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 a Cas protein and a guide RNA, 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.

Description

[0001] METHOD FOR PACKAGING PROTEIN, RNA OR A COMPLEX OF PROTEIN WITH RNA 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 proteins, including the Cas protein, and RNA, including guide RNA (guide RNA, single-guide RNA, crRNA, tracrRNA), separately or together in the form of ribonucleoprotein complexes, for packaging the Cas protein and guide RNA of any class, type, kind, origin and modification.

[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 signature protein is Cas10. 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, Cas12, Cas13 and Cas14, 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 together 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 on” or “switching off”, or by combined action [Wright, A. V, Sternberg, S. H., Taylor, D. W., Staahl, B. T., Bardales, J. A., Kornfeld, I. E., & Doudna, I. 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.1501698112] [ 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 the components of CRISPR / Cas systems can significantly alter their properties and adapt them to perform new functions, such as editing nucleotide bases, changing the sequence of DNA and RNA, 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. I. (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 β-thalassemia, N. Engl. J. Med. 384 (2021) 252-260] [A. Sharma, J. -J. Boelens, M. Cancio, J. S. Hankins, P. Bhad, M. Azizy, A. Eewandowski, 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, CRIS PR-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 an 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 encoding DNA is accompanied by difficult-to-control expression, which is fraught with the induction of side reactions, including off-target cutting [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 RNA guide, or RNP) have the highest activity, the highest speed of action, their use is associated with the lowest probability of affecting 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 CRIS PR 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 CRIS PR Cas9 and Cas 12a therapeutics." Wiley Interdisciplinary Reviews: Systems Biology and Medicine 10.1 (2018): e1408.]. 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 (1000-10000 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 Chemo Therapeutics." 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].The key characteristics of biological nanoparticles include the highest 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, reduced clearance by RES macrophages. Based on the set of essential features, the method specified in the article by Oieni, Jacopo, et al. is the closest to the proposed invention and was chosen as a prototype.

[0013] The disadvantage of this method is that it:

[0014] (1) does not provide efficient packaging of Cas proteins;

[0015] (2) does not provide efficient co-packaging of guide RNAs;

[0016] (3) is not universal and requires optimization for a specific class, type, species and variant of the CRISPR / Cas system;

[0017] (4) does not allow packaging of chemically modified RNA guides or toxic RNA guides, the expression of which in producer cells is impossible, reduces cell viability, worsens the characteristics of nanoparticles, or reduces or completely disrupts the characteristics of the resulting product in the form of nanoparticles and / or CRISPR / Cas complexes;

[0018] (5) use immunogenic, toxic and non-biocompatible nanocarriers;

[0019] (6) are technically complex and expensive to produce;

[0020] (7) require the use of methods that induce the formation of pores in nanoparticles for loading therapeutic molecules, which can lead to partial aggregation of the nanoparticles and their contents, leakage of nanoparticles, decreased biocompatibility, targeting properties and the ability to overcome biological barriers and, as a result, decreased product quality.

[0021] Thus, the objectives of the present invention are: to create a new method for packaging protein, RNA and their complexes into micro- and nanoparticles.

[0022] Technical results:

[0023] - increasing the efficiency of packaging of proteins, RNA and their complexes; - eliminating or reducing the toxicity of the method for producer cells and reducing side reactions in producer cells by eliminating the need for expression of proteins, RNA in producer cells;

[0024] - expansion of the spectrum of molecules subject to packaging into micro- and nanoparticles;

[0025] - expansion of the arsenal of methods for packaging proteins, RNA and their complexes into a particle.

[0026] 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 protein of interest and / or RNA is expressed in the original cells using genetically engineered constructs delivered to the cells by viral or non-viral methods,

[0027] b) the cells obtained in step (a) are subjected to serial extrusion through membrane filters, followed by removal of cellular debris by centrifugation, c) the mixture obtained in step (b) is subjected to extrusion through membrane filters with a pore diameter comparable to the required final particle diameter, d) the particles obtained in step (c) are purified by chromatography to remove free proteins and contaminants.

[0028] According to preferred embodiments, the said technical result is also achieved by the fact that:

[0029] - the particles obtained according to the mentioned method are nanoparticles;

[0030] - the particles obtained according to the above are microparticles; the cells are HEK293, HT1080, HeLa, PER.C6, CHO cells, mesenchymal stromal / stem cells (MSCs), induced pluripotent (iPSCs), totipotent, multipotent cells of various origins or their derivatives, NIH-3T3, BHK-21, bEnd.3, COS-7, HB54, HB55, HCA2, HMEC, HeEa and other cell lines, including those of tumor origin or non-tumor origin, as well as cultures of primary cells and tissues of various origins and morphologies, humans, mammals or plants;

[0031] - HEK293 cells are HEK293 cells of any modification selected from the group consisting of HEK293, HEK293T, HEK293F, HEK293FT, HEK Exp293E;

[0032] - mesenchymal stem cells (MSCs) are MSCs of various origins, selected from a group that includes placental, adipose, bone marrow, MSCs from umbilical cord blood, amniotic fluid, peripheral blood, synovial fluid, dental pulp, endometrium, skin, muscle tissue, salivary glands, obtained from iPSCs;

[0033] - in step (d), 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 using a combination of these methods or other methods;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 and their fragments, 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), hybrid proteins, chimeric proteins, chemically modified and / or stabilized proteins, meganucleases, protein complexes, protein aggregates, human proteins, animal proteins, plant proteins, viral proteins, bacterial proteins, archaeal proteins, bacterial proteins, chimeric proteins;- 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;

[0034] - RNA is mRNA, long non-coding RNA, short RNA, small interfering RNA, small hairpin RNA, piwiRNA, RNA of RNA interference systems, 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 contain pseudouridine, or are modified by sugar or nucleoside residues, or contain cap structures at the 5' end, a polyA sequence or modified polyA sequences in combination with other nucleotides at the 3' end, or contain fluorescent labels, sites of interaction with proteins, be a hybrid of RNA with DNA, contain additional pins, elements for dimerization, dissociation, interaction, destruction,stabilization of proteins, changes in their interaction with the target, contain mutations and nucleotide substitutions, consist entirely of one or different modified nucleotides, or contain other additional effector elements;

[0035] - RNA and / or proteins that are packaged into the particle are delivered to the original cells before step (a) by a method selected from the group including nucleofection, surfactant permeabilization, freeze-thaw method, hypotonic permeabilization, ultrasonic permeabilization;

