 8.6) is added to the resulting tablet, the suspension is mixed and sonicated. The suspension is centrifuged (6000g, 15 minutes), the supernatant is removed, the tablet is resuspended in a solution of 0.25 M sucrose in TM buffer (pH 8.6), the washing procedure is repeated twice. The supernatant is discarded, the tablet is resuspended in a solution of phosphate-buffered saline.In parallel, the CHO cell line expressing the Cas9 nuclease from the Streptococcus thermophilus microorganism with a His-tag is grown in a bioreactor to an optimal density, the cells are washed with a phosphate-buffered saline solution and lysed. The lysate is centrifuged, the protein is isolated from the lysate by sequential purification on a Ni-chelating resin and ion-exchange resin. The isolated protein is mixed in a reactor with the RNA conductor obtained by the in vitro transcription method and treated with alkaline phosphatase, the mixture is stirred and incubated for 10 minutes at a temperature of 20 ° C. The resulting mixture is added to the membrane suspension in an optimal ratio and mixed, then extruded using a liposomal extruder under nitrogen pressure at a temperature of 37°C through hydrophilic polycarbonate membranes with pore diameters of 1 μm (15 times), 0.4 μm (9 times), 0.1 μm (15 times).The resulting suspension is purified using Sepharose CL-4B gel exclusion resin and fractions corresponding to the target particles are collected.

[0112] In advantageous embodiments of the invention, cell membranes purified from cytoplasmic components, intracellular components and cell nuclei can be fragmented by ultrasound after hypotonic lysis of cells or without lysis, or fragmentation can be carried out by another chemical or physical method, and used for extrusion into a liquid containing finished proteins, protein-RNA complexes or CRISPR / Cas RNPs.

[0113] In advantageous embodiments of the invention, the membranes can be modified to change their charge or affinity for proteins, protein-RNA complexes, or CRISPR / Cas RNPs for more efficient packaging, such as by mixing with cationic or ionizable lipids as indicated in and for more efficient binding to negatively charged molecules. Proteins, protein-RNA complexes, or CRISPR / Cas RNPs can also be modified to change the charge, size, and / or affinity for the membranes for more efficient packaging, or multifactorial modifications can be made to the membranes and the packaged cargo to ensure more efficient packaging into nanoparticles.

[0114] In preferred embodiments of the invention, spherical particles can be formed by extrusion, freeze-thaw, ultrasonic treatment, nitrogen cavitation, pressure molding, and other methods.

[0115] In advantageous embodiments of the invention, the extrusion step can occur either using a manual extruder or using automated options for various volumes (from 0.5 ml to >100 l) of membrane suspension in any solvent or buffer solution, which may or may not affect the production of nanoparticles, or change or not change the properties of cell membranes, in particular, their fluidity.

[0116] In preferred embodiments of the invention, extrusion can be carried out under the pressure of inert gases (nitrogen and others) or by mechanical extrusion, centrifugal extrusion or vacuum filtration, with or without the use of ultrasonic treatment.

[0117] Standard or track membranes made of polycarbonate, polyethylene terephthalate (polyester), polypropylene, regenerated cellulose or cellulose ethers, polytetrafluoroethylene, polyvinylidene fluoride, nitrocellulose, nylon, polyethersulfone or other materials with hydrophilic or hydrophobic coatings can be used as membranes for extrusion. Membranes with different pore diameters can be used during extrusion, including 10 μm, 8 μm, 5 μm, 2 μm, 1.2 μm, 0.8 μm, 0.45 μm, 0.4 μm, 0.22 μm, 0.2 μm, 0.1 μm, 0.05 μm, 0.03 μm, 0.01 μm, with or without the use of drainage discs, or membranes with a pore diameter in the range of 0.01-50 μm can be used.

[0118] In advantageous embodiments of the invention, the obtained particles (NG) are used for the purposes of genetic editing, genome modification, correction of genetic mutations, epigenome modification, epitranscriptome modification, RNA editing, DNA destruction, RNA destruction, modification of nucleotide bases, nucleotide modification, DNA or RNA deamination, integration of DNA or RNA sequences into the human genome or mitochondria, DNA visualization, studying the spatial structure of chromatin, studying RNA metabolism, protein modification, protein editing, monitoring biological processes, monitoring physiological processes, for the purposes of vaccine prophylaxis or the creation of therapeutic vaccines based on RNA, proteins, peptides, mixtures or combinations thereof, the creation of medicinal or diagnostic preparations, cosmetic products, use in regenerative medicine, correction of drug resistance mutations, reconstructive medicine, oncology,treatment of autoimmune diseases, hereditary diseases, genetic defects, metabolic diseases and other diseases, in vitro or ex vivo for modification of cell lines, primary cultures or individual cells, including embryos, obtaining genetically modified animals, cell lines, for use in veterinary medicine and for other purposes.

[0119] Cells removed from the culture substrate can serve as a source of membranes for packaging Cas proteins and / or other proteins or peptides at the stage of obtaining nanoparticles using Avoska technology. The cells are subjected to gentle lysis in a hypotonic buffer or gentle homogenization, with or without sonication, then the membranes are washed from components of the cell nuclei and cytoplasm. In a parallel technical process, the RNA guide obtained by the in vitro transcription method or chemically synthesized is mixed with the recombinant Cas protein, the mixture is incubated at 20 °C until ribonucleoprotein complexes are formed. Then, a suspension of membranes in a neutral isoosmotic buffer (e.g., phosphate buffered saline) is mixed with the obtained ribonucleoprotein complexes.The resulting mixture is forced through a series of membranes using an extruder to form spherical particles of the required size containing the components to be packaged (in this case, CRISPR-Cas ribonucleoprotein complexes), after which the particles (nano-glands (NG)) are purified from unpackaged contents, for example, using gel exclusion chromatography.

[0120] Similarly, the above approaches can be used to package Cas nucleases, cytidine or adenine Cas base editors, systems for CRISPR activation, CRISPR interference or CRISPR-based epigenome editing, systems for CRISPR visualization, epitranscriptome editing, Prime Editing systems, systems for changing the three-dimensional structure of chromatin based on CRISPR-Cas and other Cas proteins.Similarly, using the above approaches, it is possible to package any proteins and peptides of any amino acid sequence, including chemically modified or stabilized proteins containing chemically modified amino acids or amino acid derivatives, or protein complexes, recombinant proteins, genetically modified proteins or their complexes, enzymes or enzymatic complexes, antigen fragments, as well as protein complexes with RNA or DNA, including Cas proteins of various classes, types, species, origins and modifications, Cas proteins from various types of organisms, Cas-like proteins, zinc-finger nucleases, transcription activator-like effector nucleases (TALENs), meganucleases, human proteins, proteins of animal origin, proteins of viruses, bacteria, archaea, bacteria, chimeric proteins.Proteins associated with or used in combination with low molecular weight compounds, polymers, adjuvants, materials of organic or inorganic origin.

[0121] In preferred embodiments of the invention, the target sizes of the final particles may be 0.01-50 μm, using appropriate membrane filters when using extrusion or appropriate parameters when forming particles using other methods (for example, using a series of freeze-thaw cycles, nitrogen cavitation, ultrasonic treatment, etc.).

[0122] In advantageous embodiments of the invention, the following can be used as initial cells for obtaining particles: HEK293 cells of any modification (HEK293, HEK293T, HEK293F, HEK293FT, HEK Expi293F and others), HT 1080, HeLa, PER.C6, CHO, mesenchymal stem cells (MSC) of various origins (placental, adipose, bone marrow, MSC from umbilical cord blood, amniotic fluid, peripheral blood, synovial fluid, dental pulp, endometrium, skin, muscle tissue, salivary glands and others), iPSCs of various origins, NIH-3T3, BHK-21, bEnd.3, COS-7, HB54, HB55, HCA2, HMEC, HeLa and other cell lines, including those of tumor origin, as well as cultures primary cells and tissues of various origins and morphologies.

