Liver regeneration method through liver-specific delivery of dedifferentiation gene

Abstract

The present invention relates to novel lipid nanoparticles capable of liver-specific delivery of mRNA, a manufacturing method therefor, and a liver disease therapeutic agent capable of regenerating the liver by liver-specific delivery of the bio-reprogramming factor mRNA using same. The lipid nanoparticles according to the present invention allow for liver-specific delivery of mRNA at very high efficiency, and the lipid nanoparticles that encapsulate the mRNA-based bio-reprogramming factor mRNA are specifically delivered to liver tissues of an individual requiring liver regeneration to induce short-term expression, thereby easily setting the dose and expression period of a therapeutic agent, leading to a significant reduction in side effects of a therapeutic agent for liver regeneration using an existing cell reprogramming factor.

Description

A method for liver regeneration through liver-specific delivery of induced differentiation genes

[0001] The present invention relates to a method for liver regeneration through liver-specific delivery of a dedifferentiation gene, and more particularly, to a method for treating liver disease by inducing liver regeneration in liver damage by liver-specifically delivering mRNA of a dedifferentiation transcription factor capable of inducing cell reprogramming.

[0002] Most mammalian organs undergo tissue regeneration in very limited areas after damage. To overcome this limited tissue regeneration, regenerative medicine approaches, such as adult stem cell transplantation and inducing the activation of endogenous stem cells, are being extensively explored. However, the development of pluripotent stem cell-based cell therapies has limited application, with only a few successful cases, due to technical challenges such as the characteristics of pluripotent stem cells (tumorigenic potential, genetic instability), immature functionality, immune rejection, and low engraftment rates. Consequently, the need for new regenerative medicine methodologies is emerging.

[0003] Accordingly, attempts are ongoing to apply a method of inducing cell reprogramming (in vivo reprogramming) by delivering specific genes through viral delivery methods such as AAV as a way to promote the activation of endogenous stem cells to regenerative medicine (US 2021 / 0024895 A1). Compared to existing cell therapy methods, this method is cost- and time-efficient because the 'in vitro cell culture and transplantation' process is omitted, and it has the advantage of being easier to set the dosage and administer than cell therapy due to the nature of gene therapy. However, in the case of delivery using AAV, there may be unexpected side effects due to long-term expression of reprogramming factor genes (e.g., Yamanaka factors), and there may also be side effects due to the innate immunity response of AAV itself.

[0004] Uncontrolled overexpression of reprogramming factors in the body has been reported to cause adverse effects such as the development of teratoma and pluripotent stem cells in the body (Abad et al, Nature volume 502, 340-345 (2013)), the development of renal cancer due to epigenetic changes (Kotaro et al., Cell 156, 663-677 (2014)), and death due to dysfunction of the liver and small intestine (Parras et al, Nature aging, 3, 1509-1520 (2023)).

[0005] In other words, since bioreprogramming is a technology that can cause major changes in the body, its control is important, and if reprogramming proceeds uncontrolled, it can cause tumor development or organ failure.

[0006] Meanwhile, the mammalian liver is a representative organ with regenerative capacity, capable of restoring its original state through active tissue regeneration after a certain level of damage. However, in cases of severe liver damage, such as acute liver failure or cirrhosis due to chronic liver disease, treatment options are significantly limited. This situation is fueling the growing need for regenerative medicines that leverage methods to enhance the liver's inherent regenerative capacity.

[0007] Recently, it has been reported that dedifferentiation of specific cells is induced during the regeneration phase following tissue damage in various organs such as the liver, intestines, and muscles. In particular, in the liver, dedifferentiation of hepatocytes occurs after acute and chronic injury, transforming into a population of cells with increased dividing and differentiating potential, which contributes to tissue regeneration. These research results suggest that the formation of progenitor cells is induced during the liver regeneration process, and this phenomenon is defined as "adaptive cellular reprogramming."

[0008] In the present invention, we aimed to develop a method for inducing liver tissue regeneration with minimal side effects by delivering reprogramming factors specifically to the liver and inducing selective expression in the liver for a short period of time, and focused on lipid nanoparticles as a liver-specific delivery method.

[0009] When general lipid nanoparticles are administered intravenously in a living body, they are adsorbed to specific serum proteins and mostly accumulate in the liver and enter liver cells. However, due to the characteristics of nanometer-sized particles, they may accumulate in the spleen, lymph nodes, etc. other than the liver due to the mononuclear phagocyte system, or may accumulate in organs such as the lungs during circulation in the blood, which may cause side effects due to off-target effects.