[0036] - surfactants are ionic surfactants (cationic, anionic, amphoteric) or nonionic surfactants (alkyl polyglucosides, alkyl polyethoxylates), such as saponins, and other compounds with surfactant properties; - RNAs of CRISPR-Cas systems are short RNAs (crRNA), tracrRNA, or single guide RNAs (sgRNA) or any RNA that provides recognition, cleavage or recruitment of molecules consisting of protein, RNA, DNA or combinations thereof to the target RNA, DNA, proteins or combinations thereof;

[0037] - modifications of Cas proteins are complete Cas nucleases or proteins based on catalytically inactive dead-Cas (dCas), containing mutations in the domains, Cas nicases (nCas), Cas proteins binding to DNA, RNA or proteins;

[0038] - 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 genetically engineered construct;

[0039] - the Cas, dCas or nCas proteins are Cas9, Cas12a / b or similar proteins, or improved variants selected from Cas9-HF, eSpCas9, HypaCas9, xCas9, SpRY / SpG, Fokl-fused dCas9;

[0040] - 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;

[0041] - 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;

[0042] - 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;

[0043] - domains for transcription suppression are KRAB, EZH2, KRAB-MeCP2, DNMT3A, DNMT3A-3L, ESDI domains, or their modifications or combinations thereof; - domains for editing DNA or RNA bases are rAPOBEC1, APOBECA3A, AID, TadA, ADAR2 domains, as well as their orthologs, their homologs and / or their modified variants obtained by mutagenesis or directed evolution;

[0044] - 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;

[0045] - DNA visualization systems are selected from the group including CRISPR-FISHer,

[0046] CRISPR-SIRIUS, CRISPRainbow modified CRISPR systems with fluorescent protein domains attached to and / or recruited to Cas protein and / or guide RNA;

[0047] - 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;

[0048] - packaging of proteins, RNA or their complexes is carried out intracellularly either at the stage of obtaining the mentioned particles or after the isolation of the mentioned particles;

[0049] - protein molecules, RNA or their complex optionally contain additional elements, including endosomolytic proteins, which provide or enhance interaction with membranes of biological particles and thereby increase the packaging efficiency or improve the biological properties of the resulting nanoparticles, for example, enhance endosomal release in target cells, wherein the endosomolytic proteins optionally include proteins or peptides selected from the group consisting of HA2, GALA, INF7, JTS1, ESCA, Mellitin and its analogs, LL37 and its analogs, Tat, R8, FBI, Mellitin, KALA, SAP, H5WYG, ppTGl, LAH4;

[0050] - the complex of protein and RNA is the ribonucleoprotein CRISPR / Cas; - proteins are produced in cells due to coding sequences of RNA, DNA, in the state of episomal forms, integrated forms, are used as genetically modified cell lines, producer lines;

[0051] - human and mammalian cells are used as source cells, including genetically and / or chemically modified cells, cell lines, transiently transfected cell lines, or cells or cell lines stably expressing Cas proteins and other components;

[0052] - Cas proteins are expressed either by themselves or together with RNA guides, or together with other proteins, chimeric Cas proteins, modified Cas, or components of Cas proteins, or proteins are expressed for packaging into nanoparticles;

[0053] - Cas proteins and / or other target proteins or RNA are expressed in cells constantly, or their expression can be triggered by stimuli;

[0054] - proteins are proteins of animals, plants, humans, bacteria, fungi, prions, viruses;

[0055] - proteins are fibrillar and / or globular proteins, can be simple (for example, glutelins, histones, etc.) and / or complex (metalloproteins, chromoproteins, phosphoproteins, glycoproteins, lipoproteins, etc.), according to the function they perform, they can be structural, nutritional, storage, contractile, transport, catalytic, protective, receptor, regulatory, protein-enzymes, etc.;

[0056] - proteins are used as nutrients, components of prophylactic and / or therapeutic vaccines, probiotics, prebiotics, synbiotics, are toxic proteins, their complexes or compounds, are used for the purpose of modifying RNA, DNA, proteins, their derivatives, modified forms, their complexes, can be of natural origin, can be modified, or have artificial origin;

[0057] - the stimulus for initiating expression is the introduction of an inducer (for example, a tetracycline antibiotic) into cells expressing the Cas protein under the control of an inducible promoter (for example, of the tet-on or tet-off type); the extrusion stage is carried out using a manual extruder, an automated extruder of cell mass or a suspension of cells or particles in a solvent;

[0058] - from 0.1 ml to more than 100,000 l of cell mass or suspension of cells or particles are subjected to extrusion;

[0059] - 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;

[0060] - extrusion is carried out under the pressure of inert gases or by mechanical extrusion, or by centrifugal extrusion or vacuum filtration;

[0061] - at the extrusion stage or immediately before extrusion, additional ultrasonic treatment is carried out;

[0062] - for extrusion, membranes made of polycarbonate, polyethylene terephthalate, polypropylene, regenerated cellulose or cellulose ethers, polytetrafluoroethylene, polyvinylidene fluoride, nitrocellulose, nylon, polyethersulfone or other material are used;

[0063] - the membrane for extrusion is standard or track;

[0064] - the membrane for extrusion has a hydrophilic, hydrophobic, PTFE-like, carbon coating, sliding coating, anti-adhesive, barrier, metallic or with the application of metals, bonded or amphiphilic coating;

[0065] - the extrusion membrane has pore diameters selected from the range from 100 to 0.001 µm;

[0066] - the extrusion membrane has pore diameters selected from the group comprising

[0067] 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,

[0068] 0.05 µm, 0.03 µm, 0.01 µm;

[0069] - extrusion is carried out using drainage discs;

[0070] - particles containing Cas proteins and / or other proteins or RNA are obtained by nitrogen cavitation; cells are treated with high-molecular and / or low-molecular compounds that can influence the production of nanoparticles, protein expression and

[0071] RNA, including proteins and RNA for packaging into nanoparticles; coding genetically engineered constructs contain promoters, enhancers, selectable genes, markers, and other regulatory elements, including those affecting the localization of proteins and RNA, their expression levels, stability, and translation.

[0072] The stated problem is also solved, and the technical result is achieved by creating a particle obtained in the mentioned way.