[0123] In preferred embodiments of the invention, iPSCs differentiated by transduction of viral vectors encoding the Oct3 / 4, Sox2, c-Myc and Klf4 genes or Oct4, Sox2, Nanog and Lin28 or some of these genes into somatic cells, including keratinocytes or fibroblasts, HUVEC and NHEK cell lines, can be used as cells.

[0124] In preferred embodiments of the invention, iPSCs are obtained by daily transfection of somatic cells with mRNA encoding the Oct3 / 4, Sox2, c-Myc and Klf4 genes (with one type of mRNA or a combination thereof).

[0125] In preferred embodiments of the invention, iPSCs are obtained by induction from somatic cells with a cocktail of recombinant proteins Oct4, Sox2, Klf4 and c-Myc associated with endosomolytic and / or penetrating peptides.

[0126] In preferred embodiments of the invention, iPSCs are obtained by transient transfection into somatic cells of plasmids or minicircles encoding the genes Oct3 / 4, Sox2, c-Myc and Klf4, Nanog, Lin28, or combinations thereof.

[0127] In preferred embodiments of the invention, iPSCs are obtained from somatic cells, including fibroblasts, keratinocytes, HUVEC and NHEK cell lines, by treating these cells with low-molecular-weight substances that affect the epigenetic state of the cellular genome, including 5-azacytidine, RG108, RSC133, SAHA, TSA, sodium hydroxybutyrate, VPA, Tranylcypromine, BIX, CHIR, Kenpaullone, Compound B6, LiCl, E-616452, A83-01, LY-364947, SB431542, PD0325901, AMI-5, N-oxaloylglicine, Compound B4, Dasatinib, iPY razine, PPI, Rapamycin, Compound B8, Compound BIO, D4476, BayK, FSK, PGE2, Rolipram, 2-Me-5HT, 5-(4-chlorophenyl)-3- phenylpent-2-enoic acid, 8-Br-cAMP, Fructose 2,6-bisphosphate, Quercetin, DZnep, DNP, TTNPB, Oxysterol, Purmorphamine, Shh and other substances.

[0128] In advantageous embodiments of the invention, the following can be used as a substrate for culturing cells: T25, T75, T150, T175, T225 culture flasks, multilayer culture flasks, roller-type cell bioreactors, full-fiber reactors, a perfusion reactor with or without microspheres, bioreactor flasks, multi-parallel bioreactors, tabletop bioreactors, stainless steel bioreactors, disposable bioreactors, rotary bioreactors and other types of bioreactors or culture containers.

[0129] In preferred embodiments of the invention, the cell mass can be grown to a confluence of 30-100% (adherent cultures) or a density of 30-100% (suspension cultures).

[0130] In preferred embodiments of the invention, extrusion can be carried out in a temperature range of 0-100°C.

[0131] In advantageous embodiments of the invention, NG can be packaged with both CRISPR-Cas nucleases and catalytically inactive variants (Dead-Cas) or proteins with one inactivated domain (Cas-nickase, nCas) in the following variants: dCas-VP48, dCas-VP64, dCas-VP160, dCas-VP192, dCas-p300, dCas-VPR, dCas-VP64 / sgRNA-p65-HSFl (SAM), dCas-TETl, dCas-DNMT3A, dCas-DNMT3A-3L, dCas-sgRNA-MS2 / MCP-TETl, dCas-SunTag-TETl, dCas-SunTag-VP64, dCas-SunTag-p300, dCas-KRAB, dCas-KRAB-MeCP2, dCas-EZH2, dCas-sgRNA-Casilio-VP64, dCas-LSDl, UGI-dCas-AS, UGI-nCas-AS, UGI-dCas-sgRNA-MS2 / MCP-AID, UGI-nCas-sgRNA-MS2 / MCP-AS, UGI-dCas-rAPOBECl, UGI-nCas-rAPOBECl, UGI-dCas-rAPOBEC3A, UGI-nCas-garovesza, UGI-dCas-TadA, UGI-nCas-TadA, dCas-ADAR2, dCas-METTL3, dCas-METTL14, dCas-METTL3-METTL14, dCas-FTO, dCas-ALKBH5, dCas-WTAP, nCas-RT-pegRNA (Reverse Transcriptase), dCas-RT-pegRNA and / or other CRISPR-Cas based systems.

[0132] In preferred embodiments of the invention, systems based on Cas9, Casl2a, Casl2b, Casl2c, Casl2d, Casl2e, Casl3a, Casl3b, Casl3c, Casl4, Cas-X, Cas3 and / or other CRISPR-Cas based systems can be used.

[0133] In advantageous embodiments of the invention, any proteins and peptides of any amino acid sequence can be expressed in cells for packaging, including chemically modified or stabilized proteins containing chemically modified amino acids or amino acid derivatives, or protein complexes, recombinant proteins, genetically modified proteins or their complexes, enzymes or enzymatic complexes, antigen fragments, as well as protein complexes with RNA or DNA, including Cas proteins of various classes, types, species, origins and modifications, Cas proteins from various types of organisms, Cas-like proteins, zinc-finger nucleases, transcription activator-like effector nucleases (TALENs), meganucleases, human proteins, animal proteins, proteins of viruses, bacteria, archaea, bacteria, chimeric proteins.Proteins associated with or used in combination with low molecular weight compounds, polymers, adjuvants, materials of organic or inorganic origin.

[0134] In preferred embodiments of the invention, cells can be detached from the substrate with a solution of isotonic or hypertonic citrate buffer or another buffer containing non-cytotoxic compounds that chelate Ca ions. 2+ and M 2+ (for example, with Versen solution), as well as using other reagents (Accutase, Hyaluronidase, Collagenase, Dispase, Trypsin, Papain, Accumax) and others.

[0135] In preferred embodiments of the invention, the cells can be resuspended in a solution of isotonic Tris-hydrochloride buffer, saline or other buffer solutions with a concentration of 10 4 -10 15 cells / ml.

[0136] In advantageous embodiments of the invention, during resuspension of cells or purified membranes and during the process of particle extrusion, substances that change cell fluidity (DMSO, SDS and other substances with surface-active properties, lipids with saturated or unsaturated fatty acid residues, etc.), membrane permeability, or endosomal release of the resulting particles in target cells can be added, including cationic endosomolytic peptides (HA2, GALA, INF7, JTS1, ESC A, Mellitin and its analogs, LL37 and its analogs), anionic endosomolytic peptides (Tat, R8, EB1, Mellitin, KALA, SAP, H5WYG, ppTGl, LAH4), polycationic polymers and oligomers (spermine, spermidine), low molecular weight compounds (chloroquine, hydroxychloroquine, mefloquine, their analogs and derivatives).

[0137] In preferred embodiments of the invention, extrusion can be carried out using a manual or screw extruder, an automatic liposomator, a centrifugal extrusion method, under nitrogen pressure or by mechanical extrusion or using other methods based on extrusion through filters with a certain pore diameter, by nitrogen cavitation, by vacuum filtration, with or without the use of ultrasonic treatment and / or cell lysis by other methods, including hypotonic lysis.

[0138] In preferred embodiments of the invention, standard or track membranes made of polycarbonate, polyethylene terephthalate (polyester), polypropylene, regenerated cellulose or cellulose ethers, polytetrafluoroethylene, polyvinylidene fluoride, nitrocellulose, nylon, polyethersulfone or other materials with hydrophilic or hydrophobic coatings can be used as membranes for extrusion.

[0139] In preferred embodiments of the invention, debris removal may be accomplished by filtration through filters with pore sizes greater than or equal to the pores used in the final extrusion, precipitation, or other methods.

