Method for actively packaging short RNA molecules into exosomes

Abstract

In particular, the invention relates to a method for actively packaging short RNA molecules into exosomes.

Description

[0001] METHOD FOR ACTIVELY PACKAGING SHORT RNA MOLECULES INTO EXOSOMES

[0002] Technical Field

[0003] In particular, the invention relates to a method for actively packaging short RNA molecules into exosomes.

[0004] State of the Art

[0005] Short RNAs are now packaged in different methods and used in in-vivo and in-vitro research, as well as in drug designs. There are FDA-approved short RNA-based drugs (Jing et al.

[0006] 2023). The most common method used industrially is the chemical synthesis of short RNA molecules. This method is fairly costly. Another method used in the research area is to synthesize the RNA backbone sequence by giving it to the cells as a gene. There is a lot of research on the subject (Kaykas ve Moon 2004, Fan et al. 2020). This method is less costly than the first method but more difficult to control. Short RNAs are transmitted to the cells both in-vivo and in-vitro by different packaging methods. If used in the chemically synthesized RNA backbone, in-vitro packaging methods include liposomal packaging, exosomal packaging, or nanoparticle packaging methods. These methods are also costly methods, and their efficiency is very low. Short RNA molecules can also be synthesized by giving them to cells in the DNA backbone. In this case, RNA molecules are given to the cells as plasmids or viruses with basic transfection methods in the DNA backbone and it is ensured that the cell produces the RNA molecule itself. Although this method is inexpensive, RNA amount control is a difficult method.

[0007] Exosomes are extracellular vesicles with a known size of around 40-150 nm secreted by each living cell (Gurunathan et al. 2019). The most important features of exosomes are that they can be taken into the cells with a very high efficiency compared to all known transfection and transduction methods (Batrakova and Kim 2015). For this reason, exosomes are tried to be used not only in research but also in industrial medicine and treatment methods and are carried out in many studies aimed at development (Salunkhe et al. 2020). Short RNAmolecules can be packaged into exosomes in basically 2 different ways. As mentioned above, we can divide them into in-vitro packaging and in-vivo packaging. In the in-vitro packaging method, after the RNAs are chemically synthesized, they are placed in exosomes isolated from any desired organism and tissue. These placement methods include electroporation and basic transfection methods (Kooijmans et al. 2013, Usman et al. 2018). In the in-vivo packaging method, RNAs are synthesized by cells and packaged into exosomes within the same cell. This method is very advantageous because it is both cheap and fast. There are published studies in which long RNA molecules are synthesized by cells with different methods (Si et al. 2023, Kojima et al. 2018, Wang et al. 2018, patent #US20150093433Al). Similar systems used for long RNAs were also used for short RNA molecules (Hung and Leonard 2016). All of these methods involve directing proteins that bind to certain RNA sequences to the exosome membrane and attaching these specific sequences as extensions to the RNA chain. The biggest problem in these methods is that the RNAs remain attached to the exosome membrane after packaging and if the isolated exosomes are given to another creature, their function decreases due to being connected to the membrane. It was also reported that the exosomes previously prepared with these methods were mostly degenerated in the recipient cells (Hung and Leonard 2016). Since RNA molecules attached to the membrane will not perform their functions due to the limitation of their mobility within the cell, their functions decrease. In other words, during the in-vivo packaging of the RNAs to the exosomes, their retention with RNA-binding proteins to the exosome membrane affects the RNA function in the cells, which is the next target of the exosomes. When the RNAs remaining attached to the exosome membrane are taken to the targeted cell, they are more likely to be targeted and transformed through endosomal pathways. It was previously reported that the RNA exosomes prepared with these methods had very little function in the targeted cells (Hung and Leonard 2019). Current methods include keeping RNAs in the exosome membrane and releasing them with random unbound reactions that may occur within the cell and thus ensuring their functions. Short RNAs can also be packaged with similar methods, but since the integrity and length of their sequences are much more important than long RNAs, it is fairly mysterious whether they work when taken into the cell. For much shorter RNA molecules such as miRNA and siRNA, in-vitro methods, which are methods of post-loading to exosomes, are more often preferred instead of these methods (Zou et al. 2019,0 ’Brien et al.