[0010] Accordingly, the present inventors have made diligent efforts to develop a method capable of effectively inducing liver tissue regeneration while minimizing side effects by effectively delivering reprogramming factors specifically to the liver and inducing the formation of 'regenerative stem cells' through selective expression in the liver in a short period of time, thereby completing the present invention.

[0011] The purpose of the present invention is to provide a novel lipid nanoparticle capable of delivering mRNA specifically to the liver, a method for producing the same, and a liver disease treatment agent capable of regenerating the liver by delivering a biological reprogramming factor mRNA specifically to the liver using the same.

[0012] To achieve the above object, the present invention provides a lipid nanoparticle comprising an ionized lipid comprising one or more tertiary amine structures, a helper phospholipid, cholesterol or a cholesterol analogue, a polyethylene glycol-linked lipid, and galactosyl ceramide.

[0013] In the present invention, the lipid nanoparticle may be characterized as being for mRNA delivery.

[0014] In the present invention, the lipid nanoparticle may be characterized as being for liver-specific delivery of mRNA.

[0015] In the present invention, the lipid nanoparticle may be characterized by comprising an ionized lipid containing at least one tertiary amine structure: a helper phospholipid: cholesterol or a cholesterol analogue: a lipid linked to polyethylene glycol: galactosyl ceramide in a molar ratio of 20 to 50: 10 to 30: 30 to 60: 0.5 to 2: 0.5 to 2.

[0016] The present invention also provides an mRNA-lipid nanoparticle complex comprising mRNA and the lipid nanoparticle.

[0017] The present invention also provides a method for preparing the mRNA-lipid nanoparticle complex, comprising the following steps:

[0018] (a) a step of preparing an organic phase lipid nanoparticle by dissolving an ionized lipid containing one or more tertiary amine structures, a helper phospholipid, cholesterol or a cholesterol analogue, a polyethylene glycol-linked lipid, and galactosyl ceramide in alcohol; and

[0019] (b) A step of mixing the organic lipid nanoparticles with mRNA in an aqueous phase to encapsulate the mRNA in the lipid nanoparticles.

[0020] In the present invention, the manufacturing method may additionally include, after step (b), a step (c) of dialyzing and / or filtering the lipid nanoparticles in which the mRNA is encapsulated.

[0021] In the present invention, the manufacturing method may be characterized in that, in step (b), the organic phase containing the lipid nanoparticles and the aqueous phase containing mRNA are mixed in a volume ratio of 1:1.5 to 5.

[0022] In the present invention, the manufacturing method may be characterized in that in step (b), the ionized lipid containing one or more tertiary amine structures and mRNA are mixed at a mass ratio of 5 to 20:1.

[0023] The present invention also relates to a pharmaceutical composition for preventing or treating liver disease, comprising a cell reprogramming factor mRNA and the lipid nanoparticle.

[0024] In the present invention, the cell reprogramming factors may be characterized as Oct4, Sox2, Klf4, and C-Myc.

[0025] In the present invention, the liver disease may be characterized as being selected from the group consisting of acute liver damage, chronic liver damage, hepatitis, cirrhosis, liver failure, cirrhosis, and liver cancer.

[0026] In the present invention, the composition may be administered once or multiple times, and when administered multiple times, it may be characterized in that it is administered at intervals of 6 to 48 hours.

[0027] The present invention also relates to the use of lipid nanoparticles encapsulated with cell reprogramming factor mRNA for the prevention or treatment of liver disease.

[0028] The present invention also relates to the use of lipid nanoparticles encapsulating cell reprogramming factor mRNA in the manufacture of drugs for the prevention or treatment of liver diseases.

[0029] The present invention also relates to a method for preventing or treating liver disease, comprising a step of administering a lipid nanoparticle encapsulated with a cell reprogramming factor mRNA to a subject in need thereof.

[0030] The present invention also relates to lipid nanoparticles encapsulated with cell reprogramming factor mRNA for use in the prevention or treatment of liver diseases.

[0031] The lipid nanoparticle according to the present invention can deliver mRNA specifically to the liver with very high efficiency, and the lipid nanoparticle encapsulating the mRNA-based biological reprogramming factor mRNA is specifically delivered to the liver tissue of an individual requiring liver regeneration and induces expression in a short period of time, thereby making it easy to set the dosage and expression period of the therapeutic agent, thereby significantly improving the side effects of existing liver regeneration therapeutic agents using cell reprogramming factors.

[0032] Figure 1a is a schematic diagram showing the operation of a transgenic mouse (OSKM Tet-On inducible mouse) having the ROSA26-rtTA; Col1a1-TetO-OSKM genotype used in the present invention.

[0033] Figure 1b shows the results of quantitative real-time PCR analysis of Yamanaka factor expression in liver cells after oral administration of doxycycline to OSKM Tet-On induced mice.

