Hybrid particles, method for producing same and use

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 any RNA, DNA, or a mixture thereof into biological nanoparticles of any origin and composition, inter alia nanoparticles enriched with a protein of interest.

Description

[0001]A METHOD FOR PRODUCING HYBRID PARTICLES, PARTICLES PRODUCED BY THE SAID METHOD, THEIR USE, AND A PHARMACEUTICAL COMPOSITION BASED THEREON. DESCRIPTION. Field of technology. 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 to package any RNA, DNA, or mixtures thereof into biological nanoparticles of any origin and composition, including those enriched with the protein of interest. State of the art CRISPR / Cas systems are tools for controlling and modifying DNA or RNA, which allow for the introduction of 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 is 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] 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 network of various proteins active against single-stranded DNA, and these proteins can exhibit collateral activity (the ability to cleave or modify surrounding nucleic acids). Type III systems target both DNA and RNA, and the effector complex consists of a network of proteins, of which Cas10 is the signature protein. For type IV, the signature protein is Csf1, which is part of a multi-subunit complex. In class 2, the main types of systems are II, V and VI, 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 with various characteristics are described, which can differ significantly in properties, composition, targeting principles (target site recognition and action on the target site), enzymatic activity and size. In addition, using directed evolution methods, rational engineering, in silico design methods, machine learning technologies, 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 the introduction or removal of a stimulus, which allows their functioning to be regulated by turning them on or off, or by a combined action [Wright, AV, Sternberg, SH, Taylor, DW, Staahl, BT, Bardales, JA, Kornfeld, JE, & Doudna, JA (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 DNA and RNA sequences, introducing epigenetic modifications, epitranscriptomic modifications, visualizing DNA or RNA sequences, editing proteins, changing the three-dimensional structure of chromatin, attracting proteins and RNA with enzymatic activity to specified loci of DNA, RNA, proteins or components of cells, tissues, organs and organ systems. CRISPR / Cas systems can be used to introduce specific modifications into the body's biomolecules for the purpose of treating hereditary diseases, metabolic disorders, correcting genetic mutations, starting or blocking genes, modulating gene activity, changing the three-dimensional localization of chromatin, changing the composition and properties of DNA and RNA molecules,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 introduce beneficial changes into 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 oncological diseases, correcting tumor resistance genes to drugs, overcoming microorganism resistance to antibacterial drugs, increasing the sensitivity of tumors and healthy tissues to the action of drugs,improving the absorption of drugs, nutrients, high-molecular compounds, low-molecular compounds, nanocarriers and their combinations [Mir, A., Edraki, A., Lee, J., & Sontheimer, E.J. (2018). Type II-C CRISPR-Cas9 biology, mechanism, and application. ACS chemical biology, 13(2), 357–365]. Certain methods for correcting 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, M.D. Cappellini, Y.-S. Chen, J. Domm, B. K. 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. Lewandowski, X. Zhao, S. Chitnis,R. Peddinti, CRISPR-Cas9 Editing of the HBG1 and HBG2 Promoters for the Treatment of Sickle Cell Disease, N. Engl. J. Med.389 (2023) 820–832.] [ JD Gillmore, E Gane, J Taubel, J Kao, M Fontana, ML Maitland, J Seitzer, D O'Connell, KR Walsh, K Wood, CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis, N Engl. J. Med. (2021).][ L. Xu, J. Wang, Y. Liu, L. Xie, B. Su, D. Mou, L. Wang, T. Liu, X. Wang, B. Zhang, CRISPR-edited stem cells in a patient with HIV and acute lymphocytic leukemia, N. Engl. J. Med. 381 (2019) 1240–1247.][ D. Kostyushev, A. Kostyusheva, S. Brezgin, N. Ponomareva, NF Zakirova, A. Egorshina, D. V Yanvarev, E. Bayurova, A. Sudina, I. Goptar by CRISPR-Cas9, Mol. Ther. Acids.31(2023) 482–493.]. Welcome to the snowflake snowflake snowflakes and snowflakes в здоровье человека,and for the creation of new animal species and breeds. 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, or in other variants that involve one of the above-mentioned uses [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 whose replication occurs both with the integration of genetic material into the cell genome and in episomal form), such as adeno-associated viral vectors (AAV) of various serotypes, from different types of organisms and various genetic modifications, adenoviruses (AdV), herpes viruses (e.g., HSV-1), lentiviruses, retroviruses,and also using 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. - Vol. 10. - P. 895-713.] 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 the expression of proteins and, in particular, CRISPR / Cas systems with coding DNA is accompanied by complexly controlled 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 duration of expression and requires synchronization of protein synthesis with mRNA in the presence of a guide RNA. Moreover, mRNA / RNA themselves are unstable in cells and require chemical stabilization, which significantly increases the cost of their production and 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 off-target effects on non-target molecules, while RNP complexes are short-lived and, as a rule,The duration of their existence inside cells is no more than 24 hours, but can be increased or decreased artificially [Chen, Sean, et al. "Highly efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes." Journal of Biological Chemistry 291.28 (2016): 14457-14467.]. In addition, delivery in the form of RNPs does not require the synthesis of a Cas protein foreign to humans by the cellular translational apparatus, which significantly reduces the likelihood of T-cell immune clearance of the cells into which the RNPs were delivered [Chew, Wei Leong. "Immunity to CRISPR Cas9 and Cas12a therapeutics." Wiley Interdisciplinary Reviews: Systems Biology and Medicine 10.1 (2018): e1408.]. Thus, delivery in the form of RNPs is the most preferred option for using CRISPR / Cas. However, effective methods for packaging CRISPR / Cas RNP complexes into micro- and nanoparticles are currently lacking. Micro- and nanoparticles are carriers,which 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, and 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 through rational design of nanoparticles, modification of RNPs using chemical (treatment with surface-active compounds, etc.), physical (electroporation, freezing, ultrasound treatment, 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 the glycoprotein of the vesicular stomatitis virus (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 also 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 response to immunogenic viral particles results in 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 RNPs, and also, often,the impossibility of simultaneous packaging of Cas protein and guide RNA. Indeed, it has been 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 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.]. Biological nanoparticles are the general name for nanocarriers,created on the basis of or with the help of 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 nanoparticles (EMNV) [Brezgin, Sergey,et al. "Hydroxychloroquine Enhances Cytotoxic Properties of Extracellular Vesicles and Extracellular Vesicle–Mimetic Nanovesicles Loaded with Chemotherapeutics." Pharmaceutics 15.2 (2023): 534.], which are obtained by one of the technologies associated with cell extrusion through membranes with different pore diameters, as well as nanoghosts (NG), which are nanoparticles created from components or fragments of biological membranes of human cells obtained by hypotonic lysis, sonication, purification using gradient centrifugation, and subsequent extrusion [Oieni, Jacopo, et al. "Nano-ghosts: Novel biomimetic nano-vesicles for the delivery of antisense oligonucleotides." Journal of Controlled Release 333 (2021): 28-40.]. In addition, it is possible to create hybrid nanoparticles and biomimetics based on inorganic and organic components,and components of biological nanoparticles. The key characteristics of biological nanoparticles include high biocompatibility; the ability to overcome biological barriers; safety; lack of immunogenicity; the ability to program the properties of biological nanoparticles using chemical, physical and biological (genetic) methods, high loading capacity, reduced clearance by macrophages of the RES. Thus, the disadvantages of the known technical solutions are that they: (1) do not provide efficient co-packaging of guide RNA or any other target RNA or DNA into particles; (2) are not universal and require optimization for a specific class, type, kind and variant of the CRISPR / Cas system; (3) do not provide the ability to package chemically modified guide RNA or toxic guide RNA (or other target RNA), the expression of which in producer cells is impossible,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; (4) use immunogenic, toxic and non-biocompatible nanocarriers; (5) are technically complex and expensive to produce. (6) Loading of CRISPR-Cas systems is provided by electroporation, which causes damage and / or aggregation of particles and / or their contents. Methods are known for packaging proteins and, in particular, Cas proteins in EVs using chemical dimerization-dissociation systems [Gee, P., Lung, MSY, Okuzaki, Y. et al. Extracellular nanovesicles for packaging of CRISPR-Cas9 protein and sgRNA to induce therapeutic exon skipping. Nat Commun 11, 1334 (2020), doi 10.1038 / s41467-020-14957-y] based on the FKBP12-FRB dimerization system, where dimerization is carried out by the chemical agent rapamycin. The disadvantages of this system are: (1) The need to use rapamycin, an immunosuppressant,which has a toxic effect on EV-producing cells. As a result, the use of rapamycin may cause the death of EV-producing cells, as well as packaging of by-products associated with cell death into EVs. In addition, the transfer of rapamycin and cell death products to target cells is possible, which reduces the safety profile and may lead to severe side effects; (2) the possibility of using this system only for EVs; (3) the impossibility of simultaneous co-packaging of guide RNAs. In this system for packaging guide RNAs, a complex system of packaging and release of guide RNAs is used, based on flanking the guide RNA with hepatitis D virus ribozyme sequences, as well as an encapsidation signal. The dependence of the release of guide RNAs on ribozyme sequences makes the process of self-excision of ribozyme regions in EVs random,and not only in the cytoplasm of cells. The presence of a complex construct also increases the number of coding plasmids required for transfection to 4-5. (4) Use of this system only for EVs. A method for packaging proteins into EV EXPLORs is known based on the dimerization domains of CIBN-CRY2 under irradiation with blue spectrum light [Yim, Nambin, et al. "Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein–protein interaction module." / / Nature communications 7.1 (2016): 12277]. The disadvantages of this method are: (1) the need to use blue spectrum light, which is toxic to cells; (2) the possibility of using the method only for packaging proteins into EVs; (3) the lack of the possibility of RNA co-packaging. A method for packaging RNA into EVs is known based on the CIBN-CRY2 dimerization system by creating constructs encoding one of the dimerization domains, which is attached to the constitutive EV protein, and the other to the MCP domain [Hung, Michelle E.,and Joshua N. Leonard. "A platform for actively loading cargo RNA to elucidate limiting steps in EV-mediated delivery." / / Journal of extracellular vesicles 5.1 (2016): 31027]. The RNA for packaging was modified with an MS2 hairpin. Thus, when a stimulus was applied (blue spectrum light, with a wavelength of 450-488 nm), it ensured dimerization of MCP with the second dimerization domain, and the MS2 hairpin interacted with MCP, thereby ensuring the loading of MS2-modified RNA into EVs. The disadvantages of the system are: (1) The need to use blue spectrum light, but this light is toxic to producer cells, causes the formation of reactive oxygen species, cell death, which can lead to co-packaging of toxic compounds in EVs and cause side reactions in target cells. (2) The possibility of using this system only for EVs. (3) The possibility of using this system only for RNA packaging,Lack of information on the possibility of co-packaging with the protein of interest. (4) The possibility of using CRISPR / Cas systems or analogs for packaging guide RNAs has not been demonstrated; it has only been shown for short interfering RNAs. (5) Potential lack of dissociation between MS2 and MCP upon entry into the target cell, which complicates or makes impossible the release of packaged RNA. A method for irreversible packaging of RNA into vesicles, microvesicles, and EVs using the TAMEL platform is known [Hung ME, Leonard JN A platform for actively loading cargo RNA to elucidate limiting steps in EV-mediated delivery. J Extracell Vesicles. 2016 May 13;5:31027, doi: 10.3402 / jev.v5.31027], based on the interaction of high-affinity groups of RNA hairpins with affinity domains linked to the CD63 or Lamp2b protein. The disadvantage of this method is the irreversible (i.e., without dissociation and release of active RNA into the vesicle lumen) interaction of RNA with the CD63 protein,which cancels the possibility of exhibiting functional activity of RNA, as well as the lack of possibility of use for EMNV, NG and hybrids. Based on the combination of essential features, the method for mixing EVs with RNA-loaded liposomes indicated in the article by M. Piffoux [Piffoux, M., Silva, AKA, Wilhelm, C., Gazeau, F., & Tareste, D. (2018). Modification of Extracellular Vesicles by Fusion with Liposomes for the Design of Personalized Biogenic Drug Delivery Systems. ACS nano, 12(7), 6830–6842] is the closest to the proposed invention and is selected as a prototype. The disadvantages of this method include (1) the absence of an extrusion step with the production of nanoparticles of a given size optimal for a specific use; (2) the lack of the possibility of packaging chemically and genetically modified RNAs; (3) the lack of the possibility of packaging RNA into protein-enriched micro- and nanoparticles,with the formation of ribonucleoprotein complexes; (4) the absence of the possibility of forming hybrid micro- and nanoparticles and loading RNA into EV, EMNV and NG. At the same time, in the prototype of the present invention and in other analogues in that: (1) they do not provide the possibility of packaging RNA or DNA into EV, EMNV and NG; (2) they do not provide the possibility of forming hybrid micro- and nanoparticles with EV, EMNV and NG; (3) they do not provide the possibility of packaging chemically and genetically modified RNA, hybrid and chimeric molecules, such as RNA-DNA; (4) they do not provide the possibility of packaging RNA into micro- and nanoparticles enriched in the protein of interest with or without the formation of ribonucleoprotein complexes; (5) they do not provide the ability to control the sizes of the resulting EV, EMNV, NG and hybrid micro- and nanoparticles (EV, EMNV, NG hybrids) using physical methods (extrusion, homogenization, sonication), which is necessary for various types of delivery,such as systemic delivery; (6) they do not provide the possibility of using the developed technologies for the purposes of genetic editing, for the purposes of vaccination and therapeutic vaccines, for the purposes of gene therapy, genetic engineering, delivery of therapeutic and prophylactic RNA sequences and their modified analogs, delivery of therapeutic and prophylactic proteins, their complexes, protein and RNA complexes, their medical, biomedical, veterinary use, creation of reagents and methods for delivering proteins, RNA and their complexes into human and animal cells. Thus, the objective of the present invention is to create a new method for packaging protein, RNA and their complexes in hybrid micro- and nanoparticles. Technical results: - expansion of the spectrum of molecules subjected to packaging in