[0008] 2020). As mentioned earlier, these methods are very costly and challenging.In summary, even in the most commonly used methods of RNA packaging to exosomes, the RNAs synthesized by the cells remain bound to the exosome membrane with proteins. This situation affects the function of RNA after it is taken into the next cell with the help of an exosome (Hung and Leonard 2016, Si et al. 2023, Kojima et al. 2018). This problem causes the RNAs attached to the exosome membrane to be recycled within the cell before they can perform their functions. Current solutions are based on the possibility of random connection within the cell, ignoring the connection of RNA to the membrane. Although this allows the RNAs synthesized by the cells to be packaged into exosomes, it takes control of its function in the next cell.

[0009] Due to these disadvantageous situations, new methods need to be developed especially for packaging short RNA molecules into exosomes.

[0010] References:

[0011] Alvarez- Erviti, L. et al. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat. BiotechnoL https: / / doi.org / 10.1038 / nbt.18Q7 (2011).

[0012] Fan J, Feng Y, Zhang R, Zhang W, Shu Y, Zeng Z, Huang S, Zhang L, Huang B, Wu D, Zhang B, Wang X, Lei Y, Ye Z, Zhao L, Cao D, Yang L, Chen X, Liu B, Wagstaff W, He F, Wu X, Zhang J, Moriatis Wolf J, Lee MJ, Haydon RC, Luu HH, Huang A, He TC, Yan S. A simplified system for the effective expression and delivery of functional mature microRNAs in mammalian cells. Cancer Gene Ther. 2020 Jun;27(6):424-437. doi: 10.1038 / s41417-019-0113-y. Epub 2019 Jun 20. PMID: 31222181; PMCID: PMC6923634.

[0013] Gurunathan S, Kang M-H, Jeyaraj M, Qasim M, Kim J-H. Review of the isolation, characterization, biological function, and multifarious therapeutic approaches of exosomes. Cells 2019;8(4):307.

[0014] 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. PMID: 27189348; PMCID: PMC4870355.

[0015] Jing X, Arya V, Reynolds KS, Rogers H. Clinical Pharmacology of RNA Interference-Based Therapeutics: A Summary Based on Food and Drug Administration-Approved Small Interfering RNAs. Drug Metab Dispos. 2023 Feb;51(2): 193-198. doi: 10.1124 / dmd.122.001107. Epub 2022 Nov 4. PMID: 36332914; PMCID: PMC9900864.Kamerkar, S. et al. Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer. Nature https: / / doi.org / 10.1038 / nature22341 (2017).

[0016] Kaykas A, Moon RT. A plasmid-based system for expressing small interfering RNA libraries in mammalian cells. BMC Cell Biol. 2004 Apr 30;5:16. doi: 10.1186 / 1471-2121-5-16. PMID: 15119963; PMCID: PMC416474.

[0017] Kojima, R. et al. Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson’s disease treatment. Nat. Commun. https: / / doi.org / 10.1038 / s41467-018- 03733-8 (2018).

[0018] Kooijmans, S. A. A. et al. Electroporation- induced siRNA precipitation obscures the efficiency of siRNA loading into extracellular vesicles. J. Control. Rel. https: / / doi.Org / 10.1016 / j.jconrel.2013.08.014 (2013).

[0019] Lamichhane, T. N. et al. Oncogene knockdown via active loading of small RNAs into extracellular vesicles by sonication. Cell. Mol. Bioeng. https: / / doi.org / 10.1007 / sl2195-016-0457-4 (2016).