[0140] In preferred embodiments of the invention, the particle suspension after removal of debris and / or production of hybrids can be subjected to extrusion or filtration under pressure or vacuum through pores with diameters of 0.01 μm, 0.03 μm, 0.05 μm, 0.1 μm, 0.2 μm, 0.22 μm, 0.4 μm, 0.45 μm or through filters with other pore diameters in the range of 0.01-50 μm.

[0141] In preferred embodiments of the invention, purification of particles can be accomplished by other methods, including ultracentrifugation, gradient ultracentrifugation, precipitation, ultrafiltration, tangential filtration, asymmetric flow fractionation, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and other methods.

[0142] In advantageous embodiments of the invention, the particle suspension after removal of debris can be processed and / or purified by ultracentrifugation, gradient ultracentrifugation, precipitation, ultrafiltration, tangential filtration, fractionation in asymmetric flows, ion exchange chromatography, hydrophobic chromatography, affinity chromatography or other methods, or the production of hybrids can be carried out without preliminary purification of the obtained particles.

[0143] In one embodiment of the invention, the guide RNA synthesized by in r / Uro transcription may not be treated with alkaline phosphatase; RNA synthesized by in r / Uro transcription may be used after additional processing steps, such as adding a cap structure, a poly-A tail, and / or other structural modifications.

[0144] In advantageous embodiments of the invention, the guide RNA or other RNAs can be obtained by chemical synthesis, including using nucleotides modified at the nucleoside residues of sugars or phosphate residues, the list of modifications includes, but is not limited to 2'-O-methyl (M), 2'-O-methyl 3 'phosphorothioate (MS), or 2 '-Om ethyl 3 'thioPACE (MSP), Pseudouridine T, N1- methylpseudouridine m I, N1 -methylpseudouridine m5C, 5-hydroxymethylcytosine 5hmC, 5- methyluridine m5U, 2-thiouridine s2U, BNANC[N-Me], Locked nucleic acids (LNA), 2'-O- methyl-3 '-phosphonoacetate (MP), cEt, F, PS, tetraethylene glycol (TEG), phosphonoacetate (PACE) or combinations thereof.RNA may also contain or not contain cap structures at the 5' end, a poly A sequence or modified polyA sequences in combination with other nucleotides (including non-canonical ones) at the 3' end, contain fluorescent labels, sites of interaction with proteins, be a hybrid of RNA with DNA, contain additional hairpins for dimerization, dissociation, interaction, destruction, stabilization of proteins and other molecules, and recruitment of additional effector elements.

[0145] In one embodiment of the invention, any hypotonic buffers that do not significantly change the membrane structure and integrity of membrane proteins can be used for hypotonic lysis.

[0146] In one embodiment of the invention, instead of ultrasonic treatment, homogenization can be carried out by freezing-thawing, mechanical homogenization, high-pressure homogenization, liquid homogenization, nitrogen cavitation and other methods.

[0147] In one embodiment of the invention, washing and purification of membrane fragments can be carried out by other methods, including filtration, tangential filtration, precipitation, sedimentation and other methods.

[0148] In one embodiment of the invention, the purified membrane tablet can be resuspended in any physiological solution, including saline, isotonic tris-hydrochloride buffer solution, and others.

[0149] In advantageous embodiments of the invention for packaging into membrane particles, any proteins and peptides of any amino acid sequence can be added to the membrane suspension, including chemically modified or stabilized proteins containing chemically modified amino acids or amino acid derivatives, or protein complexes, recombinant proteins, genetically modified proteins or their complexes, enzymes or enzymatic complexes, antigen fragments, as well as protein complexes with RNA or DNA, including Cas proteins of various classes, types, species, origins and modifications, Cas proteins from various types of organisms, Cas-like proteins, zinc-finger nucleases, transcription activator-like effector nucleases (TALENs), meganucleases, human proteins, proteins of animal origin, proteins of viruses, bacteria, archaea, bacteria, chimeric proteins, proteins associated with or used together with low molecular weight compounds, polymers, adjuvants,materials of organic or inorganic origin,

[0150] In one embodiment of the invention, the formation of particles in a membrane suspension can be carried out by closing the membrane as a result of freezing-thawing, ultrasonic treatment, nitrogen cavitation, high-temperature treatment and other methods.

[0151] Table 1. Examples of filter materials for extrusion.

[0152] Table 2. Examples of methods for mechanical extrusion

[0153]

[0154] Table 3. Examples of agents for enhancing RNA packaging.

[0155] Table 4. Examples of proteins for loading into micro- and nanoparticles.

[0156] Table 5. Examples of RNA modifications (caps)

[0157]

[0158] Table 6. Examples of protein modifications.

[0159]

[0160] Table 7. Examples of compounds for improving the process of cargo packaging into micro- and nanoparticles.

[0161] Table 8. Examples of drugs for simultaneous administration into micro- and nanoparticles.

[0162]

[0163] The following are examples of the implementation of the invention, which, however, do not cover all possible embodiments of the invention and do not limit the claimed invention.

[0164] Examples of implementation of the invention

[0165] Example 1. Obtaining nano-granules (NG) according to the present method

[0166] HEK293T cells or human mesenchymal stromal cells were subjected to hypotonic lysis and ultrasound homogenization. Cell membranes were sedimented from the resulting cell lysate and washed with phosphate buffer by centrifugation. Purified cell membranes were mixed with assembled complexes consisting of recombinant StCas9 protein and StlO guide RNA obtained by in vitro transcription, and serial extrusion was performed. The resulting nanoparticles in a mixture with unpackaged StCas9 / Stl 0 complexes were purified by chromatography.

[0167] The obtained NG nanoparticles were used in nanoparticle characterization tests by cryo-electron transmission microscopy (Fig. 2A) and dynamic light scattering (Fig. 2B) or NTA (Fig. 2C), as well as subsequent tests for the content of Cas protein, guide RNA.

[0168] The nanoparticle isolate loaded with 81Cas9 / guide RNA complexes using the developed technology was characterized by assessing the size distribution of the nanoparticles using the Nanoparticle Tracking Assay (NTA) method (Fig. 2A). As part of the development of methods for loading proteins into nanoparticles, the NG technology was created and nanoparticles were produced, which were used to assess protein loading levels (Table 1, Fig. 3). As an example, we used StCas9 protein loading; the loading analysis was performed using the developed polyclonal rabbit aHTn-StCas9 antibodies, which recognize all forms of StCas9 proteins regardless of the presence of additional elements. Signal detection was performed on equal amounts of nanoparticles (the amounts were counted using the NTA technology), under optimized nanoparticle lysis conditions, using Western blotting and dot blot. As a result, NG-based technologies enabled loading of up to 202 copies of StCas9 protein per nanoparticle.In this case, it is also possible to improve this indicator by introducing more concentrated mixtures of protein / RNA / protein and RNA, by changing the fluidity of cell membranes, the conditions of membrane formation (physical - changes in temperature, etc., chemical - introduction of chemical reagents, stabilizers, etc.), changing the method and conditions of extrusion or another type of assembly of membranes into particles (pressure conditions, the nature and diameter of the pores of membrane filters, ultrasonic treatment, homogenization, disintegration), or modifying the packed cargo by changing the charge, nature or conditions of interaction of cell membranes with the cargo.

[0169] Moreover, using this technology, any domain that allows expanding the functional capabilities of the system can be linked to the Cas protein, such as cytidine / adenine deaminase (base editor), epigenome remodeling enzyme (CRISPRa / CRISPRi and epigenome remodeling systems), reverse transcriptase (Prime Editing systems), factors for removing / adding epitranscriptome marks and other domains. Thus, the present method can be used not only for packaging Cas nucleases, but also for packaging any CRISPR / Cas-based systems, regardless of the type, type, structure and origin, as well as, in principle, any proteins of any structure and amino acid sequence.