[0073] The task at hand 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 epigenome modification, for epitranscriptome modification, for RNA editing, for destruction

[0074] DNA, for RNA degradation, for nucleotide base modification, for nucleotide modification, for DNA or RNA deamination, for sequence integration

[0075] DNA or RNA into the human genome or mitochondria, for DNA visualization, for studying the spatial structure of chromatin, for changing RNA metabolism, for modifying proteins, for editing proteins, for monitoring biological processes, for monitoring physiological processes, for the purposes of vaccination, for creating therapeutic vaccines based on RNA, proteins, peptides, their mixtures or combinations, for creating drugs, for creating diagnostic drugs, for cosmetics, for use in regenerative medicine, for correcting or introducing 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 influencing 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 the mentioned particles and at least one pharmaceutically acceptable excipient.

[0076] The stated task is also solved, and the technical result is achieved by using the said particle 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 nucleotide modification, for DNA or RNA deamination, 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 monitoring biological processes, for monitoring physiological processes, for vaccination purposes, for creating therapeutic vaccines based on RNA, proteins, peptides, their mixtures or combinations, for creating medicinal products, for creating diagnostic products, for cosmetic products,for use in regenerative medicine, for correcting 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.,

[0077] According to preferred embodiments, the said technical result is also achieved by the fact that:

[0078] - 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;

[0079] - 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 subcutaneous, intramuscular administration, or administration into specific organs and tissues; local administration into the body is application to the surface of the skin and mucous membranes, the surface of organs, or washing of organs;

[0080] - 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 intra-peritoneal 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 dosed 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,

[0081] Brief description of the drawings

[0082] The invention is illustrated by the following drawings.

[0083] Fig. 1 shows general schemes of protein and RNA packaging into EMNV nanoparticles. Protein and / or RNA packaging into EMNV from human or animal cells expressing the protein and / or RNA of interest by serial extrusion followed by purification from cellular debris by centrifugation or filtration, extrusion of the resulting mass through pores with a diameter of <200 nm (for systemic administration) or >30 nm but <5000 nm for local application or direct administration into tissue (bone, muscle, cartilage, etc.). Following this, the resulting nanoparticles are purified using methods such as chromatography (affinity, gel filtration, anion exchange, etc., as well as tangential filtration, ultrafiltration, etc.), concentrated, and ready, purified EMNV loaded with protein and / or RNA are obtained.

[0084] Fig. 2 shows cryoelectron images of biological nanoparticles.

[0085] Fig. 3 shows the results of the analysis of the nanoparticle size distribution for different types of biological nanoparticles loaded with Cas / RNA-conductor complexes using one of the developed technologies. All the obtained nanoparticles have a similar size with a peak in the region of 100 nm. emNV - exosome-like nanoparticles obtained by the described method from HEK293T cells; StCas9 EMNV - exosome-like nanoparticles containing the StCas9 protein and obtained by the described method from HEK293T cells expressing StCas9; E 8 - *10 8 particles / ml.

[0086] Fig. 4 shows the results of the analysis of Cas protein packaging in biological nanoparticles using the developed technologies in calculating the number of copies of the Cas protein per nanoparticle. emNV Neg cntrl is the background value of the Cas protein signal in nanoparticles without the Cas protein. StCas emNV are exosome-like nanoparticles containing the StCas9 protein and obtained by the described method from HEK293T cells expressing StCas9 StCas- mCh emNV are exosome-like nanoparticles containing the StCas9-mCherry protein and obtained by the described method.

[0087] Error bars represent standard deviations.

[0088] Figure 5 shows the results of packaging of dCas9 protein (dead Cas9, nucleolytically inactive) from Streptococcus pyogenes, linked to EGFP fluorescent protein (dCas9-EGFP), BEP protein (dCas9-BFP), or simultaneous intracellular expression of both proteins, into EMNV, measured by fluorimetry. (A) Packaging of dCas9-BFP protein into EMNV upon simultaneous expression of dCas9-EGFP and dCas9-BFP (dCas9-EGFP / BFP). EMNV are control nanoparticles without Cas proteins. (B) Packaging of EGFP-positive proteins into EMNV upon expression of CD63-EGFP protein or co-expression of dCas9-EGFP and dCas9-BFP proteins (dCas9-EGFP / BFP). EMNV - negative control, a sample of EMNV particles obtained from cells without fluorescent proteins.

[0089] Fig. 6 shows the results of packaging the RNA guide for working with the StCas9 protein into nanoparticles using various developed technologies in calculating the number of RNA copies per equal isolate of nanoparticles. EVs is the stochastic packaging of RNA into EVs, emNVs is the packaging of RNA into emNVs using the described method.

[0090] Figure 7 shows the results of internalization of nanoparticles loaded with the StCas9-mCherry protein into HEK293T cells. EMNV particles obtained from cells transfected with constructs encoding the StCas9-mCherry protein were added to HEK293T cells. Nanoparticles were labeled with a green fluorescent dye. The StCas9-mCherry protein fluoresces in the red range. Cell nuclei were stained with Hoechst33342 dye (blue fluorescence). Confocal microscopy was performed 1 hour after adding nanoparticles to the cells.

[0091] Preferred embodiments of the invention

[0092] The authors of the invention have created a method for packaging proteins, RNA and protein and RNA complexes in EMNV (exosome-mimetic nanovesicles). The general scheme of packaging proteins, RNA and their complexes is shown in Fig. 1.

[0093] In one embodiment of the invention, the packaging of proteins, RNA or their complexes in the case of EMNV is from human or animal cells expressing the protein and / or RNA of interest. The animal or human cells may be primary cells, transplanted, transformed or tumor cells, genetically modified cells or cells modified by 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, contacts with the surface of cells).In particular, mesenchymal stromal / stem cells, derivatives of mesenchymal stromal / stem cells, induced pluripotent cells or embryonic stem cells, adult, terminally or non-terminally differentiated human or animal cells, such as immune system cells (peripheral mononuclear cells, leukocytes, macrophages, monocytes, natural killers, etc.), skeletal or smooth muscle cells, cardiac tissue, epithelium, brain, etc., or cells derived from any human or animal germ layers can serve as cells for EMNV production. Protein molecules, RNA or their complexes can contain additional elements that can provide or enhance interaction with the membranes of biological nanoparticles to enhance the efficiency of packaging. Packaging of protein, RNA or their complexes is performed according to the general scheme shown in Fig. 1.In the case of EMNV, the protein, RNA or their complexes can be packaged intracellularly or after particle production. Intracellular packaging can involve the use of cells encoding the protein, RNA or their complexes, which are subjected to serial extrusion, followed by filtration or centrifugation to remove cellular debris, and the resulting mass is extruded through filters with a pore diameter of ≤200 nm (for systemic administration) or ≥10 nm but ≤5000 nm (for topical use or administration directly into tissues, organs and cavities of the body). The next step is the purification of the nanoparticles by chromatography with pre- and / or post-concentration by TFF or chromatography, filtration through cut-off pores (ultrafiltration, UF), sedimentation or precipitation, or without concentration.Extrusion can be performed using a manual, semi-industrial or industrial extruder with different pressures applied to the mass of cells to be extruded. Extrusion can be performed manually, by inert gas or gas mixture pressure or mechanical pressure (e.g. centrifugal force during centrifugation or using presses). The membrane material for extrusion can be different, hydrophilic or hydrophobic, such as polycarbonate, polyethylene terephthalate, polyimide, etc.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.

