Exosome extracellular vesicles and methods for using them

Abstract

Exosomes comprising modified RNA are disclosed. Aspects of the present disclosure further relate to methods and compositions using exosomes comprising modified RNA. In certain aspects, the exosomes disclosed herein are useful for delivering modified RNA to cells. In certain aspects, the exosomes disclosed herein may be useful for treating or preventing disorders in a subject. Methods and compositions for producing the disclosed exosomes are also provided. [Selection diagram] None

Description

[Technical Field]

[0001] This disclosure relates to exosomes containing modified RNA. This disclosure also relates to methods and compositions for using the exosomes disclosed herein. In certain embodiments, the exosomes disclosed herein may be useful for delivering modified RNA to cells. In certain embodiments, the exosomes disclosed herein may be useful for treating or preventing disorders in subjects. Methods and compositions for generating the disclosed exosomes are also provided. [Background technology]

[0002] RNA is a promising therapeutic molecule for treating a variety of diseases, including cancer, infectious diseases, and hereditary genetic disorders (Non-Patent Literature 1; Non-Patent Literature 2). RNA therapy generally involves suppressing pathogenic genes using RNA interference (RNAi, e.g., siRNA) or expressing therapeutic proteins by delivering mRNA to cells. To obtain regulatory approval, the vehicle used for RNA delivery must be safe (i.e., non-immunogenic and non-toxic) and capable of efficiently delivering therapeutic RNA into the cytoplasm of recipient cells.

[0003] Known drug carriers, such as lipid nanoparticles (LNPs), facilitate the targeting and site-specific delivery of drugs to tissues and cells, thereby increasing their bioavailability. LNPs containing ionized lipids represent one platform for RNA delivery (Non-Patent Literature 3; Non-Patent Literature 4; Non-Patent Literature 5). However, an obstacle to developing LNPs and other such carriers is the potential immunogenicity response associated with the components of their formulations.

[0004] Despite efforts to minimize LNP-related immunogenic responses, such as adding polyethylene glycol shields to avoid recognition by mononuclear phagocytic cells, LNPs can still induce immune responses (Non-Patent Literature 6). For example, certain LNP formulations have been shown to be partially toxic to recipient cells and to provoke undesirable immune responses in the host (Non-Patent Literature 7; Non-Patent Literature 8).

[0005] The immunostimulatory effects of LNPs continue to hinder their use for the safe and effective delivery of therapeutic RNA. Therefore, there is a need for the development of alternative vehicles that can effectively deliver RNA to target tissues and cells without triggering an immune response.

[0006] Extracellular vesicles (EVs) are heterogeneous nano- and micro-sized vesicles secreted by almost all cell types (Non-Patent Document 9; Non-Patent Document 10; Non-Patent Document 11). EVs can be detected in most biological fluids and in the supernatants of cultured cells (Non-Patent Document 12; Non-Patent Document 13; Non-Patent Document 14). During EV development, EVs can acquire diverse cytoplasmic components, such as lipids and proteins, as well as coding and non-coding RNAs (Non-Patent Document 15; Non-Patent Document 16; Non-Patent Document 17). Cells can also transmit these messages to each other by packaging RNA messages within EVs (Non-Patent Document 18). Thus, EVs can act as endogenous carriers for intercellular nucleic acid transport. The most extensively described EVs are exosomes, which originate from endosomes and are secreted via the exocytosis pathway.

[0007] Exosomes are nanoscale extracellular molecules (EVs) secreted by virtually all types of cells and stably present in virtually all types of bodily fluids (Non-Patent Literature 19). Exosomes can transmit a variety of signaling molecules, including nucleic acids (e.g., mRNA and microRNA), functional proteins, and lipids (Non-Patent Literature 20; Non-Patent Literature 21; Non-Patent Literature 22). Due to their small size, exosomes can escape rapid phagocytosis by mononuclear phagocytic cells, enabling stable transport and delivery of drugs in circulation and targeting cells by crossing vascular endothelium (Non-Patent Literature 23). In addition, exosomes can cross tough biological barriers such as the blood-brain barrier and the placental barrier (Non-Patent Literature 24; Non-Patent Literature 25; Non-Patent Literature 26). All of these features make exosomes a promising carrier for RNA delivery.

[0008] Several exosome-based delivery systems have been described for therapeutic applications, including the exosome-liposome hybrid nanoparticles reported in Non-Patent Document 27. However, none of these systems have been developed to address the potential toxic effects of specific RNA delivery agents. Therefore, there is still a need for an effective RNA delivery system that minimizes side effects and avoids excessive toxicity in cells and tissues. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Pardi et al. (2018) Nat Rev Drug Discov.17(4):261-79 [Non-Patent Document 2] Dunbar et al.(2018)Science 359(6372):eaan4672 [Non-Patent Document 3] Semple et al. (2010) Nat Biotechnol. 28(2):172-6 [Non-Patent Document 4] Patel et al.(2017)Nano Lett.17(9):5711-8

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Non-licensed literature 9

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[0010] In various embodiments, this disclosure provides, in part, exosomes (EVs) for the delivery of therapeutic RNA. In various embodiments, the exosomes described herein can deliver extragenic RNA, including high molecular weight mRNA, to target cells and tissues. In various embodiments, the exosomes described herein can protect the extragenic RNA during in vitro or in vivo transport and deliver the RNA to the cytoplasm of recipient cells. In various embodiments, the delivered RNA is functional and can be translated to produce therapeutic proteins. In various embodiments, the exosomes described herein may be non-immunogenic or may induce a lower immune and / or inflammatory response compared to the immune and / or inflammatory response observed with alternative delivery vehicles (e.g., LNPs). In various embodiments, the exosomes described herein have an ionized lipid:modified RNA nucleotide molar ratio of about 1:1 or less than about 1:1, for example, about 0.1:1, about 0.2:1, about 0.3:1, about 0.4:1, about 0.5:1, about 0.6:1, about 0.7:1, about 0.8:1, or about 0.9:1. In various embodiments, exosomes having an ionized lipid:modified RNA nucleotide molar ratio of about 1:1 or less than about 1:1 may be non-toxic or less toxic in host cells and tissues compared to alternative delivery vehicles having an ionized lipid:modified RNA nucleotide molar ratio greater than about 1:1 (e.g., LNP). In various embodiments, the exosomes and alternative delivery vehicles contain the same ionized lipid (e.g., DLin-MC3-DMA or DLin-DMA) and the same modified RNA, but have different ionized lipid:modified RNA nucleotide molar ratios.

[0011] In various embodiments, the Disclosure provides isolated exosomes containing modified RNA, prepared by a process comprising: (a) providing one or more lipid nanoparticles (LNPs) containing modified RNA; (b) contacting one or more cells with the LNPs under conditions that allow for LNP uptake by cells; and (c) isolating exosomes produced by one or more cells, wherein at least one isolated exosome contains modified RNA.

[0012] In some embodiments, the LNP comprises at least one ionized lipid, phospholipid, structural lipid, and / or PEG lipid.

[0013] In some embodiments, at least one ionized lipid contained in the LNP is DLin-MC3-DMA and / or DLin-DMA. In some embodiments, at least one ionized lipid contained in the LNP is DLin-MC3-DMA. In some embodiments, at least one ionized lipid contained in the LNP is DLin-DMA. In some embodiments, at least one phospholipid contained in the LNP is DSPC. In some embodiments, at least one structural lipid contained in the LNP is cholesterol. In some embodiments, at least one PEG lipid contained in the LNP is PEG-DMPE. In some embodiments, the molecular weight of PEG may be about 2,000 Da (PEG2000). In some embodiments, at least one PEG lipid contained in the LNP is PEG2000-DMPE.

[0014] In some embodiments, LNP contains an ionized lipid that is DLin-MC3-DMA or DLin-DMA, a phospholipid that is DSPC, a structural lipid that is cholesterol, and / or a PEG lipid that is PEG-DMPE or PEG2000-DMPE. In some embodiments, LNP contains an ionized lipid that is DLin-MC3-DMA, a phospholipid that is DSPC, a structural lipid that is cholesterol, and a PEG lipid that is PEG-DMPE or PEG2000-DMPE. In some embodiments, LNP contains an ionized lipid that is DLin-DMA, a phospholipid that is DSPC, a structural lipid that is cholesterol, and a PEG lipid that is PEG-DMPE or PEG2000-DMPE.

[0015] In some embodiments, the LNP has an ionized lipid:modified RNA nucleotide molar ratio of about 2:1 to about 4:1, or about 4:1, about 3:1, or about 2:1. In some embodiments, the LNP has an ionized lipid:modified RNA nucleotide molar ratio of about 3:1.

[0016] In some embodiments, the exosomes produced by contacting one or more cells with LNPs contain at least one ionized lipid, phospholipid, structural lipid, and / or PEG lipid. In some embodiments, the at least one ionized lipid, phospholipid, structural lipid, and / or PEG lipid contained in the exosomes are derived from the LNPs.

[0017] In some embodiments, at least one ionized lipid contained in the exosome is DLin-MC3-DMA and / or DLin-DMA. In some embodiments, at least one ionized lipid contained in the exosome is DLin-MC3-DMA. In some embodiments, at least one ionized lipid contained in the exosome is DLin-DMA. In some embodiments, at least one phospholipid contained in the exosome is DSPC. In some embodiments, at least one structural lipid contained in the exosome is cholesterol.

[0018] In some embodiments, the exosome contains an ionized lipid that is DLin-MC3-DMA or DLin-DMA, a phospholipid that is DSPC, and / or a structural lipid that is cholesterol. In some embodiments, the exosome contains an ionized lipid that is DLin-MC3-DMA, a phospholipid that is DSPC, and a structural lipid that is cholesterol. In some other embodiments, the exosome contains an ionized lipid that is DLin-DMA, a phospholipid that is DSPC, and a structural lipid that is cholesterol.

[0019] In some embodiments, the exosome contains an ionized lipid:modified RNA nucleotide molar ratio of about 1:1 to about 3:1, or about 3:1, about 2:1, about 1:1, or less than about 1:1. In some embodiments, the exosome contains an ionized lipid:modified RNA nucleotide molar ratio of about 1:1 or less than about 1:1, for example, about 0.1:1, about 0.2:1, about 0.3:1, about 0.4:1, about 0.5:1, about 0.6:1, about 0.7:1, about 0.8:1, or about 0.9:1. In some embodiments, the exosome contains an ionized lipid:modified RNA nucleotide molar ratio lower than the ionized lipid:modified RNA molar ratio contained in the LNP. In some embodiments, the exosome contains an ionized lipid:modified RNA nucleotide molar ratio of about 1:1 or less than about 1:1, and the LNP contains an ionized lipid:modified RNA nucleotide molar ratio of about 3:1.

[0020] In some embodiments, the exosomes have a diameter of approximately 30 nm to approximately 300 nm. In some embodiments, the exosomes have a diameter of approximately 40 nm to approximately 150 nm. In some embodiments, the exosomes have a diameter of approximately 40 nm to approximately 120 nm.

[0021] In some embodiments, cells to be contacted with LNPs to produce exosomes are obtained from a subject. In some embodiments, the cells are epithelial cells, immune cells, progenitor cells, or stem cells. In some embodiments, the cells are B lymphocytes, T lymphocytes, or monocytes.

[0022] In some embodiments, the modified RNA encodes the polypeptide of interest. In some embodiments, the modified RNA encodes a polypeptide effective in treating a disease. In some embodiments, the modified RNA encodes an erythropoietin polypeptide. In some embodiments, the modified RNA encodes a human erythropoietin polypeptide. In some embodiments, the modified RNA encodes the erythropoietin polypeptide of SEQ ID NO: 1.

[0023] In some embodiments, exosomes are prepared by a process performed in vitro. In some embodiments, the step of contacting one or more cells with LNPs is performed in the presence of human serum. In some embodiments, the human serum is present in an amount of about 0.5 vol%, about 1 vol%, or about 1.5 vol%. In some embodiments, the human serum is present in an amount of about 1 vol%. In some embodiments, the step of contacting one or more cells with LNPs includes contacting one or more cells with at least two, at least three, or at least four different doses of LNPs. In some embodiments, the step of contacting one or more cells with LNPs includes contacting one or more cells with at least three different doses of LNPs. In some embodiments, the step of isolating exosomes includes isolating exosomes from a sample of in vitro cell culture medium.

[0024] In various embodiments, the Disclosure also provides a method for delivering modified RNA to cells by contacting cells with an effective amount of exosomes or a pharmaceutical composition containing at least one exosome (e.g., either of the exosomes or pharmaceutical compositions described herein), thereby delivering the modified RNA to the cells. In some embodiments, the step of contacting cells includes adding the exosomes or pharmaceutical composition to an in vitro cell culture or administering the exosomes or pharmaceutical composition to a subject.

[0025] Furthermore, in various embodiments, the use of a pharmaceutical composition (for example, either the exosome or pharmaceutical composition described herein) containing an effective amount of exosomes or at least one exosome for the delivery of modified RNA to cells is also provided herein.

[0026] In some embodiments of the methods and uses described herein, the cells are present in an in vitro cell culture medium. In some embodiments, the cells are obtained from a subject. In some embodiments, the cells are present in a subject. In some embodiments, the cells are epithelial cells, immune cells, progenitor cells, or stem cells. In some embodiments, the cells are B lymphocytes, T lymphocytes, or monocytes.

[0027] In some embodiments of the methods and uses described herein, the delivery of modified RNA to cells does not alter the cell generation time. In some embodiments, the delivery of modified RNA to cells does not alter the total cellular protein content by weight.

[0028] Furthermore, in various embodiments, pharmaceutical compositions comprising at least one exosome (e.g., any of the exosomes described herein) and a pharmaceutically acceptable carrier are provided herein.

[0029] Furthermore, in various embodiments, therapeutic methods and uses of the exosomes and pharmaceutical compositions disclosed herein, for example, in the treatment of disorders, are also provided herein.

[0030] For example, in certain embodiments, the Disclosure provides a method for treating or preventing a disorder in a subject by administering to the subject an effective amount of exosomes or a pharmaceutical composition comprising at least one exosome (e.g., either of the exosomes or pharmaceutical compositions described herein), wherein the exosomes contain modified RNA effective in treating the disorder. In some embodiments, the exosomes are isolated from cells obtained from the subject.

[0031] In certain other embodiments, the Disclosure provides the use of a pharmaceutical composition (e.g., either of the exosomes or pharmaceutical compositions described herein) comprising an effective amount of exosomes or at least one exosome in the treatment or prevention of a disorder in a subject, wherein the exosomes contain modified RNA effective in treating the disorder. In some embodiments, the exosomes are isolated from cells obtained from a subject.

[0032] In yet another embodiment, the Disclosure provides a method for treating or preventing a disorder in a subject by: (a) providing one or more cells obtained from a subject; (b) contacting one or more lipid nanoparticles (LNPs) containing modified RNA with one or more cells under conditions that allow LNP uptake by the cells; (c) isolating exosomes produced by one or more cells, wherein at least one isolated exosome contains modified RNA; and (d) administering an effective amount of isolated exosomes to the subject, wherein the modified RNA is effective in treating the disorder. In some embodiments, the cells are epithelial cells, immune cells, progenitor cells, or stem cells. In some embodiments, the cells are B lymphocytes, T lymphocytes, or monocytes.

[0033] In yet another embodiment, the Disclosure provides the use of an effective amount of isolated exosomes in treating or preventing a disorder in a subject, by: (a) obtaining one or more cells from a subject; (b) contacting one or more lipid nanoparticles (LNPs) containing modified RNA with one or more cells under conditions that allow LNP uptake by the cells; (c) isolating exosomes produced by one or more cells, wherein at least one isolated exosome contains modified RNA; and (d) administering an effective amount of isolated exosomes to a subject, wherein the modified RNA is effective in treating the disorder. In some embodiments, the cells are epithelial cells, immune cells, progenitor cells, or stem cells. In some embodiments, the cells are B lymphocytes, T lymphocytes, or monocytes.

[0034] In some embodiments of the methods and uses disclosed herein, the disorder is selected from anemia, spinal cord malformations, immune or inflammatory diseases, monogenic diseases, and complex diseases. In some embodiments, the disorder is anemia. In some embodiments, the disorder is spinal cord malformations. In some embodiments, the disorder is an immune or inflammatory disease. In some embodiments, the immune or inflammatory disease is inflammatory bowel disease. In some embodiments, the disorder is a monogenic disease. In some embodiments, the monogenic disease is a neurodegenerative disease. In some embodiments, the disorder is a complex disease. In some embodiments, the complex disease is myocardial infarction. In some embodiments, the complex disease is cancer.

[0035] In some embodiments of the methods and uses disclosed herein, the modified RNA encodes a polypeptide effective in treating the disorder. In some embodiments, the modified RNA encodes an erythropoietin polypeptide. In some embodiments, the modified RNA encodes a human erythropoietin polypeptide. In some embodiments, the modified RNA encodes the erythropoietin polypeptide of SEQ ID NO: 1.

[0036] In some embodiments of the methods and uses disclosed herein, subjects have a reduced immune and / or inflammatory response to treatment in the presence of exosomes compared to treatment in the absence of exosomes. In some embodiments, the reduced immune and / or inflammatory response includes an increase or decrease in the level of at least one cytokine. In some embodiments, the reduced immune and / or inflammatory response includes a decrease in the level of at least one cytokine, where the cytokine is selected from IL-6, IP-10, RANTES, MCP-1, and KC.