[0170] Table 9. Packaging methods and description of nanoparticles with different options for loading proteins into biological nanoparticles using the example of the StCas9 protein.

[0171] Following this, the efficiency of packing RNA guides into nanoparticles according to the present method (in NG) and stochastic packing in EV was studied (Fig. 4).

[0172] A quantitative comparison of the levels of RNA guide packaging using stochastic loading of EVs and packaging according to the present method in NGs is shown in Fig. 4. As a result, stochastic packaging provides loading of up to 0.1 RNA copies in EVs, and the Avoska technology up to 9.3 RNA copies per NG particle. It is important to note that for Avoska technologies, an increase in RNA packaging is possible by changing the packaging conditions, concentrations of RNA, particles, cell membranes, changing the physical parameters for obtaining nanoparticles, adding chemical and physical agents, changing the membrane production technology (extrusion, ultrasound, etc.) or the constituent components of these technologies.

[0173] Therefore, a method for packaging proteins, RNA and ribonucleoprotein complexes using the CRISPR / Cas9 RNP as an example has been developed. The possibility of packaging any proteins and any RNA, including genetically and chemically modified ones, has been demonstrated. It has been shown that the packaged complexes, proteins, RNA and RNP retain pronounced activity, and the nanoparticles effectively deliver cargo to target cells. Technological approaches and technologies for packaging cargo in NG have been developed.

[0174] Example 2. Cultivation of HEK293T cells and mesenchymal stromal cells

[0175] Human HEK293T cells were cultured in DMEM (4.5 g / L glucose) with 10% fetal bovine serum, 2 μM L-glutamine, and 1% penicillin / streptomycin. Human bone marrow- or adipose-derived mesenchymal stromal cells were cultured in complete DMEM (1 g / L glucose) with 10% fetal bovine serum, 2 μM L-glutamine, and 1% penicillin / streptomycin. Mouse TC1 cells were cultured in RPMI-1640 with 10% fetal bovine serum, 2 μM L-glutamine, and 1% penicillin / streptomycin. To obtain EVs, a complete medium was prepared for a specific cell line, with fetal bovine serum previously purified from the presence of EVs by ultrafiltration.

[0176] Example 3. Transcription and purification of guide RNA in vitro

[0177] The PCR product encoding the guide RNA under the T7 promoter was synthesized using Q5 high-fidelity polymerase. Then, the T7-1GR product was used as a template for in vitro transcription using the High-Efficiency In Vitro RNA Synthesis Kit (Bioinlabs) according to the manufacturer's protocol. The in vitro transcription reaction was incubated overnight and then treated with DNase type I (NEB) for 15 min at 37°C, followed by RNA purification by the isopropanol precipitation method. Briefly, isopropanol and 5 M NaCl were added to the resulting mixture and centrifuged for 30 min at 4°C. The resulting pellet was washed twice with 70% and then 95% ethanol. The dried pellet was dissolved in RNase-free water and stored at -80°C.

[0178] Example 4. Obtaining recombinant protein StCas9

[0179] The pLysS strain of E. coli BL21 (DE3) (Novagen) was used for protein expression. E. coli cells were grown in LB medium (supplemented with antibiotic, 0.5% sucrose, 0.5% glycerol, 1 mM magnesium chloride, 50 mM Na2HPO4, 50 mM KH2PO4, and 25 mM (NEU^SC ) at 30°C to OD600. 1.2. Expression was induced for another 16 h by adding 0.1 mM isopropyl-PDl -thiogalactopyranoside at 18°C. Cells were resuspended in 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 1 mM PMSF, 0.2% Triton X-100, and 0.1% Tween 20, sonicated, and centrifuged at 15,000*g for 40 min. The cell lysate was treated with 0.05% polyethylenenimin for 30 min at 4°C, centrifuged at 15,000*g for 40 min, the supernatant was collected, and the supernatant was then coupled with Ni-chelating sepharose (GE Healthcare).The resin was washed with 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 0.05%, and Igepal CA-630; the bound protein was eluted in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.3 M imidazole, and 10% glycerol, then the protein was bound to SP-Sepharose (GE Healthcare) in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.01% Triton X-100, and 2 mM DTT, eluting with a linear gradient from 150 mM to 1 M NaCl. Example 5. Molecular Cloning.

[0180] Molecular cloning of the plasmids used in the work was performed using the Gibson assembly (NEB) technology according to the manufacturer's protocol with primers synthesized by Lumiprobe (Russia). The primers were designed in the SnapGene program. SPR products for the reaction were obtained with high-precision polymerase Q5 (NEB). Purification of SPR products from the gel was performed using a kit for DNA extraction from gel (Eurogen).

[0181] Example 6. Isolation of nucleic acids

[0182] RNA loaded into nanoparticles was isolated using the ExtractRNA reagent (Eurogen) according to the manufacturer's protocol. The isolated RNA was subjected to reverse transcription using AmpliSens Reverta-FL reagents (AmpliSens Biotechnologies) for 30 min at 37°C for subsequent PCR analysis.

[0183] Example 7. PCR analysis

[0184] Quantitative assessment of loaded guide RNAs was performed by amplification of the obtained cDNAs and standards for constructing a calibration curve on a QuantStudio5 device (Applied Biosystems). Calibration standards were genetically engineered constructs encoding the StlO guide RNA with a known concentration. Genta TaqF DNA polymerase reagents (GenTerra), as well as primers and a probe synthesized by Eurogen and Lumiprobe were used for the analysis.

[0185] Example 8. Obtaining NG according to the present method

[0186] ~8* 10 7The cells were removed using Versen solution (PanEco, Russia), washed with phosphate-buffered saline and resuspended in 10 ml of ice-cold TM buffer (0.01 M Tris and 0.001 M MgCl2) containing protease inhibitor cocktail. The suspension was incubated on ice for 15 minutes, then sonicated on a disintegrator (model) (5 sec, amplitude 27%). Then a solution of 60% sucrose in TM buffer (pH 7.4) was added to a concentration of 0.25 M sucrose, mixed, and the membranes were sedimented at 4 ° C and 6000 g (15 minutes). The supernatant was discarded, a solution of 0.25 M sucrose in TM buffer was added and washing was repeated 2 more times. After washing, the membrane pellet was resuspended in a 0.25 M sucrose solution in TM buffer (pH 8.6), sonicated (5 sec, 27% amplitude), the membranes were sedimented (4°C, 6000g, 15 minutes), and then the membranes were washed twice with a 0.25 M sucrose solution in TM buffer (pH 8.6). After the last wash, the supernatant was discarded, and the membrane pellet was resuspended in phosphate-buffered saline.In OptiMem medium, 160 μg of recombinant StCas9 protein and 25.2 μg of guide RNA were mixed and incubated for 10 minutes. The resulting RNP solution was added to the membrane suspension and gently mixed by pipetting. The resulting mixture was sequentially passed through hydrophilic polycarbonate membranes soaked in PBS-HAT buffer solution using a manual extruder: pore diameters of 1 μm (15 times), 0.4 μm (11 times), 0.1 μm (15 times). The resulting vesicles were purified using gel-exclusion chromatography on a Sepharose CL-4B resin column using a phosphate buffer solution as a mobile phase. The resulting sample was added to a 1.6x10 cm column, 40 fractions of 500 μl were collected. Fractions containing biological nanoparticles (Fractions 11–16) were pooled and supplemented with 10x PBS-HAT buffer to a concentration of 1x (0.2% human serum albumin, 25 mM trehalose, 25 mM HEPES).