[0034] Figure 1c shows the results of immunohistochemical staining for OCT4 expression in liver tissue after oral administration of doxycycline to OSKM Tet-On induced mice. CV = hepatic vein / PV = portal vein

[0035] Figure 1d shows the results of Western blot analysis of OCT4 expression in liver tissue over time after oral administration of doxycycline to OSKM Tet-On-induced mice. D1: 24 hours, D2: 48 hours, D3: 72 hours.

[0036] Figure 1e shows the results of quantitative real-time PCR confirming that liver cell-specific gene expression was reduced in liver cells after oral administration of doxycycline to OSKM Tet-On induced mice.

[0037] Figure 1f shows the results of immunohistochemical staining showing a decrease in liver cell-specific gene expression in liver tissue after oral administration of doxycycline to OSKM Tet-On induced mice.

[0038] Figure 1g shows the results of confirming the pattern of decreased glycogen storage capacity in liver cells after oral administration of doxycycline to OSKM Tet-On-induced mice. D2: 48 hours, D4: 96 hours

[0039] Figure 1h shows the results of immunofluorescence staining to confirm the cell division ability of liver cells after oral administration of doxycycline to OSKM Tet-On induced mice.

[0040] Figure 1i shows the results of confirming the cell division ability of liver cells by BrdU staining after negative administration of doxycycline to OSKM Tet-On induced mice.

[0041] Figure 1j shows the results of confirming changes in liver cell-specific drainage after negative administration of doxycycline to OSKM Tet-On induced mice.

[0042] Figure 1k shows the results of quantitative real-time PCR to confirm that liver cell-specific gene expression is reduced following doxycycline treatment after isolating liver cells from OSKM Tet-On induced mice.

[0043] Figure 2a is a schematic diagram showing an experimental plan to determine whether expression of OSKM can induce enhanced liver regeneration in an acetaminophen-induced liver injury mouse model (AILI mouse model).

[0044] Figure 2b shows the results of H&E staining to confirm whether OSKM expression can reduce liver damage in the AILI mouse model.

[0045] Figure 2c shows the results of western blot analysis to confirm whether OSKM expression can reduce apoptotic cell death in liver cells in an AILI mouse model.

[0046] Figure 2d shows the results of TUNEL staining to confirm whether OSKM expression can reduce necrotic cell death in liver cells in the AILI mouse model.

[0047] Figure 2e is a schematic diagram of two doses (moderate / severe) of AILI mouse models to confirm the mechanism of liver regeneration by bio-reprogramming.

[0048] Figure 2f shows the results of comparing the degree of tissue necrosis by H&E staining after 24 hours (300 mg / kg, moderate AILI, Day 1) or 96 hours (500 mg / kg, severe AILI, Day 4) of APAP administration in the AILI mouse model.

[0049] Figure 2g shows the results of checking AST and ALT levels by collecting blood 48 hours after a moderate AILI mouse model (administered with 300 mg / kg APAP).

[0050] Figure 2h shows the results of confirming the Ki-96 expression level 48 hours after a moderate AILI mouse model (administration of 300 mg / kg APAP).

[0051] Figure 2i shows the results of checking AST and ALT levels by collecting blood 96 hours after a severe AILI mouse model (administered with 500 mg / kg APAP).

[0052] Figure 2j shows the results of confirming the Ki-96 expression level after 96 hours in a severe AILI mouse model (administered with 500 mg / kg APAP).

[0053] Figure 3a shows the results of immunofluorescence staining of the liver for SOX9 / HNF4A 48 hours after APAP administration in a moderate AILI mouse model (administered 300 mg / kg APAP). CV = hepatic vein / PV = portal vein.

[0054] Figure 3b shows the quantification of SOX9+ liver cells in the hepatic portal vein and periphery of the hepatic vein at 24, 48, 72, and 96 hours after APSP administration in a moderate AILI mouse model (administered 300 mg / kg APAP). CV = hepatic vein / PV = portal vein

[0055] Figure 3c shows the results of quantitative real-time PCR analysis of the expression of liver progenitor cell-specific markers after oral administration of doxycycline to OSKM Tet-On induced mice.

[0056] Figure 3d shows the results of immunohistochemical staining for the expression of EpCAM after oral administration of doxycycline to OSKM Tet-On induced mice.

[0057] Figure 3e shows the results of immunohistochemical staining to confirm whether SOX9 expression is specific to the periphery of the hepatic vein after negative administration of doxycycline to OSKM Tet-On induced mice.

[0058] Figure 4a is a schematic diagram showing whether the dedifferentiation of liver cells by bio-reprogramming is mediated by an immune response.