[0037] In some embodiments of the methods and uses disclosed herein, the step of contacting one or more cells with LNPs is carried out in the presence of human serum. In some embodiments, the human serum is present in an amount of about 0.5% by volume, about 1% by volume, or about 1.5% by volume. In some embodiments, the human serum is present in an amount of about 1% by volume. In some embodiments, the step of contacting one or more cells with LNPs includes contacting one or more cells with at least two, at least three, or at least four different doses of LNPs. In some embodiments, the step of contacting one or more cells with LNPs includes contacting one or more cells with at least three different doses of LNPs. In some embodiments, the step of isolating exosomes includes isolating exosomes from a sample of in vitro cell culture medium. [Brief explanation of the drawing]

[0038] [Figure 1A] This shows the amount of hEPO mRNA detected in human epithelial (HTB-177) cells after LNP delivery. LNPs containing DLin-DMA ionized lipids (DD-LNPs) and LNPs containing DLin-MC3-DMA ionized lipids (MC3-LNPs), each containing 100 μg of hEPO mRNA, were transferred into HTB-177 cells in independent experiments. Untreated cells and cells treated with LNPs without hEPO mRNA were used as controls. 96 hours after LNP treatment, hEPO mRNA was quantified in recipient cells by RT-qPCR. The amount of hEPO mRNA detected in cells was normalized to the total number of cells collected 96 hours after LNP treatment. [Figure 1B] This shows the intracellular levels of hEPO protein detected in HTB-177 cells after LNP delivery. 100 μg of hEPO mRNA was delivered to HTB-177 cells via LNP. 96 hours after LNP treatment, hEPO protein from extracellularly delivered mRNA was quantified in recipients by ELISA. The amount of hEPO protein was normalized to the total number of cells collected 96 hours after LNP treatment. [Figure 1C]This shows the extracellular amount of hEPO protein detected in the supernatant of HTB-177 conditioned medium after LNP delivery. 100 μg of hEPO mRNA was delivered to HTB-177 cells via LNP. 96 hours after LNP treatment, the secreted hEPO protein was quantified in the supernatant of cell culture conditioned medium by ELISA. The amount of hEPO protein was normalized to the total number of cells collected 96 hours after LNP treatment. [Figure 1D] This shows the percentage of hEPO mRNA detected in the cytoplasm of LNP-treated cells relative to the total amount of hEPO mRNA (100 μg) administered via LNP. [Figure 1E] A schematic diagram illustrating the exodus of hEPO mRNA from endosomes into the cytoplasm following endocytosis of LNPs, and the translation of hEPO mRNA into protein, is shown. Alternatively, hEPO mRNA may be packaged in endosome-derived EVs (endo-EVs) and secreted via exocytosis. [Figure 1F] This shows the total amount of hEPO mRNA quantified in extracellular viable cells (EVs) isolated from LNP-treated cells. After detecting LNP-delivered hEPO mRNA in the cytoplasm of the cells, the remaining hEPO mRNA was evaluated in EVs secreted from these cells. EVs were isolated, and the hEPO mRNA in the EVs was quantified by RT-qPCR (absolute quantification). [Figure 1G] This shows the molar concentrations of ionized lipids and hEPO mRNA nucleotides (ionized lipids per unit of hEPO mRNA) in LNPs. LNPs used for cell delivery (MC3-LNP and DD-LNP) were analyzed for the molar concentrations of ionized lipids and hEPO mRNA nucleotides using gradient high-performance liquid chromatography (UPLC). Generally, LNPs contained approximately three times more ionized lipids (μmol) than hEPO mRNA nucleotides (μmol). [Figure 1H-1I]This shows the molar concentrations of ionized lipids and hEPO mRNA nucleotides in extracellular viable cells (EVs) (ionized lipids per unit of hEPO mRNA). EVs were isolated from LNP-treated cells, and the molar concentrations of ionized lipids and hEPO mRNA nucleotides in the EVs were analyzed using gradient high-performance liquid chromatography (UPLC). Generally, MC3-EV (Figure 1H) and DD-EV (Figure 1I) contained nearly equal concentrations of ionized lipids (μmol) and hEPO mRNA nucleotides (μmol). [Figure 1J] This section compares the molar ratio (mol / mol) of ionized lipids per hEPO mRNA nucleotide between LNP and EV. EV generally contained a 1:1 molar ratio (1 mol of ionized lipids per 1 mol of mRNA nucleotides), while LNP generally contained a 3:1 molar ratio (3 mol of ionized lipids per 1 mol of mRNA nucleotides). [Figure 2A] This study demonstrates the delivery of Cy5-EGFP-mRNA to human epithelial (HTB-177) cells via extracellular viable cells (EVs). DD-LNP containing 76 μg of fluorescent cyanine 5 (Cy5)EGFP-mRNA was administered to HTB-177 cells. 96 hours after LNP treatment, EVs were isolated from DD-LNP-treated cells (DD-EVs). DD-EVs containing 78 μg of Cy5-EGFP-mRNA were transferred into autologous HTB-177 cells. Cy5-EGFP-mRNA delivery was evaluated by fluorescence-based FACS at 5, 24, and 48 hours after EV delivery. Data are presented as the percentage of HTB-177 cells containing EGFP-mRNA after EV transfer at each time point. Data are presented as mean values ​​with standard deviations (SD) of at least three biological replicates. Parametric independent two-group two-tailed t-tests were used to compare the MC3-LNP and DD-LNP groups. P-values: **P<0.01, ****P<0.0001, and ns = not significant. Abbreviations: UNT(w / o EV) - untreated cells; Cells + EV(w / o Cy5-mRNA) - DD-LNP or MC3-LNP transferred without mRNA; Cells + EV(containing Cy5-mRNA) - DD-LNP or MC3-LNP transferred with mRNA. [Figure 2B-2D]This study demonstrates the delivery of Cy5-EGFP-mRNA to human peripheral blood mononuclear cells (PBMCs) via extracellular viable cells (EVs). Peripheral blood mononuclear cells (PBMCs) were isolated from healthy human buffy coat and stained with specific surface markers for CD19 (B cells), CD3 (T cells), and CD14 (monocytes). 78 μg of DD-EV containing Cy5-EGFP-mRNA was transferred to recipient cells. FACS analysis was performed 5, 24, and 48 hours after EV delivery to detect Cy5-EGFP-mRNA in recipient immune cells. Cy5-EGFP-mRNA delivery was estimated based on Cy5-fluorescence and antibody-fluorescence in B cells (Figure 2B), T cells (Figure 2C), and monocytes (Figure 2D). Data are presented as the percentage of cells containing Cy5-EGFP-mRNA after EV transfer at each time point. Data are presented as mean values ​​with standard deviations (SD) of at least three biological replicates. A parametric independent two-group two-sided t-test was used to compare the MC3-LNP and DD-LNP groups. P-values: **P<0.01, ****P<0.0001, and ns = not significant. Abbreviations: UNT(w / o EV) - untreated cells; Cells + EV(w / o Cy5-mRNA) - DD-LNP or MC3-LNP transferred without mRNA; Cells + EV(containing Cy5-mRNA) - DD-LNP or MC3-LNP transferred with mRNA. [Figure 3]This study demonstrates the detection of hEPO protein in mouse blood after hEPO mRNA delivery via EV. Mice were intravenously injected with 100 μL of MC3-EV containing 1.5 μg of hEPO mRNA (per mouse). The concentration of hEPO protein in mouse plasma was determined by human erythropoietin ELISA at 0 hours (untreated), 2 hours, 5 hours, and 24 hours after EV injection. hEPO protein was detectable in mouse blood 2 hours after EV injection. Control mice were injected with an equal volume of PBS, and mouse plasma was assayed for hEPO protein by ELISA. Data are presented from 8 replicates (n=8) at each time point, except for 2 hours (n=4). Each point in the scatter plot represents each replicate (mouse). A parametric independent two-group two-tailed t-test was used to compare plasma hEPO protein levels after MC3-EV delivery between 2 and 5 hours (ns: not significant). [Figure 4A-4B] This report shows the quantification of hEPO mRNA (Figure 4A) and hEPO protein (Figure 4B) in mouse hearts after in vivo delivery of hEPO mRNA via EV. Mice were intravenously injected with 100 μL of MC3-EV containing 1.5 μg of hEPO mRNA (per mouse). At 5, 24, and 96 hours after EV injection, the levels of hEPO mRNA and hEPO protein were quantified by RT-qPCR and ELISA, respectively. Mice injected with PBS were used as controls. Data are presented as scatter plots, with the mean values ​​at each time point being the standard deviation (SD) of four replicates (n=4). Parametric independent two-group two-tailed t-tests were used to compare the EV-treated and untreated groups at each time point. Significant values ​​are shown as P-values: *P<0.05, **P<0.01, ***P<0.001. [Figure 4C-4D]This report shows the quantification of hEPO mRNA (Figure 4C) and hEPO protein (Figure 4D) in mouse lungs. At 5, 24, and 96 hours after EV injection (1.5 μg of hEPO mRNA per mouse), hEPO mRNA levels in the lungs were quantified by RT-qPCR, and hEPO protein levels were quantified by ELISA. The data are presented as a scatter plot, with the mean values ​​(SD) of four replicates (n=4) at each time point. Parametric independent two-group two-tailed t-tests were used to compare the EV-treated and untreated groups at each time point. Significant values ​​are shown as P-values: *P<0.05, **P<0.01, ***P<0.001. [Figure 4E-4F] This section shows the quantification of hEPO mRNA (Figure 4E) and hEPO protein (Figure 4F) in mouse liver. At 5, 24, and 96 hours after EV injection (1.5 μg of hEPO mRNA per mouse), the levels of hEPO mRNA in the liver were quantified by RT-qPCR, and the levels of hEPO protein were quantified by ELISA. The liver contained the highest levels of hEPO protein compared to all other organs evaluated. The data are presented as a scatter plot, with the mean values ​​at each time point representing four replicates (n=4) with standard deviations (SD). Parametric independent two-sided t-tests were used to compare the EV-treated and untreated groups at each time point. Significant values ​​are shown as P-values: *P<0.05, **P<0.01, ***P<0.001. [Figure 4G-4H] This report shows the quantification of hEPO mRNA (Figure 4G) and hEPO protein (Figure 4H) in mouse spleens. At 5, 24, and 96 hours after EV injection (1.5 μg of hEPO mRNA per mouse), the levels of hEPO mRNA in the spleen were quantified by RT-qPCR, and the levels of hEPO protein were quantified by ELISA. The highest levels of hEPO mRNA were detected in the spleen compared to all other organs evaluated. The data are presented as a scatter plot, with the mean values ​​at each time point representing four replicates (n=4) with standard deviations (SD). Parametric independent two-sided t-tests were used to compare the EV-treated and untreated groups at each time point. Significant values ​​are shown as P-values: *P<0.05, **P<0.01, ***P<0.001. [Figure 4I-4J] This report shows the quantification of hEPO mRNA (Figure 4I) and hEPO protein (Figure 4J) in mouse kidneys. At 5, 24, and 96 hours after EV injection (1.5 μg of hEPO mRNA per mouse), the levels of hEPO mRNA in the kidneys were quantified by RT-qPCR, and the levels of hEPO protein were quantified by ELISA. The data are presented as a scatter plot, with the mean values ​​(SD) of four replicates (n=4) at each time point. Parametric independent two-group two-tailed t-tests were used to compare the EV-treated and untreated groups at each time point. Significant values ​​are shown as P-values: *P<0.05, **P<0.01, ***P<0.001. [Figure 4K-4L] This report shows the quantification of hEPO mRNA (Figure 4K) and hEPO protein (Figure 4L) in the mouse thymus. At 5, 24, and 96 hours after EV injection (1.5 μg of hEPO mRNA per mouse), the levels of hEPO mRNA in the thymus were quantified by RT-qPCR, and the levels of hEPO protein were quantified by ELISA. The data are presented as a scatter plot, with the mean values ​​(SD) of four replicates (n=4) at each time point. A parametric independent two-group two-tailed t-test was used to compare the EV-treated and untreated groups at each time point. Significant values ​​are shown as P-values: *P<0.05, **P<0.01, ***P<0.001. [Figure 4M-4N] This report shows the quantification of hEPO mRNA (Figure 4M) and hEPO protein (Figure 4N) in mouse pancreas. At 5, 24, and 96 hours after EV injection (1.5 μg of hEPO mRNA per mouse), the levels of hEPO mRNA in the pancreas were quantified by RT-qPCR, and the levels of hEPO protein were quantified by ELISA. The data are presented as a scatter plot, with the mean values ​​(SD) of four replicates (n=4) at each time point. Parametric independent two-group two-tailed t-tests were used to compare the EV-treated and untreated groups at each time point. Significant values ​​are shown as P-values: *P<0.05, **P<0.01, ***P<0.001. [Figure 4O-4P]This report shows the quantification of hEPO mRNA (Figure 4O) and hEPO protein (Figure 4P) in mouse brains. At 5, 24, and 96 hours after EV injection (1.5 μg of hEPO mRNA per mouse), the levels of hEPO mRNA in the brain were quantified by RT-qPCR, and the levels of hEPO protein were quantified by ELISA. The data are presented as a scatter plot, with the mean values ​​(SD) of four replicates (n=4) at each time point. Parametric independent two-group two-tailed t-tests were used to compare the EV-treated and untreated groups at each time point. Significant values ​​are shown as P-values: *P<0.05, **P<0.01, ***P<0.001. [Figure 5] This report shows the levels of hEPO protein in mouse blood and organs after delivery of MC3-EV and MC3-LNP. After delivering equal amounts of hEPO mRNA (1.5 μg per mouse) to mice, MC3-EV and MC3-LNP were compared for hEPO protein production. In organs, the amount of hEPO protein from LNP was generally equivalent to that from EV, except in the spleen, where a significant difference in protein production was observed, followed by the heart (showing a smaller significant difference in protein). The most significant difference was observed in plasma levels of hEPO protein, which were higher after MC3-LNP delivery compared to MC3-EV delivery. Data are presented as mean values ​​with the standard deviation (SD) of replicates (points representing each replicate in the scatter plot) at each time point. Parametric independent two-group two-tailed t-tests were used to compare the EV-treated and LNP-treated groups for each organ or plasma at each time point. Significant values ​​are shown as P-values: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 6A]This study shows the levels of the cytokine IL-6 in mouse plasma after delivery of MC3-EV and MC3-LNP. Mice were intravenously injected with 100 μL of MC3-EV or MC3-LNP containing 1.5 μg of hEPO mRNA (per mouse). The concentration of IL-6 in mouse plasma was determined 5 and 24 hours after injection of MC3-EV, MC3-LNP, or PBS using the MILLIPLEX® MAP Mouse Cytokine Magnetic Bead Kit. Data are presented from 4 mice (n=4) at each time point. Each point in the scatter plot represents each replicate (mouse). One-way ANOVA was performed, followed by Sidak's multiple comparison test. Significant values ​​are shown as P-values. [Figure 6B] This shows the levels of the cytokine IP-10 in mouse plasma after delivery of MC3-EV and MC3-LNP. Mice were intravenously injected with 100 μL of MC3-EV or MC3-LNP containing 1.5 μg of hEPO mRNA (per mouse). The concentration of IP-10 in mouse plasma was determined 5 and 24 hours after injection of MC3-EV, MC3-LNP, or PBS. Data are presented from 4 mice (n=4) at each time point. Each point in the scatter plot represents each replicate (mouse). One-way ANOVA was performed, followed by Sidak's multiple comparison test. Significant values ​​are shown as P-values. [Figure 6C] This study shows the levels of the cytokine RANTES in mouse plasma after delivery of MC3-EV and MC3-LNP. Mice were intravenously injected with 100 μL of MC3-EV or MC3-LNP containing 1.5 μg of hEPO mRNA (per mouse). RANTES concentrations in mouse plasma were determined 5 and 24 hours after injection of MC3-EV, MC3-LNP, or PBS. Data are presented from 4 mice (n=4) at each time point. Each point in the scatter plot represents each replicate (mouse). One-way ANOVA was performed, followed by Sidak's multiple comparison test. Significant values ​​are shown as P-values. [Figure 6D]This study shows the levels of the cytokine MCP-1 in mouse plasma after delivery of MC3-EV and MC3-LNP. Mice were intravenously injected with 100 μL of MC3-EV or MC3-LNP containing 1.5 μg of hEPO mRNA (per mouse). MCP-1 concentrations in mouse plasma were determined 5 and 24 hours after injection of MC3-EV, MC3-LNP, or PBS. Data are presented from 4 mice (n=4) at each time point. Each point in the scatter plot represents each replicate (mouse). One-way ANOVA was performed, followed by Sidak's multiple comparison test. Significant values ​​are shown as P-values. [Figure 6E] This study shows the levels of cytokine KC in mouse plasma after delivery of MC3-EV and MC3-LNP. Mice were intravenously injected with 100 μL of MC3-EV or MC3-LNP containing 1.5 μg of hEPO mRNA (per mouse). KC concentrations in mouse plasma were determined 5 and 24 hours after injection of MC3-EV, MC3-LNP, or PBS. Data are presented from 4 mice (n=4) at each time point. Each point in the scatter plot represents each replicate (mouse). One-way ANOVA was performed, followed by Sidak's multiple comparison test. Significant values ​​are shown as P-values. [Figure 6F] This study shows the levels of the cytokine IL1-β in mouse plasma after delivery of MC3-EV and MC3-LNP. Mice were intravenously injected with 100 μL of MC3-EV or MC3-LNP containing 1.5 μg of hEPO mRNA (per mouse). The concentration of IL1-β in mouse plasma was determined 5 and 24 hours after injection of MC3-EV, MC3-LNP, or PBS. Data are presented from 4 mice (n=4) at each time point. Each point in the scatter plot represents each replicate (mouse). One-way ANOVA was performed, followed by Sidak's multiple comparison test. Significant values ​​are shown as P-values. [Figure 6G]This study shows the levels of the cytokine TNF-α in mouse plasma after delivery of MC3-EV and MC3-LNP. Mice were intravenously injected with 100 μL of MC3-EV or MC3-LNP containing 1.5 μg of hEPO mRNA (per mouse). TNF-α concentrations in mouse plasma were determined 5 and 24 hours after injection of MC3-EV, MC3-LNP, or PBS. Data are presented from 4 mice (n=4) at each time point. Each point in the scatter plot represents each replicate (mouse). One-way ANOVA was performed, followed by Sidak's multiple comparison test. Significant values ​​are shown as P-values. [Figure 6H] This shows the levels of the cytokine IFN-γ in mouse plasma after delivery of MC3-EV and MC3-LNP. Mice were intravenously injected with 100 μL of MC3-EV or MC3-LNP containing 1.5 μg of hEPO mRNA (per mouse). The concentrations of IFN-γ in mouse plasma were determined 5 and 24 hours after injection of MC3-EV, MC3-LNP, or PBS. Data are presented from 4 mice (n=4) at each time point. Each point in the scatter plot represents each replicate (mouse). One-way ANOVA was performed, followed by Sidak's multiple comparison test. Significant values ​​are shown as P-values. [Figure 7A] A schematic diagram illustrating the hypothetical fate of human LNPs is shown. [Figure 7B] This indicates that hEPO mRNA is not released from LNPs in an acidic environment (pH 5.8 or 6.6). In contrast, at physiological pH (7.4), the hEPO mRNA and lipid components of LNPs dissociate. [Figure 7C] A schematic diagram illustrating the hypothetical fate of LNP-endosomes is shown. [Figure 8A] The structures of exemplary lipid nanoparticles (LNPs) containing two different ionized lipids, DLin-MC3-DMA and DLin-DMA, are shown. [Figure 8B]This study demonstrates the effect of LNPs on cell proliferation. In independent experiments, recipient cells were transferred with either