[0094] In one embodiment 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.

[0095] In one embodiment of the invention, proteins can be packaged in free form, in the form of sections of cell cytoplasm, in the form of complexes, or in a bound form together with low-molecular compounds, polymers, adjuvants, and materials of organic or inorganic origin.

[0096] In one embodiment of the invention, the RNA may be 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 may include sequences of ribozymes, aptamers, ribosome entry 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.RNA can be chemically modified, for example, contain pseudouridine, can be modified by sugar or nucleoside residues, contain cap structures at the 5' end, a polyA sequence or modified polyA 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.A method has been developed for packaging proteins, RNA or ribonucleoprotein complexes into EMNV, which is based on the extrusion of cells expressing the protein of interest, in particular Cas proteins or any other proteins of any type, kind, origin and modification, or RNA, including mRNA, capped RNA, polyadenylated RNA (including those with canonical or non-canonical polyA sequences), chemically modified, stabilized RNA, truncated RNA, RNA-DNA hybrids containing either no additional elements or with removed elements, or protein and RNA complexes through membrane filters with different pore diameters. Expression of proteins and RNA can occur constantly (constitutively) or be induced by stimuli (for example, tetracycline antibiotics using a regulated tet-on promoter). The general scheme of the present method is shown in Fig. 1.The protein or proteins of interest, including genetically modified, chemically modified, split proteins, improved protein variants, protein complexes, chimeric proteins, etc., can be expressed by human or animal cells due to genetic constructs, including integrated and / or episomal elements, as part of cell lines or producer lines. These cells within the framework of the present method are passed through serial extrusion, which can be carried out both with a manual extruder and with semi-automated or automated options for various volumes (from 0.5 ml to >100 l) of cell mass or cell suspension in any solvent that may or may not affect the production of nanoparticles, change or not change the properties of cell membranes, in particular, their fluidity, affect or not affect the viability of producer cells.Extrusion can be carried out under the pressure of inert gases (e.g., nitrogen) or gas mixtures, by centrifugal extrusion or vacuum filtration, with or without ultrasonic or mechanical treatment. 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 membrane material for extrusion. Membranes with different pore diameters can be used for 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 drainage discs. Also, EMNV containing Cas proteins and / or other proteins or RNA can be obtained by nitrogen cavitation.During EMNV production, either immediately prior to production or during culture, cells may be treated with high-molecular and / or low-molecular compounds that alter EMNV production, protein and RNA expression, including proteins and RNA for packaging into EMNV. Depending on the protocol and production conditions used, EMNVs may have a micellar structure, a bilayer lipid membrane with proteins incorporated into it, and sizes in the range of 20 nm to 50 μm.

[0097] In one embodiment of the invention, a method for producing a particle by packaging proteins, RNA and protein and RNA complexes, in particular CRISPR / Cas RNP, consisting of using EMNV. Packaging of proteins, RNA or their complexes can be carried out intracellularly either at the stage of obtaining the above-mentioned nanoparticles or after isolating the nanoparticles. 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.

[0098] In one embodiment of the invention, 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 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 compounds, polymers, adjuvants, materials of organic or inorganic origin. In one embodiment of the invention, the Cas proteins are obtained from bacteria or archaea, or fungi, or protozoa, or algae.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] In one embodiment of the invention, the following bacteria are used to obtain Cas proteins: 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.

[0103] In one embodiment of the invention, Chlamydomonas reinhardtii, Phaeodactylum tricornutum, Nannochloropsis oceanica are used as algae.

[0104] In one embodiment of the invention, Sulfolobus, Pyrococcus, and Methanococcus are used as archaea.

[0105] In one embodiment of the invention, Tetrahymena thermophila and Trypanosoma brucei are used as protozoa.

[0106] In one embodiment of the invention, Saccharomyces cerevisiae, th Fusarium oxysporum, Candida albicans are used as fungi. In one embodiment of the invention, 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.

[0107] In one embodiment of the invention, the Cas protein comprises additional domains, including domains for transcription activation (VP48, VP64, VP160, VP192, 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, ESDI, their modifications or combinations), for editing DNA or RNA bases (rAPOBEC1, APOBECA3A, AID, 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 the method of directed evolution or mutagenesis, dominate RNA demethylases and RNA methyltransferases (METTL3, METTL14, METTL16, FTO, ALKHB5, their combinations or modified variants obtained by mutagenesis or directed evolution), DNA visualization systems (CRISPR-FISHer,CRIS PR-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.

[0108] 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.

[0109] In one embodiment of the invention, the RNA can be chemically modified, for example, at RNA nucleotide (nucleoside) residues or sugar residues, including 2'-O-methyl (M), 2'-O-methyl 3'phosphorothioate (MS), or 2'-O-methyl 3'thioPACE (MSP), Pseudouridine ψ, N1 -methylpseudouridine m1ψ, 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.contain cap structures of NNAA at the 5' end, a polyA 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 attraction of additional effector elements.

[0110] In one embodiment of the invention, any human or mammalian cells can be used as producer cells, including genetically and / or chemically modified cells, cell lines, primary cell cultures, transiently transfected cell lines, or cells or cell lines stably expressing Cas proteins and other components.

[0111] In one embodiment of the invention, the Cas proteins are derived from bacteria or archaea or fungi or protozoa or algae.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] In one embodiment of the invention, the Cas protein comprises additional domains, including domains for transcription activation (VP48, VP64, VP160, VP192, 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, ESDI, their modifications or combinations), for DNA or RNA base editing (rAPOBEC1, APOBECA3A, AID, 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 and RNA- methyltransferases (METTL3, METTL14, METTL16, FTO, ALKHB5, their combinations or modified variants obtained by mutagenesis or directed evolution), DNA visualization systems (CRISPR-FISHer, CRIS PR-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.