[0020] O'Brien K, Breyne K, Ughetto S, Laurent LC, Breakefield XO. RNA delivery by extracellular vesicles in mammalian cells and its applications. Nat Rev Mol Cell Biol. 2020 Oct;21(10):585-606. doi: 10.1038 / s41580-020-0251-y. Epub 2020 May 26. PMID: 32457507; PMCID: PMC7249041.

[0021] Pomatto, M. A. C. et al. Improved loading of plasmaderived extracellular vesicles to encapsulate antitumor miRNAs. Mol. Ther. Methods Clin. Dev. https: / / doi.org / 10.1016 / j.omtm.2019.01.001 (2019).

[0022] Shtam, T. A. et al. Exosomes are natural carriers of exogenous siRNA to human cells in vitro. Cell Commun. Signal. (2013). 10.1186 / 1478-811X-11-88

[0023] Si K, Dai Z, Li Z, Ye Z, Ding B, Feng S, Sun B, Shen Y, Xiao Z. Engineered exosome-mediated messenger RNA and single-chain variable fragment delivery for human chimeric antigen receptor T-cell engineering. Cytotherapy. 2023 Jun;25(6):615-624. doi: 10.1016 / j .jcyt.2023.01.005. Epub 2023 Feb 23. PMID: 36828738.

[0024] Usman, W. M. et al. Efficient RNA drug delivery using red blood cell extracellular vesicles. Nat. Commun. htps: / / doi.org / 10.1038 / s41467-018-Q4791-8 (2018).

[0025] Wang, Q. et al. ARMMs as a versatile platform for intracellular delivery of macromolecules. Nat. Commun. htps: / / doi.org / 10.1038 / s41467-018- 03390- x (2018).

[0026] Zou X, Yuan M, Zhang T, Wei H, Xu S, Jiang N, Zheng N, Wu Z. Extracellular vesicles expressing a single-chain variable fragment of an HIV-1 specific antibody selectively targetEnv+tissues. Theranostics. 2019 Jul 29;9(19):5657-5671. doi: 10.7150 / thno.33925. PMID: 31534509; PMCID: PMC6735399.

[0027] Brief Description of the Invention

[0028] In the invention, it is aimed to develop a new method for the active packaging of short RNA molecules into exosomes. For this purpose, it is ensured that short RNA molecules can be packaged into exosomes with fast and low cost.

[0029] The invention allows easy packaging of short RNAs with the help of secondary cells.

[0030] The invention presented herein includes the method of packaging the exosome in the same cell by having the cells synthesize RNA. The invention solves the problem of RNA remaining attached to the exosome membrane and allows the mature short RNAs (miRNA siRNA, etc.) that are not in the hairpin position to be functionally packaged into exosomes. In this way, short RNA molecules will be packaged into exosomes much easier, faster and less costly.

[0031] With the invention, the problem of adherence of RNA molecules to the cells was solved by allowing the RNAs synthesized by the cells to break away from the exosome membrane only with the help of heat exchange after they were packaged into the exosomes.

[0032] The invention is based on the use of thermally sensitive self-cleaving ribozymes. Ribozymes are functional short RNA molecules. Thermally working ribozyme molecules were previously reported (Saragliadis et al. 2013). These ribozymes were mostly discovered in living creatures living at different temperatures. There are ribozymes that are naturally found in the cells, which have been shown to work thermally, and even artificial ribozymes that people create by combining different RNA parts. These ribozymes are ribozymes that naturally play a role in translation and transcription in different functions within the cell. Artificially created ones were also previously used in in-vitro translation imaging. Apart from this, there are no areas of use in the production of any product.

[0033] On the other hand, in addition to the frequently used RNA labeling and RNA binding proteins, thermally sensitive ribozyme is added to the desired RNA, and the labeled RNA molecule is synthesized at 37 °C and carried to the exosome with the help of the sign. ThisRNA molecule carried to the exosome leaves the membrane by cooling the isolated exosomes to 20°C and becomes soluble in the exosome. In this way, the exosome containing the RNA molecule, which is much more functional, can be produced quickly and in one step in cell culture.