micro- and nanoparticles; - increasing the efficiency of packaging protein, RNA, DNA and their complexes; - expansion of the arsenal of methods for packaging protein, RNA,DNA and their complexes into a particle. The stated problem is solved and the technical result is achieved by creating a method for producing hybrid particles, which includes the following stages: a) the synthesized target RNA, DNA, or RNA with DNA is mixed with a lipid or a mixture of lipids, then incubated to form liposomes containing the target RNA, DNA, or RNA with DNA; b) the liposomes obtained in stage (a) are mixed with at least one type of particles selected from the group consisting of exosomes (EV), exosome-like nanoparticles (emNV), nanoparticles (NG), and the mixture is incubated; c) the mixture obtained in stage (b) after incubation is extruded by forcing through at least one extruder membrane, which has a pore diameter comparable to the required diameter of the particles, to obtain hybrid particles; d) the particles obtained in stage (c) are purified from unpackaged components using chromatography. According to preferred embodiments, the said technical result is also achieved by the fact thatthat: - the obtained particles are nanoparticles; - the target RNA is synthesized chemically or by in vitro transcription; - the EV, emNV or NG particles are loaded with a protein and / or a complex of protein and / or RNA and / or DNA of interest; - the cells for obtaining the initial EV, emNV and / or NG are HEK293, HT1080, HeLa, PER.C6, CHO cells, mesenchymal stem cells (MSCs), induced pluripotent (iPSCs), totipotent, multipotent cells of various origins, NIH-3T3, BHK-21, bEnd;3, COS-7, HB54, HB55, HCA2, HMEC, HeLa and other cell lines, including those of tumor origin, as well as cultures of primary cells and tissues of various origins and morphologies; - HEK293 cells are HEK293 cells of any modification, selected from the group including HEK293, HEK293T, HEK293F, HEK293FT, HEK Expi293F; - mesenchymal stem cells (MSCs) are MSCs of various origins, selected from the 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; - at step (d) the particles were purified from unpackaged complexes using gel-exclusion chromatography, ion-exchange chromatography, hydrophobic chromatography, affinity chromatography ultracentrifugation, density gradient ultracentrifugation, ultrafiltration, tangential filtration, precipitation, fractionation in asymmetric flows, or using a combination of these methods or other methods; - the lipid is selected from the group consisting of dilinoleoylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE), dimyristoylglycerol phosphate (DMPA), dipalmitoylglycerol phosphate (DPPA), dioleoylglycerol phosphate DOPA,dimyristoylphosphoglycerol (DMPG), dipalmitoylphosphoglycerol (DPPG), dioleoylphosphoglycerol (DOPG), dimyristoylphosphatidylserine (DMPS), dipalmitoylphosphatidylserine (DPPS), dioleoylphosphatidylserine (DOPS), distearoylphosphatidylethanolamine (DSPE), cardiolipin, dioleoyltrimethylammonium propane (DOTAP), 2,3-dioleoyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanamine hydrochloride (DOSPA), cholesteryl hemisuccinate (CHEMS), ceramide phosphoethanolamine (CPE), diacylglycerol pyrophosphate (DGPP), GT-11, azidoethylsphingomyelin, sphingosine and its derivatives, C14-carnitine, derivatives of cholesterol, cholic and lithocholic acids, taurocholates, gangliosides, ceramides, ALC-0315, ALC-0159, 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl ethyl phosphate (DOCPe), bis(oleoyloxy)propyl)dimethylammonio)ethyl hydrogen phosphate (DOCP), phosphatidylinositol and its derivatives, cholesterol and its esters, dimethyldioctadecylammonium bromide (DDAB), N-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-amine (DOBAQ), 1,2-dimyristoyl-3-dimethylammonium propane (DAP), as well as their derivatives, their combinations and their conjugates; - conjugates of lipids with polyethyleneglycol, polyethyleneimine and / or peptides are used as conjugates; - a combination of DOPE and DOSPA in a ratio of 1:3 is used as a lipid; - the protein loaded into the EV, emNV or NG is a protein or peptide of any amino acid sequence, including chemically modified or stabilized proteins containing chemically modified amino acids or amino acid derivatives, recombinant proteins, genetically modified proteins, enzymes, antigenic proteins or fragments thereof, Cas proteins of various classes, types, species, origins and modifications, Cas proteins from various species of organisms, Cas-like proteins, zinc-finger nucleases, transcription activator-like effector-based nucleases (TALENs), meganucleases, human proteins, animal proteins, proteins of viruses, bacteria, archaea, bacteria,chimeric proteins; - a protein complex loaded into EV, emNV or NG is a complex of a recombinant protein, a genetically modified protein, an enzymatic complex, an antigen fragment, a protein-RNA complex, a cDNA protein complex, a Cas protein, a Cas-like protein, a zinc-finger nuclease, a transcription activator-like effector-based nuclease (TALENs), meganucleases, human proteins, animal proteins, proteins of viruses, bacteria, archaea, bacteria, chimeric proteins; - proteins or protein complexes loaded into EV, emNV or NG can be linked to or used together with low molecular weight compounds, polymers, adjuvants, materials of organic or inorganic origin; - the target RNA is selected from a group including mRNA, long non-coding RNA, RNA of the CRISPR-Cas systems, hybrid RNA consisting of various elements, optionally containing ribozyme sequences, aptamers, ribosome entry sites, encapsidation signals,signals for translation initiation and termination, sequences that alter RNA stability, RNA localization in cells, RNA translation, RNA splicing, wherein chemically modified RNAs 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 hairpins for dimerization, dissociation, interaction, destruction, stabilization of proteins, or additional effector elements; - the CRISPR-Cas RNA system is a short RNA (crRNA), tracrRNA, or a single guide RNA (sgRNA); - the target DNA is selected from a group of linear or circular DNA molecules, plasmid, phasmid, bacmid DNA, DNA minicircles (minicircle technology),synthetic oligonucleotides and antisense nucleotides (including those with modified nucleotides), fragments of the human, animal or plant genome, artificial chromosomes; - modifications of Cas proteins are complete Cas nucleases or proteins based on catalytically inactive dead-Cas (dCas) containing inactivating mutations in the nuclease domains, or Cas nickases (nCas) containing a mutation in only one domain; - Cas, dCas, nCas proteins are wild-type proteins, or improved or modified variants of Cas, dCas, nCas proteins obtained by random or directed evolution, mutagenesis with or without optimization of protein-coding codons, and optionally have additional elements in the coding construct; - 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, FokI-fused dCas9; - Cas, dCas or nCas are a single protein,or consisting of several components that assemble into a single protein in target cells, producer cells, or inside particles; - Cas, dCas, nCas proteins contain additional domains, including transcription activation domains, transcription repression domains, DNA or RNA base editing domains, 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; - transcription activation domains are VP48, VP64, VP160, VP192, p65, p65-HSF1, p300, VPR, TET1 domains, as well as their modifications according to the Scaffold, Casilio, SAM, TREE, SunTag principles or similar principles; - domains for transcription suppression are KRAB, EZH2, KRAB-MeCP2, DNMT3A, DNMT3A-3L, LSD1, or their modifications or combinations thereof; - domains for editing DNA or RNA bases are rAPOBEC1, APOBEC3A,AID, TadA, ADAR2, as well as their orthologues, their homologues and / or modified variants thereof obtained by mutagenesis or directed evolution; - the domains of RNA demethylases or RNA methyltransferases are the domains of METTL3, METTL14, METTL16, FTO, ALKHB5, or their combinations or modified variants thereof obtained by mutagenesis or directed evolution; - DNA visualization systems are selected from the group comprising CRISPR-FISHer, CRISPR-SIRIUS, CRISPRainbow modified CRISPR systems with fluorescent protein domains grafted and / or recruited to Cas protein and / or guide RNA; - RNA is chemically modified at nucleoside residues, RNA nucleotides, or sugar residues, including 2′-O-methyl derivatives (M), 2′-O-methyl-3′-phosphorothioate derivatives (MS), or 2′-O-methyl-3'-thiophosphonoacetate (MSP), pseudouridine Ψ, N1-methylpseudouridine (m1Ψ), N1-methylpseudouridine (m5C), 5-hydroxymethylcytosine (5hmC), 5-methiouridine (m5U), 2-thiouridine (s2U),aminomethylene-containing nucleotides (BNANC[N-Me]), locked nucleic acid (LNA), 2′-O-methyl-3′-phosphonoacetate (MP), ethyl group (cEt), fluoro group (F), phosphorothioate (PS), tetraethylene glycol (TEG), phosphonoacetate (PACE) or combinations thereof; - that the RNA contains cap structures at the 5′ end, a polyA sequence or modified polyA sequences in combination with other nucleotides at the 3′ end, or contains fluorescent labels, sites of interaction with proteins, or is a hybrid of RNA with DNA, or contains additional hairpins for dimerization, dissociation, interaction, destruction, stabilization of proteins and other molecules, as well as for the attraction of additional effector elements; - packaging of RNA or RNA complexes with protein is carried out at the stage of obtaining the said particles or after isolation of the said particles; - for packaging DNA or co-packaging RNA with DNA, DNA is used that is selected from a group that includes linear or circular DNA molecules, plasmids,phasmid, bacmid DNA, minicircle DNA, synthetic oligonucleotides and antisense nucleotides (including those with modified nucleotides), fragments of human, animal or plant genomes, artificial chromosomes; - DNA chemically modified at nucleoside or nucleotide residues of DNA, including fluorescently labeled nucleotides, biotinylated nucleotides, 2'F (2'-fluoro); 2'OMe (2'-O-methyl); LNA (locked nucleic acid); FANA (2'-fluoroarabinonucleic acid), HNA (hexitol nucleic acid), 2'MOE (2'-O-methoxyethyl); ribuloNA ((1'-3')-β-L-ribulonucleic acid); TNA (α-L-threose nucleic acid; tPhoNA (3'-2' phosphonomethylthreosyl nucleic acid); dXNA (2'-deoxyxylonucleic acid); PS (phosphorothioate); phNA (alkylphosphonate nucleic acid); PNA (peptide nucleic acid); - a protein molecule, or a complex of protein, RNA or DNA optionally contains additional elements,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 said additional elements are endosomolytic proteins and / or other elements; - endosomolytic proteins are proteins or peptides selected from the group consisting of HA2, GALA, INF7, JTS1, ESCA, melittin and its analogs, LL37 and its analogs, Tat, R8, EB1, Mellitin, KALA, SAP, H5WYG, ppTG1, LAH4; - the protein complex is the CRISPR / Cas ribonucleoprotein; - human and mammalian cells, including genetically and / or chemically modified cells, cell lines, transiently transfected lines and other components of a cell or cell line, are used as cells for producing the initial EVs, emNVs and / or NG; - 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; - from 0.5 ml to more than 100 liters of cell mass or a suspension of cells or particles are extruded; - the solvent affects the production of particles 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; - extrusion is carried out under the pressure of inert gases or by mechanical extrusion, or by centrifugal extrusion or vacuum filtration; - ultrasonic treatment is additionally carried out at the extrusion stage; - membranes made of polycarbonate, polyethylene terephthalate, polypropylene, regenerated cellulose or cellulose ethers, polytetrafluoroethylene, polyvinylidene fluoride, nitrocellulose, nylon are used for extrusion,polyethersulfone; - the membrane for extrusion is standard or track; - the membrane for extrusion has a hydrophilic or hydrophobic coating; - the membranes for extrusion have pore diameters selected from the range from 10 to 0.01 μm; - 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; - extrusion is carried out using drainage discs; - 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 are capable of influencing the production of nanoparticles, the expression of proteins and RNA, including proteins and RNA for packaging into nanoparticles; - to obtain hybrid particles, instead of liposomes, other particles in the size range from 2 nm to 50 μm of inorganic or organic nature, or components of particles, are used,loaded or associated with RNA guides; - the inorganic particles are tubes, vesicles, inorganic quantum dots made of metals and their compounds, including iron and its compounds, gold and its compounds, mesoporous silica and its compounds, carbon and its compounds; - the organic particles are selected from the group consisting of tubes, vesicles, polymer nanoparticles, polymer micelles, polyelectrolyte ion complexes, lipid nanoparticles, nanovesicles, quantumsomes, protein conjugates, virus-like particles, dendrimers, micelles, DNA nanoclaws; - the components of the particles loaded or bound to RNA guides are selected from the group including cholesterol, lipids, lipoprotein complexes, polymers, in particular PLGA, PEG, PEI and their lipid derivatives, components of viral particles, pH- or redox-sensitive linkers, pH-sensitive elements, endosomal release inducers, active materials (smart materials),light-sensitive and enzyme-sensitive and responsive elements; - organic and inorganic particles and their components are fused directly with the original cells and these cells are used to produce hybrid EVs, emNVs and NGs; - hybrid particles can be formed by other methods without extrusion, including simple, high- or low-temperature incubation, PEG-induced fusion, ultrasound-induced fusion, freeze-thaw fusion and others; - liposomes can be produced by self-assembly in solution, lipid film hydrogenation, detergent removal, double emulsion freeze-thaw, solvent injection, reverse-phase evaporation, lyophilization, spray drying, supercritical fluid methods, microfluidic methods and other methods,using technologies for reducing the size of the obtained liposomes; - the technology for reducing the size of the obtained liposomes is selected from extrusion, ultrasonic treatment, homogenization under high pressure. The stated problem is also solved, and the technical result is achieved by creating a particle obtained by the said method. The stated problem is also solved, and the technical result is achieved by creating a pharmaceutical composition for genetic editing, for modifying the genome, for correcting genetic mutations, for modifying the epigenome, for modifying the epitranscriptome, for editing RNA, for destroying DNA, for destroying RNA, for modifying nucleotide bases, for modifying nucleotides, for deaminating DNA or RNA, for integrating DNA or RNA sequences into the human genome or mitochondria, for visualizing DNA, for studying the spatial structure of chromatin, for changing RNA metabolism, for modifying proteins, for editing proteins,for the control of biological processes, for the control of physiological processes, for the purposes of vaccination, for the creation of therapeutic vaccines based on RNA, proteins, peptides, their mixtures or combinations, for the creation of drugs, for the creation of diagnostic drugs, for cosmetology products, for use in regenerative medicine, for the correction or introduction of mutations, including drug resistance mutations, in reconstructive medicine, for the treatment of oncological diseases, for the treatment of tumor metastases, for the treatment of autoimmune diseases, for the treatment of hereditary diseases, for the treatment of metabolic diseases, for the treatment of dysbacteriosis, for 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 theranostics 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 the production, 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, containing an effective amount of the said particles and at least one pharmaceutically acceptable excipient. The stated problem is also solved, and the technical result is achieved by using the said particle for genetic editing, for genome modification, for the correction of genetic mutations, for the modification of the epigenome, for the modification of the epitranscriptome, for RNA editing, for the destruction of DNA, for the destruction of RNA,for modifying nucleotide bases, for modifying nucleotides, for deaminating DNA or RNA, for integrating DNA or RNA sequences into the human or mitochondrial genome, for visualizing DNA, 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 