[0059] Figure 4b shows the results of western blot analysis of the expression levels of HNF4A, SOX9, pSTAT3, and PCNA after administration of doxycycline to ROSA26-rtTA mice or OSKM Tet-On induced mice.

[0060] Figure 4c shows the results of western blot analysis of the expression levels of pSTAT3, SOX9, and PCNA over time after administration of APAP or doxycycline to OSKM Tet-On induced mice.

[0061] Figure 4d shows the results of immunofluorescence staining of F4 / 80 protein in liver tissue after administration of APAP or doxycycline to OSKM Tet-On induced mice.

[0062] FIG. 5a is a schematic diagram showing the configuration of C12-SPM-GAL lipid nanoparticles according to one embodiment of the present invention.

[0063] Figure 5b shows the results of comparing the mRNA intracellular delivery efficiency of C12-SPM-GAL lipid nanoparticles with that of Lipofectamine using luciferase mRNA.

[0064] Figure 5c shows the results of confirming the cytotoxicity of C12-SPM-GAL lipid nanoparticles.

[0065] Figure 6a shows the results of comparing the mRNA in vivo delivery efficiency of C12-SPM-GAL lipid nanoparticles with that of DLin-MC3-DMA lipid nanoparticles using luciferase mRNA.

[0066] Figure 6b shows the results of confirming the liver toxicity according to the mRNA in vivo delivery of C12-SPM-GAL lipid nanoparticles by ALT levels.

[0067] Figure 6c shows the results of confirming the liver toxicity according to the mRNA in vivo delivery of C12-SPM-GAL lipid nanoparticles using AST levels.

[0068] Figure 7a shows the results of in vitro transcription (IVT) of mRNAs synthesized from plasmid DNA encoding each OSKM factor, and agarose gel electrophoresis.

[0069] Figure 7b shows the results of manufacturing four types of C12-SPM-GAL LNPs, each encapsulating the mRNA of the OSKM factor, and evaluating the encapsulation efficiency using a ribogreen assay.

[0070] Figure 7c shows the results of treating mouse-derived fibroblasts with four types of C12-SPM-GAL LNPs, each containing the mRNA of the OSKM factor, and confirming protein expression by western blot.

[0071] Figure 7d shows the results of treating mouse-derived fibroblasts with four types of C12-SPM-GAL LNPs, each containing the mRNA of the OSKM factor, and confirming protein expression by immunofluorescence staining.

[0072] Figure 8a is a schematic diagram showing a mouse experimental plan to confirm in vivo liver cell reprogramming by introducing OSKM mRNA lipid nanoparticles.

[0073] Figure 8b shows the results of quantitative real-time PCR analysis of OSKM expression in liver tissue after introducing OSKM mRNA lipid nanoparticles into normal mice.

[0074] Figure 8c shows the results of H&E staining and MT staining to confirm whether necrosis or fibrosis occurred in liver tissue after introducing OSKM mRNA lipid nanoparticles into normal mice.

[0075] Figure 8d shows the results of quantitative real-time PCR analysis of Sox 9 and Ki-67 expression in liver tissue after introducing OSKM mRNA lipid nanoparticles into normal mice.

[0076] Figure 8e shows the results of immunofluorescence staining to confirm the expression of Sox 9 and Ki-67 in liver tissue after introducing OSKM mRNA lipid nanoparticles into normal mice.

[0077] Figure 8f shows the results of confirming the change in the expression levels of Sox 9, pHH3, and PCNA in liver tissue after introducing OSKM mRNA lipid nanoparticles into normal mice.

[0078] Figure 9a is a schematic diagram showing a mouse experimental plan to confirm the effect of promoting liver regeneration by administering OSKM mRNA lipid nanoparticles in a liver damage mouse model.

[0079] Figure 9b shows the degree of liver damage confirmed by inducing a mouse model of liver damage using a photograph of the entire liver and H&E staining of liver tissue.

[0080] Figure 9c shows the results confirming the increased survival rate following administration of OSKM mRNA lipid nanoparticles in a liver damage mouse model.

[0081] Figure 9d shows the results of confirming the effect of reducing liver necrosis following administration of OSKM mRNA lipid nanoparticles in a liver damage mouse model using H&E staining of liver tissue.

[0082] Figure 9e shows the results of immunofluorescence analysis of liver tissue confirming the effect of reducing cell death following administration of OSKM mRNA lipid nanoparticles in a liver damage mouse model.

[0083] Figure 9f shows the results of confirming the effect of reducing cell death by administering OSKM mRNA lipid nanoparticles in a liver damage mouse model using western blot of liver cells.