[0034] The targeted packaging of RNAs by cells is a method known and frequently used by different groups. This method is frequently used especially in long RNA molecules such as mRNA. By integrating thermally sensitive ribozymes into this method, short RNA molecules can be produced quickly and cheaply in mature forms in secondary cells. In other words, these thermally sensitive ribozymes are used in exosome packaging in the invention. Interestingly, in the invention, it was observed that cell activities increased with the use of such ribozymes in RNA exosome packaging. The same method can be used in long RNAs. In this case, it is predicted that the activities of longer RNAs in the next cell may increase.

[0035] References:

[0036] Athanasios Saragliadis, Stefanie S. Krajewski, Charlotte Rehm, Franz Narberhaus & Jorg S. Hartig (2013) Thermozymes, RNA Biology, 10:6, 1009-1016, DOI: 10.4161 / ma.24482 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. PMID: 27189348; PMCID: PMC4870355.

[0037] The original element of the invention is the use of "thermally sensitive self-cleaving ribozymes" during the packaging of RNAs into exosomes. In this way, it eliminates the step of first isolating the exosomes, which is the most common exosome packaging method of mature short RNAs, and then loading them with RNA. In this way, both extra steps will be reduced and toxic situations caused by this method will be eliminated. The exosomes produced by the cells will be designed to contain the desired RNA molecule and the isolated exosomes will be directly containing the RNA molecule. This will increase the likelihood that exosomes will be used without recycling back RNAs in the cells where they are given. The method, which is the subject of the invention, can be used in all RNA packaging, as well as offering great innovation, especially in the packaging of mature short RNAs.The invention is based on thermally sensitive self-cleaving ribozymes offered for different purposes. These ribozymes can mostly be discovered in living creatures living at different temperatures or can be obtained by combining RNA fragments taken from living creatures living at different temperatures in known ribozymes (Waldminghaus et al. 2007, Saragliadis et al. 2013). Although there are many areas of use, it has never been used in exosome packaging before. An example of one of these ribozymes is the article published by Saragliadis et al. (2013). In the article, ribozymes are used for methods that have nothing to do with exosome packaging.

[0038] The method used for exosome packaging is based on the frequently used RNA labeling and RNA-binding protein relationship. There are many examples for this section (Hung and Leonard 2016, Si et al. 2023, Kojima et al. 2018, Wang et al. 2018, patent #US20150093433Al). However, the invention is not based on the originality of this part.

[0039] In summary, there are no packaging methods using the method of the invention for short RNA molecules. Although the method, which is the subject of the invention, can be used for long RNA molecules, it is very effective, especially in the packaging of short RNAs.

[0040] References:

[0041] Athanasios Saragliadis, Stefanie S. Krajewski, Charlotte Rehm, Franz Narberhaus & Jorg S. Hartig (2013) Thermozymes, RNA Biology, 10:6, 1009-1016, DOI: 10.4161 / ma.24482 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. PMID: 27189348; PMCID: PMC4870355.

[0042] Kojima, R. et al. Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson’s disease treatment. Nat. Commun. https: / / doi.org / 10.1038 / s41467-018- 03733-8 (2018).

[0043] Si K, Dai Z, Li Z, Ye Z, Ding B, Feng S, Sun B, Shen Y, Xiao Z. Engineered exosome-mediated messenger RNA and single-chain variable fragment delivery for human chimeric antigen receptor T-cell engineering. Cytotherapy. 2023 Jun;25(6):615-624. doi: 10.1016 / j .jcyt.2023.01.005. Epub 2023 Feb 23. PMID: 36828738.

[0044] Wang, Q. et al. ARMMs as a versatile platform for intracellular delivery of macromolecules.

[0045] Nat. Commun. https: / / doi.org / 10.1038 / s41467-018- 03390- x (2018).Waldminghaus T, Heidrich N, Brand S, Narberhaus F. FourU: a novel type of RNA thermometer in Salmonella. Mol Microbiol. 2007 Jul;65(2):413-24. doi: 10.1111 / j.1365-2958.2007.05794.x. PMID: 17630972.