prevention, 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 treating cancer, for treating autoimmune diseases, for treating hereditary diseases, for treating metabolic diseases, for 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 veterinary use. According to preferred embodiments, said technical result is also achieved by the fact that: - 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 injection 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, injection, irrigation, parenteral administration, oral administration, using a powder inhaler, transbuccally, using a tracheal tube,using vascular access; - systemic administration into the body is intravenous, intra-arterial, bolus, intraperitoneal, subarachnoid, epidural or intrasternal administration, or administration into the sinuses, into the cavities of the body, or administration subcutaneously, intramuscularly, or administration into specific organs and tissues; - local administration into the body is application to the surface of the skin and mucous membranes, the surface of organs, or organ lavage; - 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,gel for application to the oral mucosa, dental gel, intestinal gel, nasal gel, periodontal gel, rectal gel, dental gel, transdermal gel, urethral gel, ear gel, 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 inhalations, drops for local use,Oral drops, oral drops, dental drops, nasal drops, sublingual drops, ear drops, capsules, vaginal capsules, intrauterine capsules, chewable capsules, enteric-coated capsules, prolonged-release enteric-coated capsules, sublingual capsules, rectal capsules, modified-release capsules, inhalation powder capsules, prolonged-release capsules, concentrate, 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, liniment endocervical, lyophilisate for preparation, dispersion, drops, concentrate, solution, spray, suspension, emulsion, vaginal ointment,eye ointment, inhalation ointment, topical ointment, oral mucosal ointment, external ointment, nasal ointment, rectal ointment, ear ointment, inhalation oil, topical oil, external oil, internal oil, tincture, inhalation tincture, topical tincture, external tincture, dental sticks, periodontal sticks, nasal sticks, urethral sticks, ear sticks, gum paste, external paste, oral suspension paste, oral paste, oral paste, medicinal dental paste, vaginal foam, intrauterine foam, external foam, rectal foam, patch, oral mucosal patch mouth, transdermal patch, eye films, films for sticking to the gums, cheek films,orally dispersible films, periodontal films, sublingual films, dosed inhalation powder, powder for external use, powder for the preparation of gel, dispersion, drops, solution, paste, syrup, spray, suspension, powder for oral administration, nasal powder, periodontal powder, ear powder, effervescent powder, vaginal solution, intrauterine solution, solution for intra-arterial administration, solution for 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 topical use, solution for cutaneous scarification application, solution for application to the gums, solution for external use, solution for periarticular administration, solution for gastric irrigation, solution for bladder irrigation, solution for parabulbar administration, solution for peritoneal dialysis, solution for subcutaneous administration, solution for rinsing, solution for oral administration, solution for prick test, solution for eye wash, solution for nasal cavity wash, solution for oral wash, solution for ear canal wash, solution for oral mucosa, solution for subconjunctival administration, solution for extra-amniotic administration, solution for endo-sinusial administration, solution for endo-tracheal administration, dental solution, rectal solution, transdermal solution, syrup, vaginal therapeutic system,therapeutic intrauterine system, spray for topical use, spray for external use, spray for the oral mucosa, nasal spray, sublingual metered-dose spray, transdermal spray, ear spray, vaginal suppositories, rectal suppositories, vaginal suspension, suspension for intradermal administration, suspension for intramuscular administration, suspension for intra-articular administration, suspension for gastrointestinal administration, suspension for injection, suspension for injection 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 the oral mucosa, suspension for endo-sinusial administration, suspension for endotracheal administration, suspension dental, rectal suspension,tablets, vaginal tablets, effervescent vaginal tablets, intrauterine tablets, orodispersible tablets, dispersible tablets, tablets for implantation, tablets for inhalation, tablets for drops, solution, suspension, lozenges, chewable tablets, buccal tablets, mucoadhesive buccal tablets, enteric-coated tablets, prolonged-release enteric-coated tablets, lyophilisate tablets, sublingual 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 application, 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 pastilles, medicinal plates, tiles, medicinal absorbable wipes, medicinal shampoo, elixir, pills. Brief description of the drawings The invention is explained by the following drawings. Fig. 1 shows general schemes for packaging RNA in EMNV (A), NG (B) and EV (C) by the method of producing hybrid particles. (A) In the first step, EMNVs are obtained, loaded or not loaded with the protein(s) of interest. Following this, the obtained EMNVs are mixed with micro- and nanoparticles (particle size can vary from 1 nm to 2-5 µm) loaded with RNA, DNA, their mixture or other molecules,including molecules of low-molecular-weight compounds. In addition to low- and high-temperature incubation, hybridization can be carried out using ultrasonic treatment, serial freezing-thawing, PEG-induced fusion, microfluidics, and other methods. Micro- and nanoparticles can be liposomes, polyplexes, polymeric compounds, or other types of micro- and nanoparticles of organic or inorganic nature. The resulting mixture forms hybrid nanoparticles, which are the result of the fusion of EMNV with micro- and nanoparticles of a different nature, loaded with RNA, DNA, their mixture, or other molecules, including molecules of low-molecular-weight compounds. In this case, micro- and nanoparticles of a different organic or inorganic nature serve to load RNA, DNA, their mixture, or other molecules, including molecules of low-molecular-weight compounds, into EMNV. EMNV can be used per se to impart new,preferred properties of the obtained micro- and nanoparticles or for the simultaneous packaging of the protein(s) of interest. The next step is the extrusion of hybrid particles to obtain micro- and nanoparticles of a given size. The last step is strictly necessary to obtain particles of a given size for systemic administration, but may be optional for local use or direct introduction of micro- and nanoparticles into tissues. (B) Creation of hybrid micro- and nanoparticles from NG and organic or inorganic micro- and nanoparticles loaded with RNA, DNA, their mixture, or other molecules, including low-molecular-weight molecules. (C) Creation of hybrid micro- and nanoparticles from EV and organic or inorganic micro- and nanoparticles loaded with RNA, DNA, their mixture, or other molecules, including low-molecular-weight molecules. The step of loading RNA, DNA, their mixture, or other molecules,including molecules of low molecular weight compounds may be optional if it is necessary to use micro- and nanoparticles of organic or inorganic nature not for the purpose of packaging RNA, DNA, their mixture or other molecules, including molecules of low molecular weight compounds, but for the purpose of imparting new properties to hybrid micro- and nanoparticles, such as better efficiency of internalization into cells, improved endosomal release rates, import into cell nuclei or other cell organelles (e.g. mitochondria), avoidance of the immune response (innate or adaptive), the ability to controlled distribution (e.g. under the influence of a magnetic field), obtaining properties of stimulus-induced release, for example, when decreasing pH or changing the redox potential, coating particles with polyethylene glycol (PEG) by conjugating PEG with lipophilic anchors,microfluidics methods, etc. Figure 2 shows the characterization of four types of biological nanoparticles. (A) Cryoelectron images, (B) z-potential, and (C) size distribution of RNA-loaded hybrid nanoparticles. All the resulting nanoparticles have a similar size with a peak near 100 nm. Error bars represent standard deviations. Figure 3 shows the results of the analysis of Cas protein packaging in biological nanoparticles using the developed technologies in calculating the number of Cas protein copies per nanoparticle. Neg cntrl emNV is the background value of the Cas protein signal in nanoparticles without Cas protein. Hybrids is the packaging of StCas9 protein into hybrid nanoparticles. Error bars represent standard deviations. Figure 4 shows the results of RNA packaging using different loading methods. The data are presented as the number of RNA copies in the nanoparticle isolate (A, B),or in terms of RNA copies per nanoparticle (B). (A) RNA packaging using hybrid nanoparticle formation technology. The use of hybrid nanoparticle technology provides packaging from 2×10, 6English: copies of RNA into nanoparticles, while increasing the ratio of RNA for loading or liposomes / nanoparticles of another nature leads to a proportional increase in the levels of RNA packaged into hybrid nanoparticles. H 1× is a single dose of liposomes for creating hybrid nanoparticles; H 3× is a three-fold dose of liposomes for creating hybrid nanoparticles. (B) Evaluation of the levels of RNA packaging into hybrid nanoparticles obtained from two different emNV samples containing the Cas protein (emNV1, emNV2) using a liposome dose of 1× (H) or 2× (H 2×) for forming hybrids. (C) Packaging of RNA into different types of nanoparticles: cntrl is a negative control without RNA; EV is stochastic packaging in EV; Hybrids is packaging into hybrid nanoparticles. Error bars correspond to standard deviations. In Fig. 5 presents the results of the evaluation of the internalization of nanoparticles loaded with RNA using hybrid nanoparticle technology,in human and mouse cell lines. Delivery of DIR-labeled nanoparticles and FAM-labeled RNA. Data are presented as the percentage of cells positive for RNA (green bars) or nanoparticles (purple bars) signal in (A) mouse TC1 cells with (B) representative histograms of signal distribution in the FITC-A channel (FAM-RNA) and APC Cy7-A (DIR-NPs) or in (C) human HepG2 cells with (D) representative histograms of signal distribution in the FITC-A channel (FAM-RNA) and APC Cy7-A (DIR-NPs). Cntrl is the control sample treated with unlabeled nanoparticles; RNA-NPs are hybrid nanoparticles labeled with DIR dye and loaded with FAM-RNA. Error bars correspond to standard deviations. In Fig. 6 shows the results of confocal microscopy of TC1 cells treated or not treated with DIR-labeled nanoparticles loaded with FAM-labeled RNA. TC1 negative – negative control cells treated with unlabeled nanoparticles. NP – TC1 cells treated with DIR-labeled nanoparticles,loaded with FAM-labeled RNA. Green fluorescence – FAM-RNA; blue staining – internalization of DIR-labeled nanoparticles by cells; brightfield – bright field microscopy. Merge – overlay. Fig. 7 shows the results of confocal microscopy of HepG2 cells treated or not treated with DIR-labeled nanoparticles loaded with FAM-labeled RNA. HepG2 negative – negative control of cells treated with unlabeled nanoparticles. NP – HepG2 cells treated with DIR-labeled nanoparticles loaded with FAM-labeled RNA. Green fluorescence – FAM-RNA; blue staining – internalization of DIR-labeled nanoparticles by cells; brightfield – bright field microscopy. Merge – overlay. Fig. 8 shows the results of a test for the nucleolytic activity of nanoparticles loaded with various StCas9 complexes with guide RNAs. For the test, HEK293T cells were transfected with the EGxxFP plasmid,and on day 2 post-transfection, they were treated once with nanoparticles at a single dose of nanoparticles. The results were analyzed on day 3 after the addition of nanoparticles using flow cytometry in the FITC-A channel (Green fluorescence, Ex / Em = 488 / 517 nm). Shown is the EGFP signal (detected in the FITC-A channel) in the EGxxFP assay with Hybr nanoparticles obtained from two different EV samples containing the StCas9 protein (EV1, EV2) and loaded with the St10 guide RNA using hybrid particle technology using liposomes at three different doses (1 / 4×, 1×, 4×). Error bars represent standard deviations. The results confirm high nucleolytic activity, while the use of nanoparticles leads to saturation of the EGFP signal in the range of 40-60% of EGFP-positive (FITC-positive) cells, which does not allow a semi-quantitative comparison of nanoparticles for nucleolytic activity. Fig. 9 shows the results of the nucleolytic activity test of nanoparticles,loaded with different StCas9-guide RNA complexes. (A) Results obtained with EV nanoparticles and Hybr hybrid nanoparticles prepared from four different emNV samples (emNV1-4) loaded with StCas9 protein and loaded with st10 guide RNA using hybrid particle technology. (B) Results obtained with NG nanoparticles or Hybr NG hybrids or EV nanoparticles or Hybr EV hybrids with different hybrid nanoparticle formulations depending on the ratio of liposomes with RNA (1×, 2×, 4×) and loaded with Cas protein. (B) Results obtained with EMNV hybrid nanoparticles or EV hybrid particles loaded with StCas9 protein using two different EV samples (EV1, EV2) and two emNV samples (emNV1, emNV2) and loaded with St10 guide RNA or non-targeting Nc guide RNA using hybrid particle technology. Error bars represent standard deviations. The results confirm high nucleolytic activity,Moreover, the use of nanoparticles leads to saturation of the EGFP signal in the range of 40-60% of EGFP-positive cells, which does not allow for a semi-quantitative comparison of the nanoparticles for nucleolytic activity. Preferred embodiments of the invention A method for packaging RNA or DNA into hybrid nanoparticles obtained from EMNV, EV or NG, including those enriched with the protein of interest. General packaging schemes are shown in Fig. 1. In one embodiment of the invention, emNV, EV or NG loaded with the StCas nuclease from the Streptococcus thermophilus microorganism are used to obtain hybrid particles. In parallel, the RNA guide obtained by in vitro transcription and treated with alkaline phosphatase and a lipid mixture containing a DOSPA:DOPE mixture in a ratio of 3:1 are mixed in a reactor and incubated at a temperature of 20°C for 10 minutes. The resulting liposome mixture is mixed with a concentrate of biological nanoparticles, previously purified by the TFF method, and incubated at TFF temperature,and incubated at 37°C for 15 minutes. The resulting particle mixture is extruded using a liposomal extruder under nitrogen pressure through a hydrophilic polycarbonate membrane with a pore diameter of 0.1 μm (7 times) and passed through a column containing Sepharose CL-4B gel-exclusion resin, after which fractions corresponding to the target particles are collected. Micro- and nanoparticles of organic or inorganic nature, other than the nature of biological nanoparticles, can be organic particles ranging in size from 2 nm to 50 μm, such as liposomes, cationic lipids, polymeric nanoparticles, polyplexes, dendrimers, micelles, hydrogels, protein particles, particles from components of viruses or bacteria, (nano)emulsions, solid-lipid nanoparticles, etc., including PLGA, PEG, PEI, liposomes, or inorganic nanoparticles ranging in size from 2 nm to 50 μm, such as nanoparticles based on iron, carbon, gold, silver, quantum dots, mesoporous silica, lanthanides,upconversion nanoparticles; organic or inorganic micro- and nanoparticles may or may not contain additional components, including pH-sensitive linkers and elements, endosomal exit inducers, smart materials, light-sensitive and enzyme-sensitive and responsive elements, ribozymes loaded or linked to RNA guides, to biological nanoparticles, or to proteins. In one embodiment of the invention, the RNA may be any sequences of ribonucleic acids, their derivatives, ribonucleotides, RNA components, including RNA guides of the CRISPR / Cas systems or similar systems, their derivatives due to genetic modification, addition or removal of RNA guide elements, hybrid RNAs linked to 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. In one embodiment of the invention, 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, recruitment of additional effector elements. RNA can be RNA for translation and protein production or for the 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. 