[0187] Example 9. Cell transfection

[0188] Transfection of HEK293T cells was performed using our described technology [Kostyusheva, A. P, Kostyushev, DS, Brezgin, SA, Zarifyan, DN, Volchkova, E. V, & Chulanov, VP (2019). Molekuliarnaia biologiia, 53(2), 311–323. https: / / doi.org / 10.1134 / S0026898419010Q751 using polyethyleneimine. The day before transfection, the cells were seeded at a density of ~70%, the next day they were transfected with DNA using polyethyleneimine (7.5 mM) with NaCl (150 mM). After 24 hours, the medium was removed, the cells were washed with a phosphate-buffered saline solution and complete medium was added. Transfection of mesenchymal stromal cells was performed using our own technology.

[0189] Example 10. Screening fluorescence microscopy

[0190] Microscopy of 96-well plastic plates was performed using an LCI Imager ExFluorer.

[0191] Example 11. Confocal microscopy

[0192] Confocal images of transfected cells and cells treated with different nanoparticles were performed on an FV3000 microscope (Olympus). Example 12. Flow cytometry

[0193] Flow cytometry was performed using a LongCyte instrument (ChallengeBio).

[0194] Example 13. Western blotting and dot blotting

[0195] Nanoparticles were lysed according to the developed protocol and used for SDS-PAGE, followed by transfer to polyvinylidene fluoride membranes. Membranes were blocked in 5% skim milk in PBST buffer (80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, 0.1% Tween 20) at room temperature for 1 hour. Membranes were incubated with primary anti-Cas polyclonal rabbit antibodies at 4 °C overnight on a slow shaker. Membranes were washed 3 times for 10 minutes in PBST, then incubated with anti-rabbit IgG secondary antibodies conjugated with horseradish peroxidase. Subsequently, the membranes were washed 3 times for 10 minutes in PBST buffer. The signal was developed using the ECL reagent (Thermo Fisher) and detected using a Fusion-FX6.Edge V.070 gel and chemidocumenting system.

[0196] Example 14. Transmission electron microscopy Sguo-TEM

[0197] Cryoelectron micrographs of nanoparticles were obtained using a transmission electron microscope (TEM Tecnai G212 SPIRIT, FEI, USA). A copper grid on a carbon substrate was used for this purpose. 3 μl of the nanoparticle sample was applied to the grid. Excess sample was removed by blotting the grid for 1 s and then immersing it in liquid ethane (Vitrobot FEI, USA). Then the grid with the sample was transferred in liquid nitrogen to the TEM.

[0198] Example 15. Measurement of ζ-potential by dynamic light scattering DLS The Malvern Zetasizer NanoZS instrument (Malvern, UK) was used to analyze the average size and charge of all biological and hybrid nanoparticles. Each sample was diluted 1000-fold with PBS, filtered through a 0.2 μM filter (Coming) and analyzed 5 times; 1.5 ml of the diluted samples were loaded into polystyrene cuvettes (DTS0012; Malvern). The analysis was carried out at 25 °C (100 measurements) using a 20 mW He / Ne laser (633 nm). The data were analyzed using Zetasizer 8.01.4906 software (Malvern). The ζ-potential was analyzed in U-type cuvettes (DTS1070; Malvern) with gold electrodes. Z-potential measurements were performed at 25 °C at least 5 times. The background signal was estimated using filtered PBS.

[0199] Example 16. Measurement of the number of nanoparticles using nanoparticle trajectory analysis (NTA)

[0200] Nanoparticles were analysed using a Nanosight LM10 HS instrument (NanoSight Ltd., UK) equipped with a 405 wavelength laser. Particle tracking videos were recorded at room temperature with passive temperature readout and the following camera settings optimised for nanoparticles: camera shutter 1500, camera gain 500, lower threshold 195 and upper threshold 1885. Videos were processed using Nanoparticle Tracking Analysis software version 2.3 (NanoSight Ltd., UK) with a detection threshold of 5. At least 12 individual videos of 60 s duration were recorded and processed. Data from multiple videos were combined to produce a histogram of particle size and mean total concentration corrected for the dilution factor.

[0201] Example 17. Statistical analysis

[0202] Values ​​were expressed as mean ± standard deviation of triplicate experiments in GraphPad Prism software. Student's t-test with Tukey's HSD post hoc test was used to compare variables and calculate p-values ​​to detect statistically significant differences in means. To determine whether each percentage of off-target indels in the deep sequencing assay was significant compared to mock-treated controls, a two-tailed p-value was calculated using Fisher's exact test.

[0203] Example 18.

[0204] Induced pluripotent stem cells were seeded in a rotary perfusion bioreactor, the cell mass was grown in the culture medium until 100% confluency was achieved. After reaching confluency, the cells were washed with a phosphate-buffered saline solution and detached from the reactor substrate with a Versene solution. The cell mass was washed twice with a phosphate-buffered saline solution and resuspended in a hypotonic buffer TM solution (0.01 M Tris-HCl, 0.001 M MgCh) containing an inhibitor cocktail for proteases, with a density of up to 2x10 6cells / ml. The cell suspension was incubated at 0°C for 20 minutes and then sonicated. A 60% sucrose solution in TM buffer was added to the reactor containing the lysed cell suspension to a concentration of 0.25 M sucrose and the mixture was centrifuged (6000g, 15 minutes). The supernatant was removed, a 0.25 M sucrose solution in TM buffer (pH 7.4) was added to the membrane tablet and mixed, the procedure of washing and removing the supernatant was repeated twice. A 0.25 M sucrose solution in TM buffer (pH 8.6) was added to the resulting tablet, the suspension was mixed and sonicated. The suspension was centrifuged (6000g, 15 minutes), the supernatant was removed, the tablet was resuspended in a solution of 0.25 M sucrose in TM buffer (pH 8.6), the washing procedure was repeated twice. The supernatant was discarded, the tablet was resuspended in a solution of phosphate-buffered saline.In parallel, the CHO cell line expressing the His-tagged Cas9 nuclease from Streptococcus thermophilus was grown in a bioreactor to an optimal density, the cells were washed with a phosphate-buffered saline solution and lysed. The lysate was centrifuged, and the protein was isolated from the lysate by sequential purification on a Ni-chelating resin and an ion-exchange resin. The isolated protein was mixed in a reactor with the RNA guide obtained by the in (' / / / «-transcription method and treated with alkaline phosphatase, the mixture was stirred and incubated for 10 minutes at a temperature of 20°C. The resulting mixture was added to the membrane suspension in an optimal ratio and stirred, then extruded using a liposome extruder under nitrogen pressure at a temperature of 37°C through hydrophilic polycarbonate membranes with pore diameters of 1 μm (15 times), with pore diameters of 0.4 μm (9 times), with pore diameters of 0.1 μm (15 times).The resulting suspension was purified using Sepharose CL-4B gel exclusion resin and fractions corresponding to the target NG particles were collected.

[0205] Example 19.

[0206] 1. Take the nanoparticle preparation obtained according to Example 8, in a dose of 1 x 10 15 particles per kilogram of patient weight loaded with CRISPR / Cas base editor complexes to correct a genetic mutation in the SMN1 gene in spinal muscular atrophy. The drug is administered intravenously, CRISPR / Cas is delivered to the motor neurons of the spinal cord, which leads to CRISPR / Cas-mediated correction of the mutation and elimination of clinical symptoms of the disease, stopping disease progression and restoring motor activity.

[0207] 2. Take the nanoparticle preparation obtained according to Example 8, in a dose of 1 * 10 15particles per kilogram of patient weight loaded with CRISPR / Cas antiviral gene transcription activation complexes based on dCas9-p300 with RNA guides to intracellular antiviral factors, which leads to activation of intracellular antiviral signaling with suppression or elimination of pathogenic viruses.

[0208] Example 20.

[0209] 1. Take a nanoparticle preparation based on a vaginal gel with a concentration of 1 x 10 18 particles per milliliter of gel containing nanoparticles loaded with CRISPR / Cas complexes and directed to the human papillomavirus genome. The resulting gel is used to treat papillomavirus infection.