[0117] 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.

[0118] 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, HH55WWYYGG, ppTGl, LAH4), low molecular weight compounds (chloroquine, hydroxychloroquine, mefloquine, their analogs and derivatives).In one embodiment of the invention, Cas proteins can be expressed by themselves or together with RNA guides, or together with other proteins, chimeric Cas proteins, modified Cas, or components of Cas proteins, or any proteins for packaging into nanoparticles can be expressed. Cas proteins and / or other target proteins or RNA can be expressed in cells constitutively (constantly), or their expression can be triggered by the action of stimuli (for example, by introducing doxycycline into cells expressing Cas protein under the control of a tet-on type promoter).

[0119] In one embodiment of the invention, the coding vectors may contain promoters, enhancers and other regulatory elements that affect the localization of proteins and RNA, their expression levels, stability and translation, encode RNA with modified 5'- and 3'-ends, for example, contain internal ribosome entry sites (IRES) or WPRE hairpins, encode autocatalytic peptides (T2A, P2A, etc.), etc. The following are examples of the invention, which, however, do not cover all possible embodiments of the invention and do not limit the claimed invention.

[0120] Examples of implementation of the invention

[0121] Example 1. Production of exosome-like nanoparticles (EMNV)

[0122] At 72 hours post transfection, ≈4x10 7The cells were removed using Versen solution (PanEco, Russia), washed with phosphate-buffered saline and resuspended in phosphate-buffered saline solution containing protease inhibitor cocktail (cat.). Using a manual extruder at 37°C, the cell suspension was passed sequentially through hydrophilic polycarbonate membranes soaked in PBS-HAT buffer solution (0.2% human serum albumin, 25 mM trehalose, 25 mM HEPES): pore diameters of 10 μm (9 times), 5 μm (9 times), 1 μm (9 times). The resulting suspension was centrifuged at 4°C for 5 minutes (2000g) to remove cell debris. The supernatant was passed using an extruder through a hydrophilic polycarbonate membrane with a pore size of 0.1 μm, soaked in PBS-HAT buffer solution (11 times). Exosome-like nanoparticles were purified using gel exclusion chromatography on a Sepharose CL-4B resin column using phosphate buffered saline as the mobile phase.The obtained sample was loaded onto a 1.6x10 cm column, and 40 fractions of 500 μl were collected. Fractions containing biological nanoparticles (Fractions 11-16) were pooled and 10x PBS-HAT buffer was added to a concentration of 1x (0.2% human serum albumin, 25 mM trehalose, 25 mM HEPES).

[0123] Example 2. Cultivation of HEK293T cells and mesenchymal stromal cells

[0124] 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.

[0125] Example 3. Transcription and purification of guide RNA in vitro

[0126] The PCR product encoding the guide RNA under the T7 promoter was synthesized using Q5 high-fidelity polymerase. Then, the T7 PCR 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.

[0127] Example 4. Obtaining recombinant protein StCas9

[0128] The pLysS E. coli BL21 (DE3) strain (Novagen) was used for protein expression. E. coli cells were grown in EB medium (with the addition of antibiotic, 0.5% sucrose, 0.5% glycerol, 1 mM magnesium chloride, 50 mM at 30°C to OD600. 1.2. Expression was induced for an additional 16 h by adding 0.1 mM isopropyl-β-Dl-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 x g for 40 min. Cell lysate was treated with 0.05% polyethyleneimine for 30 min at 4°C, centrifuged at 15,000 x g for 40 min, and the supernatant was collected, followed by binding of the supernatant to 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.

[0129] Example 5. Molecular cloning

[0130] Molecular cloning of the plasmids used in the work was performed using the Gibson assembly technology (NEB) according to the manufacturer's protocol with primers synthesized by Lumiprobe (Russia). The primers were designed in the SnapGene program. The PT JP products for the reaction were obtained with high-precision polymerase Q5 (NEB). Purification of the PT JP products from the gel was performed using a kit for DNA isolation from gel (Eurogen).

[0131] Example 6. Isolation of nucleic acids

[0132] The RNA loaded into the 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.

[0133] Example 7. PCR analysis

[0134] Quantitative assessment of loaded guide RNAs was performed by amplification of the obtained cDNAs and standards for constructing a calibration curve on a QuantS tudio5 instrument (Applied Biosystems). Calibration standards were genetically engineered constructs encoding the S t 10 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.

[0135] Example 8. Cell transfection

[0136] Transfection of HEK293T cells was performed using polyethyleneimine. The day before transfection, the cells were seeded at a density of ≈70%, the next day DNA was transfected 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.

[0137] Example 9. Screening fluorescence microscopy

[0138] Microscopy of 96-well plastic plates was performed using an LCI Imager ExFluorer.

[0139] Example 10. Confocal microscopy

[0140] Confocal images of transfected cells and cells treated with different nanoparticles were performed on an FV3000 microscope (Olympus).

[0141] Example 11. Flow cytometry

[0142] Flow cytometry was performed using a LongCyte instrument (ChallengeBio).

[0143] Example 12: Western blotting and dot blotting

[0144] 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.

[0145] Example 13. Transmission electron microscopy Sguo-TEM

[0146] Cryoelectron micrographs of the 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.

[0147] Example 14. Measurement of Z-potential by dynamic light scattering DLS The Malvern Zetasizer NanoZS (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 (Corning) and analyzed 5 times; 1.5 ml of the diluted samples were loaded into polystyrene cuvettes (DTS0012; The analysis was performed at 25 °C (100 measurements) using a 20 mW (633 nm) He / Ne laser. Data were analyzed using Zetasizer 8.01.4906 software (Malvern). Z-potential was analyzed in U-type cuvettes (DTS1070; With GOLD electrodes. Z-potential measurements were performed at 25 °C at least 5 times. The background signal was estimated using filtered PBS.

[0148] Example 15. Measurement of the number of nanoparticles using nanoparticle trajectory analysis (NTA)

[0149] Nanoparticles were analysed using a Nanosight EM 10 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.

[0150] Example 16. Statistical analysis of results

[0151] Values ​​are 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.