[0084] Figure 9g shows the results of immunofluorescence analysis of liver tissue to confirm the effect of liver cell dedifferentiation following administration of OSKM mRNA lipid nanoparticles in a liver injury mouse model.

[0085] Figure 9h shows the results of confirming the effect of liver cell dedifferentiation following administration of OSKM mRNA lipid nanoparticles in a liver injury mouse model using western blot of liver cells.

[0086]

[0087] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0088]

[0089] In the numerical ranges described herein, “within” is used to mean including (more than and less than) both critical ranges, and when both critical ranges are not included, the numerical ranges are described as “more than” and “less than”. The term “about” used in the numerical values ​​herein is used to mean including a range that is expected to exhibit substantially the same effect as the numerical values ​​described by those of ordinary skill in the art, and may be, for example, ±20%, ±10%, ±5%, etc. of the described numerical value, but is not limited thereto.

[0090]

[0091] In the present invention, an mRNA-based therapeutic agent was developed that specifically and briefly expresses a cell reprogramming factor in the form of mRNA in liver tissue to induce regeneration of damaged liver tissue. Specifically, in the present invention, a lipid nanoparticle capable of effectively delivering mRNA specifically to liver tissue was developed, and mRNA of Oct4, Sox2, Klf4, and c-Myc (Yamanaka factor, OSKM), which are transcription factors capable of inducing cell reprogramming, were encapsulated in the lipid nanoparticle and delivered to liver tissue, thereby inducing liver cell-specific reprogramming, promoting the production of liver progenitor cells, and forming a large number of 'regenerative stem cells' without tissue damage, thereby confirming that effective liver regeneration can be induced for severe liver damage.

[0092]

[0093] Accordingly, the present invention relates, in one aspect, to a lipid nanoparticle comprising an ionizable lipid comprising one or more tertiary amine structures, a helper phospholipid, cholesterol or a cholesterol analog, a polyethylene glycol-linked lipid, and galactosyl ceramide.

[0094] In the present invention, the 'ionizable lipid comprising one or more tertiary amine structures' may be selected from the group consisting of C12-SPM, SM-102 and ALC-0315, but is not limited thereto.

[0095] In the present invention, the 'ionizable lipid comprising one or more tertiary amine structures' may be an 'ionizable lipid comprising a polyamine structure', and the structure of each compound is disclosed in Advanced Healthcare Materials, 5(22), 2931-2941, or corresponds to a known compound, so a separate description is omitted.

[0096] In the present invention, the helper phospholipid is dioleoylphosphatidylethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), It may be, but is not limited to, 1,2-distearoyl-sn-glycero-3-phospho-L-serine (PS), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE).

[0097] In the present invention, the cholesterol or cholesterol analogue may be, but is not limited to, cholesterol, 20α-hydroxycholesterol, 7α-hydroxycholesterol, β-sitosterol, campesterol, fucosterol, or stigmastanol.

[0098] In the present invention, the lipid linked to the polyethylene glycol may be, but is not limited to, PEG-ceramide (e.g., C16-PEG2000 ceramide), DMG-PEG (e.g., 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000, DMG-PEG2000), or ALC-0159 (Methoxypolyethyleneglycoloxy(2000)-N,N-ditetradecylacetamide).

[0099] In the present invention, the lipid nanoparticles contain an ionized lipid containing one or more tertiary amine structures and galactosyl ceramide, thereby exhibiting a particularly high expression effect in liver tissue compared to existing lipid nanoparticles.

[0100] In the present invention, the lipid nanoparticle may be characterized by comprising an ionized lipid (e.g., C12-SPM) containing one or more tertiary amine structures: galactosyl ceramide in a molar ratio of 20 to 50:0.5 to 2.

[0101] In one embodiment, the lipid nanoparticle may include C12-SPM, dioleoylphosphatidylethanolamine (DOPE), cholesterol or a cholesterol analog, polyethylene glycol-ceramide (PEG-ceramide), or galactosyl ceramide.

[0102] In the present invention, the lipid nanoparticle may be characterized as being for mRNA delivery, and the lipid nanoparticle may be characterized as being for liver-specific delivery of mRNA.

[0103] In the present invention, it may be characterized in that the ionized lipid including at least one tertiary amine structure: helper phospholipid: cholesterol or cholesterol analog: polyethylene glycol-linked lipid: galactosyl ceramide is included in a molar ratio of 20 to 50: 10 to 30: 30 to 60: 0.5 to 2: 0.5 to 2.

[0104] For example, in the present invention, the lipid nanoparticle may be characterized by containing C12-SPM: dioleoylphosphatidylethanolamine (DOPE): cholesterol: polyethylene glycol-ceramide (PEG-ceramide): galactosyl ceramide in a molar ratio of 20 to 50: 10 to 30: 30 to 60: 0.5 to 2: 0.5 to 2.