DD-LNPs or MC3-LNPs containing 100 μg of hEPO mRNA, respectively. Cells were also treated with control LNPs that did not contain hEPO mRNA. 96 hours after LNP treatment, cells were collected and counted. The cell generation time (time until the cell population doubles (hr)) was calculated based on the difference in cell number between the start and end of the LNP treatment interval (defined as ΔN (change in cell number)). The number (n) represents the biological replicates evaluated for each treatment. Significant differences between groups were assessed using one-way ANOVA followed by Tukey's multiple comparison test (significant P-value < 0.05). Only significant P-values ​​are shown: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 8C] This study demonstrates the effect of LNP on total cellular RNA. Total cellular RNA was quantified 96 hours after LNP treatment and normalized to ΔN. The number (n) represents the biological replicates evaluated for each treatment. Significant differences between groups were assessed using one-way ANOVA followed by Tukey's multiple comparison test (significant P-value < 0.05). Only significant P-values ​​are shown: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 8D-8E] The effects of LNP on total intracellular proteins (Figure 8D) and total secretory proteins (Figure 8E) are shown. Total intracellular and secretory proteins were quantified 96 hours after LNP treatment and normalized to ΔN. The number (n) represents the biological replicates evaluated for each treatment. Significant differences between groups were evaluated using one-way ANOVA followed by Tukey's multiple comparison test (significant P-value < 0.05). Only significant P-values ​​are shown: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 8F]This study demonstrates the effect of LNP on total RNA in extracellular viable cells (EVs). Total RNA in EVs was quantified 96 hours after LNP treatment and normalized to ΔN. The number (n) represents the biological replicates evaluated for each treatment. Significant differences between groups were assessed using one-way ANOVA followed by Tukey's multiple comparison test (significant P-value < 0.05). Only significant P-values ​​are shown: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 8G] This paper demonstrates the effect of LNP on all proteins in extracellular matrix (EV). Total proteins in EV were quantified 96 hours after LNP treatment and normalized to ΔN. The number (n) represents the biological replicates evaluated for each treatment. Significant differences between groups were assessed using one-way ANOVA followed by Tukey's multiple comparison test (significant P-value < 0.05). Only significant P-values ​​are shown: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 9]This study evaluates the direct transfer of hEPO mRNA from LNPs to extracellular organisms (EVs). MC3-LNPs and DD-LNPs containing hEPO mRNA were incubated with EVs that had not undergone any prior treatment (in the absence of cells) in 30 mL of PBS at 37°C for 2 hours. Two different ratios of EVs and LNPs were incubated: 200 μg of EV + 300 μL of LNP (1×), and 50 μg of EV + 300 μL of LNP (4×). After 2 hours of incubation, EVs were again isolated by ultracentrifugation. Total RNA was isolated from EVs, and hEPO mRNA was quantified by qPCR to evaluate the direct transfer of hEPO mRNA from LNPs to EVs. As a positive control, equal volumes of DD-LNP or MC3-LNP were incubated in PBS in the absence of EVs, followed by ultracentrifugation. Total RNA was isolated, and hEPO mRNA was quantified by qPCR. In parallel experiments, MC3-LNP and DD-LNP containing hEPO mRNA were transferred into cells, extracellular viable cells (EVs) were isolated from the cells, total RNA was isolated from the EVs, and hEPO mRNA was quantified by qPCR to evaluate the hEPO mRNA in EVs from LNP-treated cells. Abbreviations: dd-EV or mc3-EV, cell-derived - EVs produced by cells treated with DD-LNP or MC3-LNP containing hEPO mRNA; mixed EV + LNP (×1) - EVs (200 μg) mixed with 300 μL of LNP; mixed EV + LNP (×4) - EVs (50 μg) mixed with 300 μL of LNP; LNP, w / oEV - only LNP containing hEPO mRNA was not mixed with EVs. Experiments were conducted using biological triple repeats. The data is presented as the percentage of hEPO mRNA detected in EVs after LNP treatment of cells or after incubation (direct mixing) of LNP and EVs, relative to the initial amount of mRNA delivered to cells by LNPs or the amount of mRNA directly mixed with EVs. The mean value with the standard deviation (SD) of the repeats is shown. [Figure 10A]This study demonstrates the delivery of hEPO mRNA to cells by extracellular viable cells (EVs) and the detection of the translated protein in the cytoplasm. MC3-EVs (derived from MC3-LNP-treated cells) incorporating hEPO mRNA, or DD-EVs (derived from DD-LNP-treated cells) incorporating hEPO mRNA, were transferred into HTB-177 recipient cells. EVs without hEPO mRNA (obtained from untreated cells) and LNPs without hEPO mRNA were delivered as controls. 96 hours after hEPO mRNA delivery, hEPO protein was quantified in cell lysates by human erythropoietin-specific ELISA. The amount of hEPO protein in cell lysates was normalized for ΔN (defined as the change in cell number, i.e., the difference in the number of cells at the start and end of the LNP treatment interval). Data are presented from two (n=2) replicates at each time point. Each point in the scatter plot represents one replicate. [Figure 10B] This report demonstrates the delivery of hEPO mRNA to cells and the detection of secreted proteins by EV. 96 hours after hEPO mRNA delivery, the level of secreted hEPO protein was quantified in the supernatant of the cultured conditioned medium using a human erythropoietin-specific ELISA. The amount of hEPO protein in the supernatant was normalized to ΔN. Data are presented from two (n=2) replicates at each time point. Each point in the scatter plot represents one replicate. [Figure 10C] The total amount of hEPO protein quantified in cells and supernatant, and normalized to ΔN, is shown. Data are presented from two (n=2) replicates at each time point. Each point in the scatter plot represents one replicate. [Figure 10D] This shows the generation time of recipient cells after 96 hours of culture with EV-treated material. Data is presented from two (n=2) replicates at each time point. Each point in the scatter plot represents one replicate. [Figure 10E] The data shows total cellular proteins normalized to ΔN. The data is presented from two (n=2) replicates at each time point. Each point in the scatter plot represents one replicate. [Figure 10F]The total cellular proteins in the supernatant, normalized to ΔN, are shown. Data are presented from two (n=2) replicates at each time point. Each point in the scatter plot represents one replicate. [Figure 11] This report presents FACS analysis of Cy5-EGFP-mRNA delivery to epithelial cells and primary immune cells via extracellular matrix (EVs). Epithelial cells (column 1): Human epithelial (HTB-177) cells were transferred with DD-LNPs containing 76 μg of Cy5-EGFP-mRNA. After 96 hours of LNP treatment, EVs (DD-EVs with incorporated mRNA) were isolated from these cells. DD-EVs containing 78 μg of Cy5-EGFP-mRNA were transferred into HTB-177 cells (cultured at 2 × 10⁵ cells per well). Cells were collected at various intervals (2 hours, 24 hours, 48 ​​hours) after EV transfer, and the presence of Cy5-EGFP-mRNA in the cells was analyzed by FACS. Primary immune cells (columns 2-4): Peripheral blood mononuclear cells (PBMCs) isolated from healthy human buffy coat were cultured (2 × 10⁵ cells per well), and 78 μg of DD-EV containing Cy5-EGFP-mRNA was transferred to the PBMCs. Cells were collected at various intervals (2 hours, 24 hours, 48 ​​hours) after EV transfer and stained with monoclonal antibodies (mAbs) for surface markers against CD19 (B cells), CD3 (T cells), and CD14 (monocytes). FACS analysis was performed to detect Cy5-EGFP-mRNA in recipient cells, and the presence of Cy5-EGFP-mRNA in each cell type was estimated based on CY5-fluorescence and antibody-fluorescence specific to each cell type. The FACS dot plot represents Cy5-EGFP-mRNA (y axis) relative to FCS-A (x axis) at each time point. Cy5-EGFP-mRNA positive cells are shown in the upper right quadrant. Abbreviations: Cell w / o EV - Untreated cell; Cell + EV (w / o mRNA) - Cell treated with EV without mRNA; Cell + EV (containing mRNA) - Cell treated with EV containing mRNA. [Figure 12]This report describes the use of a human erythropoietin ELISA kit (STEMCELL Technologies) to confirm the specificity of human erythropoietin (hEPO) and to test its cross-reactivity with mouse plasma EPO protein. Plasma from untreated mice and fresh plasma from normal humans (both undiluted and diluted (2x-2x: 10x-10x)) were tested. No signal was detected for hEPO in mouse plasma. [Figure 13] This study demonstrates the detection of hEPO protein in mouse blood after delivery of hEPO mRNA via MC3-LNP. Mice were intravenously injected with either 100 μL of MC3-LNP containing 1.5 μg of hEPO mRNA (per mouse) or an equal volume of PBS as a control. The concentration of hEPO protein in mouse plasma was determined by ELISA at 0, 2, 5, and 24 hours after injection. Data are shown from 8 mice (n=8), except for the 2-hour time point (n=4). Each point in the scatter plot represents each replicate (mouse). The number of untreated mice (PBS) was n=8. A parametric independent two-tailed t-test was used to compare plasma hEPO protein from MC3-LNP delivery between 2 and 5 hours (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, and ns=not significant). [Figures 14A-14B]This report shows the quantification of hEPO mRNA (Figure 14A) and hEPO protein (Figure 14B) in mouse hearts. Mice were intravenously injected with either 100 μL of MC3-LNP containing 1.5 μg of hEPO mRNA (per mouse) or an equal volume of PBS as a control. Levels of hEPO mRNA and hEPO protein were quantified by RT-qPCR and ELISA at 5, 24, and 96 hours after injection, respectively. Data are presented as the total amount of hEPO mRNA or protein detected in all organs, normalized to organ weight. Four mice (n=4) were used for each time point and each treatment (LNP or PBS). Data are presented as the mean with standard deviation (SD) of four replicates (n=4) at each time point. Each point in the scatter plot represents one replicate. Parametric independent two-tailed t-tests were used to compare the MC3-LNP and PBS groups at each time point. Only significant p-values ​​are shown: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 14C-14D] This report shows the quantification of hEPO mRNA (Figure 14C) and hEPO protein (Figure 14D) in mouse lungs. Mice were intravenously injected with either 100 μL of MC3-LNP containing 1.5 μg of hEPO mRNA (per mouse) or an equal volume of PBS as a control. Levels of hEPO mRNA and hEPO protein were quantified by RT-qPCR and ELISA at 5, 24, and 96 hours after injection, respectively. Data are presented as the total amount of hEPO mRNA or protein detected in all organs, normalized to organ weight. Four mice (n=4) were used for each time point and each treatment (LNP or PBS). Data are presented as the mean with standard deviation (SD) of four replicates (n=4) at each time point. Each point in the scatter plot represents one replicate. Parametric independent two-tailed t-tests were used to compare the MC3-LNP and PBS groups at each time point. Only significant p-values ​​are shown: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figures 14E-14F]This report shows the quantification of hEPO mRNA (Figure 14E) and hEPO protein (Figure 14F) in mouse liver. Mice were intravenously injected with either 100 μL of MC3-LNP containing 1.5 μg of hEPO mRNA (per mouse) or an equal volume of PBS as a control. Levels of hEPO mRNA and hEPO protein were quantified by RT-qPCR and ELISA at 5, 24, and 96 hours after injection, respectively. Data are presented as the total amount of hEPO mRNA or protein detected in all organs, normalized to organ weight. Four mice (n=4) were used for each time point and each treatment (LNP or PBS). Data are presented as the mean with standard deviation (SD) of four replicates (n=4) at each time point. Each point in the scatter plot represents one replicate. Parametric independent two-tailed t-tests were used to compare the MC3-LNP and PBS groups at each time point. Only significant p-values ​​are shown: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 14G-14H] This report shows the quantification of hEPO mRNA (Figure 14G) and hEPO protein (Figure 14H) in mouse spleens. Mice were intravenously injected with either 100 μL of MC3-LNP containing 1.5 μg of hEPO mRNA (per mouse) or an equal volume of PBS as a control. Levels of hEPO mRNA and hEPO protein were quantified by RT-qPCR and ELISA at 5, 24, and 96 hours after injection, respectively. Data are presented as the total amount of hEPO mRNA or protein detected in all organs, normalized to organ weight. Four mice (n=4) were used for each time point and each treatment (LNP or PBS). Data are presented as the mean with standard deviation (SD) of four replicates (n=4) at each time point. Each point in the scatter plot represents one replicate. Parametric independent two-tailed t-tests were used to compare the MC3-LNP and PBS groups at each time point. Only significant p-values ​​are shown: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 14I-14J]This report shows the quantification of hEPO mRNA (Figure 14I) and hEPO protein (Figure 14J) in mouse kidneys. Mice were intravenously injected with either 100 μL of MC3-LNP containing 1.5 μg of hEPO mRNA (per mouse) or an equal volume of PBS as a control. Levels of hEPO mRNA and hEPO protein were quantified by RT-qPCR and ELISA at 5, 24, and 96 hours after injection, respectively. Data are presented as the total amount of hEPO mRNA or protein detected in all organs, normalized to organ weight. Four mice (n=4) were used for each time point and each treatment (LNP or PBS). Data are presented as the mean with standard deviation (SD) of four replicates (n=4) at each time point. Each point in the scatter plot represents one replicate. Parametric independent two-tailed t-tests were used to compare the MC3-LNP and PBS groups at each time point. Only significant p-values ​​are shown: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 14K-14L] This report shows the quantification of hEPO mRNA (Figure 14K) and hEPO protein (Figure 14L) in the mouse thymus. Mice were intravenously injected with either 100 μL of MC3-LNP containing 1.5 μg of hEPO mRNA (per mouse) or an equal volume of PBS as a control. The levels of hEPO mRNA and hEPO protein were quantified by RT-qPCR and ELISA at 5, 24, and 96 hours after injection, respectively. Data are presented as the total amount of hEPO mRNA or protein detected in all organs, normalized to organ weight. Four mice (n=4) were used for each time point and each treatment (LNP or PBS). Data are presented as the mean with standard deviation (SD) of four replicates (n=4) at each time point. Each point in the scatter plot represents one replicate. For each time point, the MC3-LNP and PBS groups were compared using a parametric independent two-tailed t-test. Only significant p-values ​​are shown: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 14M-14N]This report shows the quantification of hEPO mRNA (Figure 14M) and hEPO protein (Figure 14N) in mouse pancreas. Mice were intravenously injected with either 100 μL of MC3-LNP containing 1.5 μg of hEPO mRNA (per mouse) or an equal volume of PBS as a control. Levels of hEPO mRNA and hEPO protein were quantified by RT-qPCR and ELISA at 5, 24, and 96 hours after injection, respectively. Data are presented as the total amount of hEPO mRNA or protein detected in all organs, normalized to organ weight. Four mice (n=4) were used for each time point and each treatment (LNP or PBS). Data are presented as the mean with standard deviation (SD) of four replicates (n=4) at each time point. Each point in the scatter plot represents one replicate. Parametric independent two-tailed t-tests were used to compare the MC3-LNP and PBS groups at each time point. Only significant p-values ​​are shown: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 14O-14P] This report shows the quantification of hEPO mRNA (Figure 14O) and hEPO protein (Figure 14P) in mouse brains. Mice were intravenously injected with either 100 μL of MC3-LNP containing 1.5 μg of hEPO mRNA (per mouse) or an equal volume of PBS as a control. Levels of hEPO mRNA and hEPO protein were quantified by RT-qPCR and ELISA at 5, 24, and 96 hours after injection, respectively. Data are presented as the total amount of hEPO mRNA or protein detected in all organs, normalized to organ weight. Four mice (n=4) were used for each time point and each treatment (LNP or PBS). Data are presented as the mean with standard deviation (SD) of four replicates (n=4) at each time point. Each point in the scatter plot represents one replicate. Parametric independent two-tailed t-tests were used to compare the MC3-LNP and PBS groups at each time point. Only significant p-values ​​are shown: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 15A-15H]This shows organ weights after in vivo delivery of human EPO mRNA via MC3-EV or MC3-LNP. Mice were intravenously injected with 100 μL of MC3-EV or MC3-LNP containing 1.5 μg of hEPO mRNA (per mouse), or an equal volume of PBS as a control. The weights of each mouse organ—thymus (Figure 15A), kidney (Figure 15B), heart (Figure 15C), liver (Figure 15D), pancreas (Figure 15E), brain (Figure 15F), pancreas (Figure 15G), and lung (Figure 15H)—were determined 5, 24, and 96 hours after injection. No significant differences were observed between the injection groups (PBS, EV, LNP). Four mice (n=4) were used for both the treated (MC3-EV, MC3-LNP) and untreated (PBS) groups. Abbreviations: PBS - untreated mouse; EV - MC3-EV containing hEPO mRNA; LNP - MC3-LNP containing hEPO mRNA. Data are shown as mean values ​​with standard deviation (SD). Each point in the scatter plot represents each replicate (mouse). [Figures 16A-16B] The results of nanoparticle tracking analysis (NTA) of isolated extracellular molecules (EVs) from untreated or MC3-LNP-treated HTB-177 cells are shown. Figure 16A shows the particle size distribution and concentration of EVs from untreated HTB-177 cells (n=3, (i)~(iii)). Figure 16B shows the particle size distribution and concentration of EVs from MC3-LNP-treated HTB-177 cells (n=3, (i)~(iii)). The mean particle size, mode size, standard deviation (SD), D10, D50, D90, and particle concentration are shown in Tables 3~8. [Figures 17A-17B] This shows the characterization of EVs obtained from LNP-treated cells. Figure 17A shows an FAC dot plot representing Cy5-mRNA (y-axis) against CD9 (x-axis). The percentage of CD63 / CD9-positive EVs containing Cy5-mRNA is shown in the upper right quadrant. Beads incubated with PBS alone are shown as a negative control instead of EVs. One of two biological replicates is shown. Figure 17B shows the effect of RNase treatment on control pure hEPO mRNA (cell-free and EV-free (left)) or MC3-EV (right). The experiment was performed in three biological replicates (n=3), and hEPO mRNA qPCR data are presented as a scatter dot plot and mean standard deviation (SD). [Figure 18] The results of ULC-MS analysis of DLin-MC3-DMA samples prepared in ethanol, 1% (w / w) Triton X-100, and 1% (w / w) Triton X-100 with a quantitative amount of "empty" EV added are shown. [Modes for carrying out the invention]