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 killer cells, etc.), cells of skeletal or smooth muscle, cardiac tissue, epithelium, brain, etc., or cells derived from any germ layers of humans or animals. Protein molecules, RNA, DNA, or their complexes may contain additional elements that can provide or enhance interaction with the membranes of biological nanoparticles to increase the packaging efficiency. In one embodiment of the invention, extrusion can be carried out using a manual, semi-industrial, or industrial extruder with varying pressure applied to the mass of cells for extrusion. Extrusion can be performed manually, due to the pressure of inert gases or gas mixtures, or mechanical pressure (for example, centrifugal force during centrifugation or the use of presses). The material of the membranes for extrusion can differ, be 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 claimed 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. In one embodiment of the invention, instead of extrusion, the formation of hybrid micro- and nanoparticles can be achieved by other methods, including simple, high- or low-temperature incubation, PEG-induced fusion or fusion induced by other polymeric materials or cationic lipids, induced by ultrasound,using the freeze-thaw method or other methods. In the case of EVs, cells expressing protein, RNA, or ribonucleoprotein complexes, or not expressing transgene molecules, are used and are cultured in 2D or 3D culture systems under adherent, suspension, or mixed conditions, to obtain a conditioned medium containing EVs. The production of conditioned medium may be associated with the use of endogenous or exogenous stimuli to obtain the required qualitative and quantitative composition of EVs. In one embodiment of the invention, purification of EVs from the conditioned medium can be carried out using TFF, UF, chromatography, or other methods. The process of obtaining purified EVs may also include an extrusion step to standardize the obtained isolates of EV micro- and nanoparticles, obtaining homogeneous or more homogeneous EV products. General scheme for the creation of hybrid particles from EMNVs,EV and NG are shown in Fig. 1. In the case of EMNV, EV and NG, by mixing the obtained EMNV, EV or NG with micro- and nanoparticles of organic or inorganic origin, loaded or not loaded with RNA, hybrid nanoparticles are formed containing EMNV, EV and / or NG components and components of micro- and nanoparticles of organic or inorganic origin. The resulting hybrid micro- and nanoparticles can be passed through serial extrusion to obtain micro- and nanoparticles of the required size. In one embodiment of the invention, emNV, EV or NG loaded with Cas nucleases, cytidine or adenine base editors Cas, systems for CRISPR activation, CRISPR interference or CRISPR-based epigenome editing, systems for CRISPR visualization, epitranscriptome editing, Prime Editing systems,systems for changing the three-dimensional structure of chromatin based on CRISPR-Cas and other Cas proteins. In one embodiment of the invention, emNV, EV or NG loaded with any proteins and peptides of any amino acid sequence can be used to produce hybrid particles, including chemically modified or stabilized proteins containing chemically modified amino acids or amino acid derivatives, or protein complexes, recombinant proteins, genetically modified proteins or their complexes, enzymes or enzymatic complexes, antigen fragments, as well as protein complexes with RNA or DNA, including Cas proteins of various classes, types, species, origins and modifications, Cas proteins from various species of organisms, Cas-like proteins, zinc-finger nucleases, transcription activator-like effector nucleases (TALENs), meganucleases, human proteins, proteins of animal origin, proteins of viruses, bacteria, archaea, bacteria, chimeric proteins. Proteins,Associated with or used in combination with low-molecular compounds, polymers, adjuvants, and materials of organic or inorganic origin, the target particle size may be 0.01-5 µm in one embodiment of the invention, using appropriate membrane filters when extrusion is used, or appropriate parameters when forming particles using other methods (e.g., freeze-thaw cycles, nitrogen cavitation, ultrasonic treatment, etc.). In one embodiment of the invention, the following can be used as source cells for obtaining emNV, EV or NG for the formation of hybrid particles: HEK293 cells of any modification (HEK293, HEK293T, HEK293F, HEK293FT, HEK Expi293F and others), HT1080, HeLa, PER.C6, CHO, mesenchymal stem cells (MSCs) of various origins (placental, adipose, bone marrow, MSCs from umbilical cord blood, amniotic fluid, peripheral blood,synovial fluid, dental pulp, endometrium, skin, muscle tissue, salivary glands, etc.), iPSCs of various origins, NIH-3T3, BHK-21, bEnd.3, COS-7, HB54, HB55, HCA2, HMEC, HeLa, and other cell lines, including those of tumor origin, as well as cultures of primary cells and tissues of various origins and morphologies. In one embodiment of the invention, iPSCs differentiated by transduction of viral vectors encoding the Oct3 / 4, Sox2, c-Myc, and Klf4 genes, or Oct4, Sox2, Nanog, and Lin28 genes, or some of these genes, into somatic cells, including keratinocytes or fibroblasts, HUVEC, and NHEK cell lines, can be used as cells. In one embodiment of the invention, iPSCs are obtained by daily transfection of somatic cells with mRNA encoding the Oct3 / 4, Sox2,c-Myc and Klf4 (with one mRNA type or a combination thereof). In one embodiment of the invention, iPSCs are obtained by induction from somatic cells with a cocktail of recombinant proteins Oct4, Sox2, Klf4, and c-Myc linked to endosomolytic and / or penetrating peptides. In one embodiment of the invention, iPSCs are obtained by transient transfection of somatic cells with plasmids or minicircles encoding the genes Oct3 / 4, Sox2, c-Myc and Klf4, Nanog, Lin28, or a combination thereof. In one embodiment of the invention, iPSCs are obtained from somatic cells, including fibroblasts, keratinocytes, HUVEC and NHEK cell lines, by treating these cells with low-molecular substances that affect the epigenetic state of the cellular genome, including 5-azacytidine, RG108, RSC133, SAHA, TSA, sodium hydroxybutyrate, VPA, tranylcypromine, BIX, CHIR, Kenpaullone, Compound B6, LiCl, E-616452, A83-01, LY-364947, SB431542, PD0325901, AMI-5, N-oxaloylglycine, compound B4, dasatinib,iPY rasin, PP1, rapamycin, compound B8, compound B10, D4476, BayK, FSK, PGE2, rolipram, 2-Me-5HT, 5-(4-chlorophenyl)-3-phenylpent-2-enoic acid, 8-Br-cAMP, fructose-2,6-bisphosphate, quercetin, DZnep, DNP, TTNPB, oxysterol, purmorphamine, Shh and other substances. In one embodiment of the invention, extrusion can be carried out in the temperature range of 0-100°C. In one embodiment of the invention, emNV, EV and NG loaded with both CRISPR-Cas nucleases and catalytically inactive variants (Dead-Cas) or proteins with one inactivated domain (Cas-nickases, nCas) in the following variants can be used to obtain hybrid particles: dCas-VP48, dCas-VP64, dCas-VP160, dCas-VP192, dCas-p300, dCas-VPR, dCas-VP64 / sgRNA-p65-HSF1 (SAM), dCas-TET1, dCas-DNMT3A, dCas-DNMT3A-3L, dCas-sgRNA-MS2 / MCP-TET1, dCas-SunTag-TET1, dCas-SunTag-VP64, dCas-SunTag-p300, dCas-KRAB, dCas-KRAB-MeCP2, dCas-EZH2, dCas-sgRNA-Casilio-VP64, dCas-LSD1, UGI-dCas-AID, UGI-nCas-AID,UGI-dCas-sgRNA-MS2 / MCP-AID, UGI-nCas-sgRNA-MS2 / MCP-AID, UGI-dCas-rAPOBEC1, UGI-nCas-rAPOBEC1, UGI-dCas-rAPOBEC3A, UGI-nCas-rAPOBEC3A, UGI-dCas-TadA, UGI-nCas-TadA, dCas-ADAR2, dCas-METTL3, dCas-METTL14, dCas-METTL3-METTL14, dCas-FTO, dCas-ALKBH5, dCas-WTAP, nCas-RT-pegRNA (reverse transcriptase), dCas-RT-pegRNA and / or other CRISPR-Cas based systems. In one embodiment of the invention, EVs, emNVs, or NGs loaded with Cas9, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas14, Cas-X, Cas3, and / or other CRISPR-Cas-based systems can be used to produce hybrid particles. In one embodiment of the invention, during resuspension of cells or purified membranes to obtain the original EVs, emNVs, or NGs and during the particle extrusion process, substances that alter cell fluidity (DMSO, SDS, and other substances with surface-active properties, lipids with saturated or unsaturated fatty acid residues, etc.) can be added.membrane permeability or endosomal release of the resulting particles in target cells, including cationic endosomolytic peptides (HA2, GALA, INF7, JTS1, ESCA, Mellitin and its analogs, LL37 and its analogs), anionic endosomolytic peptides (Tat, R8, EB1, Mellitin, KALA, SAP, H5WYG, ppTG1, LAH4), polycationic polymers and oligomers (PEI and its modifications, spermine, spermidine), cationic and ionizable lipids (DLPC, DMPC, DPPC, DSPC, DOPC, DMPE, DPPE, DOPE, DMPA, DPPA, DOPA, DMPG, DPPG, DOPG, DMPS, DPPS, DOPS, DOPE, DSPE, Cardiolipin, DSPE, DOTAP, DOSPA, CHEMS, CPE, DGPP, GT-11, Azydoethylsphyngomyelin, Sphingosine and its derivatives, C14-carnitine, taurocholates and other derivatives of cholic and lithocholic acids, gangliosides, ceramides, ALC-0315, ALC-0159, DOCPe, DOCP, phosphatidylinositol and its derivatives, cholesterol and its esters, cholesterol esters of fatty acids, DDAB, DOBAQ, DAP, TAP, analogs of these substances, their derivatives, combinations and conjugates, for example, with polyethylene glycol,peptides and other compounds), low molecular weight compounds (chloroquine, hydroxychloroquine, mefloquine, their analogs and derivatives). In one embodiment of the invention, extrusion can be carried out using a manual or screw extruder, an automatic liposomator, a centrifugal extrusion method, under nitrogen pressure or by mechanical extrusion or using other methods based on extrusion through filters with a certain pore diameter, by nitrogen cavitation, by vacuum filtration, with or without ultrasonic treatment and / or cell lysis by other methods, including hypotonic lysis. In one embodiment of the invention, standard or track membranes made of polycarbonate, polyethylene terephthalate (polyester), polypropylene, regenerated cellulose or cellulose ethers, polytetrafluoroethylene, polyvinylidene fluoride, nitrocellulose, nylon,polyethersulfone or other materials with hydrophilic or hydrophobic coatings. In one embodiment of the invention, membranes with various 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 and combinations thereof, with or without the use of drainage disks, or other membranes with a pore diameter in the range of 0.01-50 μm can be used. In one embodiment of the invention, particle purification can be accomplished by other methods, including ultracentrifugation, gradient ultracentrifugation, precipitation, ultrafiltration, tangential filtration, asymmetric flow fractionation, ion exchange chromatography, hydrophobic chromatography, affinity chromatography and other methods. In one embodiment of the invention, the guide RNA synthesized by in vitro transcription,may not be treated with alkaline phosphatase, RNA synthesized by in vitro transcription after additional processing steps, such as the addition of a cap structure, a poly-A tail, and / or other structural modifications, may be used. In one embodiment of the invention, the guide RNA or other RNAs can be obtained by chemical synthesis, including using nucleotides modified at sugar nucleoside residues or phosphate residues, the list of modifications includes, but is not limited to, 2'-O-methyl (M), 2'-O-methyl 3'-phosphorothioate (MS) or 2'-O-methyl-3'thiophosphonoacetate (2'-O-methyl-3'thioPACE, MSP), pseudouridine Ψ, N1-methylpseuduridine m1Ψ, N5-methylcytosine m5C, 5-hydroxymethylcytosine 5hmC, 5-methyluridine m5U, 2-thiouridine s2U, BNANC[N-Me], locked nucleic acids (LNA), 2'-O-methyl-3'-phosphonoacetate (MP), cEt, F, PS, tetraethylene glycol (TEG),phosphonoacetate (PACE) or combinations thereof. RNA may also contain or not contain cap structures at the 5' end, a 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, recruitment of additional effector elements. In one embodiment of the invention, DNA of any size can be used to obtain hybrids, including episomal plasmid DNA, genomic DNA of humans and animals, bacterial or viral DNA, DNA of archaea and other organisms, bacmid, phasmid DNA, artificial chromosomes, DNA minicircles,synthetic oligonucleotides and antisense nucleotides. In one embodiment of the invention, other lipids can be used to prepare liposomes, the list of which includes, but is not limited to, DLPC, DMPC, DPPC, DSPC, DOPC, DMPE, DPPE, DOPE, DMPA, DPPA, DOPA, DMPG, DPPG, DOPG, DMPS, DPPS, DOPS, DOPE, DSPE, Cardiolipin, DSPE, DOTAP, DOSPA, CHEMS, CPE, DGPP, GT-11, azidoethylsphingomyelin, sphingosine and its derivatives, C14-carnitine, taurocholates and other derivatives of cholic and lithocholic acids, gangliosides, ceramides, ALC-0315, ALC-0159, DOCPe, DOCP, phosphatidylinositol and its derivatives, cholesterol and its esters, esters of cholesterol and fatty acids, DDAB, DOBAQ, DAP, TAP, analogs of these substances, their derivatives, combinations, and conjugates, for example, with polyethylene glycol, peptides, and other compounds. In one embodiment of the invention, RNA or DNA can be used to fuse with biological nanoparticles, forming polyplexes, for example,in complex with PEI and its derivatives, DEAE-dextran and its derivatives, other non-liposomal reagents (e.g. FuGENE), or RNA or DNA can be associated with inorganic particles (include metals and their compounds, including iron and its compounds, gold and its compounds, mesoporous silica and its compounds, upconversion compounds, quantum dots, carbon and its compounds, calcium phosphate crystals), organic particles (polymer nanoparticles, polymer micelles, polyelectrolyte ion complexes, nanovesicles, quatsomes, protein conjugates, virus-like particles, dendrimers, micelles, DNA nanoclaws, etc.) or components of liposomes (cholesterol, lipids, lipoprotein complexes, polymers, including PLGA, PEG, PEI and their lipid derivatives, components of viral particles, pH- or redox-sensitive linkers, pH-sensitive elements, endosomal release inducers, smart materials,light-sensitive and enzyme-sensitive and responsive elements). In one embodiment of the invention, liposomes can be obtained by self-assembly in solution at high or low temperature, lipid film hydrogenation, detergent removal method, freeze-thaw, double emulsion, solvent introduction method, reversed-phase evaporation method, lyophilization, spray drying, methods using supercritical fluids, microfluidic methods and other methods, with or without the use of technologies for reducing the size of the obtained liposomes (extrusion, ultrasonic treatment, high-pressure homogenization). In one embodiment of the invention, the fusion of liposomes or other particles (e.g., PEI polyplexes) with biological particles can be accomplished by PEG-induced fusion, freeze-thaw, ultrasonic treatment,incubation under pressure and other methods. In one embodiment of the invention, any biological nanoparticles can be used to produce hybrids, including extracellular vesicles of any size, as well as exosome-like micro- and nanoparticles and membrane particles characterized in paragraphs 1 and 3; in addition, hybrid particles can be obtained by fusing particles containing RNA\DNA directly with producer cells expressing or not expressing the target protein (including Cas proteins); in this case, the production of hybrid particles after the fusion of cells with RNA\DNA carriers is carried out in accordance with the claimed method. In one embodiment of the invention, biological micro- and nanoparticles EV with liposomes loaded with RNA are used. The particles are taken to each other in a ratio of 1:1 or in a range of ratios from 1:0.01 to 1:1000, or in a range of ratios from 0.01:1 to 1000:1,In one embodiment of the invention, EMNV biological particles are taken with liposomes in a 1:1 ratio, either in the ratio range from 1:0.01 to 1:1000, or in the ratio range from 0.01:1 to 1000:1, mixed in a phosphate buffer solution at 37°C, incubated for 15 minutes, and then used for extrusion through a series of polycarbonate filters with pore diameters of 2 μm, 1 μm, and 0.1 μm. In one embodiment of the invention, NG biological particles are taken with liposomes in a ratio of 1:1 or in a ratio range from 1:0.01 to 1:1000, or in a ratio range of 0.01:1 to 1000:1, mixed in a phosphate buffer solution at a temperature of 37°C,the mixture is incubated for 15 minutes and then used for extrusion through a series of polycarbonate filters with pore diameters of 2 μm, 1 μm and 0.1 μm. In one embodiment of the invention, a mixture of biological micro- and nanoparticles (EV, EMNV or NG) is used. In one embodiment