[0152] Example 17. Obtaining particles according to the present method

[0153] Induced pluripotent stem cells containing an integrated transgene encoding the Cas9 nuclease from Streptococcus thermophilus with an aptamer-binding domain of Com and simultaneously expressing a guide RNA containing a rs14 linker and a Com hairpin at the 3' end were seeded in a rotary perfusion bioreactor, the cell mass was grown in a culture medium until 100% confluency was achieved (-48-72 hours). After achieving confluency, the cells were washed with a phosphate-buffered saline solution and detached from the reactor substrate with a Versene solution.

[0154] The cell mass was washed twice with phosphate-buffered saline and resuspended in phosphate-buffered saline containing protease inhibitor cocktail at a density of 10 7 cells / ml.

[0155] The cell mass was extruded at 37°C using a liposome extruder under nitrogen pressure through hydrophilic polycarbonate membranes with pore diameters of 10 μm (9 times), then 5 μm (9 times), then 1 μm (9 times). Cell debris was removed by centrifugation at 2000g (5 minutes). The resulting particle mixture was extruded using a liposome extruder under nitrogen pressure through hydrophilic polycarbonate membranes with pore diameters of 0.1 μm (11 times) and passed through a column containing Sepharose CL-4B gel exclusion resin, then fractions corresponding to the target particles were collected.

[0156] Example 18.

[0157] 1. Take the nanoparticle preparation obtained according to Example 17, in a dose of 1x10 15particles 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.

[0158] 2. Take the nanoparticle preparation obtained according to Example 17, in a dose of 1x10 15 particles 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.

[0159] Example 19.

[0160] 1. Take a nanoparticle preparation based on a vaginal gel with a concentration of 1x10 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.

[0161] 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.

[0162] Example 20.

[0163] HEK293T cells or human mesenchymal stromal cells were transfected with plasmids encoding the Cas9 protein from Streptococcus thermophilus (StCas9) and a PCR product encoding the St10 guide RNA under the control of the U6 promoter. The resulting cells were subjected to serial extrusion followed by removal of cellular debris and washing with phosphate buffer by centrifugation. Following this, the resulting mixture was extruded through membrane filters with a pore diameter of 100 nm and purified by chromatography from free proteins and their complexes.

[0164] Each nanoparticle isolate obtained by each technology is loaded

[0165] StCas9 / guide RNA complexes of each developed technology were characterized by assessing the size distribution of nanoparticles using the Nanoparticle Tracking Assay (NTA) method (Fig. 3).

[0166] As an example, loading of StCas9 protein was used, loading analysis was performed using developed polyclonal rabbit anti-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 Nanoparticle Tracking Assay technology), under optimized conditions of nanoparticle lysis, using Western blotting and dot blot.

[0167] The present method with any variant of intracellular protein expression caused a multiple increase in protein packaging (up to 35-77 protein copies per particle). At the same time, differences in the levels of StCas9 protein packaging of different species were again observed, which may probably be associated with both the nature of the Cas protein and the levels of intracellular expression.

[0168] Within the framework of the disclosed method, the protein packaging of the catalytically inactive Cas9 protein (dCas9 - dead Cas9, nucleolytically inactive) from another species of organism (Steptococcus pyogenes), which significantly differs in size, charge and amino acid sequence from the StCas9 protein (Fig. 5), was carried out. In addition, Cas9 proteins associated with one of two fluorescent proteins were used: EGFP (green fluorescent protein) or BFP (blue fluorescent protein), which additionally introduced a difference in the properties of the proteins being packaged. In this case, the proteins were expressed separately in the cell, or co-expression of the proteins was carried out, and EMNV particles were obtained according to the present method. Detection of packaged proteins was performed using fluorimetric analysis at 380 nm excitation, 460 nm signal detection (ex380-ex460) for blue fluorescent protein, and at 470 nm excitation and 515 nm emission (ex470-515) for green fluorescent protein.In all cases, using the present method, a strong fluorescence signal corresponding to the packaged fluorescently labeled Cas9 proteins was detected, which was >1000-fold higher than the background signal of EMNV particles obtained from cells without Cas9 expression.

[0169] In addition to the EGFP / BFP fluorescent proteins, any domain that allows for expansion of the system's functionality 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), epitranscriptome mark removal / addition factors (RNA methyltransferases and demethylases), and other domains.

[0170] Thus, the present method can be used not only for packaging nucleases.

[0171] Cas, but also for packaging any CRISPR / Cas-based systems, regardless of type, type, structure and origin.

[0172] Table L. Packaging methods and description of nanoparticles with different options for loading proteins into biological nanoparticles using the example of the StCas9 protein.

[0173] The next step was to demonstrate that, in addition to the actual cargo packaging, the particles are capable of delivering functionally active cargo to target cells. For this purpose, at the first step, fluorescently labeled EMNV nanoparticles loaded with fluorescently labeled StCas9-mCherry protein were added to HEK293T cells (Fig. 7). The possibility of internalization of nanoparticles by target cells with the release of StCas9 protein and its distribution throughout the cells was demonstrated. Consequently, a method for packaging proteins, RNA, and ribonucleoprotein complexes using CRISPR / Cas9 RNP as an example was developed. It was 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 cargo packaging in EMNV were developed.

[0174] Thus, the declared technical solution ensures:

[0175] (a) packaging from 2.5 to >73 copies of Cas proteins per nanoparticle, while the amount of packaged Cas proteins per nanoparticle can be regulated by changing the expression levels of Cas protein in human producer cells;

[0176] (b) simultaneous packaging, co-packaging or post-packaging of guide RNAs into nanoparticles together with, before or after packaging of Cas proteins into nanoparticles, wherein the number of copies of the loaded guide RNA(s) can be regulated by changing the intensity of RNA expression in producer cells;

[0177] (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'-OMe, phosphorothioates, LNA, UNA, 2'F-ANA, butane linkers, 4'-Ome, 2',4'-diOme, 2'F,4'-OMe, the use of DNA-RNA hybrids, their combinations, 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. In this case, the packaging of chemically modified RNAs should be carried out by electroporation, permeabilization with surfactants

[0178] (saponins), by freezing-thawing, hypotonic permeabilization, ultrasonic permeabilization or other methods directly into producer cells to obtain emNV from them;

[0179] (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 size of Cas proteins and their complexes, is not determined by the composition of RNA guides, the presence or absence of additional elements, or the removal of any elements in the composition

[0180] CRISPR / Cas or using additional components, is not determined by charge

[0181] Cas proteins, guide RNAs and the resulting RNPs are not determined by the use of full-length, truncated, or split proteins, domains or combinations thereof, and also provides for loading of any guide RNAs in the form of 1 guide RNA, their combinations, extended 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;

[0182] (d) is a universal system for packaging any proteins, RNA, as well as protein-RNA complexes, including in the form of RNP.