[0039] To make this disclosure more accessible, certain terms are defined throughout this detailed description. Unless otherwise defined herein, all scientific and technical terms used in connection with this disclosure have the same meaning as those generally understood by those skilled in the art.

[0040] As used herein, the term “extracellular vesicle” or “EV” refers to a nano- or micro-sized membrane vesicle secreted or released from a cell. EVs are encapsulated by a lipid bilayer and have a particle size in the range of approximately 30 nm to 10,000 nm. In certain embodiments, EVs are exosomes.

[0041] As used herein, the terms “exosome” and “endo-EV” are interchangeable herein and refer to endosome-derived EVs. While not bound by any particular theory, it is thought that substances phagocytosed by a cell or other cellular component may be fractionated into endosomal compartments to form intermembryonic vesicles (multivesicular endosomes or multivesicles (MVBs)). These vesicles may then be released into the extracellular environment upon fusion of the MVBs with the cell membrane. The released vesicles may be referred to herein as exosomes. In some embodiments, exosomes are secreted or released from a cell after phagocytosis of an LNP. In some embodiments, exosomes secreted or released from a cell in contact with or treated with an LNP may contain one or more LNP components (e.g., one or more ionized lipids and / or one or more modified RNAs). In some embodiments, exosomes are secreted via the exocytosis pathway. In some embodiments, exosomes have a diameter of approximately 30 nm to approximately 300 nm. In some embodiments, the exosomes have a diameter of about 30 nm to about 150 nm. In some embodiments, the exosomes have a diameter of about 40 nm to about 150 nm. In some embodiments, the exosomes have a diameter of about 40 nm to about 120 nm. Exemplary exosomes include MC3-EV and DD-EV, as described and illustrated herein.

[0042] As used herein, the term “exit from endosomes” refers to RNA that exits the endosomal pathway and enters the cytoplasm after endocytosis. The endocytosis pathway is considered the primary cellular uptake mechanism for biological agents such as DNA, RNA, or proteins. These substances are confined within endosomes and degraded by specific enzymes in lysosomes. Therefore, in some embodiments, a limiting step in achieving effective biological therapy is to facilitate the exitation of therapeutic agents (e.g., modified RNA, e.g., mRNA) from endosomes while ensuring their cytoplasmic delivery. In some embodiments, less than 1% of the total modified RNA delivered to the cell via LNPs may be released into the cytoplasm via exit from endosomes. In some embodiments, some LNP components (e.g., non-exit modified RNA and / or some LNP lipid components) ultimately reach exosomes secreted from the cell after LNP endocytosis.

[0043] As used herein, the term “lipid nanoparticles” or “LNP” refers to nanoparticles comprising one or more lipids and one or more therapeutic agents (e.g., one or more modified RNAs). Nanoparticles are typically about or less than a micrometer in size. In some embodiments, the LNPs disclosed herein have an average particle size of about 200 nm or less, about 150 nm or less, about 100 nm or less, about 75 nm or less, or about 50 nm or less. In some embodiments, the LNPs disclosed herein have a high electron-density nanostructured core generated by microfluidic mixing of a lipid-containing solution in ethanol with an aqueous solution containing modified RNA (e.g., mRNA). In some embodiments, the LNPs disclosed herein do not have more than 50% by volume of continuous aqueous regions, thus excluding conventional liposomes such as single lamellar vesicles.

[0044] In various embodiments, isolated exosomes can be prepared by a process comprising: (a) providing one or more lipid nanoparticles (LNPs) containing modified RNA; (b) contacting one or more cells with the LNPs under conditions that allow for LNP uptake by cells; and (c) isolating exosomes produced by one or more cells, wherein at least one isolated exosome contains modified RNA.

[0045] In various embodiments, the LNP has an average particle size of about 50 nm to about 100 nm, for example, about 60 nm to about 90 nm, about 70 nm to about 80 nm, or about 80 nm to about 90 nm. In some embodiments, the LNP has an average particle size of about 70 nm, about 71 nm, about 72 nm, about 73 nm, about 74 nm, about 75 nm, about 76 nm, about 77 nm, about 78 nm, about 79 nm, about 80 nm, about 81 nm, about 82 nm, about 83 nm, about 84 nm, about 85 nm, about 86 nm, about 87 nm, about 88 nm, about 89 nm, or about 90 nm. In some embodiments, the LNP has an average particle size of about 83 nm, about 84 nm, about 85 nm, about 86 nm, about 87 nm, or about 88 nm.

[0046] In various embodiments, LNPs have a polydispersity index (PDI) of about 0.01 to about 0.15, for example, about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.10, about 0.11, about 0.12, about 0.13, about 0.14, or about 0.15. As used herein, the terms “polydispersity index” or “PDI” refer to a measure of the distribution of nanoparticles in a nanoparticle sample (see NIST Special Publication 1200-6, “Measuring the Size of Nanoparticles in Aqueous Media Using Batch Mode Dynamic Light Scattering”).

[0047] In various embodiments, the LNPs have an encapsulation efficiency (%EE) of modified RNA of about 80% or more, about 85% or more, about 90% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more. As used herein, the terms “encapsulation efficiency” or “%EE” refer to the ratio of modified RNA encapsulated in the LNP to the total modified RNA content in the composition, which is measured by dissolving the LNP with a surfactant, for example, Triton X-100.

[0048] In various embodiments, the LNP comprises a lipid component and at least one modified RNA. In various embodiments, one or more exosomes produced by contacting one or more cells with the LNP (e.g., any of the LNPs disclosed herein) comprise a lipid component and at least one modified RNA. In various embodiments, the lipid component of the exosome may be at least partially derived from the lipid component of the LNP. In various embodiments, the lipid component of the exosome may contain the same one or more lipids as the lipid component of the LNP.

[0049] As used herein, the term “lipid component” refers to a component of an LNP and / or exosome that contains one or more lipids. For example, the lipid component of an LNP and / or exosome disclosed herein may include one or more ionized lipids, one or more phospholipids, one or more structural lipids, and / or one or more PEG lipids. In various embodiments, the lipid component of an LNP and / or exosome may include a lipid monolayer and / or a lipid bilayer.

[0050] In various embodiments, the lipid components of the LNP may consist of any material used in conventional nanoparticle technology, such as ionized lipids, phospholipids, structural lipids, and / or PEG lipids. In various embodiments, at least one lipid present in the lipid components of the LNP is also present in the lipid components of the exosome, in which case the exosome is generated by contacting one or more cells with the LNP.

[0051] In various embodiments, the lipid components of the LNP and / or exosomes disclosed herein may comprise one or more ionized lipids.

[0052] Non-limiting examples of ionized lipids include, for example, lipids that contain a positive charge at an acidic pH, such as 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA; see, e.g., U.S. Patent No. 8,158,601 incorporated herein by reference), 2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), Merck-32 (see, e.g., International Publication No. 2012 / 018754 incorporated herein by reference), Acuitas-5 (see, e.g., International Publication No. 2015 / 199952 incorporated herein by reference), KL-10 (see, e.g., U.S. Patent Application Publication No. 2012 / 0295832 incorporated herein by reference), C12-200 (see, e.g., Love et al. Examples include al. (2009) PNAS 107(5):1864-69). In various embodiments, the ionized lipid is DLin-MC3-DMA. In various embodiments, the ionized lipid is DLin-DMA.

[0053] In some embodiments, the LNP contains at least one ionized lipid. In some embodiments, the at least one ionized lipid is present in the LNP in an amount ranging from about 5% to about 90%, about 10% to about 80%, about 25% to about 75%, or about 40% to about 60%, for example, about 50% (mol percent), relative to the total lipids present in the LNP. In some embodiments, the at least one ionized lipid is present in the LNP in an amount of about 50% (mol percent), relative to the total lipids present in the LNP. In some embodiments, the at least one ionized lipid is DLin-MC3-DMA. In some embodiments, the at least one ionized lipid is DLin-DMA.

[0054] As used herein, the term "MC3-LNP" may be used to refer to LNPs that contain the majority of ionized lipids: DLin-MC3-DMA ionized lipids.

[0055] As used herein, the term "DD-LNP" may be used to refer to LNPs that contain a majority of ionized lipids: DLin-DMA ionized lipids.

[0056] In some embodiments, the exosome contains at least one ionized lipid. In some embodiments, the at least one ionized lipid is DLin-MC3-DMA. In some other embodiments, the at least one ionized lipid is DLin-DMA.

[0057] As used herein, the term "MC3-EV" may be used to refer to exosomes (EVs) isolated from cells that have been in contact with or treated with MC3-LNP.

[0058] As used herein, the term "DD-EV" may be used to refer to exosomes (EVs) isolated from cells that have been in contact with or treated with DD-LNP.

[0059] In various embodiments, the lipid components of LNPs and / or exosomes disclosed herein may comprise one or more phospholipids. In various embodiments, phospholipids may be assembled into one or more lipid bilayers. Generally, phospholipids may comprise a phosphate moiety and one or more carbon chains, e.g., saturated fatty acid chains. In various embodiments, phospholipids may comprise one or more multiple (e.g., double or triple) bonds (i.e., one or more unsaturated bonds). In various embodiments, phospholipids may facilitate fusion with a membrane. In various embodiments, fusion of phospholipids to a membrane may allow one or more elements of the lipid-containing composition to pass through the membrane, thereby enabling, for example, delivery of one or more elements to a cell.

[0060] Examples of phospholipids include, but are not limited to, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dipalmito Il-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 dietherPC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexade Sil-sn-glycero-3-phosphocholine (C16LysoPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-difytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2 Examples include -dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleyl-sn-glycero-3-phospholac-(1-glycero)sodium salt (DOPG), and sphingomyelin. In various embodiments, the phospholipid is DSPC.

[0061] Examples of phospholipids include, for example, neutral lipids that have zero effective charge at physiological pH. Non-limiting examples of neutral lipids include lipids that exist in an uncharged or neutral zwitterionic form at physiological pH, such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-dimiristoyl-sn-glycero-phosphocholine (DMPC). In various embodiments, the phospholipid is DSPC.

[0062] In some embodiments, the LNP contains at least one phospholipid. In some embodiments, the at least one phospholipid may be present in the LNP in amounts ranging from about 1% to about 50%, about 5% to about 20%, about 7.5% to about 12.5%, for example, about 10% (mol percent), relative to the total lipids present in the LNP. In some embodiments, the at least one phospholipid is present in the LNP in amounts of about 10% (mol percent), relative to the total lipids present in the LNP. In some embodiments, the at least one phospholipid is a DSPC.

[0063] In some embodiments, the exosome contains at least one phospholipid. In some embodiments, the at least one phospholipid is a DSPC.

[0064] In various embodiments, the lipid components of LNPs and / or exosomes disclosed herein may comprise one or more structural lipids. As used herein, the term “structural lipid” refers to molecules that can stabilize and / or maintain the structure or fluidity of lipid components, such as sterols (e.g., animal sterols, plant sterols, fungal sterols). An exemplary structural lipid is cholesterol.

[0065] Non-limiting examples of structural lipids include, for example, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, α-tocopherol, and mixtures thereof. In various embodiments, the structural lipid is cholesterol.

[0066] In some embodiments, the LNP contains at least one structural lipid. In some embodiments, the at least one structural lipid may be present in the LNP in amounts ranging from about 10% to about 90%, about 20% to about 50%, about 35% to about 45%, for example, about 38.5% (mol percent), relative to the total lipids present in the LNP. In some embodiments, the at least one structural lipid is present in the LNP in amounts of about 38.5% (mol percent), relative to the total lipids present in the LNP. In some embodiments, the at least one structural lipid is cholesterol.

[0067] In some embodiments, the exosome contains at least one structural lipid. In some embodiments, the at least one structural lipid is cholesterol.

[0068] In various embodiments, the lipid components of the LNP and / or exosomes disclosed herein may include one or more PEG or PEG-modified lipids. Such lipids may also be referred to as PEGylated lipids. In various embodiments, the PEG lipid includes both a lipid moiety and a polyethylene glycol moiety.

[0069] Examples of PEG lipids include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. For example, in various embodiments, the PEG lipid may be PEG-c-DOMG, PEG-DMG (1,2-dimyristoyl-OT-glycerol methoxypolyethylene glycol, available from Avanti Polar Lipids), PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE. In various embodiments, the molecular weight of PEG may be about 2,000 Da (PEG2000). In various embodiments, the PEG lipid may be PEG2000-DMPE, PEG2000-DPPE, PEG2000-DMG, PEG2000-DPG, PEG2000-c-DOMG, or PEG2000-C-DOPG. In various embodiments, the PEG lipid is PEG-DMPE or PEG2000-DMPE. In various embodiments, the molecular weight of PEG may be in the range of about 500 to about 10,000 Da, or about 1,000 to about 5,000 Da.

[0070] In some embodiments, the LNP contains at least one PEG lipid. In some embodiments, the at least one PEG lipid is present in the LNP in amounts ranging from about 0% to about 20%, about 0.5% to about 5%, about 1% to about 2%, for example, about 1.5% (mol percent), relative to the total lipids present in the LNP. In some embodiments, the at least one PEG lipid is present in the LNP in amounts of about 1.5% (mol percent), relative to the total lipids present in the LNP. In some embodiments, the at least one PEG lipid is PEG-DMPE or PEG2000-DMPE.

[0071] In some embodiments, the exosome contains at least one PEG lipid. In some embodiments, the at least one PEG lipid is PEG-DMPE or PEG2000-DMPE.

[0072] In various embodiments, the LNPs disclosed herein can be prepared by combining multiple lipid components. For example, in various embodiments, LNPs can be prepared by combining one or more of ionized lipids, phospholipids, structural lipids, and PEG lipids. In various embodiments, LNPs can be prepared by combining ionized lipids, phospholipids, structural lipids, and PEG lipids, where the PEG lipid is present in the LNP at a molar percentage of about 1.5% relative to the total lipids present in the LNP. In various embodiments, LNPs can be prepared by combining ionized lipids (e.g., DLin-MC3-DMA or DLin-DMA), phospholipids (e.g., DSPC), structural lipids (e.g., cholesterol), and PEG lipids (e.g., PEG-DMPE) in a molar ratio of about 50:10:38.5:1.5 (mol / mol) relative to the total lipids present.

[0073] The selection of ionized lipids, phospholipids, structural lipids, and / or PEG lipids containing LNPs disclosed herein, and the relative molar ratios of these lipids to each other, can be determined by the characteristics of the selected lipids, the properties of the intended target cells, and the characteristics of the modified RNA (e.g., mRNA) to be delivered. For example, in some embodiments, the molar ratio of ionized lipids in LNPs may be greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, or greater than about 70% relative to the total lipids present. In some embodiments, the molar percentage of phospholipids in LNPs may be greater than about 5%, greater than about 10%, greater than about 20%, greater than about 30%, or greater than about 40% relative to the total lipids present. In some embodiments, the molar percentage of structural lipids in LNPs may be greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, or greater than about 50% relative to the total lipids present. In some embodiments, the molar percentage of PEG lipids in the LNP may be more than about 0.25%, for example, more than about 1%, more than about 1.5%, more than about 2%, more than about 5%, or more than about 10%, relative to the total lipids present.

[0074] In various embodiments, the LNPs disclosed herein may comprise each of ionized lipids, phospholipids, structural lipids, and / or PEG lipids in any desired useful orientation. In some embodiments, the core of the LNP may comprise ionized lipids and structural lipids, followed by one or more layers comprising phospholipids and / or PEG lipids surrounding the core. For example, in some embodiments, the LNP may comprise a core containing ionized lipids (e.g., DLin-MC3-DMA or DLin-DMA) and structural lipids (e.g., cholesterol) in any particular ratio, which are surrounded by a phospholipid monolayer (e.g., DSPC) of any particular thickness, and further surrounded by an outer PEG lipid monolayer of any particular thickness. In some embodiments, modified RNA (e.g., mRNA) can be incorporated into either the core or one of the subsequent layers, depending on the properties of the intended target cell and the characteristics of the modified RNA to be delivered, e.g., mRNA. In some embodiments, the core and outer layers may further comprise other components typically incorporated into LNPs known in the art.

[0075] In addition, in some embodiments, the molar percentage of ionized lipids, phospholipids, structural lipids, and / or PEG lipids containing LNPs may be selected to provide specific physical parameters for the LNP as a whole, such as the surface area of ​​one or more lipids. For example, in some embodiments, the molar percentage of ionized lipids, phospholipids, structural lipids, and / or PEG lipids containing LNPs may be selected to achieve a surface area per phospholipid (e.g., DSPC). In some embodiments, the molar percentage of ionized lipids, phospholipids, structural lipids, and / or PEG lipids may be selected to achieve a surface area of ​​approximately 1.0 nm per phospholipid (e.g., DSPC). 2 ~about 2.0nm 2 You may decide to achieve this.

[0076] In addition to lipid components, in various embodiments, the LNPs and / or exosomes disclosed herein further comprise at least one modified RNA (e.g., mRNA).

[0077] As used herein, the term “modified RNA” refers to any ribonucleic acid (RNA) sequence that has been modified relative to a reference (unmodified) RNA sequence (e.g., a naturally occurring equivalent). In some embodiments, modified RNA includes one or more nucleoside / nucleotide substitutions, deletions, and / or insertions compared to the reference RNA. In some embodiments, modified RNA includes one or more chemical modifications to nucleosides / nucleotides compared to the reference RNA. In some embodiments, for example, modified RNA may include at least one uridine monophosphate (UMP) modified to form N1-methyl-psoid-UMP. In some embodiments, all UMPs in the modified RNA are substituted with N1-methyl-psoid-UMP. Modified RNA does not necessarily require physical manipulation of the reference RNA. Modified RNA is considered “modified” as long as it contains at least one modification compared to the reference RNA sequence, regardless of how it was synthesized. In some embodiments, modified RNA is low molecular weight modified RNA. In some embodiments, modified RNA is high molecular weight modified RNA. In some embodiments, the modified RNA is modified messenger RNA (mRNA). In some embodiments, the modified RNA is low molecular weight modified mRNA. In some other embodiments, the modified RNA is high molecular weight modified mRNA. In some embodiments, the modified RNA encodes an erythropoietin polypeptide. In some embodiments, the modified RNA encodes a human erythropoietin polypeptide. In some embodiments, the modified RNA encodes the erythropoietin polypeptide of SEQ ID NO: 1. Exemplary RNAs and amino acid sequences are shown in Table 1.