[0105] In one embodiment, the lipid nanoparticle may include C12-SPM: dioleoylphosphatidylethanolamine (DOPE): cholesterol: polyethylene glycol-ceramide (PEG-ceramide): galactosyl ceramide in a molar ratio of 20 to 30: 20 to 30: 45 to 55: 1.2 to 1.8: 1.2 to 1.8.

[0106] In another embodiment, the lipid nanoparticle may include C12-SPM: dioleoylphosphatidylethanolamine (DOPE): cholesterol: polyethylene glycol-ceramide (PEG-ceramide): galactosyl ceramide in a molar ratio of about 20: about 26.5: about 50.5: about 1.5: about 1.5.

[0107] In the present invention, the lipid nanoparticles can be used for drug delivery purposes, such as as mRNA-based drug delivery vehicles, and specifically, as drug delivery vehicles targeting the liver. In the present invention, the lipid nanoparticles exhibit significantly improved efficiency in delivering mRNA specifically to liver tissue without cytotoxicity or hepatotoxicity.

[0108] Therefore, the present invention relates to the liver-specific delivery use of mRNA of the lipid nanoparticles from another perspective,

[0109] From another perspective, it relates to a method for delivering mRNA to the liver tissue of an individual in need thereof using the above lipid nanoparticles.

[0110] From another perspective, the present invention relates to the use of the lipid nanoparticles for the preparation of liver-specifically delivered mRNA drugs.

[0111]

[0112] In another aspect, the present invention relates to an mRNA-lipid nanoparticle complex comprising mRNA and the lipid nanoparticle.

[0113] The above mRNA may be encapsulated inside the lipid nanoparticle.

[0114]

[0115] In another aspect, the present invention relates to a method for producing the mRNA-lipid nanoparticle complex, wherein the method may include the following steps:

[0116] (a) a step of preparing an organic phase lipid nanoparticle by dissolving an ionized lipid containing one or more tertiary amine structures, a helper phospholipid, cholesterol or a cholesterol analogue, a polyethylene glycol-linked lipid, and galactosyl ceramide in alcohol; and

[0117] (b) A step of mixing the organic lipid nanoparticles with mRNA in an aqueous phase to encapsulate the mRNA in the lipid nanoparticles.

[0118] In one embodiment, the method for preparing the mRNA-lipid nanoparticle complex may include the following steps:

[0119] (a) a step of preparing C12-SPM-GAL lipid nanoparticles in an organic phase by dissolving C12-SPM, dioleoylphosphatidylethanolamine (DOPE), cholesterol, polyethylene glycol-ceramide (PEG-ceramide), and galactosyl ceramide in alcohol; and

[0120] (b) A step of mixing the organic C12-SPM-GAL lipid nanoparticles with the aqueous phase mRNA to encapsulate the mRNA into the C12-SPM-GAL lipid nanoparticles.

[0121] In the present invention, the alcohol may be methanol, ethanol, isopropanol or butanol, but is not limited thereto.

[0122] In the present invention, the aqueous solution may be a weakly acidic buffer having a pH of 4.0 to 4.5, for example, a sodium citrate buffer (pH of about 4.0 to about 4.5) or a sodium acetate buffer (pH of about 4.0 to about 4.5) of about 10 to about 20 mM, but is not limited thereto.

[0123] In the present invention, the manufacturing method may further include, after step (b), a step (c) of dialyzing and / or filtering the lipid nanoparticles (e.g., C12-SPM-GAL lipid nanoparticles) in which the mRNA is encapsulated.

[0124] In the present invention, the dialysis is a step for purifying lipid nanoparticles, and various methods known in the art can be used, and for example, it can be performed using a method of Tubular Dialysis, Spin Column Dialysis, Dynamic Dialysis, intrafiltration, and Dialysis Combo.

[0125] In the present invention, the filtration is a step for concentrating lipid nanoparticles, and various methods known in the art can be used, for example, ultracentrifugation, vacuum centrifugation, TFF (Tangential Flow Filtration, Precipitation Methods, Lyophilization).

[0126] In the present invention, in the step (b), the organic phase containing the lipid nanoparticle (e.g., C12-SPM-GAL lipid nanoparticle) and the aqueous phase containing mRNA may be mixed in a volume ratio of 1:1.5 to 5.

[0127] In the present invention, it may be characterized in that in step (b), the ionizable lipid (e.g., C12-SPM) containing one or more tertiary amine structures and mRNA are mixed at a mass ratio of 5 to 20:1.