[0210] 2. A cream containing nanoparticles loaded with tissue regeneration factors is taken and applied to the skin. The cream is applied daily, internalization of nanoparticles into the cells of the dermis occurs, which has an anti-inflammatory and pro-regenerative effect.

[0211] Thus, the declared technical solution ensures:

[0212] (a) packaging of up to >202 copies of Cas proteins per nanoparticle, while the amount of packaged Cas proteins per nanoparticle can be controlled by increasing the concentration of CRISPR / Cas RNP complexes and / or membrane fragments in the loading solution;

[0213] (b) simultaneous packaging and co-packaging of guide RNA into nanoparticles with an efficiency of <0.2 copies to >9 copies of guide RNA per nanoparticle, wherein the number of copies of loaded guide RNA(s) can be controlled by changing the concentration of the loading solution and changing the concentration of the components (loaded RNA and cell membranes) in the packaging mixture;

[0214] (c) packaging of chemically modified, including stabilized, guide RNAs, such as guide RNAs with chemical modifications, including but not limited to 2'-deoxy, 2'-F, 2'-0Me, phosphorothioates, LNA, UNA, 2'F-ANA, butane linkers, 4'-0me, 2',4'-diOme, 2'F,4'-OMe, the use of DNA-RNA hybrids, combinations thereof, which can be used to change the stability of RNA, the efficiency of CRISPR / Cas action, the specificity of CRISPR / Cas action, the safety of RNP action;

[0215] (d) eliminating the need for intracellular expression of the guide RNA together with Cas proteins, which may be associated with modification of the target gene(s), off-target CRISPR / Cas activity, the occurrence of genetic mutations, aberrations, genome instability, anoikis, changes in the properties of producer strains, changes in the characteristics of the resulting nanoparticles, packaging of potentially dangerous (toxic, carcinogenic) compounds into nanoparticles, or changes in the surface of nanoparticles with a violation of tropism, the ability to overcome biological barriers, and stability;

[0216] (d) is a universal system for packaging CRISPR / Cas systems in the form of RNPs of any class, type, kind, origin, variant and modification, since the packaging does not require optimization for specific proteins, is not limited by the sizes of Cas proteins and their complexes, is not determined by the composition of guide RNAs, the presence or absence of additional elements, the removal of any elements in the CRISPR / Cas composition or the use of additional components, is not determined by the charge of Cas proteins, guide RNAs and the resulting RNPs, is not determined by the use of full-length, truncated, or split proteins, domains or combinations thereof, and also ensures the loading of any guide RNAs in the form of 1 guide RNA, their combinations, elongated or truncated sequences that, when assembled or processed (including cutting), cause the formation of RNA that can be used to interact with the Cas protein or other components;

[0217] (e) is a universal system for packaging any proteins, RNA, as well as protein-RNA complexes, including in the form of RNP.

Claims

The AMENDED CLAUSE OF THE INVENTION was received by the International Bureau on July 25, 2025 (25.07.2025) 1. A method for packaging a protein, RNA, or protein-RNA complex into a particle, comprising the following steps: a. the original cells are lysed to obtain a cell lysate; b. from the cell lysate obtained in step (a), sedimentation of the cell membranes is carried out, followed by washing to obtain a suspension of cell membranes; c. the suspension of cell membranes obtained in step (b) is mixed with proteins, RNA, and assembled ribonucleoprotein complexes, then the resulting mixture is extruded by pressing through at least one membrane of the extruder to form spherical particles of the required size; d. the particles obtained in step (c) are purified from unpackaged components to obtain a particle comprising protein, RNA, or a protein-RNA complex, and having a size of 50 to 0.01 μm, where the particles are purified using chromatography, ultracentrifugation, filtration, precipitation, fractionation, or a combination thereof.

2. The method according to item 1, characterized in that the particles obtained according to said method are microparticles or nanoparticles.

3. The method according to item 1, characterized in that the particles obtained according to the said method are nanoparticles (NG).

4. The method according to claim 1, characterized in that at step (c) the closure of membrane fragments into spherical particles can be carried out as a result of a series of freeze-thaw cycles, ultrasonic treatment, microfluidics.

5. The method according to 1, characterized in that the cells are HEK293, HT 1080, HeLa, PER.C6, CHO cells, mesenchymal stem cells (MSCs), induced pluripotent (iPSCs), totipotent, multipotent cells of various origins, NIH-3T3, BHK-21, bEnd.3, COS-7, HB54, HB55, HCA2, HMEC, HeLa and other cell lines, including those of tumor origin, as well as cultures of primary cells of various origins and morphologies.

6. The method according to item 5, characterized in that the HEK293 cells are HEK293 cells of any modification selected from the group including HEK293, HEK293T, HEK293F, HEK293FT, HEK Exp293E.

7. The method according to item 3, characterized in that the mesenchymal stem cells (MSCs) are MSCs of various origins, selected from a group including placental, adipose, bone marrow, MSCs from umbilical cord blood, amniotic fluid, peripheral blood, synovial fluid, dental pulp, endometrium, skin, muscle tissue, and salivary glands.

8. The method according to claim 1, characterized in that in step (e) the particles are purified from unpacked complexes using gel-exclusion chromatography, ion-exchange chromatography, hydrophobic chromatography, affinity chromatography ultracentrifugation, ultracentrifugation in a density gradient, ultrafiltration, tangential filtration, precipitation, fractionation in asymmetric flows, or using a combination of these methods.

9. The method according to claim 1, characterized in that the protein is a protein or peptide of any amino acid sequence, including chemically modified or stabilized proteins containing chemically modified amino acids or amino acid derivatives, recombinant proteins, genetically modified proteins, enzymes, antigenic proteins or fragments thereof, Cas proteins of various classes, types, species, origins and modifications, Cas proteins from various types of organisms, Cas-like proteins, zinc-finger nucleases, transcription activator-like effector-based nucleases (TALENs), meganucleases, human proteins, animal proteins, proteins of viruses, bacteria, archaea, chimeric proteins.

10. The method according to claim 1, characterized in that the protein complex is a complex of a recombinant protein, a genetically modified protein, an enzymatic complex, an antigen fragment, a complex of a protein with RNA or DNA, a Cas protein, a Cas-like protein, a zinc-finger nuclease, a nuclease based on an effector similar to a transcription activator (TALENs), meganucleases, human proteins, proteins of animal origin, proteins of viruses, bacteria, archaea, chimeric proteins, proteins associated with or used together with low-molecular compounds, polymers, adjuvants, materials of organic or inorganic origin.

11. The method according to item 1, wherein to form a protein-RNA complex, RNA molecules are chemically synthesized or obtained using in vitro transcription, then RNA mixed with the protein of interest, the mixture is incubated until the ribonucleoprotein complex is formed.

12. Method by and. 1, characterized in that the RNA is mRNA, long non-coding RNA, RNA of the CRISPR-Cas systems, hybrid RNA consisting of various elements, optionally containing ribozyme sequences, aptamers, ribosome entry sites, encapsidation signals, translation initiation and termination signals, sequences that change RNA stability, RNA localization in cells, RNA translation, RNA splicing, wherein the chemically modified RNA optionally contains pseudouridine, or is modified by sugar or nucleoside residues, or contains cap structures at the 5'-end, a polyA sequence or modified polyA sequences in combination with other nucleotides at the 3'-end, or contains fluorescent labels, sites of interaction with proteins, is a hybrid of RNA with DNA, contains additional hairpins for dimerization, dissociation, interaction, destruction, stabilization of proteins, or additional effector elements.

13. The method according to item 12, characterized in that the RNA of the CRISPR-Cas systems are short RNA (crRNA), tracrRNA, or single guide RNA (sgRNA).