Claims

The amended claims were received by the International Bureau on July 22, 2025. CLAUSES OF THE INVENTION (as amended in accordance with Article 19 of the PCT) 1. A method for packaging a protein of interest or a complex of the protein of interest with RNA into a particle, comprising the following steps: a. the protein of interest or the protein of interest with RNA is expressed in the original cells using genetically engineered constructs delivered into the cells by viral or non-viral methods, b. the cells obtained in step (a) are subjected to serial extrusion through membrane filters, followed by removal of cellular debris by centrifugation, c. the mixture obtained in step (b) is subjected to extrusion through membrane filters with a pore diameter comparable to the desired final particle diameter, d. the particles obtained in step (c) are purified from free proteins and contaminants to obtain a particle comprising a protein or a protein-RNA complex and having a size of 100 to 0.001 μm, where the particles are purified using chromatography, ultracentrifugation, filtration, precipitation, fractionation, or a combination thereof.

2. The method according to any one of claim 1, characterized in that the particles obtained according to said method are microparticles or nanoparticles.

3. The method according to any one of claim 1, characterized in that the cells are HEK293, HT1080, HeLa, PER.C6, CHO cells, mesenchymal stem cells (MSCs), induced pluripotent (iPSCs), totipotent, multipotent cells of various origins or their derivatives, NIH-3T3, BHK-21, bEnd.3, COS-7, HB54, HB55, HCA2, HMEC, HeLa and other cell lines, including those of tumor origin or non-tumor origin, as well as cultures of primary cells and tissues of various origins and morphologies, humans, mammals or plants.

4. The method according to item 3, characterized in that the HEK293 cells are HEK293 cells of any modification selected from the group including HEK293, HEK293T, HEK293F, HEK293FT, HEK Exp1293E.

5. 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, salivary glands, obtained from iPSCs.

6. The method according to i.1, characterized in that in step (d) 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.

7. 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, viral proteins, bacterial proteins, archaeal proteins, prion proteins, chimeric proteins.

8. The method of 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 transcription activator-like effector-based nuclease (TALENs), hybrid proteins, chimeric proteins, chemically modified and / or stabilized proteins, meganucleases, protein complexes, protein aggregates, human proteins, animal proteins, plant proteins, viral proteins, bacterial proteins, archaeal proteins, bacterial proteins, prion proteins.

9. Method by and. 1, characterized in that the RNA is mRNA, long non-coding RNA, short RNA, small interfering RNA, small hairpin RNA, piwiRNA, RNA of RNA interference systems, 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, and 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, , contain additional hairpins, elements for dimerization, dissociation, interaction, destruction,stabilization of proteins, changes in their interaction with the target, contain mutations and nucleotide substitutions, consist entirely of one or different modified nucleotides, or additional effector elements.

10. The method according to i.1, characterized in that the proteins or proteins with RNA that are packaged into the particle are delivered to the original cells before step (a) by a method selected from the group including nucleofection, permeabilization with surfactants, the freeze-thaw method, hypotonic permeabilization, and ultrasonic permeabilization.

11. The method according to claim 10, characterized in that the surfactants are ionic surfactants or non-ionic surfactants, such as saponins.

12. The method according to claim 9, characterized in that the RNA of the CRISPR-Cas systems are short RNA (crRNA), tracrRNA, or single guide RNA (sgRNA) or any RNA that provides recognition, cutting or attraction of molecules consisting of protein, RNA, DNA or combinations thereof to the target RNA, DNA, proteins or combinations thereof.

13. 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 domains, Cas nicases (nCas), Cas proteins binding to DNA, RNA or proteins.

14. The method according to claim 13, 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 genetically engineered construct.

15. The method according to claim 13, wherein 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.

16. The method according to item 13, characterized in that 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.

17. The method according to claim 1, 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.

18. The method according to claim 17, 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.

19. The method according to claim 17, characterized in that the transcription suppression domains are KRAB, EZH2, KRAB-MeCP2, DNMT3A, DNMT3A-3L, ESDI domains, or modifications thereof, or combinations thereof.

20. The method according to claim 17, 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.

21. The method according to item 17, characterized in that the domains of RNA demethylases or RNA methyltransferases are the domains METTL3, METTL14, METTL16, FTO, ALKHB5, or their combinations or their modified variants obtained by mutagenesis or directed evolution.

22. The method according to item 17, characterized in that the DNA visualization systems are selected from the group including CRISPR-FISHer, CRISPR-SIRIUS, CRISPRainbow modified CRISPR systems with fluorescent protein domains attached to and / or recruited to a Cas protein and / or guide RNA.

23. 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 contains additional hairpins for dimerization, dissociation, interaction, destruction, stabilization of proteins and other molecules, as well as for attracting additional effector elements.

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

25. The method according to claim 1, characterized in that the protein molecules, RNA or their complex optionally contain additional elements, including endosomolytic proteins, which provide or enhance interaction with the membranes of biological particles and thereby increase the packaging efficiency or improve the biological properties of the resulting nanoparticles, for example, enhance endosomal release in target cells, wherein the endosomolytic proteins optionally include proteins or peptides selected from the group consisting of HA2, GALA, INF7, JTS1, ESCA, Mellitin and its analogs, LL37 and its analogs, Tat, R8, FBI, Mellitin, KALA, SAP, H5WYG, ppTGl, LAH4.

26. The method according to any one of paragraphs 1 or 2, characterized in that the complex of protein and RNA is a CRISPR / Cas ribonucleoprotein.

27. The method according to claim 1, characterized in that the proteins are produced in cells due to coding sequences of RNA, DNA, in the state of episomal forms, integrated forms, and are used as genetically modified cell lines, producer lines.

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, or cells or cell lines stably expressing Cas proteins and other components.

29. The method according to claim 28, characterized in that the Cas proteins are expressed on their own, or together with RNA guides, or together with other proteins, chimeric Cas proteins, modified Cas, or components of Cas proteins, or proteins are expressed for packaging into nanoparticles.

30. The method according to item 28, characterized in that Cas proteins and / or other target proteins or RNA are constantly expressed in cells, or their expression can be triggered by the action of stimuli.