[0046] Descriptions of the Figures

[0047] Figure 1. A flow diagram of the inventive method.

[0048] Figure 2. A theoretical representative view of the inventive method.

[0049] Description of References in Figures

[0050] The corresponding numbers in the figures are given below in order to better understand the invention:

[0051] 100. Method

[0052] 101. Cloning of at least one RNA molecule sequence containing at least one marker RNA sequence and at least one thermal sensitive ribozyme molecule at the 3' or 5' end according to the ribozyme type

[0053] 102. Cloning of plasmid containing at least one other protein capable of binding RNA adjacent to at least one exosome membrane protein, labelled or unlabelled with any at least one fluorescent protein

[0054] 103. Expression of at least one first plasmid and at least one second plasmid containing at least one other protein capable of binding RNA adjacent to at least one exosome membrane protein labeled or unlabeled with any at least one fluorescent protein and allowing the transport of the RNA molecule to the exosome membrane and at least one second plasmid containing the desired short RNA molecule to at least one secondary or primary cell lines

[0055] 104. Collecting exosomes from the medium of cells expressing at least one first plasmid and at least one second plasmid at the same time and preferably cooling them to 25°C-15°C, more preferably 20°C, and incubating said exosomes at least 1 hour, preferably 1 to 2 hours, in any thermal block or any incubator, preferably at 25°C-15°C, more preferably 20°C, and obtaining exosomes functionally enriched in the desired RNA

[0056] 105. Exosome isolation from cell mediumDetailed Description of the Invention

[0057] The invention is based on the separation of short RNA molecules carried to the exosomes with the help of ribozymes from the exosome membrane.

[0058] The ribozyme molecule called hammerhead, which is the most suitable of the thermally sensitive ribozyme molecules, was used in the invention. If this molecule is used, it is mandatory to place the marker and thermal sensitive ribozyme molecule at the 5' end of the RNA sequence. Otherwise, the active molecule cannot be obtained. However, there are also ribozymes such as HDV. However, thermal ones of these ribozymes are not yet known, but they are also open to discovery. If there is a thermal enzyme such as HDV derivative, it is also possible to add it to the 3' end. Although there are different ribozyme molecules, according to the ribozyme type, some can be cleaved at the 5' end (or added to the 5' end), and some can be cleaved at the 3' end (or added to the 3' end). In the event that the method (100) given below is cleaved at the 5' end in step 101, the important thing is to cleave the complete structure containing the functional RNA molecule. This is very important only for short RNAs. However, this does not matter in the long RNA part.

[0059] The invention is a method (100) for the active packaging of short RNA molecules into exosomes, comprising the following steps:

[0060] Cloning of at least one RNA molecule sequence containing at least one marker RNA sequence and at least one thermal sensitive ribozyme molecule at the 3' or 5' end according to the ribozyme type (101)

[0061] Cloning of plasmid containing at least one other protein capable of binding RNA adjacent to at least one exosome membrane protein, labelled or unlabelled with any at least one fluorescent protein (102)

[0062] - Expression of at least one first plasmid and at least one second plasmid containing at least one other protein capable of binding RNA adjacent to at least one exosome membrane protein labeled or unlabeled with any at least one fluorescent protein and allowing the transport of the RNA molecule to the exosome membrane and at least one second plasmid containing the desired short RNA molecule to at least one secondary or primary cell lines (103)Collecting exosomes from the medium of cells expressing at least one first plasmid and at least one second plasmid at the same time and preferably cooling them to 25°C- 15°C, more preferably 20°C, and incubating said exosomes at least 1 hour, preferably 1 to 2 hours, in any thermal block or any incubator, preferably at 25°C-15°C, more preferably 20°C, and obtaining exosomes functionally enriched in the desired RNA (104)

[0063] Exosome isolation from cell medium (105)

[0064] The RNA molecule cloned in step 101 is plasmid containing the desired short RNA molecule. Although the exosome membrane proteins mentioned in step 102 may be proteins specific to the exosome membrane such as CD63, CD9, Lamp2b, they may also be soluble proteins known to be specific in exosomes. CD9 is preferably used as the exosome membrane protein in the invention.