[0128] In the present invention, the mixing may be microfluidic mixing that mixes the organic phase and the aqueous solution through a micrometer-sized channel, and this may be mixing using a microfluidic mixing device.

[0129] In another embodiment, the mixing may be bulk mixing, and the bulk mixing may be pipette mixing or vortex mixing. In the present invention, pipette mixing may be a method of mixing by pipetting an aqueous solution into an organic phase using a pipette for 20-30 seconds, and vortex mixing may be a method of mixing by adding the organic phase into the aqueous solution at once using a pipette while vortexing the aqueous solution with a vortex mixer turned on at the lowest intensity and vortexing for about 20-30 seconds.

[0130] In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating liver disease, comprising a cell reprogramming factor mRNA and the lipid nanoparticle.

[0131] The above cell reprogramming factor mRNA is encapsulated inside the lipid nanoparticle, which may be an mRNA-lipid nanoparticle complex, and the lipid nanoparticle may be a carrier that specifically delivers the cell reprogramming factor mRNA to liver tissue.

[0132] In the present invention, the cell reprogramming factor may specifically be a cell differentiation factor, and may be any combination selected from the group consisting of Oct4, Sox2, Klf4, C-Myc, Nanog, Lin28, Esrrb (Estrogen-related receptor beta), Glis1, and L-Myc, for example, a combination of Oct4, Sox2, Klf4, and Nanog, or a combination of Oct4, Sox2, Klf4, c-Myc, and Lin28.

[0133] In the present invention, the cell reprogramming factor may be, but is not limited to, a Yamanaka factor.

[0134] In the present invention, Yamanaka factors are key transcription factors used to revert mature cells to early-stage induced pluripotent stem cells (iPSCs), and may include OCT4 (Octamer-binding transcription factor 4), SOX2 (SRY-box transcription factor 2), KLF4 (Kruppel-like factor 4), and c-MYC (Myelocytomatosis viral oncogene homolog).

[0135] In one embodiment, the cell reprogramming factor may be a combination of Oct4, Sox2, Klf4 and C-Myc.

[0136] In the present invention, the liver disease includes all liver diseases in which liver tissue is damaged and tissue regeneration is required, for example, all liver diseases in which liver transplantation is considered.

[0137] In the present invention, the liver disease may include, but is not limited to, acute liver damage, chronic liver damage, hepatitis, cirrhosis, liver failure, liver cirrhosis, liver tumor, and liver cancer.

[0138] For example, the liver disease may be characterized by being selected from the group consisting of, but not limited to, acute liver failure, chronic liver failure, drug-induced liver damage, liver damage due to exposure to toxic substances, viral acute hepatitis, ischemic liver damage, traumatic liver damage, alcoholic liver disease, metabolic fatty liver disease, metabolic steatohepatitis, chronic viral hepatitis, autoimmune cirrhosis, hepatitis, cirrhosis, liver tumor, and liver cancer.

[0139] In the present invention, the term "reprogramming" may refer to the process of altering or reversing the differentiation state of a somatic cell. The cell may be partially or terminally differentiated prior to reprogramming. Reprogramming may involve completely converting the differentiation state of a somatic cell to a pluripotent cell. Such complete reversal of differentiation may produce induced pluripotent (iPS) cells. As used herein, reprogramming may also include partial reversal of a cell's differentiation state, such as to a pluripotent state or to a somatic cell that is neither pluripotent nor pluripotent but has lost one or more specific characteristics of the differentiated cell from which it originated, such as directly reprogramming a differentiated cell into another somatic cell type. Reprogramming may involve altering, for example, reversing, at least some of the heritable patterns of nucleic acid modifications (e.g., methylation), chromatin condensation, epigenetic changes, and genomic imprinting that occur during cell differentiation as a zygote develops into an adult.

[0140] In the present invention, the term "reprogramming factor" may refer to a molecule associated with cellular "reprogramming," i.e., differentiation and / or dedifferentiation and / or transdifferentiation, which allows cells to transform into a different cell type or phenotype. Reprogramming factors generally affect the expression of genes associated with cell differentiation, dedifferentiation, and / or transdifferentiation. Transcription factors are examples of reprogramming factors.

[0141] In the present invention, the pharmaceutical composition may include a pharmaceutically acceptable carrier.

[0142] In the present invention, the term "pharmaceutically acceptable" may mean a compound, material, composition, and / or formulation that is suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio within the scope of sound medical judgment.

[0143] As used herein, the term "pharmaceutically acceptable carrier" may mean any pharmaceutically acceptable substance, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or stearic acid), or solvent encapsulating material, which is involved in carrying or transporting the subject compound from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient.

[0144] In the present invention, the composition may be administered once or multiple times.