of the invention, organic particles from Table 1 can be used instead of liposomes. In one embodiment of the invention, inorganic particles from Table 2 can be used instead of liposomes. In one embodiment of the invention, components of organic origin from Table 3 can be used instead of liposomes. In one embodiment of the invention, instead of extrusion through a series of polycarbonate filters,the filter material from Table 4 can be used. In one embodiment of the invention, the pore diameter of the polycarbonate filters can be in the range from 10 nm to 20 μm. In one embodiment of the invention, the methods from Table 5 can be used instead of mechanical extrusion. In one embodiment of the invention, the particle size is not controlled, and their natural fusion occurs to form hybrid particles. In one embodiment of the invention, the particle size is not controlled, and their natural fusion occurs to form hybrid particles. In one embodiment of the invention, agents for endolysosomal release can be added to the particle mixture or to the particle composition, as indicated in Table 6. In one embodiment of the invention, agents for enhancing RNA packaging can be added to the particle mixture or to the particle composition,as indicated in Table 7. In one embodiment of the invention, the biological particles can be loaded with the proteins indicated in Table 8. In one embodiment of the invention, the RNA can contain modifications as indicated in Table 9. In one embodiment of the invention, the proteins can contain modifications as indicated in Table 10. In one embodiment of the invention, compounds can be introduced into the particle mixture or into the particle composition to improve the cargo packaging process, as indicated in Table 11. In one embodiment of the invention, drugs can be introduced into the particle mixture or into the particle composition to simultaneously act on one or more targets; examples are indicated in Table 12. Table 1. Examples of organic micro- and nanoparticles and micro- and nanocapsules for creating hybrid micro- and nanoparticles. Table 2. Examples of inorganic micro- and nanoparticles for creating hybrid micro- and nanoparticles. Table 3. Examples of components of organic origin. Table 4. Examples of filter materials for extrusion. Table 5. Examples of methods for mechanical extrusion Table 6. Examples of agents for inducing endolysosomal release. Table 7. Examples of agents for enhancing RNA packaging. Table 8. Examples of proteins for loading into micro- and nanoparticles. Table 9. Examples of RNA modifications Table 10. Examples of protein modifications. Table 11. Examples of compounds for improving the process of cargo packaging into micro- and nanoparticles. Table 12. Examples of drugs for simultaneous administration into micro- and nanoparticles. In one embodiment of the invention, the RNA may be any RNA sequences, mRNA, long non-coding RNA, RNA of various types of CRISPR-Cas systems (crRNA, tracrRNA, sgRNA), oligo- and pre-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.In one embodiment of the invention, the RNA may be chemically modified, for example, at RNA nucleotide(s) residues or sugar residues, including 2′-O-methyl (M), 2′-O-methyl 3′phosphorothioate (MS), or 2′-O-methyl 3′thioPACE (MSP), Pseudouridine 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 their combinations.contain cap structures 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 the attraction of additional effector elements. In one embodiment of the invention, the DNA may be linear or circular DNA molecules, plasmid, phasmid, bacmid DNA, DNA minicircles, synthetic oligonucleotides (including those containing modifications such as fluorescently labeled nucleotides, biotinylated nucleotides, 2′F; 2′OMe; LNA; FANA; HNA; 2′MOE; ribuloNA; TNA; tPhoNA; dXNA; PS; phNA; PNA, etc.), antisense oligonucleotides, fragments of the human, animal, or plant genome, artificial chromosomes, etc.In one embodiment of the invention, elements for improving the biological properties of the resulting nanoparticles, in particular endosomal release, include substances capable of enhancing the destabilization of the endosome and the release of contents into the cytosol by various mechanisms, including the formation of pores, fusion with the endosomal membrane, the “proton sponge” mechanism, and destabilization of the endosomal membrane.The list of these substances includes, but is not limited to, cationic peptides (HA2, GALA, INF7, JTS1, ESCA, Mellitin and its analogs, LL37 and its analogs), anionic peptides (Tat, R8, EB1, Mellitin, KALA, SAP, H5WYG, ppTG1, LAH4), polycationic polymers and oligomers (PEI and its modifications, spermine, spermidine), cationic and ionizable lipids (the list includes, but is not limited to DLPC, DMPC, DPPC, DSPC, DOPC, DMPE, DPPE, DOPE, DMPA, DPPA, DOPA, DMPG, DPPG, DOPG, DMPS, DPPS, DOPS, DOPE, Cardiolipin, DSPE, DOTAP, DOSPA, CHEMS, their analogs and combinations), low molecular weight compounds (chloroquine, hydroxychloroquine, mefloquine, their analogues and derivatives).In one embodiment of the invention, the technology for forming hybrid nanoparticles involves fusing liposomes or other nanoparticles, nanotubes and nanovesicles, or particles, tubes and vesicles (ranging in size from 2 nm to 50 μm) of inorganic or organic origin loaded with or linked to guide RNA, with biological nanoparticles (EV, EMNV, NG) loaded with Cas proteins, followed by extrusion of the resulting hybrids through pores of varying pore diameters or formation of hybrid particles by other methods without the use of extrusion. By fusing the two types of nanoparticles, larger nanoparticles are formed that contain both the Cas protein and the guide RNA. A subsequent extrusion step can ensure crushing of the resulting hybrids to hybrid nanoparticles of the required size.On the one hand, this solves the problem of packaging any given amount of RNA guides into nanoparticles with Cas proteins, and, on the other hand, ensures the required size of nanoparticles, optimal for a specific type of delivery (systemic or local). In one embodiment of the invention, the inorganic particles are metals and their compounds, including iron and its compounds, gold and its compounds, mesoporous silica and its compounds, upconversion compounds, quantum dots, carbon and its compounds, etc. In one embodiment of the invention, the organic particles are polymer nanoparticles, polymer micelles, polyelectrolyte ion complexes, lipid nanoparticles (including liposomes), nanovesicles, quantumsomes, protein conjugates, virus-like particles, dendrimers, micelles, DNA nanoclaws, etc.) or their components (cholesterol, lipids, lipoprotein complexes, polymers, including PLGA, PEG, PEI, etc., viral particle components, pH- or redox-sensitive linkers, pH-sensitive elements, endosomal release inducers, smart materials, light-sensitive and enzyme-sensitive and responsive elements. In one embodiment of the invention, liposomes can be obtained by self-assembly in solution, lipid film hydrogenation, detergent removal, double emulsion freezing and thawing, solvent introduction, reverse-phase evaporation, lyophilization, spray drying, supercritical fluid methods, microfluidic methods, and other methods, with or without the use of liposome size reduction technologies (extrusion, ultrasonic treatment, high-pressure homogenization).In one embodiment of the invention, the RNA may be any RNA sequences, mRNA, long non-coding RNA, RNA of various types of CRISPR-Cas systems (crRNA, tracrRNA, sgRNA), oligo- and pre-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.In one embodiment of the invention, the RNA may be chemically modified, for example, at RNA nucleotide(s) residues or sugar residues, including 2′-O-methyl (M), 2′-O-methyl 3′phosphorothioate (MS), or 2′-O-methyl 3′thioPACE (MSP), Pseudouridine 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 their combinations.contain cap structures 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 the attraction of additional effector elements. In one embodiment of the invention, the DNA may be linear or circular DNA molecules, plasmid, phasmid, bacmid DNA, DNA minicircles, synthetic oligonucleotides (including those containing modifications such as fluorescently labeled nucleotides, biotinylated nucleotides, 2′F; 2′OMe; LNA; FANA; HNA; 2′MOE; ribuloNA; TNA; tPhoNA; dXNA; PS; phNA; PNA, etc.), antisense oligonucleotides, fragments of the human, animal, or plant genome, artificial chromosomes, etc.In one embodiment of the invention, in order to improve the biological properties of the resulting nanoparticles, in particular, endosomal release, during the production of the particles, substances are added that are capable of enhancing the destabilization of the endosome and the release of contents into the cytosol by various mechanisms, including the formation of pores, fusion with the endosomal membrane, the “proton sponge” mechanism, and destabilization of the endosomal membrane.The list of these substances includes, but is not limited to, cationic peptides (HA2, GALA, INF7, JTS1, ESCA, Mellitin and its analogs, LL37 and its analogs), anionic peptides (Tat, R8, EB1, Mellitin, KALA, SAP, H5WYG, ppTG1, LAH4), polycationic polymers and oligomers (PEI and its modifications, spermine, spermidine), cationic and ionizable lipids (the list includes, but is not limited to DLPC, DMPC, DPPC, DSPC, DOPC, DMPE, DPPE, DOPE, DMPA, DPPA, DOPA, DMPG, DPPG, DOPG, DMPS, DPPS, DOPS, DOPE, Cardiolipin, DSPE, DOTAP, DOSPA, CHEMS, their analogs and combinations), low molecular weight compounds (chloroquine, hydroxychloroquine, mefloquine, their analogues and derivatives).In one embodiment of the invention, the guide RNA or other RNAs can be obtained by in vitro transcription or chemical synthesis, including using nucleotides modified at sugar nucleoside residues or phosphate residues, the list of modifications includes, but is not limited to 2′-O-methyl (M), 2′-O-methyl 3′phosphorothioate (MS), or 2′-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.RNA may also contain or not contain cap structures 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, attraction of additional effector elements. In one embodiment of the invention, liposomes or other nanoparticles, nanotubes and nanovesicles or particles, tubes and vesicles (in the size range from 2 nm to 50 μm) of inorganic or organic nature, loaded with or associated with RNA guides, other RNA or DNA are fused directly with the original cells, which serve as a source for the production of EVs, emNVs or NGs, after which the hybrid particles are obtained according to the protocols used for the production of EVs, emNVs or NGs.The protocol for producing hybrid EVs involves isolating hybrid particles from the conditioned medium of cells, followed by purification. The protocol for producing hybrid emNVs involves removing cells from the culture substrate by extruding them through a series of membrane filters (typically 10, 5, and 1 μm). The protocol for producing hybrid NGs involves removing cells from the culture substrate, gently lysing them using hypotonic solutions and / or ultrasonic treatment, sedimentation and purification of cell membranes, and forming spherical particles using extrusion, centrifugation, freezing-thawing, incubation, or other methods. In one embodiment, the method for producing hybrid particles includes simple, high- or low-temperature incubation, PEG-induced fusion, ultrasound-induced fusion, freeze-thawing fusion, and other methods.In one embodiment of the invention, the extrusion step can be performed using either a manual extruder or automated extruders for various volumes (from 0.5 ml to >100 l) of particle suspension in any solvent or buffer solution, which may or may not affect nanoparticle production and may or may not alter the properties of cell membranes, particularly their fluidity. In one embodiment, extrusion can be performed under inert gas pressure (nitrogen and others) or by mechanical extrusion, centrifugal extrusion, or vacuum filtration, with or without ultrasonic treatment.In one embodiment of the invention, standard or track membranes made of polycarbonate, polyethylene terephthalate (polyester), polypropylene, regenerated cellulose or cellulose ethers, polytetrafluoroethylene, polyvinylidene fluoride, nitrocellulose, nylon, polyethersulfone or other materials with hydrophilic or hydrophobic coatings can be used as membranes for extrusion. During extrusion, membranes with various pore diameters can be used, including 10 μm, 8 μm, 5 μm, 2 μm, 1.2 μm, 0.8 μm, 0.45 μm, 0.4 μm, 0.22 μm, 0.2 μm, 0.1 μm, 0.05 μm, 0.03 μm, 0.01 μm, with or without the use of drainage discs, or membranes with a pore diameter in the range of 0.01-50 μm can be used.In one embodiment of the invention, natural or synthetic neutral, anionic, cationic or ionizable lipid compounds are used as lipids, including phospholipids, sphingolipids, glycolipids, ceramides, cholesterol and its derivatives and other lipid compounds, the list of which includes but is not limited to DLPC, DMPC, DPPC, DSPC, DOPC, DMPE, DPPE, DOPE, DMPA, DPPA, DOPA, DMPG, DPPG, DOPG, DMPS, DPPS, DOPS, DOPE, DSPE, Cardiolipin, DSPE, DOTAP, DOSPA, CHEMS, CPE, DGPP, GT-11, azidoethylsphingomyelin, sphingosine and its derivatives, C14-carnitine, taurocholates and other derivatives of cholic and lithocholic acids, gangliosides, ceramides, ALC-0315, ALC-0159, DOCPe, DOCP, phosphatidylinositol and its derivatives, cholesterol and its esters, cholesterol esters of fatty acids, DDAB, DOBAQ, DAP, TAP, analogs of these substances, their derivatives, combinations and conjugates (for example, with PEG or PEI) and others.In one embodiment of the invention, a method for packaging chemically modified guide RNA into nanoparticles containing Cas proteins by forming hybrid nanoparticles involves preparing inorganic or organic nanoparticles (e.g., liposomes) loaded with chemically modified guide RNA(s) and biological nanoparticles (EV, EMNV, NG) loaded with Cas protein, followed by fusion of the two types of nanoparticles to form hybrids. The hybrids can then undergo an extrusion step to form nanoparticles of a given size. Chemical modifications of guide RNAs may include modifications of sugar residues (e.g., replacing hydroxyl groups with hydroxymethyl, sulfhydryl, or fluorine residues), modification of the phosphate backbone (changing phosphates to phosphorothioates), modification of the 5' and 3' ends, hairpins of guide RNAs, creation of additional secondary structures, attachment of fluorophores or other molecules, and other modifications.