[0065] The RNA-binding proteins mentioned in step 102 include all proteins that have been shown to be able to bind by recognizing specific RNA sequences in the literature. Viral MCP (MS2 coat protein) PCP (PP7 coat protein) proteins can be given as examples of the RNA-binding proteins mentioned in step 102. Preferably, the RNA labeling extension PP7 stem-loop sequence and the PCP protein specifically bound to it are used in the invention.

[0066] All fluorescent proteins or peptides presented in the literature can be used as the fluorescent labeling protein mentioned in step 102. Preferably, GFP is used for fluorescent labeling in the invention.

[0067] Any RNA labeling molecule presented in the literature can be used as the RNA labeling sequence mentioned in step 101. Viral-derived step-loop structures such as PP7, MS2, Qb can be given as examples, and RNA sequences found in all RNA labeling methods presented in the literature can be used. The use of these sequences with more than one repetition will also increase the likelihood of the short RNA molecule moving to the exosome. In addition, the RNA-binding protein used in step 102 and the RNA used in RNA labeling should be able to interact with each other.

[0068] All of the hammerhead ribozyme sequences found to work in living things that can live at temperatures greater than 37°C or all of the sequences created by the addition of hairpinsequences that enable these ribozyme sequences to be low-functioning can be used as the sequence of the RNA molecule containing the ribozyme molecule in step 101. Other ribozyme molecules that have been or will be shown to function by folding correctly at temperatures greater than 37°C can also be used as an alternative to hammerhead ribozymes. The point to be considered here is that the nucleotide cleaved by the ribozyme molecule does not disrupt the structure of the mature RNA molecule.

[0069] Any transfection method in the literature can be used to create cells expressing the protein structure that carries the RNAs mentioned in step 103 to the exosomes.

[0070] The structure formed in step 101 and containing 3 different RNA molecules can be synthesized behind RNA promoter regions such as the U6 promoter region, or it can be expressed to coincide with the 3' UTR end of a fluorescent sign protein behind protein promoter regions, that is, plasmid is given to the cell in the DNA structure, the cell takes DNA, transcribes it first and then translates it.

[0071] The temperature is 20°C for the correct folding process mentioned in step 104, but it may also be at lower temperatures. A rapid drop in temperature can also prevent ribozyme from being cleaved, as it can prevent it from folding correctly. 20°C is the most optimum temperature value.

[0072] In order to be sure of separation, the time mentioned in step 104 is maximum 2 hours. RNAs can also start to break down if said time is as long as 5-6 hours. Therefore, 1 to 2 hours is an optimum time.

[0073] The theoretical description of the method is given in Figure 2. Accordingly, Figure-2 A shows the situation of the structure in the exosome membrane at 37°C and 20°C in detail. CD9, an exosome membrane protein, is given as an example, and GFP is given for fluorescent labeling. The RNA labeling extension PP7 stem-loop sequence and the PCP protein specifically bound to it are given as representative. Although miRNA packaging is shown as a representative, the method can be used for all RNA molecules. In Figure-1 B, it includes the general description of the active RNA molecules separated from the membrane when they are incubated at 20°C after exosomal packaging and isolated exosomes. As mentioned in the text, thermal sensitive ribozymes (shown in red as HHR), which cannot be folded correctly at37°C, will function by folding correctly when taken to 20°C and will release the miRNA molecule from the membrane by cleaving itself. When the temperature is lowered, the RNA molecule is released from the membrane.

[0074] Step 101 is the most important and necessary step of the invention. The thermal sensitive ribozyme structure in the vector to be cloned in this step is the most specific step.