[0078] [Table 1]

[0079] [Table 2]

[0080] As used herein, the term “low molecular weight” refers to an RNA molecule with a nucleotide length of less than approximately 750, less than approximately 650, less than approximately 550, less than approximately 450, less than approximately 350, less than approximately 250, or less than approximately 150, when referring to modified RNA.

[0081] As used herein, the term “high molecular weight” refers to an RNA molecule with a nucleotide length greater than approximately 550, 650, 750, 850, 950, 1000, 1500, or 2000 when referring to modified RNA. An example of high molecular weight RNA is human erythropoietin mRNA (SEQ ID NO: 2; coding region, SEQ ID NO: 3), as described and illustrated herein.

[0082] As used herein, the term “external” refers to a substance or molecule that originates from or is produced outside of a cell or organism. In some embodiments, modified RNA delivered to a cell or administered to a subject is external RNA (e.g., external mRNA).

[0083] In some embodiments, the LNP and / or exosome comprises multiple modified RNAs (e.g., mRNA). In some embodiments, the LNP and / or exosome comprises one or more modified RNAs (e.g., mRNA) encoding the polypeptide or protein of interest.

[0084] In some embodiments, the LNP and / or exosomes contain an ionized lipid:modified RNA nucleotide molar ratio of about 1:1 to about 4:1, or about 4:1, about 3:1, about 2:1, about 1:1, or less than about 1:1. In some embodiments, the LNP and / or exosomes contain an ionized lipid:modified RNA nucleotide molar ratio of about 1:1. In some embodiments, the LNP and / or exosomes contain an ionized lipid:modified RNA nucleotide molar ratio of about 1:1, for example, about 0.1:1, about 0.2:1, about 0.3:1, about 0.4:1, about 0.5:1, about 0.6:1, about 0.7:1, about 0.8:1, or less than about 0.9:1. As used herein, the phrase “ionized lipid:modified RNA nucleotide molar ratio” refers to the approximate molar ratio of ionized lipids to modified RNA nucleotides in the LNP and / or exosomes disclosed herein.

[0085] In some embodiments, the LNP contains an ionized lipid:modified RNA nucleotide molar ratio of about 2:1 to about 4:1, or about 4:1, about 3:1, or about 2:1. In some embodiments, the LNP contains an ionized lipid:modified RNA nucleotide molar ratio of about 3:1. In some embodiments, the exosome contains an ionized lipid:modified RNA nucleotide molar ratio of about 1:1 to about 3:1, or about 3:1, about 2:1, about 1:1, or less than about 1:1. In some embodiments, the exosome contains an ionized lipid:modified RNA nucleotide molar ratio of about 1:1, or less than about 1:1. In some embodiments, the exosome contains an ionized lipid:modified RNA nucleotide molar ratio lower than the ionized lipid:modified RNA nucleotide molar ratio contained in the LNP. In some embodiments, the exosome contains an ionized lipid:modified RNA nucleotide molar ratio of about 1:1, or less than about 1:1, and the LNP contains an ionized lipid:modified RNA nucleotide molar ratio of about 3:1. In some embodiments, exosomes containing an ionized lipid:modified RNA nucleotide molar ratio of about 1:1 or less than about 1:1 may be non-toxic or less toxic in host cells and tissues compared to LNPs or alternative delivery vehicles containing an ionized lipid:modified RNA nucleotide molar ratio greater than about 1:1. In some embodiments, exosomes and LNPs or alternative delivery vehicles contain the same ionized lipid (e.g., DLin-MC3-DMA or DLin-DMA) and the same modified RNA nucleotide, but with different ionized lipid:modified RNA nucleotide molar ratios. In some embodiments, uncharged (i.e., containing an ionized lipid:modified RNA nucleotide molar ratio of about 1:1) modified RNA and / or exosomes can pass through the endosomal membrane, while positively charged (i.e., containing an ionized lipid:modified RNA nucleotide molar ratio greater than about 1:1) modified RNA and / or LNPs cannot pass through the endosomal membrane.

[0086] composition In various embodiments, this disclosure provides pharmaceutical compositions comprising at least one exosome (e.g., any of the exosomes described herein). In some embodiments, the pharmaceutical composition comprises multiple exosomes. As used herein, the term “pharmaceutical composition” means a preparation of at least one exosome, in addition to other components suitable for administration to a subject, e.g., pharmaceutically acceptable carriers and / or excipients. The pharmaceutical compositions provided herein are in a form that enables administration, subsequently provides the intended biological activity of the active ingredient, and / or achieves a therapeutic effect. The pharmaceutical compositions provided herein preferably do not contain any additional components that are toxic to the subject to whom the formulation is administered.

[0087] The term "pharmaceutically acceptable" is used herein to indicate, within the bounds of sound medical judgment, a compound, substance, composition, and / or dosage form that is suitable for use in contact with human and animal tissues without causing excessive toxicity, irritation, allergic reactions, or other problems or complications, and that provides a reasonable benefit-risk ratio. Drug approval agencies (e.g., EMA, US-FDA) provide guidance for approving pharmaceutically acceptable compounds, substances, compositions, and / or dosage forms.

[0088] As used herein, the term “pharmaceutically acceptable carrier” refers to a carrier or diluent that does not cause significant irritation to a subject and does not impair the biological activity and properties of the exosomes administered in the composition. A pharmaceutically acceptable carrier can enhance or stabilize the composition or be used to facilitate the preparation of the composition. Examples of pharmaceutically acceptable carriers include solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, which are physiologically compatible. The carrier may be selected to minimize adverse side effects in a subject and / or degradation of the active ingredient. Adjuvants may also be included in any of these formulations.

[0089] As used herein, the term “pharmaceutically acceptable excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of the active ingredient. Parenteral formulations may contain excipients such as sterile water or saline solution, polyalkylene glycols such as polyethylene glycol, vegetable oils, or naphthalene hydrogenation. Other exemplary excipients include, but are not limited to, calcium bicarbonate, calcium phosphate, various sugars and starches, cellulose derivatives, gelatin, ethylene-vinyl acetate copolymer particles, and surfactants such as polysorbate 20.

[0090] The pharmaceutical compositions of this disclosure can be administered by various methods known in the art. The route and / or method of administration may vary depending on the desired outcome. In some embodiments, administration is intravitreous, intravenous, intramuscular, intraperitoneal, or subcutaneous. The pharmaceutically acceptable carrier must be suitable for intravitreous, intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). In some embodiments, a pharmaceutical composition comprising at least one exosome and a pharmaceutically acceptable carrier or excipient may be a form suitable for parenteral administration. In some embodiments, the pharmaceutical composition may be in the form of a sterile injectable aqueous solution or suspension, which can be formulated according to known procedures. The sterile injectable formulation may also be a sterile injectable suspension in a parenterally acceptable non-toxic buffer.

[0091] To produce a single dosage form, the amount of modified RNA in the exosome and / or the number of exosomes containing modified RNA combined with one or more carriers or excipients will inevitably vary depending on the subject being treated and the specific route of administration. Typically, a therapeutically effective amount or effective dose of modified RNA and / or exosomes containing modified RNA is used in the pharmaceutical composition of this disclosure. Exosomes containing modified RNA can be formulated into pharmaceutically acceptable dosage forms by conventional methods well known to those skilled in the art.

[0092] The exosome administration regimen may be modified to provide the desired optimal response (e.g., therapeutic response). For example, the exosome may be administered as a single bolus, in multiple divided doses over a predetermined period, or the dose may be gradually reduced or increased depending on the requirements of the treatment situation. For any particular subject, the specific administration regimen may be modified over time according to the individual needs and the professional judgment of the clinician in charge. The parenteral composition may be formulated into unit dosage forms for ease of administration and uniformity of administration. As used herein, unit dosage forms refer to physically separate units suitable as unitary dosages for subjects receiving treatment; each unit contains a predetermined amount of the active ingredient (i.e., exosomes) calculated to produce the desired therapeutic effect, along with the necessary formulation carrier.

[0093] The dose value of a composition containing at least one exosome can be selected based on the unique properties of the active ingredient and the specific therapeutic effect to be achieved. A physician or veterinarian may start with a dose of the exosome or pharmaceutical composition of this disclosure at a level lower than the level required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is obtained. In general, for the treatment of disorders, the effective dose of the exosome or pharmaceutical composition of this disclosure may vary depending on many different factors, such as the means of administration, the target site, the physiological state of the patient, whether the patient is human or animal, other drugs being administered, and whether the treatment is prophylactic or therapeutic. The selected dose level may also vary depending on various pharmacokinetic factors, such as the activity of the specific exosome or pharmaceutical composition used, the route of administration, the time of administration, the excretion rate, the duration of treatment, other drugs, compounds and / or substances used in combination with the specific exosome or pharmaceutical composition used, and the age, sex, weight, condition, overall health status and medical history of the patient receiving treatment. A treatment dose may be determined to optimize safety and efficacy.

[0094] The toxicity and therapeutic effects of the exosomes and pharmaceutical compositions provided herein can be determined by standard pharmaceutical methods in cell culture or animal models. For example, the LD50, ED50, EC50, and IC50 can be determined, and the dose-to-toxicity ratio (LD50 / ED50) can be calculated as a therapeutic index. Data obtained from in vitro and in vivo assays can be used to estimate or formulate dose ranges for human use.

[0095] In various embodiments, kits for use in the therapeutic applications described herein are also included in the scope of this disclosure. In various embodiments, this disclosure provides a kit comprising at least one exosome. In various embodiments, the kit comprises multiple exosomes. In various embodiments, the kit further comprises one or more additional components, including, but not limited to, instructions for use; other reagents, e.g., a second therapeutic agent; apparatus, container, or other device for preparing exosomes for administration; a pharmaceutically acceptable carrier; and apparatus, container, or other device for administering exosomes to a subject. Instructions for use, either as an insert or label, may include guidance for therapeutic applications, including proposed doses and / or methods of administration. In various embodiments, the kit includes an effective amount of exosomes or a pharmaceutical composition comprising at least one exosome (e.g., any of the exosomes or pharmaceutical compositions described herein) and instructions for using the exosomes or pharmaceutical composition to treat or prevent a disorder.

[0096] method In various embodiments, the Disclosure provides a method for delivering modified RNA to cells by contacting cells with a pharmaceutical composition containing an effective amount of exosomes or at least one exosome (e.g., any of the exosomes or pharmaceutical compositions described herein), thereby delivering the modified RNA to the cells.

[0097] This disclosure further provides the use of a pharmaceutical composition comprising an effective amount of exosomes or at least one exosome (e.g., either of the exosomes or pharmaceutical compositions described herein) in the delivery of modified RNA to cells in various embodiments.

[0098] As used herein, the term “deliver” means to provide an entity to a destination. For example, in some embodiments, the delivery of modified RNA to a cell may include the step of bringing the cell into contact with at least one exosome containing modified RNA, or a pharmaceutical composition comprising at least one exosome containing modified RNA. In some embodiments, the delivery of modified RNA to a subject may include the step of administering to the subject at least one exosome containing modified RNA, or a pharmaceutical composition comprising at least one exosome containing modified RNA. The administration of at least one exosome or a pharmaceutical composition comprising at least one exosome to mammalian tissue or to a subject may include the step of bringing one or more cells into contact with the exosome or pharmaceutical composition.

[0099] As used herein, the term “administering” means the placement of at least one exosome and / or a pharmaceutical composition comprising at least one exosome into mammalian tissue or into a subject’s body by a method or route that achieves at least partial localization of the exosome and / or components of the pharmaceutical composition to a desired site or tissue location.

[0100] The terms “subject” and “patient” are used interchangeably herein and refer to any human or non-human animal to which treatment is provided, including prophylactic treatment, using the methods and compositions herein. For the treatment of a condition or disease specific to a particular animal, such as a human subject, the term “subject” refers to the specific animal. Non-human animals include all vertebrates (e.g., mammals and non-mammals), such as any mammal. Non-limiting examples of mammals include humans, mice, rats, rabbits, dogs, monkeys, and pigs. In some embodiments, the subject is human.

[0101] In some embodiments, the step of bringing an effective amount of exosomes or a pharmaceutical composition into contact with cells includes adding the exosomes or pharmaceutical composition to an in vitro cell culture medium or administering the exosomes or pharmaceutical composition to a subject.

[0102] In some embodiments, the cells are present in an in vitro cell culture medium. In some embodiments, the cells are obtained from a subject. In some embodiments, the cells are present in a subject. In some embodiments, the cells are epithelial cells, immune cells, progenitor cells, or stem cells. In some embodiments, the cells are B lymphocytes, T lymphocytes, or monocytes.

[0103] In some embodiments, delivery of modified RNA to cells does not alter cell generation time. In other embodiments, delivery of modified RNA to cells alters cell generation time. In some embodiments, delivery of modified RNA to cells does not alter total cellular protein content per unit weight. In other embodiments, delivery of modified RNA to cells alters total cellular protein content per unit weight.

[0104] In various other embodiments, the disclosure also provides therapeutic methods and uses thereof relating to the exosomes and pharmaceutical compositions disclosed herein, for example, in the treatment of diseases. In some embodiments, exosomes are isolated from cells obtained from a subject who has a disorder and / or requires treatment.

[0105] For example, in certain embodiments, the Disclosure provides a method for treating or preventing a disorder in a subject by administering to the subject an effective amount of exosomes or a pharmaceutical composition comprising at least one exosome (e.g., any of the exosomes or pharmaceutical compositions described herein), wherein the exosomes contain modified RNA effective in treating the disorder.

[0106] In certain other embodiments, the Disclosure provides the use of a pharmaceutical composition comprising an effective amount of exosomes or at least one exosome (e.g., any of the exosomes or pharmaceutical compositions described herein) in the treatment or prevention of a subject injury, wherein the exosomes comprise modified RNA effective in treating the injury.

[0107] In certain other embodiments, the Disclosure provides a method for treating or preventing a disorder in a subject by: (a) providing one or more cells obtained from a subject; (b) contacting one or more lipid nanoparticles (LNPs) containing modified RNA with one or more cells under conditions that allow LNP uptake by the cells; (c) isolating exosomes produced by one or more cells, wherein at least one isolated exosome contains modified RNA; and (d) administering an effective amount of isolated exosomes to the subject, wherein the modified RNA is effective in treating the disorder. In some embodiments, the cells are epithelial cells, immune cells, progenitor cells, or stem cells. In some embodiments, the cells are B lymphocytes, T lymphocytes, or monocytes.

[0108] As used herein, the terms “to treat” and its cognates mean improvement of a disease, disorder, or condition (e.g., anemia, spinal malformation, immune or inflammatory disease, monogenic disease (e.g., neurodegenerative disease), or complex disease (e.g., myocardial infarction or cancer)), or at least one recognizable symptom thereof. In some embodiments, “to treat” means improvement of at least one measurable physical parameter, which is not necessarily recognizable by the patient. In some embodiments, “to treat” means either or both physically (e.g., stabilization of recognizable symptoms) and / or physiologically (e.g., stabilization of physical parameters) the progression of a disease, disorder, or condition. In some embodiments, “to treat” means slowing or reversing the progression of a disease, disorder, or condition. As used herein, “to treat” and its cognates further include delaying the onset of a given disease, disorder, or condition, or reducing the risk of acquiring it.

[0109] The terms “disease” or “disorder” are used interchangeably herein and refer to any change in the condition of the body or any organ that interrupts or interferes with the functioning of a person who is affected or who has come into contact with a particular person, and / or causes symptoms such as discomfort, dysfunction, pain, or death in such person. Disease or disorder may also relate to irritability, frailty, discomfort, illness, malaise, illness, dissatisfaction, mild illness, or illness. In some embodiments, the disorder is selected from anemia, spinal malformations, immune or inflammatory diseases, monogenic diseases (e.g., neurodegenerative diseases), and complex diseases (e.g., myocardial infarction or cancer). In some embodiments, the disorder is anemia. In some embodiments, the disorder is spinal malformation. In some embodiments, the disorder is inflammatory bowel disease. In some embodiments, the modified RNA encodes an erythropoietin polypeptide. In some embodiments, the modified RNA encodes a human erythropoietin polypeptide. In some embodiments, the modified RNA encodes the erythropoietin polypeptide of SEQ ID NO: 1.

[0110] As used herein, the term “effective amount” refers to a number of exosomes containing modified RNA sufficient to reduce at least one symptom of a disease or disorder, or to provide a desired effect, or to an amount of a pharmaceutical composition containing exosomes containing modified RNA. For example, this could be an amount that induces a clinical marker associated with a therapeutically significant reduction of symptoms or wound healing. In relation to exosomes, or pharmaceutical compositions containing at least one exosome, the term “effective amount” refers to an amount of exosomes or pharmaceutical composition sufficient to deliver the modified RNA and to modulate protein expression in a target tissue and / or cell type. In some embodiments, the effective amount of exosomes or pharmaceutical composition is sufficient to treat a disease or disorder associated with a protein expressed by the modified RNA.

[0111] In some embodiments, the exosomes and pharmaceutical compositions disclosed herein may reduce and / or inhibit the manifestation or activity of at least one biomarker or symptom of an immune response, for example, an immune response observed by an alternative treatment (e.g., LNP). In some embodiments, the therapeutic methods disclosed herein reduce and / or inhibit the production or amount of pro-inflammatory markers (e.g., cytokines, chemokines) compared to treatment methods using an alternative delivery vehicle (e.g., LNP). Non-limiting examples of pro-inflammatory markers include cytokines and chemokines, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IP-10, IL-12(p40), IL-12(p70), IL-13, IL-15, IL-16, IL-17, KC, MCP-1, exotaxin, FGF (basic), G-CSF, GM-CSF, LIF, MIG, MIP-1, MIP-2, MCP-1, INF-γ, INFα2, RANTES, TNFα, and IL-1β.

[0112] As used herein, the phrase “reduce and / or inhibit” means a change (plus or minus) of about 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more compared to a control level. As used herein, the term “control level” means either an untreated sample or subject, or a sample or subject treated in the absence of exosomes. In some embodiments, the control level is the level of expression or activity (e.g., the level of expression of one or more pro-inflammatory markers) in a control sample or subject treated in the absence of exosomes.