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. Examples of the invention Example 1. Production of hybrid nanoparticles according to the present method EVs were produced by 2-4 days of culturing HEK293T cells or mesenchymal stromal cells in a medium supplemented with fetal bovine serum depleted of EVs. The resulting volume of conditioned medium was used to purify EVs by ion-exchange chromatography. Purified EV, EMNV, or NG particles were used to create hybrid nanoparticles by mixing with cationic liposomes loaded with RNA guides. By mixing and incubating, the liposomes formed hybrids with EVs, EMNV, and NG, which were then extruded through membrane filters with a pore diameter of 100 nm. The resulting hybrid particles were purified by gel filtration (see Fig. 1).The resulting EV, EMNV, and NG nanoparticles, as well as the hybrid particles, were used in nanoparticle characterization tests using cryo-electron transmission microscopy (Fig. 2A) and dynamic light scattering or NTA (Figs. 2B, 2C), as well as subsequent assays for guide RNA content, internalization, and activity. To monitor the integrity of the prepackaged protein during hybrid particle formation, StCas9 protein loading 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 (quantities were quantified using NanoSight technology) under optimized nanoparticle lysis conditions, and by Western blotting and dot blot.The creation of hybrid particles by fusing EMNV loaded with StCas9 protein with liposomes ensured the preservation of the pre-packaged protein after the formation of hybrid particles; in the final isolate, the content was up to 109 protein copies per particle. Following this, the efficiency of packaging of guide RNA into biological particles loaded with Cas protein was studied using the technology of forming hybrid nanoparticles by fusing with RNA-loaded liposomes. The possibility of a multiple increase in the levels of packaging of guide RNA into nanoparticles was demonstrated by shifting the ratio of liposomes and / or guide RNA for loading to a higher value. Indeed, with a multiple increase in the amount of guide RNA (Fig. 4A) and guide RNA with liposomes (Fig. 4B), a multiple increase in the amount of guide RNA packaged into nanoparticles occurred.Moreover, using two different Cas protein loading technologies (Tech1 and Tech2), it was demonstrated that the loading of the guide RNA does not depend on the nature and characteristics of the Cas protein within the nanoparticles (Fig. 4B). A quantitative comparison of the levels of guide RNA packaging using stochastic EV loading and the hybrid nanoparticle technology is shown in Fig. 4C. As a result, stochastic packaging ensures loading of up to 0.1 RNA copies per EV, while hybrids, under the selected conditions, provide loading of up to 65.3 RNA copies per particle. It is important to note that for hybrids, an increase in RNA packaging is possible by changing the packaging conditions, concentrations of RNA, particles, and cell membranes, changing the physical parameters of nanoparticle production, adding chemical and physical agents, changing the membrane production technology (extrusion, ultrasound, etc.) or the constituent components of these technologies.The next step was to demonstrate that, in addition to cargo packaging, the particles were capable of delivering functionally active cargo to target cells. Hybrid nanoparticles were obtained from fluorescently labeled EMNV particles with fluorescently labeled FAM-RNA loaded into liposomes. The hybrid nanoparticles were added to mouse TC1 cells and human HepG2 cells (Fig. 5). Thus, nanoparticle-based delivery of not only protein but also RNA, and a chemically modified RNA guide, was demonstrated. The efficiency of nanoparticle internalization was 70-98%, and the efficiency of RNA delivery into cells was 83-90%, depending on the cell line. Human hepatoma HepG2 cells grow in "clamps," clusters of cells with some cells layered on top of each other; This explains the lower values ​​of internalization of nanoparticles for this cell line compared to monolayer TC1.Representative fluorescence micrographs of cells with and without treatment with the loaded RNA nanoparticles are shown in Fig. 6 for TC1 cells and in Fig. 7 for HepG2 cells. Next, the nucleolytic activity (ability to cut target DNA) of the CRISPR / Cas9 ribonucleoprotein complexes within EV, EMNV, NG, and hybrid nanoparticles was analyzed (Fig. 8). To analyze the functioning of the cloned genetic constructs, transfection with the EGxxFP construct was performed (Fig. 8). EGxxFP is designed to analyze the nucleolytic activity of nucleases in cells, where xx is the target of the guide RNA. When xx is cut by nuclease proteins, the EGFP reading frame is restored with the emission of green fluorescence (Fig. 8A). On the 2nd day after transfection of the EGxxFP reporter construct, nanoparticles loaded with CRISPR / Cas9 RNPs using different packaging technologies (Tech1, Tech2) were added to HEK293T cells, and the guide RNA was packaged using different RNA concentrations (1 / 4x, 1x, 4x).On the 4th day after the addition of any of the used nanoparticles, green fluorescence emission of EGFP was detected, indicating high nucleolytic activity of the nanoparticles (Figs. 8 and 9). The upper limit of the percentage of EGFP-positive cells of 40-70%, observed for all types of nanoparticles, is apparently associated with saturation of the system and cleavage of all EGxxFP targets. Therefore, methods and approaches to packaging RNA and DNA have been developed using the example of packaging guide RNA into particles preloaded with Cas protein to form CRISPR / Cas9 RNPs. The possibility of packaging any RNA, including genetically and chemically modified ones, has been demonstrated. It has been shown that the packaged complexes, proteins, RNA, and RNPs retain pronounced activity, and the nanoparticles effectively deliver cargo to target cells. Technological approaches and technologies for packaging cargo into hybrid nanoparticles have been developed. Example 2.Cultivation of HEK293T cells and mesenchymal stromal cells. 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 mesenchymal stromal cells obtained from bone marrow or adipose tissue 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 first purified to remove any EVs by ultrafiltration. Example 3. Transcription and purification of guide RNA in vitro. The PCR product encoding guide RNA under the T7 promoter was synthesized using high-fidelity polymerase Q5.The T7 PCR product was then used as a template for in vitro transcription using a 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 type I DNase (NEB) for 15 min at 37°C, followed by RNA purification by isopropanol precipitation. 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. Example 4. Molecular Cloning. Molecular cloning of the plasmids used in this study was performed using Gibson assembly (NEB) technology according to the manufacturer's protocol with primers synthesized by Lumiprobe (Russia). Primers were designed using SnapGene. PCR products for the reaction were obtained using high-fidelity polymerase Q5 (NEB).Purification of PCR products from the gel was performed using a DNA gel extraction kit (Eurogen). Example 5. Nucleic acid isolation. RNA loaded into nanoparticles was isolated using the ExtractRNA reagent (Eurogen) according to the manufacturer's protocol. The isolated RNA was reverse transcribed using AmpliSens Reverta-FL reagents (AmpliSens Biotechnologies) for 30 min at 37°C for subsequent PCR analysis. Example 6. PCR analysis. Quantification of the loaded guide RNAs was performed by amplifying the obtained cDNAs and standards for constructing a calibration curve on a QuantStudio5 instrument (Applied Biosystems). The calibration standards were genetically engineered constructs encoding the St10 guide RNA with a known concentration. Genta TaqF DNA polymerase reagents (GenTerra), as well as primers and probe synthesized by Eurogen and Lumiprobe, were used for the analysis. Example 7. Obtaining EMNV ≈4×10. 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 cellular debris. The supernatant was extruded through a hydrophilic polycarbonate membrane with a 0.1 µm pore size soaked in PBS-HAT buffer (11 times). The exosome-like nanoparticles were purified by gel-exclusion chromatography on a Sepharose CL-4B resin column using a phosphate buffer solution as the mobile phase.The obtained sample was loaded onto a 1.6×10 cm column, and 40 fractions of 500 µl each were collected. Fractions containing biological nanoparticles (Fraction 11–16) were pooled and supplemented with 10× PBS-HAT buffer to a concentration of 1× (0.2% human serum albumin, 25 mM trehalose, 25 mM HEPES). Example 8. Obtaining NG ≈8×10. 7Cells were removed using Versen solution (PanEco, Russia), washed with phosphate-buffered saline and resuspended in 10 ml of ice-cold TM buffer (0.01 M Tris and 0.001 M MgCl2) containing a protease inhibitor cocktail. The suspension was incubated on ice for 15 minutes, then sonicated on a disintegrator (model) (5 sec, amplitude 27%). Next, a solution of 60% sucrose in TM buffer (pH 7.4) was added to a concentration of 0.25 M sucrose, mixed, and the membranes were sedimented at 4 °C and 6000 g (15 minutes). The supernatant was discarded, a solution of 0.25 M sucrose in TM buffer was added and washing was repeated 2 more times. After washing, the membrane pellet was resuspended in a 0.25 M sucrose solution in TM buffer (pH 8.6), sonicated (5 sec, 27% amplitude), and pelleted (4°C, 6000g, 15 min). The membranes were then washed twice more with a 0.25 M sucrose solution in TM buffer (pH 8.6). After the final wash, the supernatant was discarded, and the membrane pellet was resuspended in phosphate-buffered saline.The resulting membrane suspension was passed sequentially through hydrophilic polycarbonate membranes soaked in PBS-HAT buffer solution using a hand extruder: pore diameters of 1 µm (15 times), 0.4 µm (11 times), and 0.1 µm (15 times). The resulting vesicles were purified using gel-exclusion chromatography on a Sepharose CL-4B resin column using a phosphate buffer solution as the mobile phase. The resulting sample was loaded onto a 1.6 x 10 cm column, and 40 fractions of 500 µl each were collected. Fractions containing biological nanoparticles (Fraction 11-16) were pooled and added with 10× PBS-HAT buffer to a concentration of 1× (0.2% human serum albumin, 25 mM trehalose, 25 mM HEPES). Example 9. Preparation of hybrid nanoparticles according to the present method ≈4×10. 7Cells 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 cells were passed sequentially through hydrophilic polycarbonate membranes soaked in PBS-HAT buffer solution: pore diameters of 10 um (9 times), 5 um (9 times), 1 um (9 times). The resulting suspension was centrifuged at 4°C for 5 minutes (2000g) to remove cellular debris. 31.2 µg of guide RNA and 40 µl of Lipofectamine 3000 (ThermoFisher Scientific) were mixed in OptiMem medium, the resulting liposome mixture was incubated for 10 minutes at room temperature. The supernatant obtained after cell extrusion and removal of cellular debris was mixed with liposomes and incubated for 15 min at 37°C, the resulting suspension was passed using an extruder through a hydrophilic polycarbonate membrane with a pore size of 0.1 um,soaked in PBS-HAT buffer solution (11 times). Hybrid nanoparticles were purified using gel-exclusion chromatography on a Sepharose CL-4B resin column using a phosphate buffer solution as the mobile phase. The obtained sample was applied to a 1.6 × 10 cm column, and 40 fractions of 500 μl were collected. Fractions containing biological nanoparticles (Fraction 11-16) were pooled and added 10x PBS-HAT storage buffer to a concentration of 1× (0.2% human serum albumin, 25 mM trehalose, 25 mM HEPES). Example 10. Cell transfection HEK293T cells were transfected using the technology described by us [Kostyusheva, AP, Kostyushev, DS, Brezgin, SA, Zarifyan, DN, Volchkova, EV, & Chulanov, VP (2019). Molekuliarnaia biologiia, 53(2), 311–323. https: / / doi.org / 10.1134 / S0026898419010075] 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 phosphate-buffered saline, and complete medium was added. Transfection of mesenchymal stromal cells was performed using our own technology. Example 11. Screening fluorescence microscopy Microscopy of 96-well plastic plates was performed on an LCI Imager ExFluorer. Example 12. Confocal microscopy Confocal images of transfected cells and cells treated with various nanoparticles were performed on an FV3000 microscope (Olympus). Example 13. Flow cytometry Flow cytometry was performed on a LongCyte instrument (ChallengeBio). Example 14. Western blotting and dot blotting Nanoparticles were lysed according to the developed protocol and used for SDS-PAGE, followed by transfer to polyvinylidene fluoride membranes. The 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 three times for 10 minutes in PBST, then incubated with anti-rabbit IgG secondary antibodies conjugated to horseradish peroxidase. Subsequently, the membranes were washed three times for 10 minutes in PBST. The signal was developed using the ECL reagent (Thermo Fisher) and detected on a Fusion-FX6.Edge V.070 gel and chemidocumentation system. Example 15. Transmission Electron Microscopy Cryo-TEM Cryoelectron micrographs of nanoparticles were obtained using a transmission electron microscope (TEM Tecnai G212 SPIRIT, FEI, USA). A copper grid on a carbon backing 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). The grid with the sample was then transferred in liquid nitrogen to the TEM. Example 16. Measurement of Ζ-potential by dynamic light scattering DLS The Malvern Zetasizer NanoZS instrument (Malvern, UK) was used to analyze the average size and charge of all biological and hybrid nanoparticles. Each sample was diluted 1000-fold with PBS, filtered through a 0.2 μM filter (Corning) and analyzed 5 times; 1,Five milliliters of diluted samples were loaded into polystyrene cuvettes (DTS0012; Malvern). The analysis was performed at 25 °C (100 measurements) using a 20 mW (633 nm) helium / neon laser. Data were analyzed using Zetasizer 8.01.4906 software (Malvern). Ζ-potential was analyzed in U-type cuvettes (DTS1070; Malvern) with gold electrodes. Ζ-potential measurements were performed at 25 °C at least five times. Background signal was estimated using filtered PBS. Example 17. Nanoparticle Counting by Nanoparticle Trajectory Analysis (NTA) Nanoparticle counts were analyzed using a Nanosight LM10 HS instrument (NanoSight Ltd., UK) equipped with a 405 wavelength laser. Particle tracking videos were recorded at room temperature with passive temperature reading and the following camera settings optimized for nanoparticles: camera shutter 1500, camera gain 500,lower threshold of 195 and upper threshold of 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 60-s videos 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. Example 18. Statistical analysis Values ​​were expressed as the 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 identify statistically significant differences in means. To determine whether each percentage of off-target indels in the deep sequencing analysis was significant compared to the mock-treated controls,The two-tailed p-value was calculated using Fisher's exact test. Thus, the claimed technical solution ensures: (a) Preservation of pre-packaged proteins (using the Cas protein as an example) during nanoparticle formation; (b) simultaneous packaging, co-packaging, or post-packaging of guide RNAs into nanoparticles together with, before, or after packaging of Cas proteins into nanoparticles with an efficiency of up to >80 copies of guide RNA per nanoparticle, while the number of copies of loaded guide RNA(s) can be regulated by changing the amounts of guide RNA in the loaded non-biological carrier, improving the formation of lipid nanocarriers before fusion with biological nanoparticles; (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; (d) eliminating the need for intracellular expression of the guide RNA together with Cas proteins (as part of hybrids), which may be associated with modification of the target gene(s), off-target activity of CRISPR / Cas, the occurrence of genetic mutations, aberrations, genome instability, anoikis, changes in the properties of producer strains, changes in the characteristics of the resulting nanoparticles, packaging potentially hazardous (toxic, carcinogenic) compounds into nanoparticles or changing the surface of nanoparticles with a violation of tropism, the ability to overcome biological barriers, stability; (e) 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 guide RNA, the presence or absence of additional elements, the removal of any elements in the CRISPR / Cas composition or the use of additional components, is not determined by the charge of Cas proteins, guide RNA and the resulting RNPs, is not determined by the use of full-length, truncated, or split proteins, domains or combinations thereof, and also provides for the loading of any guide RNA in the form of 1 guide RNA, their combinations, elongated or truncated sequences that, when assembled or processed (including cutting), cause the formation of RNA that can be used to interact with the Cas protein or other components; (e) is a universal system for packaging any proteins, RNA, DNA, as well as protein and RNA complexes, including in the form of RNPs. Example 19. 1. A preparation of hybrid nanoparticles was taken,obtained according to Example 9, in a dose of 1×10, 15 particles per kilogram of patient weight loaded with CRISPR / Cas base editor complexes to correct a genetic mutation in the SMN1 gene associated with spinal muscular atrophy. The drug was administered intravenously, and CRISPR / Cas was delivered to spinal motor neurons, resulting in CRISPR / Cas-mediated mutation correction and elimination of clinical symptoms, halting disease progression, and restoring motor activity. 2. The hybrid nanoparticle preparation obtained in Example 9 was taken at a dose of 1×10 15particles per kilogram of patient weight loaded with CRISPR / Cas antiviral gene transcription activation complexes based on dCas9-p300 with RNA guides to intracellular antiviral factors, which led to the activation of intracellular antiviral signaling with the suppression or elimination of pathogenic viruses. Example 20. 1. A preparation of hybrid nanoparticles based on a vaginal gel with a concentration of 1×10 18 particles per milliliter of gel containing nanoparticles loaded with CRISPR / Cas complexes and targeting the human papillomavirus genome. The resulting gel was used to treat human papillomavirus infection. 2. A cream containing hybrid nanoparticles loaded with tissue regeneration factors was applied to the skin. The cream was applied daily, internalizing the nanoparticles into dermal cells, producing anti-inflammatory and pro-regenerative effects.