14. The method according to claim 1, characterized in that the modifications of Cas proteins are complete Cas nucleases or proteins based on catalytically inactive dead-Cas (dCas), containing inactivating mutations in the nuclease domains, or Cas-nickases (nCas), containing a mutation in only one domain.

15. The method according to claim 14, characterized in that the Cas, dCas, nCas proteins are wild-type proteins, or improved or modified variants of the Cas, dCas, nCas proteins, obtained by random or directed evolution, mutagenesis with or without optimization of protein-coding codons, and optionally have additional elements in the coding construct.

16. The method according to claim 14, wherein the Cas, dCas or nCas proteins are Cas9, Cas12a / b, Cas13a, Cas13b, Cas13c, Cas14, Cas-X or similar proteins, or improved variants selected from Cas9-HF, eSpCas9, HypaCas9, xCas9, SpRY / SpG, Fokl-fused dCas9.

17. The method according to claim 14, wherein Cas, dCas or nCas are a single protein or consist of several components that assemble into a single protein in target cells, producer cells or inside particles.

18. The method of claim 14, characterized in that the Cas, dCas, and nCas proteins contain additional domains, including domains for activating transcription, domains for repressing transcription, domains for editing DNA or RNA bases, reverse transcriptase domains of Prime Editing systems and their modifications and / or improved variants obtained by directed evolution or mutagenesis, RNA demethylase or RNA methyltransferase domains, and DNA visualization systems.

19. The method according to claim 18, characterized in that the domains for transcription activation are the domains VP48, VP64, VP160, VP192, p65, p65-HSF1, p300, VPR, TET1, as well as their modifications according to the principles of Scaffold, Casilio, SAM, TREE, SunTag or similar principles.

20. The method according to claim 18, wherein the transcription suppression domains are KRAB, EZH2, KRAB-MeCP2, DNMT3A, DNMT3A-3L, ESDI domains, or modifications thereof, or combinations thereof.

21. The method according to claim 18, characterized in that the domains for editing DNA or RNA bases are the domains rAPOBEC1, ABOVECA, AID, TadA, ADAR2, as well as their orthologs, their homologs and / or their modified variants obtained by mutagenesis or directed evolution.

22. The method according to item 18, characterized in that the domains of RNA demethylases or RNA methyltransferases are the domains of METTL3, METTL14, METTL16, FTO, ALKHB5, or their combinations or modified variants thereof obtained by mutagenesis or directed evolution.

23. The method according to item 18, characterized in that the DNA visualization systems are selected from the group including CRISPR-FISHer, CRISPR-SIRIUS, CRISPRainbow modified CCRRIISSPPRR systems with fluorescent protein domains grafted and / or recruited to the Cas protein and / or RNA guide.

24. The method according to item 1, characterized in that the RNA is chemically modified at nucleotide residues, RNA nucleotides, or sugar residues, including 2'-O-methyl derivatives (M), 2'-O-methyl-3'-phosphorothioate derivatives (MS), or 2'-O-methyl-3'-thiophosphonoacetate (MSP), pseudouridine Ψ, N1-methylpseudouridine (mlT), N 1-methylpseudouridine (sh5C), 5-hydroxymethylcytosine (5hmC), 5-methiouridine (m5U), 2-thiouridine (s2U), aminomethylene-containing nucleotides (BNANC[N-Me]), locked nucleic acid (LNA), 2'-O-methyl-3'-phosphonoacetate (MP), ethyl group (cEt), fluoro group (F), phosphorothioate (PS), tetraethylene glycol (TEG), phosphonoacetate (PACE) or their combinations.

25. The method according to claim 1, characterized in that the RNA contains cap structures at the 5'-end, a polyA sequence or modified polyA sequences in combination with other nucleotides at the 3'-end, or contains fluorescent labels, sites of interaction with proteins, or is a hybrid of RNA with DNA, or contains additional hairpins for dimerization, dissociation, interaction, destruction, stabilization of proteins and other molecules, as well as for attracting additional effector elements.

26. The method according to 1, characterized in that the packaging of proteins, RNA or their complexes is carried out at the stage of obtaining the said particles.

27. The method according to claim 1, characterized in that the complex of protein and RNA is the CRISPR / Cas ribonucleoprotein.

28. The method according to claim 1, characterized in that human and mammalian cells are used as the source cells, including genetically and / or chemically modified cells, cell lines, transiently transfected cell lines and other components of a cell or cell line.

29. The method according to claim 1, characterized in that the extrusion step is carried out using a manual extruder, an automated extruder of cell mass or cell suspension, or cell membranes, or particles in a solvent.

30. The method according to item 1, characterized in that from 0.5 ml to more than 1000 l of cell mass or suspension of cell membranes or particles. ZE Method according to claim 1, characterized in that the solvent influences the production of nanoparticles and / or changes the properties of cell or particle membranes, in particular their fluidity, and / or influences the cellular viability or integrity of the particles.

32. The method according to item 1, characterized in that the extrusion is carried out under the pressure of inert gases or by mechanical extrusion, or by centrifugal extrusion or vacuum filtration.

33. The method according to paragraph 29, characterized in that at the extrusion stage, ultrasonic treatment is additionally carried out.

34. The method according to item 1, characterized in that membranes made of polycarbonate, polyethylene terephthalate, polypropylene, regenerated cellulose or cellulose ethers, polytetrafluoroethylene, polyvinylidene fluoride, nitrocellulose, nylon, or polyethersulfone are used for extrusion.

35. The method according to item 34, characterized in that the membrane for extrusion is standard or track-type.

36. The method according to item 34, characterized in that the membrane for extrusion has a hydrophilic or hydrophobic coating.

37. The method according to item 34, characterized in that the membranes for extrusion have pore diameters selected from the range from 10 to 0.01 µm.

38. The method according to item 37, characterized in that the membranes for extrusion have pore diameters selected from the group consisting of 10 μm, 8 μm, 5 μm, 2 μm, 1.2 μm, 0.8 μm, 0.45 μm, 0.4 μm, 0.22 μm, 0.2 μm, 0.1 μm, 0.05 μm, 0.03 μm, 0.01 μm.

39. The method according to item 1, characterized in that the extrusion is carried out using drainage discs.

40. The method according to claim 1, characterized in that the cells are treated with high-molecular and / or low-molecular compounds that are capable of influencing the production of nanoparticles, the expression of proteins and / or RNA.

41. The method according to item 1, characterized in that the cell membranes, purified from cytoplasmic components, intracellular components and cell nuclei, are optionally fragmented by ultrasound after hypotonic lysis of the cells, or the fragmentation is carried out by a chemical or physical method, and is used for extrusion in a liquid containing finished proteins, protein-RNA complexes or CRISPR / Cas ribonucleoprotein.

42. The method according to claim 1, characterized in that the membranes are modified to change their charge or affinity to proteins, protein-RNA complexes or CRISPR / Cas ribonucleoprotein, for more efficient packaging.

43. The method according to claim 1, characterized in that proteins, protein-RNA complexes or RNPs CRISPR / Cas are also modified to alter charge, size, and / or membrane affinity for more efficient packaging, or introduced Multifactorial modifications of membranes and packable cargo to provide more efficient packaging into nanoparticles.