[0113] In some embodiments, subjects have a reduced immune and / or inflammatory response to treatment in the presence of modified RNA-containing exosomes compared to treatment in the absence of exosomes or in the absence of exosomes without modified RNA. In some embodiments, the reduced immune and / or inflammatory response includes an increase or decrease in the level of at least one cytokine. In some embodiments, the reduced immune and / or inflammatory response includes a decrease in the level of at least one cytokine, where the cytokine is selected from IL-6, IP-10, RANTES, MCP-1, and KC.

[0114] In some embodiments, the step of contacting one or more cells with LNPs is carried out in the presence of human serum. In some embodiments, the human serum is present in an amount of about 0.5% by volume, about 1% by volume, or about 1.5% by volume. In some embodiments, the human serum is present in an amount of about 1% by volume. In some embodiments, the step of contacting one or more cells with LNPs includes contacting one or more cells with at least two, at least three, or at least four different doses of LNPs. In some embodiments, the step of contacting one or more cells with LNPs includes contacting one or more cells with at least three different doses of LNPs. In some embodiments, the step of isolating exosomes includes isolating exosomes from a sample of in vitro cell culture medium. [Examples]

[0115] The following embodiments provide exemplary embodiments of the Disclosure. Those skilled in the art will recognize numerous modifications and changes that can be implemented without altering the spirit or scope of the Disclosure. Such modifications and changes are included within the scope of the Disclosure. The examples provided are not limiting to the Disclosure.

[0116] I. Materials and Methods Formulation and characterization of lipid nanoparticles (LNPs) Two different types of LNPs, namely DLin-MC3-DMA (MC3-LNP) and DLin-DMA LNP (DD-LNP) containing modified hEPO mRNA (858 nucleotides) (5 meC, Ψ) (Trilink), were prepared by precipitating mRNA with four different lipid components, as previously described (Yanez Arteta et al. (2018) PNAS 115(15):E3351-60). The four components included: a low-pH ionizable (cationic) ionized lipid (DLin-MC3-DMA or DLin-DMA); two helper lipids (DSPC and cholesterol); and a PEGylated lipid (PEG2000-DMPE). Aqueous solutions of hEPO mRNA were prepared by mixing mRNA dissolved in MilliQ-water, 100 mM citrate buffer (pH 3), and MilliQ-water to obtain a 50 mM citrate solution. A 99.5% lipid solution in ethanol was prepared using a composition with four lipid components (ionized lipids: DSPC: cholesterol: PEG2000-DMPE = 50:10:38.5:1.5 mol percent) and a total lipid content of 12.5 mM. The mRNA and lipid solutions were mixed in a NanoAssemblr (Precision Nanosystems) microfluidic mixing system with a volume-to-body ratio of Aq:EtOH = 3:1 and a constant flow rate of 12 mL / min. At the time of mixing, the ratio of nitrogen atoms on the ionized lipids to phosphorus atoms on the mRNA chain was 3.1. If an "empty" LNP (i.e., an LNP without mRNA) was prepared, the ethanol phase was mixed with only 50 mM citrate buffer (pH 3). The initial 0.35 mL and final 0.05 mL of the prepared LNP solution were discarded, and the remaining volume was collected as the sample fraction.

[0117] Using the same procedure as described above, MC3-LNPs and DD-LNPs containing cyanine 5-EGFP-mRNA (996 nucleotides) (5meC,Ψ) (Trilink) were prepared, but cyanine 5-EGFP-mRNA was incorporated into the LNPs instead of hEPO mRNA.

[0118] To characterize the formulated LNPs, 25 μL of the sample fraction was injected into 975 μL of 10 mM phosphate buffer (pH 7.4), and the intensity-average particle size (Z-mean) was measured using a Malvern ZetaSizer (ZetaSizer Nano ZS, Malvern Instruments Inc.). The sample fraction was immediately transferred to a Slide-a-lyzer G2 dialysis cassette (10000 MWCO, Thermo Fischer Scientific) and dialyzed overnight at 4°C in PBS (pH 7.4). The volume of PBS buffer was 650-800 × sample fraction volume. Next, the sample fraction was collected. From this volume, 25 μL was injected into 975 μL of 10 mM phosphate buffer (pH 7.4), and the particle size was measured again (post-dialysis particle size). The final mRNA concentration and encapsulation efficiency (EE) were measured using a Quant-it Ribogreen Assay Kit (Thermo Fischer Scientific).

[0119] cell culture Human epithelial HTB-177 (NCI-H460) cell line was purchased from the American Type Culture Collection (ATCC) and cultured according to the ATCC guidelines. Exosome-deficient fetal bovine serum (FBS) (Sigma Aldrich), 1% L-glutamine (2 mM) (Thermo Fisher Scientific), and 1% penicillin-streptomycin (10,000 U / mL) (Thermo Fisher Scientific) were added to RPMI-1640 growth medium (Sigma Aldrich), which contains sodium bicarbonate but does not contain sodium pyruvate or HEPES, at 37°C in the presence of 5% CO2. The thermally inactivated FBS was ultracentrifuged at 120,000 × g at 4°C for 2 hours in an Optima L-100 XP ultracentrifuge equipped with a 70Ti rotor (Beckman Coulter) to delete exosomes, and the exosome-deficient supernatant was filtered through a 0.2 μm filter.

[0120] Fresh buffy coat from healthy donors was obtained from Sahlgrenska University Hospital (Gothenburg, Sweden), and peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation. PBMCs were cultured in complete RPMI-1640 growth medium supplemented with L-glutamine, non-essential amino acids, sodium pyruvate, 1% penicillin-streptomycin, β-mercaptoethanol, and 10% FBS (exosome-deficient), and stimulated with goat anti-human IgA / IgG / IgM F(ab')2 fragment (2.5 μg / mL; Jackson ImmunoResearch Laboratories, Inc.) and phorbol myristate acetate (PMA) (1 μg / mL; InvivoGen).

[0121] LNP-mediated mRNA delivery to epithelial cells Human epithelial (HTB-177) cells were divided into 30 mL of growth medium, with 3 × 10⁶ cells per 175T flask. 6 Cells were inoculated at cell density and incubated for 24 hours. After 24 hours of incubation, cells were treated with 1 mL of DD-LNP or MC3-LNP containing 100 μg of hEPO mRNA per flask in the presence of 1% human serum (Sigma Aldrich). 100 μg of hEPO mRNA in 1 mL of LNP solution was administered in three different doses: (Day 1): 200 μL of LNP (20 μg of mRNA); (Day 2): 400 μL of LNP (40 μg of mRNA); (Day 3): 400 μL of LNP (40 μg of mRNA). Cells were collected after 96 hours. Cells treated with the same amount (200 μL, 400 μL, 400 μL) of corresponding empty DD-LNP or empty MC3-LNP (i.e., LNP without mRNA), as well as untreated cells, were used as negative controls.

[0122] Isolation and characterization of exosomal extracellular vesicles (EVs) Extracellular viable cells (EVs) were isolated from the conditioned medium of LNP-treated cells and negative controls, as previously described (El-Andaloussi et al. (2012) Nat Protoc. 7(12):2112-26). Briefly, to remove cell debris, the cells were centrifuged at 3000×g for 15 minutes at 4°C using a 4K15 centrifuge (Sigma). The resulting supernatant was collected and ultracentrifuged at 60,000×g for 35 minutes at 4°C, followed by filtration through a 0.2 μm filter to obtain EVs with a diameter of less than 200 nm. Subsequently, the filtered supernatant was ultracentrifuged at 120,000×g for 70 minutes at 4°C to pelletize the EVs. The EV pellet was resuspended in an appropriate amount (50-80 μL) of PBS (Sigma Aldrich). All ultracentrifugation steps were performed using an Optima L-100 XP ultracentrifuge with a 70Ti rotor (Beckman Coulter).

[0123] EV was quantified based on protein concentration. A 2 μL EV suspension incubated with 2 μL of M-PER Mammalian Protein Extraction Reagent (Thermo Fischer Scientific) was sonicated using an Ultrasonic cleaner (VWR) at 54°C for 5 minutes to produce an EV extract. EV protein was quantified using a Qubit 2.0 fluorometer (Thermo Fischer Scientific). 1 μL of EV, 1 μL of Qubit protein reagent, and 198 μL of Qubit buffer were mixed and incubated at room temperature for 15 minutes. Readings were recorded using the Qubit 2.0 fluorometer.

[0124] RNA isolation from cells after EV and LNP administration Total RNA was isolated from EV and LNP-treated cells using the miRCURY® RNA Isolation Kit - Cells and Plants (Exiqon) according to the manufacturer's instructions. Total RNA was quantified using a Qubit 2.0 fluorometer (Thermo Fisher Scientific). RNA quality (230 / 260 ratio) was evaluated using NanoDrop 1000 (Thermo Fisher Scientific).

[0125] Nanoparticle tracking analysis (NTA) for determining EV particle size and concentration. Using a NanoSight LM10 instrument (Malvern Panalytical) equipped with a Hamamatsu C11440-50B / A11893-02 camera, HTB-177 MC3-EV and untreated EV samples were evaluated for particle size (nm) and concentration (particles / mL). Prior to analysis, particles were diluted 500-fold in 0.1 μM filtered PBS (Sigma) to reduce the number of particles in the field of view to less than 180 particles per frame. Three independent measurements (biological replicates) were performed in scatter mode. Measurement readings for each EV sample were acquired over 60 seconds with 5 captures, at 25 frames per second (fps), with adjusted camera levels (10-16) and detection thresholds (5-15) according to individual sample and manual temperature monitoring. Blur and Max Jump Distance were set automatically. Data reading, acquisition, and analysis were performed using NanoSight Fluorescent NTA LM10 software version 3.3 (Malvern Panalytical).

[0126] Detection of LNP-derived extra-extra-hEPO mRNA in extravasation cells (EVs) and quantification by qPCR. hEPO mRNA in EVs, lysates of their parental cells after LNP administration, and in corresponding negative controls was quantified using quantitative real-time PCR (RT-qPCR). Based on RNA yield, 0.25–1 μg of total EV RNA and total cellular RNA were converted to cDNA using a large-volume cDNA kit (Thermo Fisher Scientific). Following the manufacturer's instructions, 100 ng of total cDNA was used for the quantification of hEPO mRNA using a TaqMan probe assay with a ViiA® 7 instrument (Thermo Fisher Scientific). 2 μg of pure hEPO mRNA (RNA from Trilink) was reverse transcribed, and the resulting cDNA was serially diluted (10-fold) to prepare seven standards (maximum point: 100 ng). These were then used to create a standard curve by performing three technical replicates. Subsequently, EV cDNA and cellular cDNA were used for hEPO mRNA analysis; minimum R 2 Absolute quantification was interpolated against a standard curve >0.975. GAPDH was used as the internal standard. To calculate the molar amount of hEPO mRNA, the following estimation was made: 1 mole of hEPO mRNA = 858 hEPO mRNA nucleotides.

[0127] Detection of EV markers (CD63 and CD9) and identification of exogenous mRNA in CD63 / CD9-positive EVs HTB-177 cells were treated with MC3-LNP containing 100 μg of Cy5-mRNA (Trilink), as described above. Untreated cells were included as a control. After 96 hours, total EVs were isolated from the culture media of LNP-treated cells (MC3-EVs) and untreated cells (EVs) by fractional ultracentrifugation (pre-concentration), resuspended in PBS, and quantified. After pre-concentration, CD63 / CD9-positive EVs were isolated using an affinity-based method, and the presence of Cy5-mRNA was evaluated by FACS. CD63-positive EVs were immobilized on paramagnetic Dynabeads according to the manufacturer's instructions using the exosome-human CD63 isolation / detection reagent for cell medium (Thermo Fisher Scientific). For the binding reaction, 20 μL of paramagnetic Dynabeads were incubated with 25 μg or 50 μg of total MC3-EVs. As a negative control, 20 μL of paramagnetic Dynabeads was incubated with an equal volume of PBS (without EVs). After immobilizing CD63-positive EVs, they were stained with mouse anti-human PE-CD9 antibody (BD Pharmingen, Cat. No. 555372) according to the manufacturer's instructions. After obtaining EVs using the BD FACSLyric system (BD Biosciences), CD9 and Cy5-mRNA were detected. Data were analyzed using FlowJo software (TreeStar Inc.). Experiments were performed using biological double replicates.

[0128] Analysis of direct mRNA transfer from LNPs to EVs in the absence of cells After directly mixing LNP and EV, various proportions (amounts) of LNP and EV were incubated at 37°C (in the absence of cells). Two different proportions (amounts) of EV that had not undergone any prior treatment were incubated with 300 μL of DD-LNP or MC3-LNP (39 μg of hEPO mRNA) in 30 mL of PBS at 37°C for 2 hours. In the first setup, the ratio of EV to LNP was 200 μg of EV + 300 μL of LNP (1×), while in the second setup, this ratio was 50 μg of EV + 300 μL of LNP (4×). After 2 hours of incubation, EV was again isolated by ultracentrifugation, total RNA was isolated from EV, and hEPO mRNA was quantified by qPCR to evaluate the direct transfer of hEPO mRNA from LNP to EV. As a negative control, equal amounts of DD-LNP or MC3-LNP were incubated in PBS without EV and then ultracentrifuged. As a positive control, cells were administered with DD-LNP or MC3-LNP containing hEPO mRNA, and extracellular viable cells (EVs) were isolated (DD-EV or MC3-EV, respectively). RNA was isolated, and hEPO mRNA was quantified by qPCR. The experiment was performed using biological triple replicates. Data are presented as the percentage of hEPO mRNA detected in EVs relative to the amount of hEPO mRNA delivered to cells by LNP or the amount of hEPO mRNA directly mixed with EVs. Mean values ​​are shown along with the standard deviation (SD) of the replicates.

[0129] EV-mRNA protection assay As previously mentioned, HTB-177 cells were treated with MC3-LNP containing 100 μg of hEPO mRNA. Untreated cells were included as a control. After 96 hours, extracellular viable cells (EVs) were isolated and quantified. First, to evaluate the efficiency of RNase A activity (Thermo Fisher Scientific), 280 ng of pure hEPO mRNA (Trilink) was incubated with RNase A (0.5 μg / μL) or an equal volume of PBS at 37°C for 20 minutes. Next, 200 μg of MC3-EVs were treated with RNase A under the same conditions. As a negative control, 200 μg of MC3-EVs and 150 μg of untreated EVs were incubated under the same conditions except that PBS was used instead of RNase A. After incubation, total RNA was isolated from EVs using the miRCURY™ RNA Isolation Kit-Cell and Plant (Exiqon). To evaluate the effect of RNase A on hEPO mRNA content when present outside the extracellular matrix (EV), hEPO mRNA was quantified by qPCR. The experiment was performed using biological triple replicates.

[0130] Analysis of ionized cationic lipids in EVs by gradient UPLC The presence of LNP-derived ionized lipids in extracellular vesicles (EVs) was assessed using EV fractions. 5–10 μL of each EV sample (obtained from MC3-LNP and DD-LNP-treated cells) was diluted 50-fold in PBS, and then further diluted 1+1 with a mixture of 2 w / v% Triton® X-100 in Tris / EDTA buffer. After incubation at 37°C for 30 minutes, the samples were injected into an Acquity Ultra Performance LC connected to a Single Quad Detector (SQD) (Waters). The analytical column was a Waters Acquity UPLC® CSH C18, 1.7 μm, 2.1 × 100 mm, maintained at 60°C. The flow rate was 0.50 mL / min using a mobile phase of 0.1% formic acid in water (A) and 0.1% formic acid in an equal mixture of acetonitrile and isopropyl alcohol (B). A gradient run was applied, starting with 10% B at 0.0 min, increasing to 85% B from 1.0 to 5.0 min, and then maintaining at 85% B until 7.5 min. A washing step with 99% B was added to the gradient run from 7.6 min to 9.5 min. Next, 10% B was applied for conditioning from 9.6 min to 12.0 min. Under these conditions, the elution time for DLin-DMA was 6.3 min, and the elution time for DLin-MC3-DMA was 6.5 min. Quantification was determined using external standard solutions of DLin-DMA and DLin-MC3-DMA dissolved in 99.5% ethanol. SQD was performed using electrospray, positive mode, and adjustment was made using automated adjustment with DLin-MC3-DMA solution. Cationic lipid recording was performed for each cationic lipid using M+1 Single Ion Recording (SIR).

[0131] Finally, the molar ratio of ionized lipids to hEPO mRNA nucleotides (ionized lipids per hEPO mRNA) was determined for both EV and LNP. The experiment was performed in at least six biological replicates.

[0132] hePO protein quantification After LNP treatment, the cell-conditioned supernatant was collected and used directly for hEPO protein detection. Alternatively, whole cell proteins were extracted from the cell lysate using 500 μL of M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific) in the presence of a 1% Halt protease inhibitor cocktail (Thermo Fisher Scientific). Briefly, the cells were gently agitated in a 3D Bio-rocker at 4°C for 10 minutes, and the cell debris was pelleted by centrifugation at 14,000 × g for 10 minutes. The resulting supernatant (containing protein) was transferred to a new tube. Simultaneously, the cultured supernatant was centrifuged in a 4K15 centrifuge (Sigma) at 4°C at 3000 × g for 15 minutes to remove cell debris. The resulting supernatant was transferred to a new tube. Furthermore, hEPO protein was also analyzed in the EV lysate using an erythropoietin ELISA kit (STEMCELL Technologies) according to the manufacturer's instructions. 2 μL of EV suspension was incubated with 2 μL of M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific), and the mixture was sonicated at 54°C for 5 minutes using an Ultrasonic cleaner (VWR) to produce EV extracts. Total cell proteins, total proteins, and total EV proteins from the culture supernatant were quantified using a Qubit 2.0 fluorometer (Thermo Fisher Scientific) according to the manufacturer's protocol. To detect hEPO protein, an erythropoietin ELISA kit (STEMCELL Technologies) was used according to the manufacturer's instructions. Using 50 μL of total protein solution (for both cells and culture supernatant), hEPO protein levels were calculated as mU / mL according to a relative standard curve. The concentrations were converted to fg / mL using a conversion method (119 mU = 1 ng) and normalized to the total number of cells.

[0133] Effects of LNP administration on cell proliferation, RNA, and protein content To determine the effect of LNP on cell behavior and to evaluate cell tolerance to LNP treatment (DD-LNP or MC3-LNP), cell generation time (cell proliferation), total cellular RNA, total intracellular protein content, and total secreted protein content were calculated after 96 hours of treatment with DD-LNP or MC3-LNP. The effect of LNP on extracellular proteins (EVs) was also tested by quantifying the total EV RNA and protein content after LNP treatment.