Claims

The amended claims were received by the International Bureau on August 19, 2025.

1. A method for producing hybrid particles, comprising the following steps: a. the synthesized target RNA, DNA, or RNA with DNA is mixed with a lipid or a mixture of lipids, then incubated to form liposomes containing the target RNA, DNA, or RNA with DNA; b. the liposomes obtained in step (a) are mixed with at least one type of particles selected from the group consisting of exosome-like nanoparticles (emNV), nanoparticles (NG), and the mixture is incubated; c. the mixture obtained in step (b) after incubation is extruded by pressing through at least one extruder membrane, which has a pore diameter comparable to the required particle diameter, to obtain hybrid particles; d. the particles obtained in step (c) are purified from unpackaged components to obtain particles having sizes of 0.01-5 μm, where the particles are purified using chromatography, ultracentrifugation, filtration, precipitation or fractionation.

2. The method according to any of paragraph 1, characterized in that the obtained particles are nanoparticles.

3. The method according to any of paragraph 1, characterized in that the target RNA is synthesized chemically or by in vitro transcription.

4. The method according to any of paragraph 1, characterized in that the emNV or NG particles are loaded with a protein and / or a protein complex and / or RNA and / or DNA of interest.

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

6. The method according to claim 5, characterized in that the HEK293 cells are HEK293 cells of any modification selected from the group including HEK293, HEK293T, HEK293F, HEK293FT, HEK Expi293F.

7. The method according to claim 5, characterized in that the mesenchymal stem cells (MSCs) are MSCs of various origins, selected from the group including placental, adipose, bone marrow, MSCs from umbilical cord blood, amniotic fluid, peripheral blood, synovial fluid, dental pulp, endometrium, skin, muscle tissue, and salivary glands.

8. The method according to claim 1, characterized in that in step (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.

9. Method according to p.1, characterized in that the lipid is selected from the group consisting of dilinoleoylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE), dimyristoylglycerol phosphate (DMPA), dipalmitoylglycerol phosphate (DPPA), dioleoylglycerol phosphate DOPA, dimyristoylphosphoglycerol (DMPG), dipalmitoylphosphoglycerol (DPPG), dioleoylphosphoglycerol (DOPG), dimyristoylphosphatidylserine (DMPS), dipalmitoylphosphatidylserine (DPPS), dioleoylphosphatidylserine (DOPS), distearoylphosphatidylethanolamine (DSPE), cardiolipin, dioleoyltrimethylammonium propane (DOTAP), 2,3-dioleoyloxy-N-[2-(sperminecarboxamido)ethyl]-N, N-d-methyl-1-propanamine hydrochloride (DO SPA), cholesteryl hemisuccinate (CHEMS), ceramide phosphoethanolamine (CPE), diacylglycerol pyrophosphate (DGPP), GT-11, azidoethylsphingomyelin, sphingosine and its derivatives, C.14 -carnitine, derivatives of cholesterol, cholic and lithocholic acids, taurocholates, gangliosides, ceramides, ALC-0315, ALC-0159, 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl ethyl phosphate (DOCPe), bis(oleoyloxy)propyl)dimethylammonio)ethyl hydrogen phosphate (DOCP), phosphatidylinositol and its derivatives, cholesterol and its esters, dimethyldioctadecylammonium bromide (DDAB), N-carboxybenzyl)-N,N-dimethyl-2,3- bis(oleoyloxy)propan-1-amine (DOB AQ), 1,2-dimyristoyl-3-dimethylammonium propane (DAP), as well as their derivatives, their combinations and their conjugates.

10. The method according to claim 9, characterized in that conjugates of lipids with polyethylene glycol, polyethyleneimine and / or peptides are used as conjugates.

11. The method according to claim 1, characterized in that a combination of DOPE and DOSPA in a ratio of 1:3 is used as the lipid.