[0075] Any method in the literature can be used for step 102. Only the RNA-binding protein and the RNA used in RNA labeling should be able to interact with each other.

[0076] After step 105, the exosomes are ready to use.

[0077] Industrial Applicability

[0078] The invention relates to a method for the active packaging of long or short RNA molecules into exosomes, and is applicable to the industry.

[0079] The invention has been created for use in designed exosomal packaging research, especially in the packaging of RNAs. If it is developed outside the areas of use at the research level, it is possible to be used in the medicine and treatment sector.

[0080] The content of the RNA-containing exosomes produced by the cells can be determined in detail, and the exosomes that can be used both in R&D and clinically can be produced. The point to be considered here is to determine the products that will not harm the next living thing in the content of the exosomal structures. If it is used in humans, it can be synthesized by human-derived mesenchymal cells, or if it is applied to animals, it can be synthesized by animal-derived cells, and the activity of the exosomes can be increased and their toxicity can be reduced. Since the same method can be used in plant and yeast cells, it can also allow the development of herbal exosomated products on the market.

[0081] The invention is not limited to the above explanations; however, a person skilled in the art can easily present different embodiments of the invention. They must be assessed within the scope of the protection claimed by the claims of the invention.

Claims

CLAIMS1. A method (100) for the active packaging of short RNA molecules into exosomes synthesized in living cells, characterized in that it comprises the following steps:Cloning of at least one RNA molecule sequence containing at least one marker RNA sequence and at least one thermal sensitive ribozyme molecule at the 3' or 5' end, depending on the ribozyme type (101)Cloning of plasmid containing at least one other protein capable of binding RNA adjacent to at least one exosome membrane protein, which may or may not be labeled with any at least one fluorescent protein (102)- Expression of at least one first plasmid and at least one second plasmid onto at least one secondary or primary cell line containing at least one first plasmid that enables the transport of the RNA molecule to the exosome membrane and at least one second plasmid containing the desired short RNA molecule, which contains at least one additional protein capable of binding to RNA adjacent to at least one exosome membrane protein labeled or unlabeled with at least one fluorescent protein (103) Collecting exosomes from the medium of cells expressing at least one first plasmid and at least one second plasmid at the same time and preferably cooling them to 25°C- 15°C, more preferably 20°C, and incubating said exosomes at least 1 hour, preferably 1 to 2 hours, in any thermal block or any incubator, preferably at 25°C-15°C, more preferably 20°C, and obtaining exosomes functionally enriched in the desired RNA (104)Isolation of exosomes from the cell culture medium (105)2. A method (100) according to claim 1, characterized in that the exosome membrane proteins mentioned in step 102 are proteins specifically located on the exosome membrane, such as CD63, CD9, and Lamp2b.

3. A method (100) according to claim 2, characterized in that the RNA-binding proteins mentioned in step 102 are viral MCP and PCP proteins that have been shown to be able to bind by recognizing specific RNA sequences in the literature.A method (100) according to claim 3, characterized in that it uses GFP for the fluorescent marking mentioned in step 102.

5. A method (100) according to claim 4, characterized in that all of the hammerhead ribozyme sequences found to work in living things that can live at temperatures greater than 37°C or the sequences created by the addition of hairpin sequences that enable these ribozyme sequences to be low-functioning are the sequences of the RNA molecule containing the ribozyme molecule in step 101.

6. A method (100) according to claim 5, characterized in that the RNA-binding protein and the RNA used in RNA labeling have the ability to interact with each other for the step of forming cells expressing the protein structure that carries the RNAs mentioned in step 103 to the exosomes.

7. A method (100) according to claim 6, characterized in that the structure formed in step 101 and containing 3 different RNA molecules can be synthesized behind RNA promoter regions such as the U6 promoter region, or a fluorescent sign protein can be expressed behind protein promoter regions to coincide with the 3' UTR end.

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