[0134] The cell generation time (G), defined as the time (hr) it takes for a population of cells to double, is given by the following formula: G = t / n t = LNP administration interval (h)

number

[0135] The variability of total RNA in cells and extracellular vehicles (EVs), total proteins in EVs, total proteins in cells (intracellular), and total proteins in the culture supernatant (secretion) was normalized against the corresponding ΔN (change in cell number).

[0136] hEPO mRNA delivery to human epithelial (HTB-177) cells via EV Human epithelial cells (HTB-177) 5 × 10⁶ per T175 flask 6Cells were inoculated at cell density and cultured in RPMI-1640 complete medium. 600 μg of MC3-EV (700 ng of hEPO mRNA) isolated from MC3-LNP-treated cells and 600 μg of DD-EV (1100 ng of hEPO mRNA) isolated from DD-LNP-treated cells were lysed in RPMI-1640 medium and transferred to recipient cells in independent assays over two days as follows: Day 1: 300 μg of MC3-EV, and Day 2: 300 μg of MC3-EV in two separate doses of 150 μg each, 8 hours later. Similarly, 300 μg of DD-EV (Day 1) and 300 μg (Day 2) were transferred to recipient cells in independent assays. Empty EVs (without hEPO mRNA) and EVs from untreated cells were delivered to recipient cells as controls. After 48 hours, cells and culture supernatant were collected and total RNA was isolated. hEPO mRNA and hEPO protein were evaluated by RT-qPCR and ELISA, respectively, according to the protocol described above. The experiment was performed in two independent biological replicates.

[0137] Cyanin 5-EGFP-mRNA delivery to epithelial and primary blood cells via EVs HTB-177 cells were delivered in various doses (200 μL, 400 μL, 400 μL) of 1 mL of DD-LNP containing fluorescent cyanine 5 (Cy5)-EGFP-mRNA, according to the protocol described above, except that 1 mL of LNP contained 76 μg of fluorescent Cy5-EGFP-mRNA per flask (compared to 100 μg / mL of hEPO mRNA). 96 hours after LNP administration, the conditioned medium (supernatant) and parent cells were collected and analyzed for hEPO mRNA and hEPO protein, or stored for further testing. Empty DD-LNP (without mRNA) and untreated cells were used as controls. HTB-177 cells, along with immune cells such as B cells, T cells, and monocytes purified from peripheral blood mononuclear cells (PBMCs, isolated from buffy coat as described above), were 2 × 10⁶ cells per well. 5Cells were inoculated at cell density and cultured in 200 μL of medium in a 96-well round-bottom plate, incubated overnight at 37°C and 5% CO2. After 24 hours of stimulation of the cultured cells, recipient cells were delivered with 78 μg of DD-EV containing Cy5-EGFP-mRNA (Trilink) in 25 μL of PBS solution. For control assays, empty DD-EV (without EGFP-mRNA) and native EV (EV from untreated cells) were delivered to the cells, or the cells were left untreated. After 5 hours, 24 hours, and 48 hours of EV treatment, cells were harvested and stained for surface markers against CD19 (B cells), CD3 (T cells), and CD14 (monocytes) (Becton-Dickinson Biosciences) with monoclonal antibodies (mAbs). Cells were obtained using FACSVerse (BD Biosciences). Cy5-EGFP-mRNA was detected based on fluorescence in each cell type, and the data was analyzed using FlowJo software (TreeStar Inc.).

[0138] The effect of pH on hEPO mRNA release from LNPs MC3-LNPs containing hEPO mRNA at a concentration of 0.011 mg / mL were incubated under static conditions at 37°C in 10 mM citrate-sodium2HPO4 buffer solution with 150 mM NaCl (pH 7.4, 6.6, or 5.8). The total amount of mRNA at zero was measured using 0.125 mM TritonX-100 (VWR Proteomics Grade) and 0.125 mM sodium dodecyl sulfate (Sigma) in a RiboGreen assay. To evaluate the mRNA fractions released from LNPs at various pH levels (pH 7.4, 6.6, or 5.8), free mRNA was analyzed using a Perkin Elmer LS55 Luminescence Spectrometer (e.g., 480 nm, em: 525 nm) with the Quant-iT RiboGreen RNA Reagent Assay Kit (Invitrogen, Thermo Fisher Scientific).

[0139] In vivo transfer of hEPO mRNA via EV and MC3-LNP The experimental procedure was approved by the Regional Laboratory Animal Ethics Committee of Gothenburg, Sweden (Ethics Review Application No. 83-2015). All procedures conform to the Swedish Animal Welfare Act and its regulations SJVFS 2012:26. C57BL6 / N Crl female mice (n=36), 9-10 weeks old, were purchased from Charles River Laboratory (Germany) and housed in an animal facility in AstraZeneca, Molndal, Sweden. The mice were housed in groups of four per cage under standard conditions (21°C RT, 12:12h photoperiod, 45-55% humidity) and were given free access to standard solid feed (R70, Lactamin AB) and water. Environmental enrichment was provided (carton, wooden tongue depressor, and cotton nesting pads). 100 μL of MC3-LNP-derived EV or MC3-LNP containing an equal volume of 1.5 μg hEPO mRNA was intravenously injected into mice (n=4 per group). 100 μL of PBS was injected into control mice. Blood samples were collected from the mouse groups (n=4) by saphenous vein microsampling 2, 5, 24, and 48 hours after EV and LNP injection. The collected blood samples in 35 μL EDTA-prep capillary tubes were centrifuged at 1700 × g to collect plasma and stored frozen at -86°C until time of analysis. The mouse groups were killed 5, 24, and 96 hours after injection for organ collection. The mice were sedated with isoflurane anesthesia, blood was collected from the orbital venous plexus, and the hearts were resected. Next, all organs (liver, kidneys, spleen, pancreas, heart, thymus, lungs, and brain) were collected, snap-frozen in liquid nitrogen, and stored at -86°C until time for analysis.

[0140] Detection of human EPO protein in mouse plasma To analyze hEPO protein in mouse plasma after hEPO mRNA delivery via MC3-LNP and MC3-EV, we developed an in-house hEPO assay on the Gyros platform. Using the EZ-Link Sulfo-NHS-LC-Biotin kit (Thermo Fisher Scientific, #21327), the supplement antibody (3F6, MAIIA Diagnostics) was biotinylated according to the kit's instructions. The detection antibody (7D3, MAIIA Diagnostics) was labeled with Alexa 647 using a monoclonal antibody labeling kit (Thermo Fisher Scientific, #A20186). Using our own hEPO protein, we created standard curves in Rexxip A buffer (Gyros Protein Technologies) in the range of 12.2 pg / mL to 50 ng / mL. Prior to analysis, mouse plasma samples were diluted 1:1 (v:v) with Rexxip A-max buffer (Gyros Protein Technologies). Samples were analyzed using a Gyrolab Bioaffy 1000 CD (Gyros Protein Technologies) equipped with a Gyrolab instrument (Gyrolab xP workstation, Gyros Protein Technologies). Five-parameter curve fitting was used for the standard curve. All standards and samples had a CV of less than 10%.

[0141] Detection of human EPO protein in mouse tissue Total protein was extracted from organs using M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific) in the presence of a 1% Halt protease inhibitor cocktail (Thermo Fisher Scientific), following the manufacturer's instructions. Briefly, 20–70 mg of tissue was lysed for 3–5 minutes at maximum speed (30 Hz) in 200–350 μL of lysis buffer (depending on tissue weight) with protease inhibitor (Thermo Fisher Scientific) added to Tissue Lyser II (Qiagen), and all tissue residue was removed by centrifugation at 10,000 × g for 15 minutes at 4°C. The resulting supernatant was used for protein quantification using a Qubit 2.0 fluorometer (Thermo Fisher Scientific). 50 μL of total protein was analyzed for hEPO protein detection using an erythropoietin ELISA kit (STEMCELL Technologies), following the manufacturer's instructions. The amount of hEPO protein (ng) in each organ was normalized to the relative organ weight (g).

[0142] Cytokine analysis in mouse plasma Following intravenous administration of MC3-LNP and MC3-EV, plasma concentrations of mouse cytokines were measured using the EMD Millipore's MILLIPLEX® MAP Mouse Cytokine Magnetic Bead Kit (Merck KGaA, #MCYTOMAG-70K) for simultaneous quantification of IL-6, KC, MCP-1, RANTES, TNFα, IFN-γ, IL-1β, and IP-10. First, samples were diluted 1:2 with assay buffer and then placed in a 96-well plate along with standards and quality controls. A solution containing beads was added; the beads were magnetic microspheres coated with specific antibodies. The mixture was incubated overnight at 4°C, and then incubated with streptavidin-PE conjugate to complete the reaction on the surface of each microsphere. The plates were read on a Bio Rad Luminex 200® analyzer. Each individual microsphere was identified, and its bioassay results were quantified based on the fluorescence reporter signal. The concentration was measured using the median fluorescence intensity (MFI) data obtained by fitting a 5-parameter logistic curve.

[0143] Detection of human EPO mRNA in mouse organs Total RNA was isolated from mouse organs using the RNeasy kit (Qiagen) according to the manufacturer's recommendations. 10–50 mg of tissue was lysed in 600 μL of RLT buffer in Tissue Lyser II (Qiagen) at maximum speed (30 Hz) for 3–4 minutes, and then all tissue residue was removed by centrifugation at 10,000 × g at 20°C for 3 minutes. Subsequently, the supernatant was transferred to a column and further processed. RNA was quantified using a Qubit 2.0 fluorometer (Thermo Fisher Scientific), and RNA quality was evaluated using a NanoDrop 1000 (Thermo Fisher Scientific). Based on the RNA yield, 0.5–1 μg of total RNA was retrotranscribed to cDNA, and 100 ng was used to detect hEPO mRNA by RT-qPCR according to the protocol described above. The amount of hEPO mRNA (μg) in each organ was normalized to the relative organ weight (g).

[0144] statistical analysis Statistical analysis was performed for all experiments using GraphPad Prism v.7 (GraphPad). In vitro data were analyzed using independent two-group two-tailed Student t-tests, except for the effects of LNP administration on HTB-177 proliferation, RNA content, and protein content, which were analyzed using one-way ANOVA followed by Tukey's multiple comparison test (significant P-value < 0.05). hEPO content in mouse plasma and organs was analyzed using independent two-group two-tailed Student t-tests, while cytokine levels in mouse plasma were analyzed using one-way ANOVA followed by Sidac's multiple comparison test. The significance levels of the P-values ​​were indicated as follows: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0145] II. Results Example 1: Characterization of lipid nanoparticles (LNPs) The LNPs used in the experiments described herein contained five main components: ionized lipids (DLin-MC3-DMA or DLin-DMA), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, PEGylated lipids (PEG2000-DMPE), and mRNA (hEPO mRNA). LNPs containing DLin-MC3-DMA ionized lipids were called MC3-LNPs; LNPs containing DLin-DMA ionized lipids were called DD-LNPs. Both LNP formulations were characterized with respect to several biophysical parameters, including loading efficiency, mean particle size, polydispersity index (PDI), and mole percentage ratios between individual LNP components (Table 2). Data are presented in Table 2 as mean ± standard error of mean (SEM) (MC3-LNP, n=4; DD-LNP, n=9).

[0146] The loading efficiency (defined as encapsulation efficiency (%EE)) of hEPO mRNA constructs in LNPs was in the range of 93–97%. The average measured particle size of LNPs containing mRNA constructs was in the range of 82–90 nm. The concentration of hEPO mRNA constructs in LNPs was 0.1 mg (100 μg). The mole percent ratios between the individual components in the LNPs were 50:10:38.5:1.5 (DLin-DMA:DSPC:cholesterol:PEG2000-DMPE) and 50:10:38.5:1.5 (DLin-MC3-DMA:DSPC:cholesterol:PEG2000-DMPE). The structures of exemplary LNPs containing two different ionized lipids, namely DLin-DMA and DLin-MC3, are shown in Figure 8A.

[0147] [Table 3]

[0148] Example 2: Delivery of hEPO mRNA to cells by LNP We investigated the delivery of mRNA encoding human erythropoietin (hEPO protein) to cells using two different LNP formulations. The effectiveness of mRNA delivery via DLin-DMA-LNP (referred to as DD-LNP) and DLin-MC3-LNP (referred to as MC3-LNP) was tested by determining the amount of intracellular hEPO mRNA and the amount of hEPO produced in both the intracellular and extracellular environments.

[0149] In independent experiments, 100 μg of hEPO mRNA was transferred into human epithelial (HTB-177) cells via DD-LNP or MC3-LNP. 96 hours after LNP treatment, hEPO mRNA was quantified in the lysates of recipient cells. hEPO protein was also quantified in the lysates and the supernatant of the cell culture medium. Both LNP formulations successfully delivered hEPO mRNA to the cells (Figure 1A). Furthermore, both LNP formulations achieved the production of hEPO protein from exogenously delivered mRNA, which is deficient in recipient cells (Figures 1B and 1C). MC3-LNP delivered significantly higher amounts of hEPO mRNA compared to DD-LNP. Similarly, significantly higher amounts of hEPO protein were observed after MC3-LNP delivery compared to DD-LNP delivery. The hEPO protein, which has been characterized as a secreted protein (Bettan et al. (2000) Mol Ther. 2(3):204-10; Shapir et al. (2015) Hum Gene Ther Clin Dev. 26(4):216-27), was detected in greater quantities in the extracellular fraction (Figure 1C) compared to the cytoplasm (Figure 1B).

[0150] Nanoparticles have been shown to induce cellular stress and alter cellular behavior (Panariti et al. (2012) Nanotechnol Sci Appl. 5:87-100; Halamoda Kenzaoui et al. (2012) Biochem J. 441(3):813-21; Petersen et al. (2011) Eur J Pharm Biopharm. 79(1):150-61). Nanoparticles may also induce autophagy-lysosome activation as a result of their chemical composition and / or mRNA content (Klionsky et al. (2016) Autophagy 12(1):1-222; Mizushima et al. (2011) Annu Rev Cell Dev Biol. 27:107-32; Yang and Klionsky (2010) Nat Cell Biol. 12(9):814-22; Zabilnyk et al. (2007) Autophagy 3(3):278-81).

[0151] To determine whether DD-LNP or MC3-LNP can affect cell proliferation, total RNA synthesis, and / or protein production, HTB-177 epithelial cells were harvested after being treated with LNP for 96 hours. After harvesting, cells were counted and generation time (i.e., time (hr) for the cell population to double) was calculated. Cell generation time was significantly increased after LNP treatment, particularly after treatment with LNP containing hEPO mRNA (Figure 8B). This indicates that cells may proliferate more slowly when treated with LNP containing hEPO mRNA compared to cells treated with empty LNP (without hEPO mRNA) or cells not treated with LNP at all (untreated cells).

[0152] Total RNA content increased slightly in cells treated with mRNA-free LNPs, but this was not statistically significant (Figure 8C). Total intracellular protein content remained unchanged after LNP treatment (Figure 8D). However, total secreted protein content in the extracellular environment increased significantly after treatment with DD-LNPs, particularly those containing hEPO mRNA (Figure 8E).

[0153] Quantification of total RNA and protein content of EVs: From the evaluation of the effect of hEPO mRNA-containing LNPs on cell-derived extracellular vesicles (EVs), it was found that the total RNA content of EVs from cells treated with mRNA-loaded MC3-LNPs was higher than that of EVs from untreated cells or cells treated with mRNA-loaded DD-LNPs. The total RNA content of EVs increased after treatment with MC3-LNPs, especially those containing hEPO mRNA (Figure 8F). This suggests that after treatment with MC3-LNPs, cellular RNA can ultimately remain within EVs. In addition, the total EV protein content increased after MC3-LNP treatment (Figure 8G). Taken together, these results suggest that cells may secrete more proteins when receiving LNPs, and the secreted proteins can be detected in both the culture supernatant and secreted EVs (Figure 8E and Figure 8G). Therefore, administration of LNPs induces cellular stress, which can result in changes in cell proliferation and / or the amount of proteins secreted by the cells.

[0154] EV size determination and concentration: Nanoparticle tracking analysis (NTA) was used to characterize EVs in terms of size and concentration. The mean ± SEM EV mode size (measured from triplicate samples) was 100.2 ± 3.7 nm from untreated cells and 116.4 ± 9.23 nm from MC3-LNP-treated cells (see also Figures 16A and 16B; Tables 3 - 8). The mean ± SEM EV concentration (measured from triplicate samples) was 5.81×10 11 ±2.25×10 10 particles / mL from untreated cells and 1.25×10 12 ±1.54×10 11 particles / mL from MC3-LNP-treated cells.

[0155] [Table 4]

[0156] [Table 5]

[0157] [Table 6]

[0158] [Table 7]

[0159] [Table 8]

[0160] [Table 9]

[0161] Example 3: Detection of LNP-derived ionized lipids and mRNA in EV Studies have shown that while most RNA delivered by LNPs undergoes lysosomal degradation and / or endocytosis recirculation, only a small amount escapes from the endosome; for example, the net escape rate of LNP-delivered siRNA is estimated to be less than 2% (Semple et al. (2010) Nat Biotechnol. 28(2):172-6; Sahay et al. (2013) Nat Biotechnol. 31(7):653-8; Sahay et al. (2010) J Control Release 145(3):82-95).

[0162] Consistent with these results, the experiments performed and described herein also reveal that, upon delivery of LNP-mRNA, less than 1% of the administered mRNA can be detected in the cytoplasm of LNP-treated cells (Figure 1D). mRNA delivered via LNPs containing DLin-MC3-DMA formulations exhibits approximately twice the endosomal escape rate compared to mRNA delivered via LNPs containing DLin-DMA formulations.

[0163] While not intending to be bound by any particular theory, we hypothesized a relationship between endocytosis (LNP uptake) and exocytosis, given that some LNP components taken up into cells may ultimately remain within secreted endosome-derived extracellular organisms (EVs) ("endosomes" or "endo-EVs") (Figure 1E). To investigate the fate of LNP delivery mRNA and other components of LNP after cell uptake, hEPO mRNA in EVs was quantified by qPCR 96 hours after LNP administration (100 μg of hEPO mRNA) to cells. These results suggest that endo-EVs secreted from LNP-treated cells acquired hEPO mRNA (Figure 1F). Endo-EVs obtained from MC3-LNP-treated cells contained nearly 1000 times more hEPO mRNA than endo-EVs obtained from DD-LNP-treated cells using the same dose of hEPO mRNA (Figure 1F). In particular, quantification of hEPO mRNA in both the cytoplasm and secreted extracellular viable cells (EVs) of LNP-treated cells showed that mRNA induced by DLin-MC3-DMA preparations was more abundant than mRNA induced by DLin-DMA preparations, indicating greater exodus from endosomes. More mRNA was also detected in EVs in the case of DLin-MC3-DMA preparations (Figures 1D and 1F).