12. The method according to claim 4, characterized in that the protein loaded into the emNV or NG 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), methane cleases, human proteins, animal proteins, proteins of viruses, bacteria, archaea, bacteria, chimeric proteins.

13. The method according to any of claim 4, characterized in that the complex of the protein loaded into the emNV or into the NG is a complex of a recombinant protein, a genetically modified protein, an enzymatic complex, an antigen fragment, a protein-RNA complex, a protein-cDNA complex, a Cas protein, a Cas-like protein, a zinc-finger nuclease, a transcription activator-like effector-based nuclease (TALENs), methane cleases, human proteins, animal proteins, proteins of viruses, bacteria, archaea, bacteria, chimeric proteins.

14. The method according to claim 13, characterized in that the proteins or protein complexes loaded into emNV or NG can be linked or used together with low-molecular compounds, polymers, adjuvants, materials of organic or inorganic origin.

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

16. The method according to claim 15, characterized in that the CRISPR-Cas RNA system is a short RNA (crRNA), tracrRNA, or a single guide RNA (sgRNA).

17. The method according to claim 1, characterized in that the target DNA is selected from the group of linear or circular DNA molecules, plasmid, phasmid, bacmid DNA, DNA minicircles, synthetic oligonucleotides and antisense nucleotides, fragments of the human, animal or plant genome, artificial chromosomes, etc.

18. The method according to claim 1, characterized in that the target DNA contains modified nucleotides.

19. The method according to claim 12, characterized in that the modifications of Cas proteins are complete Cas nucleases or proteins based on catalytically inactive dead-Cas (dCas), containing inactivating mutations in the nuclease domains, or Cas nicases (nCas), containing a mutation in only one domain.

20. The method according to claim 19, characterized in that the Cas, dCas, nCas proteins are wild-type proteins, or improved or modified variants of the Cas, dCas, nCas proteins, obtained by random or directed evolution, mutagenesis with or without optimization of protein-coding codons, and optionally have additional elements in the coding construct.

21. The method according to claim 19, characterized in that the Cas, dCas or nCas proteins are Cas9, Cas12a / b, Cas13a, Cas13b, Cas13c, Cas14, Cas-X or similar proteins, or improved variants selected from Cas9-HF, eSpCas9, HypaCas9, xCas9, SpRY / SpG, Fokl-fused dCas9.

22. The method according to item 19, 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.

23. The method according to claim 19, characterized in that the Cas, dCas, 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 the method of directed evolution or mutagenesis, domains of RNA demethylases or RNA methyltransferases, systems for visualizing DNA.

24. The method according to claim 23, characterized in that the domains for activating transcription 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.

25. The method according to claim 23, characterized in that the transcription suppression domains are KRAB, EZH2, KRAB-MeCP2, DNMT3A, DNMT3A-3L, LSD1 domains, or modifications thereof, or a combination thereof.

26. The method according to claim 23, characterized in that the domains for editing DNA or RNA bases are the domains rAPOBEC1, ABOVECA, ASh, TadA, ADAR2, as well as their orthologs, their homologs and / or their modified variants obtained by mutagenesis or directed evolution.

27. The method according to claim 23, characterized in that the domains of RNA demethylases or RNA methyltransferases are the domains of METTL3, METTL14, METTL16, FTO, ALKHB5, or combinations thereof or modified variants thereof obtained by mutagenesis or directed evolution.

28. The method according to claim 23, characterized in that the systems for visualizing DNA 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.

29. The method according to claim 3, characterized in that the RNA is chemically modified at nucleoside residues, RNA nucleotides or sugar residues, including 2'-O-methyl derivatives (M), 2'-O-methyl-3'-phosphorothioate derivatives (MS), or 2'-O-methyl-3'-thiophosphonoacetate (MSP), pseudouridine ψ, N1-methylpseudouridine (m1ψ), N1-methylpseudouridine (m5C), 5-hydroxymethylcytosine (5hmC), 5-methiouridine (m5U), 2-thiouridine (s2U), aminomethylene-containing nucleotides (BNANC[N-Me]), closed nucleic acid (LNA), 2'-O-methyl-3'-phosphonoacetate (MP), ethyl group (cEt), fluoro group (F), phosphorothioate (PS), tetraethylene glycol (TEG), phosphonoacetate (PACE), or combinations thereof.

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

31. The method according to claim 1, characterized in that the packaging of RNA or RNA-protein complexes is carried out at the stage of obtaining said particles or after isolating said particles.

32. The method according to claim 1, characterized in that DNA selected from a group including linear or circular DNA molecules, plasmid, phasmid, bacmid DNA, DNA minicircles, synthetic oligonucleotides and antisense nucleotides, including those with modified nucleotides, fragments of the human, animal or plant genome, and artificial chromosomes is used for packaging DNA or co-packaging RNA with DNA.

33. The method according to claim 30, characterized in that the DNA is chemically modified at the residues of nucleosides or nucleotides of DNA, including fluorescently labeled nucleotides, biotinylated nucleotides, 2T (2-fluoro); 2'OMe (2'-O-methyl); LNA (closed nucleic acid): FANA (2'-fluoroarabinonucleic acid), HNA (hexitol nucleic acid), 2' MOE (2'-O-methyl methyl); ribuloNA ((1-3')-β-L-ribulonucleic acid); TNA (al-threose nucleic acid; tPhoNA (3'-2' phosphonomethylthreosyl nucleic acid); dXNA (2-deoxyxylonucleic acid); PS (phosphorothioate); phNA (alkylphosphonate nucleic acid); PNA (peptide nucleic acid).

34. The method according to claim 4, characterized in that the protein molecule, or a complex of protein, RNA or DNA optionally contains additional elements that 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, Moreover, the mentioned additional elements are endosomolytic proteins.

35. The method according to claim 34, characterized in that ensuring or enhancing interaction with membranes of biological particles is an enhancement of endosomal release in target cells.

36. The method according to claim 34, characterized in that the endosomolytic proteins are proteins or peptides selected from the group consisting of HA2, GALA, INF7, JTS1, ESC A, melitgin and its analogs, LL37 and its analogs, Tat, R8, EB1, KALA, SAP, H5WYG, ppTG1, LAH4.

37. The method according to claim 4, characterized in that the protein complex is a CRISPR / Cas ribonucleoprotein.

38. The method according to claim 4, characterized in that human and mammalian cells, including genetically and / or chemically modified cells, cell lines, and transit-transfected lines, are used as cells for obtaining the initial emNV and / or NG.

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

40. The method according to paragraph 39, characterized in that from 0.5 ml to more than 100 l of cell mass or suspension of cells or particles is subjected to extrusion.

41. The method according to claim 39, characterized in that the solvent influences the production of particles 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.

42. The method according to paragraph 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.

43. The method according to paragraph 42, characterized in that at the extrusion stage, ultrasonic treatment is additionally carried out.

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

45. The method according to paragraph 44, characterized in that the membrane for extrusion is standard or track-type.

46. ​​The method according to claim 44, characterized in that the membrane for extrusion has a hydrophilic or hydrophobic coating.

47. The method according to claim 44, characterized in that the membranes for extrusion have pore diameters selected from the range from 20 to 0.01 μm.

48. The method according to claim 47, characterized in that the membranes for extrusion have pore diameters selected from the group consisting of 20 μm, 14 μm, 12 μm, 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.

49. The method according to any of paragraph 1, characterized in that the extrusion is carried out using drainage discs.

50. 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.

51. The method according to claim 5, characterized in that the cells are treated with high-molecular and / or low-molecular compounds that are capable of influencing the production of nanoparticles, the expression of proteins and RNA, including proteins and RNA for packaging into nanoparticles.

52. The method according to claim 1, characterized in that the liposomes can be obtained by the method of self-assembly in solution, hydrogenation of the lipid film, the method of removing the detergent, freezing and thawing of a double emulsion, the method of introducing a solvent, the method of reverse-phase evaporation, lyophilization, spray drying, methods using supercritical fluids, microfluidic methods and other methods, using technologies for reducing the size of the obtained liposomes.

53. The method according to paragraph 52, characterized in that the technology for reducing the size of the obtained liposomes is selected from eXxcttrruuzziiiiiii, ultrasonic treatment, homogenization at high pressure.

54. A particle for genetic editing, for genome modification, for correction of genetic mutations, for modification of the epigenome, for modification of the epitranscriptome, for editing RNA, for DNA destruction, for RNA destruction, for modification of nucleotide bases, for modification of nucleotides, for deamination of DNA or RNA, for integration of DNA sequences or RNA 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 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 autoimmune diseases, for the treatment of hereditary diseases, for the treatment of metabolic diseases, for in vitro or ex vivo modification of cell lines, primary cultures or individual cells, including animal embryos, to obtain genetically modified animals, to obtain animal cell lines,for use in veterinary medicine, obtained by the method according to any of paragraphs. 1-53, and having a size of 0.01-5 µm, 55. A pharmaceutical composition for genetic editing, for genome modification, for correcting genetic mutations, for modifying the epigenome, for modifying the epitranscriptome, for editing RNA, for DNA destruction, for RNA destruction, for modifying nucleotide bases, for modifying nucleotides by 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, mixtures or combinations thereof, 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 autoimmune diseases, for the treatment of hereditary diseases, for the treatment of metabolic diseases, for in vitro or ex vivo modification of cell lines, primary cultures or individual cells, including animal embryos, to obtain genetically modified, animals, for obtaining animal cell lines for use in veterinary medicine, containing an effective amount of particles according to paragraph 54 and at least one pharmaceutically acceptable excipient.

56. Use of a particle according to claim 54 for genetic editing, for genome modification, for correction of genetic mutations, for modification of the epigenome, for modification of the epitranscriptome, for editing RNA, for DNA destruction, for RNA destruction, for modification of nucleotide bases, for modification of nucleotides by deamination of DNA or RNA, for integration of DNA or RNA sequences into the human genome or mitochondria, for DNA visualization, for studying the spatial structure of chromatin, for studying RNA metabolism, for modification of proteins, for editing proteins, for control of biological processes, for control of physiological processes, 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 of drug resistance mutations, for reconstructive medicine, for the treatment of oncological diseases, for the treatment of autoimmune diseases, for the treatment of hereditary diseases, for the treatment of metabolic diseases, for in vitro or ex vivo modification of cell lines, primary cultures or individual cells, including animal embryos, for the production of genetically modified animals, for the production of animal cell lines, for use in veterinary medicine.

57. Use according to paragraph 56, characterized in that the said particles are administered systemically into the body, by local administration, by inhalation, subcutaneously, directly into organs and tissues in a certain dosage form.

58. Use according to paragraph 56, 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.

59. Use according to paragraph 56, 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.

60. The use according to claim 56, 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, a gel ear, endocervical gel, granules, granules for preparation, drops, solutions, suspensions, granules for resorption, enteric granules,enteric-coated granules with prolonged release, film-coated granules, cut-and-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, modified-release capsules,capsules with powder for inhalation, capsules with prolonged release, concentrate for preparation, dispersion, solution, suspension, emulsion, vaginal cream, eye cream, cream for local use, cream for external use, cream for application to the oral mucosa, nasal cream, rectal cream, ear cream, vaginal liniment, liniment for local use, liniment for external use, periodontal liniment, liniment, endocervical, lyophilisate for preparation, 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, tincture for inhalation, tincture for topical use, tincture for external use, dental sticks, periodontal sticks, nasal sticks, urethral sticks, ear sticks, paste for application to the gums, paste for external use, paste for preparation of suspension for internal use, paste for internal use, paste for application to the oral mucosa, medicinal dental paste, vaginal foam, intrauterine foam, foam for external use, rectal foam, patch,Oral mucosal patch, transdermal patch, eye films, films for sticking to the gums, buccal films, orally dispersible films, periodontal films, sublingual films, dosed inhalation powder, powder for external use, powder for the preparation of gel, dispersion, drops, solution, paste, syrup, spray, suspension, powder for oral administration, nasal powder, periodontal powder, ear powder, effervescent powder, vaginal solution, intrauterine solution, solution for intra-arterial administration, solution for intraperitoneal administration, solution for intravenous administration, solution for intraocular administration, solution for intradermal administration, solution for intracoronary administration, solution for intramuscular administration, solution for intracavitary administration, solution for intravesical administration, solution for intra-articular administration, solution for gastrointestinal administration, solution for hemodialysis,solution for hemodiafiltration, solution for hemofiltration, solution for inhalation, solution for intra-amniotic administration, solution for intra-lymphatic administration, solution for infusion, solution for injection, solution for topical use, solution for cutaneous scarification application, solution for application to the gums, solution for external use, solution for periarticular administration, solution for gastric irrigation, solution for bladder irrigation, solution for parabulbar administration, solution for peritoneal dialysis, solution for subcutaneous administration, solution for rinsing, solution for oral administration, solution for prick test, solution for eye wash, solution for nasal wash, oral rinsing solution, ear canal rinsing solution, oral mucosal solution, subconjunctival solution, extra-amniotic solution, endo-sinusial solution, endotracheal solution, dental solution, rectal solution, transdermal solution, syrup, vaginal therapeutic system, intrauterine therapeutic system, topical spray, topical spray, oral mucosal spray, nasal spray, sublingual metered-dose spray, transdermal spray, ear spray, vaginal suppositories, rectal suppositories, vaginal suspension, intradermal suspension, intramuscular suspension, intra-articular suspension, gastrointestinal suspension, injection suspension, extended-release injection suspension, implantable suspension, 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, effervescent vaginal 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, mucoadhesive buccal 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 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 tablets, tablets, medicated absorbable wipes, medicated shampoo, elixir, pills.

Images