31. The method according to claim 30, characterized in that the stimulus for initiating expression is the introduction of an inducer into cells expressing the Cas protein under the control of an inducible promoter.

32. The method according to item 31, characterized in that the inducer is an antibiotic of the tetracycline series.

33. The method according to claim 31, characterized in that the inducible promoter is a tet-on or tet-off type promoter.

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

35. The method according to item 34, characterized in that from 0.1 ml to more than 100,000 l of cell mass or suspension of cells or particles is subjected to extrusion.

36. The method according to item 34, characterized in that the solvent influences the production of nanoparticles and / or changes the properties of the membranes of cells or particles, in particular their fluidity, and / or influences the cellular viability or integrity of the particles.

37. 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.

38. The method according to item 34, characterized in that at the extrusion stage or immediately before extrusion, additional ultrasonic treatment is carried out.

39. 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, polyethersulfone or other material are used for extrusion.

40. The method according to item 39, characterized in that the membrane for extrusion is standard or track-type.

41. The method according to item 39, characterized in that the membrane for extrusion has a hydrophilic, hydrophobic, PTFE-like, carbon coating, sliding coating, anti-adhesive, barrier, metallic or with the application of metals, adherent or amphiphilic coating.

42. The method according to item 39, characterized in that the membranes for extrusion have pore diameters selected from the range from 100 to 0.001 µm.

43. The method according to item 39, 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.

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

45. The method according to claim 1, characterized in that the particles containing Cas proteins and / or other proteins or RNA are obtained by the nitrogen cavitation method.

46. ​​The method according to item 1, characterized in that the cells are treated with high-molecular and / or low-molecular compounds that are capable of influencing the production nanoparticles, expression of proteins and RNA, including proteins and RNA for packaging into nanoparticles.

47. The method according to claim 1, characterized in that the coding genetically engineered constructs contain promoters, enhancers, selective genes, markers, and other regulatory elements, including those influencing the localization of proteins and RNA, their expression levels, stability, and translation.

48. A particle comprising a protein or a protein-RNA complex 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 DNA visualization, for studying the spatial structure of chromatin, for studying RNA metabolism, for modifying proteins, for editing proteins, for monitoring biological processes, for monitoring physiological processes, for the purposes of vaccine prophylaxis, for creating therapeutic vaccines based on RNA, proteins, peptides, their mixtures or combinations, for creating medicinal products, for creating diagnostic products, 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 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 microbiome, 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, to obtain,modification or differentiation of stem cells, 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 1-47 and has a size from 100 to 0.001 μm., 49. A pharmaceutical composition 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 DNA or RNA sequences in the human genome or mitochondria, for DNA visualization, for studying the spatial structure of chromatin, for studying RNA metabolism, for modifying proteins, for editing proteins, for controlling biological processes, for controlling physiological processes, for the purposes of vaccination, for creating therapeutic vaccines based on RNA, proteins, peptides, their mixtures or combinations, for creating drugs, for creating diagnostic drugs, for cosmetics, for use in regenerative medicine, for correcting drug resistance mutations, for 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 influencing the microbiome, 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 theranostic purposes, 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 particles according to claim 48 and at least one pharmaceutically acceptable excipient.

50. Use of a particle according to Article 48 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 changing 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, 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 correction or introduction of mutations,including drug resistance mutations, in 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 cellular, 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 veterinary use.

51. The use according to claim 50, characterized in that the said 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 internal 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 an intravenous infusion, an intramuscular injection, a catheter, an enema, rectal administration, inhalation, instillation, insufflation, intravaginally, intranasally, by injection, by irrigation, parenteral administration, by oral administration, using a powder inhaler, transbuccally, using a tracheal tube or using vascular access.

52. Use according to paragraph 51, 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.

53. Use according to paragraph 51, 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.

54. Application by and. 51, 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 preparations, drops, solutions, suspensions, granules for resorption, enteric-coated granules, enteric-coated granules with prolonged release,film-coated granules, cut-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, drops, eye drops with prolonged release, inhalation drops, 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, capsules with prolonged release, 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 local use applications, liniment for external use, periodontal liniment, endocervical liniment, lyophilisate for preparation,dispersions, drops, concentrates, solutions, sprays, suspensions, emulsions, vaginal ointments, eye ointments, inhalation ointments, topical ointments, oral mucosal ointments, external ointments, nasal ointments, rectal ointments, ear ointments, inhalation oils, topical oils, external oils, internal oils, tinctures, inhalation tinctures, topical tinctures, external tinctures, dental sticks, periodontal sticks, nasal sticks, urethral sticks, ear sticks, gingival pastes, external pastes, pastes for oral suspensions, oral pastes, oral pastes, medicinal dental pastes, vaginal foams, intrauterine foams, foam for external use, rectal foam, patch, patch for the oral mucosa, transdermal patch,eye films, films for sticking to the gums, buccal films, films dispersible in the oral cavity, periodontal films, sublingual films, dosed inhalation powder, powder for external use, powder for the preparation of a 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 intra-lymphatic administration, solution for infusion, solution for injection, solution for local use, Solution for cutaneous scarification, 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 testing, solution for eye wash, solution for nasal cavity rinsing, solution for oral rinsing, solution for rinsing the ear canal, solution for the oral mucosa, solution for subconjunctival administration, solution for extra-amniotic administration, solution for endo-sinusial administration, solution for endo-tracheal administration, dental solution, rectal solution, transdermal solution, syrup, vaginal therapeutic system, intrauterine therapeutic system, spray for local use, spray for external use,Oral spray, nasal spray, sublingual metered-dose spray, transdermal spray, ear spray, vaginal suppositories, rectal suppositories, vaginal suspension, intradermal suspension, intramuscular suspension, intraarticular suspension, gastrointestinal suspension, injection suspension, extended-release injection suspension, implantation suspension, inhalation suspension, topical suspension, cutaneous scarification suspension, external suspension, periarticular suspension, subcutaneous suspension, oral suspension, oral mucosal suspension, endotracheal suspension, dental suspension, rectal suspension, tablets, vaginal tablets, vaginal tablets effervescent, 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, soluble tablets, modified-release tablets, prolonged-release tablets, effervescent tablets, medicated vaginal tampons, medicated tampons for inhalation, 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, medicinal sponge, dragee, medicinal pencil, medicinal nail polish, medicinal lozenges, Medicinal lozenges, medicinal tablets, bars, medicinal absorbable wipes, medicinal shampoo, elixir, pills.

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