[0164] Furthermore, the presence of LNP ionized lipids (DLin-MC3-DMA or DLin-DMA) was analyzed in EVs. MC3-LNP and DD-LNP transferred into cells contained ionized lipids:hEPO mRNA nucleotides in a 3:1 molar ratio (mol / mol) (Figure 1G). EVs secreted from LNP-treated cells were also found to contain ionized lipids. However, EVs contained fewer ionized cationic lipids per hEPO mRNA molecule compared to LNPs (Figures 1H and 1I). EVs contained ionized lipids:hEPO mRNA nucleotides in a 1:1 molar ratio (mol / mol) (Figure 1J).

[0165] For UPLC-MS analysis, DLin-MC3-DMA samples were dissolved in ethanol, 1% (w / w) Triton X-100, or 1% (w / w) Triton X-100 with quantitative "empty" EV added, and then injected into the UPLC-MS system. The response of Triton X-100 samples (with and without "empty" EV) was lower than that of samples dissolved in pure ethanol, but the quantitative error was estimated to be less than 10% (Figure 18). Considering the variability of sample preparations (mRNA and lipid quantification) and qPCR (mRNA) and UPLC-MS (lipid) analysis, mRNA and ionized lipids appear to be transported simultaneously in secreted EV as stoichiometric complex salts (1:1). Furthermore, a strong correlation was observed between nucleotide concentrations measured by qPCR and lipid concentrations determined by UPLC-MS for various EV samples (Figure 1J).

[0166] Following LNP delivery, hEPO mRNA was detected in the cytoplasm and translated into hEPO protein (Figure 1A-C), and hEPO mRNA was detected in secreted extracellular viable cells (EVs) (Figure 1F). Therefore, experiments were conducted to investigate whether EVs acquire hEPO protein from LNP-treated cells. Briefly, EVs isolated from LNP-treated cells were lysed, and the hEPO protein was analyzed by human erythropoietin ELISA. No hEPO protein was detected in the EVs, suggesting that the produced hEPO protein may originate from hEPO mRNA delivered via the EVs (data not shown).

[0167] Furthermore, we evaluated the interaction between extracellular LNPs and EVs, as well as the possibility of direct transport of LNP-mRNA to EVs. EVs were directly mixed with LNPs containing hEPO mRNA (in the absence of cells). After 2 hours of incubation, the EVs were isolated again, and the presence of hEPO mRNA in the EVs was evaluated. The results showed that EVs were negative for hEPO mRNA when directly mixed with LNPs in the absence of cells (Figure 9), which suggests that they do not directly transport hEPO mRNA to extracellular EVs. Instead, EVs may acquire mRNA via endocytosis of LNPs when cells are treated with LNPs.

[0168] Example 4: Delivery of hEPO mRNA to epithelial and immune cells via EV The aforementioned experiments suggest that extracellular hEPO mRNA can be incorporated into the endo-EV after endocytosis of LNPs (Figures 1F and 9). Next, we investigated (a) whether the EV could subsequently transport the extracellular hEPO mRNA to recipient cells by acting as a delivery vehicle, and (b) whether the hEPO mRNA delivered to the cells via the EV was functional, i.e., capable of translating mRNA to produce the hEPO protein.

[0169] MC3-EV isolated from MC3-LNP-treated cells and DD-EV isolated from DD-LNP-treated cells were transported to recipient HTB-177 epithelial cells. 96 hours after hEPO mRNA delivery, the cells and their culture supernatants were collected, and hEPO protein was evaluated by human erythropoietin ELISA. hEPO protein was detectable in the cell lysates and in the culture supernatants of recipient cells (i.e., cells that do not express hEPO protein themselves due to the lack of hEPO protein) (Figures 10A-C). Consistent with MC3-LNP cell delivery (Figures 1B and 1C), MC3-EV-mediated hEPO mRNA delivery showed higher levels of hEPO protein production compared to DD-EV-mediated hEPO delivery (Figure 10C).

[0170] LNP delivery resulted in some undesirable effects on cell behavior (Figure 8C-E). In contrast, when using either EVs containing hEPO mRNA or EVs without hEPO mRNA, EV delivery did not affect cell generation time or cellular protein content (Figure 10D-F). This suggests that cells stressed by LNP delivery may be more tolerant of EV delivery.

[0171] Next, we investigated whether EVs could deliver extracellular mRNA (i.e., hEPO mRNA) to immune cells, which are generally difficult to transfect using other delivery vehicles. Peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coat of healthy humans and inoculated. After incubation overnight in the presence of appropriate immune cell stimulation, DD-EVs (derived from DD-LNP-treated cells) containing Cy5-EGFP-mRNA were delivered to recipient cells individually in single doses over 5, 24, and 48 hours.

[0172] Cells were collected after EV-mediated Cy5-EGFP-mRNA transport at various time intervals (5 hours, 24 hours, and 48 hours) and stained with monoclonal antibodies (mAbs) for surface markers against CD19 (B cells), CD3 (T cells), and CD14 (monocytes) (Becton-Dickinson Biosciences). FACS analysis was performed to detect Cy5-EGFP-mRNA in recipient cells, and the percentage of each cell type containing Cy5-EGFP-mRNA was estimated based on antibody-fluorescence and Cy5-fluorescence. Cy5-EGFP-mRNA was detectable in recipient immune cells after 5 hours of EV-mediated mRNA delivery, indicating that EVs can deliver mRNA to immune cells (Figures 2B-D and 11). Nearly 70% of HTB-177 cells showed uptake of Cy5-EGFP-mRNA 24 hours after EV delivery (Figure 2A). At 5, 24, and 48 hours after EV delivery, 6%, 30%, and 40% of B cells, respectively, contained Cy5-EGFP-mRNA (Figure 2B). At 5 hours after EV delivery, 17% of T cells and 71% of monocytes contained Cy5-EGFP-mRNA (Figures 2C and 2D).

[0173] Example 5: Delivery of human EPO mRNA to mice via EV After determining that endo-EVs could deliver hEPO mRNA in vitro, we investigated whether these EVs could also deliver extracellular mRNA (i.e., hEPO mRNA) in vivo. Before administering hEPO mRNA in vivo, blood samples were collected from untreated healthy C57BL6 / NCrl mice and tested for hEPO protein using human erythropoietin ELISA to confirm that the recipient mice lacked and did not express human-type EPO protein (hEPO). As expected, the plasma from untreated mice was negative for hEPO protein (Figure 12).

[0174] Next, C57BL6 / NCrl mice were injected with a single intravenous dose of MC3-EV (1.5 μg of hEPO mRNA per mouse), and hEPO protein production was examined in both plasma and organs. Two hours after EV-mediated hEPO mRNA delivery, hEPO protein was detected in mouse plasma and persisted for 24 hours, indicating that EV can deliver extragenic mRNA to mice and, moreover, can lead to the production of the target protein (Figure 3). Furthermore, the presence of hEPO mRNA and hEPO protein was tested in eight organs of sacrificial mice at various time points (5 hours, 24 hours, and 96 hours). The results showed that EV not only delivers hEPO mRNA to various organs but also enables the production of hEPO protein, indicating that EV can deliver functional mRNA to organs (Figures 4A-P). hEPO mRNA and hEPO protein were detectable in the heart, lungs, liver, and spleen (Figures 4A-H). In the heart and lungs, hEPO mRNA and translated hEPO protein were detectable 5 hours after EV-mediated mRNA delivery, and the mRNA persisted for 24 hours (Figures 4A-D). In the liver, hEPO mRNA was detectable only 5 hours after EV delivery, while translated hEPO protein was detectable after 5 hours and persisted for 24 hours (Figures 4E and 4F). However, the pattern was reversed in the spleen. In the spleen, hEPO mRNA was detectable after 5 hours and persisted for 24 hours, while translated hEPO protein was detectable only after 5 hours (Figures 4G and 4H).

[0175] In particular, among the four organs that tested positive for hEPO mRNA, the liver had the highest amount of hEPO protein (Figure 4F), while the spleen had the highest amount of hEPO mRNA (Figure 4G). The amount of hEPO detected in the spleen after 5 hours was comparable to that detected in the other organs. The kidney showed relatively small amounts of hEPO mRNA, but the protein was undetectable (Figures 4I and 4J). In three of the eight organs analyzed (thymus, pancreas, and brain), hEPO mRNA and hEPO protein were undetectable (Figures 4K-P).

[0176] To test LNP-mediated hEPO mRNA delivery, parallel experiments were conducted using MC3-LNP. C57BL6 / NCrl mice were intravenously injected with a single dose of MC3-LNP (1.5 μg of hEPO mRNA per mouse), and hEPO protein production was examined in both plasma and organs. The data showed that hEPO protein was detectable in plasma 2 hours after LNP-mediated hEPO mRNA delivery and persisted for 5 hours (Figure 13). Furthermore, the presence of hEPO mRNA and hEPO protein was tested in eight organs of sacrificial mice 5, 24, and 96 hours after LNP delivery. hEPO mRNA and hEPO protein were detectable in five organs: heart, lungs, liver, spleen, and kidneys (Figures 14A-J). Of all organs positive for hEPO mRNA and hEPO protein, most hEPO mRNA was detected in the kidney (persisting for 24 hours), followed by the spleen (persisting for 96 hours). Most hEPO proteins were detected in the liver. However, both hEPO mRNA and hEPO protein were detected in the heart and lungs 5 ​​hours after injection. The thymus, pancreas, and brain were negative for both hEPO mRNA and hEPO protein (Figure 14K-P).

[0177] In summary, these data suggest that extracellular deliveries (EVs) can be used as delivery vehicles to express any desired protein from exogenously present mRNA.

[0178] Example 6: Delivery of human EPO mRNA to mice via EV or LNP To determine how efficiently EV delivers hEPO mRNA compared to MC3-LNP, hEPO protein production was compared after in vivo delivery of hEPO mRNA by LNP and EV. In organs, the amount of hEPO protein from hEPO mRNA delivery by LNP was generally equivalent to that from EV, except in the spleen, where there was a significant difference in protein production, followed by the heart (with a smaller significance) (Figure 5). The amount of hEPO protein in the blood was higher with LNP delivery compared to EV delivery (Figure 5). Furthermore, the effects of MC3-LNP and MC3-EV on tissues were tested. Neither MC3-LNP nor MC3-EV showed a significant effect on organ weight in recipient mice (Figures 15A-H).

[0179] Example 7: EV induces a lower immune / inflammatory response compared to LNP. To investigate whether EV is less immunogenic than LNP in recipient mice, single doses of MC3-LNP (1.5 μg hEPO mRNA per mouse) and MC3-EV (1.5 μg hEPO mRNA per mouse) were intravenously injected into C57BL6 / NCrl mice. The concentrations of eight different cytokines commonly involved in immune and inflammatory responses were measured in mouse plasma at two time intervals (5 hours and 24 hours). Secretion levels of several pro-inflammatory cytokines detected 5 hours after injection (particularly IL-6, IP-10, RANTES, MCP-1, and KC) were significantly higher after LNP delivery than after EV delivery (Figures 6A-E).

[0180] These results indicate that systemic delivery of LNPs can induce a high inflammatory response, while extracellular proteins (EVs) are less immunogenic and well-tolerated by recipient mice. Although LNPs may result in higher hEPO protein production than EVs (Figure 5), LNPs are thought to be more immunogenic than EVs, and therefore, EVs may be a safer vehicle for delivering therapeutic RNA to human patients.

[0181] Example 8: Detection of EV markers and exogenous mRNA in CD63 / CD9-positive EVs To confirm that LNP-mRNA was supported by EVs, HTB-177 cells were treated with MC3-LNP containing 100 μg of Cy5-mRNA, or without it. After 96 hours, all EVs were isolated from the culture media of LNP-treated cells (MC3-EVs) and untreated cells (EVs) by fractional ultracentrifugation, resuspended in PBS, and quantified. 25 μg or 50 μg of all MC3-EVs or all untreated EVs were incubated with 20 μL of paramagnetic Dynabeads conjugated with anti-CD63 antibody, and CD63-positive EVs were specifically isolated using the Exosome-Human CD63 Isolation / Detection Kit (Thermo Fisher Scientific). Next, CD63-positive EVs were stained with mouse anti-human PE-CD9 antibody (BD Pharmingen, Cat. No. 555372), and then analyzed by FACS for Cy5-mRNA detection. FACS analysis revealed that approximately 96% of immunoprecipitated extracellular vesicles (EVs) from untreated cells (50 μg assay) were positive for CD63 and CD9, but negative for mRNA. In contrast, approximately 88% of immunoprecipitated EVs from LNP-mRNA-treated cells (50 μg assay) were positive for CD63 and CD9, but 26% contained mRNA secreted after endocytosis of LNPs containing Cy5-mRNA (Cy5-mRNA) (Figure 17A). No Cy5-mRNA signal was detected in the negative controls (beads only, and CD63 / CD9-positive EVs obtained from untreated cells), indicating that mRNA is carried by the EVs (CD63 / CD9-positive EVs).

[0182] Example 9: EV-mRNA protection assay To confirm that the mRNA is located within the EV and protected, rather than bound to the EV membrane, MC3-EVs containing hEPO mRNA were exposed to RNase treatment. After RNase treatment, total RNA was isolated from the EVs, and hEPO mRNA was quantified by qPCR. Despite the efficient endonucleotide binding degradation activity of RNase (shown in EV-free RNA), RNase-treated MC3-EVs showed only a 2Ct-fold decrease in hEPO mRNA content compared to untreated MC3-EVs. The experiment was conducted in three biological replicates (n=3), and hEPO mRNA qPCR data are presented as a scatter dot plot and mean standard deviation (SD) (Figure 17B). EVs obtained from untreated cells without RNase treatment were used as a negative control. Although a small portion of the delivery mRNA may be bound to the outer membrane layer of the EV, most of the hEPO mRNA was found to be protected from endonucleotide binding degradation activity and located within the EV.

Claims

1. A pharmaceutical composition comprising an isolated exosome and a pharmaceutically acceptable carrier, wherein the isolated exosome comprises modified mRNA, and wherein the isolated exosome is as follows: (a) Providing one or more lipid nanoparticles (LNPs) containing the modified mRNA; (b) The step of bringing one or more cells into contact with the LNPs under conditions that enable LNP uptake by cells; and (c) A step of isolating exosomes produced by one or more cells, wherein at least one isolated exosome contains the modified mRNA. Prepared by a process including, A pharmaceutical composition wherein the length of the modified mRNA is 1000 nucleotides or less.

2. The pharmaceutical composition according to claim 1, wherein the LNP comprises at least one ionized lipid, phospholipid, structural lipid, and / or PEG lipid.

3. (a) The at least one ionized lipid is DLin-MC3-DMA and / or DLin-DMA; and / or (b) The at least one phospholipid is a DSPC; and / or (c) The at least one structural lipid is cholesterol; and / or (d) The pharmaceutical composition according to claim 2, wherein the at least one PEG lipid is PEG-DMPE or PEG2000-DMPE.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the LNP contains an ionized lipid which is DLin-MC3-DMA or DLin-DMA, a phospholipid which is DSPC, a structural lipid which is cholesterol, and / or a PEG lipid which is PEG-DMPE or PEG2000-DMPE.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the LNP has an ionized lipid:modified mRNA nucleotide molar ratio of 2:1 to 4:1, or 4:1, 3:1, or 2:

1.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the exosome comprises at least one ionized lipid, phospholipid, structural lipid, and / or PEG lipid.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the exosome contains an ionized lipid:modified mRNA nucleotide molar ratio of 1:1 to 3:1, or 3:1, 2:1, 1:1, or less than 1:

1.

8. The pharmaceutical composition according to claim 7, wherein the molar ratio of ionized lipid to modified mRNA nucleotide is lower than the molar ratio of ionized lipid to modified RNA nucleotide of the LNP.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the cells are obtained from a subject.

10. (a) The cells are epithelial cells, immune cells, progenitor cells, or stem cells; or (b) The cells are B lymphocytes, T lymphocytes, or monocytes, according to claim 9.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the process is carried out in vitro.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the step of isolating exosomes comprises the step of isolating exosomes from a sample of in vitro cell culture medium.

13. A pharmaceutical composition according to any one of claims 1 to 12 for use in a method for treating or preventing a disorder in a subject, wherein the method comprises the step of administering an effective amount of the pharmaceutical composition to the subject, wherein the exosome contains modified mRNA effective for treating the disorder.

14. The pharmaceutical composition according to claim 13, wherein the exosomes are isolated from cells obtained from the subject.

15. A pharmaceutical composition according to claim 1 for use in a method for treating or preventing a disorder in a subject, wherein the method is as follows: (a) Providing one or more cells obtained from the subject; (b) A step of bringing one or more lipid nanoparticles (LNPs) containing modified mRNA into contact with one or more cells under conditions that enable LNP uptake by cells; (c) a step of isolating exosomes produced by one or more cells, wherein at least one isolated exosome contains the modified mRNA; and (d) The step of administering an effective amount of the isolated exosomes to the subject. including, Herein, the modified mRNA is effective in treating the disorder, and the pharmaceutical composition herein comprises the isolated exosome.

16. The pharmaceutical composition according to claim 15, wherein the cells are epithelial cells, immune cells, progenitor cells, or stem cells, or the cells are B lymphocytes, T lymphocytes, or monocytes.

17. The pharmaceutical composition according to claim 15 or 16, wherein the step of isolating exosomes comprises isolating exosomes from a sample of in vitro cell culture medium.

18. An in vitro method for producing isolated exosomes containing modified mRNA, the following: (a) Providing one or more lipid nanoparticles (LNPs) containing the modified mRNA; (b) The step of bringing one or more cells into contact with the LNPs under conditions that enable LNP uptake by cells; and (c) A step of isolating exosomes produced by one or more cells, wherein at least one isolated exosome contains the modified mRNA. A method comprising, wherein the length of the modified mRNA is 1000 nucleotides or less.

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