44. A particle comprising a protein, RNA, or a protein-RNA complex, for genetic editing, for genome modification, for correction of genetic mutations, for modification of the epigenome, for modification of the epitranscriptome, for editing RNA, for DNA destruction, for RNA destruction, for modification of nucleotide bases, for modification of nucleotides, for deamination of DNA or RNA, for integration of DNA or RNA sequences into the human or mitochondrial genome, for DNA visualization, for studying the spatial structure of chromatin, for studying RNA metabolism, for modification of proteins, for editing proteins, for control of biological processes, for control of physiological processes, for the purposes of vaccination, for the creation of therapeutic vaccines of RNA, proteins, peptides, mixtures or combinations thereof, for the creation of medicinal products, for the creation of diagnostic products, for cosmetic products, for use in regenerative medicine,for the correction of drug resistance mutations, for reconstructive medicine, for the treatment of oncological diseases, for the treatment of autoimmune diseases, for the treatment of hereditary diseases, for the treatment of metabolic diseases, for the in vitro or ex vivo modification of cell lines, primary cultures or individual cells, including animal embryos, for the production of genetically modified animals, for the production of animal cell lines, for use in veterinary medicine, and characterized in that the said particle was obtained by the method according to any of paragraphs 1-43 and has a size from 50 to 0.01 μm., 45. A pharmaceutical composition for genetic editing, for genome modification, for correction of genetic mutations, for modification of the epigenome, for modification of the epitranscriptome, for editing RNA, for DNA destruction, for RNA destruction, for modification of nucleotide bases, for modification of nucleotides, for deamination of DNA or RNA, for integration of DNA or RNA sequences into the human genome or mitochondria, for DNA visualization, for studying the spatial structure of chromatin, for studying RNA metabolism, for modification of proteins, for editing proteins, for control of biological processes, for control of physiological processes, in for the purposes of vaccination, for the creation of therapeutic vaccines based on RNA, proteins, peptides, their mixtures or combinations, for the creation of medicinal products, for the creation of diagnostic products, for cosmetic products, for use in regenerative medicine, for the correction of drug resistance mutations, for reconstructive medicine, for the treatment of oncological diseases, for the treatment of autoimmune diseases, for the treatment of hereditary diseases, for the treatment of metabolic diseases, for the in vitro or ex vivo modification of cell lines, primary cultures or individual cells, including animal embryos, for the production of genetically modified animals, for the production of animal cell lines, for use in veterinary medicine, containing an effective amount of particles according to paragraph 44 and at least one pharmaceutically acceptable excipient, characterized in that the said particles were obtained by the method according to any of paragraphs 1-43.

46. ​​Use of a particle according to item 44 for genetic editing, for genome modification, for correction of genetic mutations, for epigenome modification, for epitranscriptome modification, for RNA editing, for DNA destruction, for RNA destruction, for modification of nucleotide bases, for modification of nucleotides, for deamination of DNA or RNA, for integration of DNA or RNA sequences into the human genome or mitochondria, for DNA visualization, for studying the spatial structure of chromatin, for studying RNA metabolism, for protein modification, for protein editing, for control of biological processes, for control of physiological processes, for the purposes of vaccination, for the creation of therapeutic vaccines based on RNA, proteins, peptides, their mixtures or combinations, for the creation of drugs, for the creation of diagnostic drugs, for cosmetic products, for use in regenerative medicine,for correction of drug resistance mutations, for reconstructive medicine, for the treatment of oncological diseases, for the treatment of autoimmune diseases, for the treatment of hereditary diseases, for the treatment of metabolic diseases, for the in vitro or ex vivo modification of cell lines, primary cultures or individual cells, including animal embryos, for the production of genetically modified animals, for the production of animal cell lines, and / or for use in, veterinary medicine, characterized in that the said particle was obtained by the method according to any of paragraphs 1-43.

47. The use of particles according to paragraph 46, characterized in that the said particles are administered systemically into the body, by local administration, by inhalation, subcutaneously, directly into organs and tissues in a certain dosage form.

48. Use according to paragraph 47, characterized in that the systemic administration into the body is intravenous, intra-arterial, bolus, intraperitoneal, subarachnoid, epidural or intrasternal administration, or administration into the sinuses, into the cavities of the body, or administration subcutaneously, intramuscularly, or administration into specific organs and tissues.

49. Use according to paragraph 47, characterized in that local administration into the body is application to the surface of the skin and mucous membranes, the surface of organs, or washing of organs.

50. The use according to claim 47, characterized in that the dosage form is selected from the group comprising an aerosol for inhalation, an aerosol for topical use, an aerosol for external use, an aerosol for application to the oral mucosa, a nasal aerosol, a sublingual aerosol, a transdermal aerosol, an ear aerosol, a vaginal gel, an eye gel, an injection gel, a gel for topical use, a gel for application to the gums, a gel for external use, a gel for subcutaneous administration, a gel for the preparation of a suspension for oral administration, a gel for oral administration, a gel for application to the oral mucosa, a dental gel, an intestinal gel, a nasal gel, a periodontal gel, a rectal gel, a dental gel, a transdermal gel, a urethral gel, an ear gel, an endocervical gel, granules, granules for preparation, drops, solutions, suspensions, granules for resorption, enteric-coated granules, enteric-coated granules with prolonged release,film-coated granules, cut-and-pressed granules, modified-release granules, prolonged-release granules, effervescent granules, dispersion for infusion, dispersion for injection, dispersion for intravenous administration, dispersion for intradermal administration, liquid for inhalation, liquid for external use, liquid for oral administration, implant, intravitreal implant, eye drops, prolonged-release eye drops, drops for inhalation, drops for topical use, drops for application to the oral mucosa, drops for oral administration, drops, dental, nasal drops, sublingual drops, ear drops, capsules, vaginal capsules, intrauterine capsules, chewable capsules, enteric-coated capsules, enteric-coated capsules with prolonged release, sublingual capsules, rectal capsules, modified-release capsules, capsules with inhalation powder, prolonged-release capsules, concentrate for the preparation of, dispersion, solution, suspension, emulsion, vaginal cream, eye cream, cream for topical use, cream for external use, cream for application to the oral mucosa, nasal cream, rectal cream, ear cream, vaginal liniment, liniment for topical use, liniment for external use, periodontal liniment, endocervical liniment, lyophilisate for the preparation of, dispersion, drops, concentrate, solution, spray, suspension, emulsion, vaginal ointment, eye ointment, ointment for inhalation, ointment for topical use,ointment for application to the oral mucosa, ointment for external use, nasal ointment, rectal ointment, ear ointment, oil for inhalation, oil for topical application, oil for external use, oil for internal use, tincture, tincture for inhalation, tincture for topical application, tincture for external use, dental sticks, periodontal sticks, nasal sticks, urethral sticks, ear sticks, paste for application to the gums, paste for external use, paste for the preparation of a suspension for oral administration, paste for internal use, paste for application to the oral mucosa, medicinal dental paste, vaginal foam, intrauterine foam, foam for external use, rectal foam, patch, patch for the oral mucosa, transdermal patch, eye films, films for gum adhesives, buccal films, films dispersible in the oral cavity, periodontal films, sublingual films,powder for inhalation, dosed powder, powder for external use, powder for the preparation of gel, dispersion, drops, solution, paste, syrup, spray, suspension, powder for oral administration, nasal powder, periodontal powder, ear powder, effervescent powder, vaginal solution, intrauterine solution, solution for intra-arterial administration, solution for intraperitoneal administration, solution for intravenous administration, solution for intraocular administration, solution for intradermal administration, solution for intracoronary administration, solution for intramuscular administration, solution for intracavitary administration, solution for intravesical administration, solution for, intra-articular administration, solution for gastrointestinal administration, solution for hemodialysis, solution for hemodiafiltration, solution for hemofiltration, solution for inhalation, solution for intra-amniotic administration, solution for intralymphatic administration, solution for infusion, solution for injection, solution for topical use, solution for cutaneous scarification application, solution for application to the gums, solution for external use, solution for periarticular administration, solution for gastric irrigation, solution for bladder irrigation, solution for parabulbar administration, solution for peritoneal dialysis, solution for subcutaneous administration, solution for rinsing, solution for oral administration, solution for prick test, solution for eye wash, solution for nasal cavity wash, solution for oral wash, solution for ear canal wash, solution for oral mucosa, solution for subconjunctival administration,solution for extra-amniotic administration, solution for endo-sinusial administration, solution for endo-tracheal administration, dental 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