[0145] In the present invention, the composition may be characterized in that it is administered at intervals of about 6 to about 48 hours when administered multiple times.

[0146] In another embodiment, the composition may be characterized in that it is administered at intervals of about 12 to about 48 hours when administered multiple times.

[0147] In another embodiment, the composition may be characterized in that it is administered at intervals of about 24 to about 48 hours when administered multiple times.

[0148] In another aspect, the present invention relates to a method for preventing or treating liver disease, comprising a step of administering the mRNA-lipid nanoparticle complex or the pharmaceutical composition to a subject in need thereof.

[0149] In another aspect, the present invention relates to the mRNA-lipid nanoparticle complex or the pharmaceutical composition for use in preventing or treating liver diseases.

[0150] In another aspect, the present invention relates to the use of the mRNA-lipid nanoparticle complex or the pharmaceutical composition for the preparation of a drug for the prevention or treatment of liver disease.

[0151] In another aspect, the present invention relates to the use of lipid nanoparticles encapsulated with cell reprogramming factor mRNA for the prevention or treatment of liver disease.

[0152] In another aspect, the present invention relates to the use of lipid nanoparticles encapsulating cell reprogramming factor mRNA in the manufacture of drugs for the prevention or treatment of liver diseases.

[0153] In another aspect, the present invention relates to a method for preventing or treating liver disease, comprising administering to a subject in need thereof a lipid nanoparticle encapsulated with a cell reprogramming factor mRNA.

[0154] In another aspect, the present invention relates to lipid nanoparticles encapsulated with cell reprogramming factor mRNA for use in the prevention or treatment of liver diseases.

[0155] The term "prevention" in the present invention refers to any action that prevents the onset of a disease or delays its progression through the administration of the composition. Furthermore, the term "treatment" as used herein refers to any action that improves, alleviates, or completely cures the symptoms of a disease through the administration of the composition.

[0156] In the present invention, “subject” may be used interchangeably with “object” and means a mammal suffering from or at risk of a condition or disease that can be alleviated, suppressed or treated by administering a composition according to the present invention, and preferably means a human.

[0157] The term “administration” in the present invention refers to an act of introducing the composition of the present invention into a subject by any appropriate method, and the route of administration may be administered through various routes, such as oral or parenteral, as long as it can reach the target tissue. Parenteral administration may be intramuscular (IM), intravenous (IV), subcutaneous (SC), intraperitoneal (IP), intratumoral (IT), intradermal (ID), or intracerebral injection, and the dosage may vary depending on the patient’s condition and weight, the degree of disease, the drug form, the route of administration, and the time of administration, but may be appropriately selected by a person skilled in the art.

[0158] In one embodiment, the composition may be for intravenous administration.

[0159] The dosage of the composition of the present invention for the human body may vary depending on the patient's age, body weight, sex, dosage form, health condition, and degree of disease.

[0160] In the present invention, when formulating the composition, it is manufactured using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc. Non-aqueous solvents and suspensions may include vegetable oils such as propylene glycol, polyethylene glycol, olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, and gelatin.

[0161]

[0162] Example

[0163] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples. Reagents not specifically indicated in the examples were from Sigma Aldrich, and antibodies were from Cell signaling technology. In cases where the same experiment was repeated, only the initial experimental method was described in detail.

[0164]

[0165] [Example 1] Discovery of liver cell dedifferentiation in a mouse model overexpressing a reprogramming factor.

[0166]

[0167] 1-1. OSKM-expressing Tet-On system transgenic mice

[0168] To observe the in vivo reprogramming phenotype, transgenic mice with the ROSA26-rtTA; Col1a1-TetO-OSKM genotype (The Jackson laboratory, #011004) were used (Fig. 1a). When doxycycline is administered to these transgenic mice, rtTA (reverse tetracycline inducible transactivator) binds to the TRE promoter, inducing the expression of Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc; OSKM).

[0169] Hereinafter, unless otherwise stated, all animal experiments in the present invention used male mice aged 8 to 12 weeks. The control group was administered high-pressure steam-sterilized drinking water not containing doxycycline, and the experimental group was administered doxycycline (0.15 mg / ml, 5% sucrose) in the drinking water. The drinking water was administered ad libitum without any restrictions on the intake amount.

[0170]

[0171] 1-2. Confirmation of OSKM gene expression

[0172] After 72 hours of drinking water administration, liver cells were isolated from control and experimental mice (n=3 per group) using the two-step collagenase perfusion method (see Charni-Natan, M. & Goldstein, I. Protocol for Primary Mouse Hepatocyte Isolation. STARProtoc,1, 100086 (2020).), RNA was extracted, and cDNA was synthesized using this as a template, followed by quantitative real-time PCR.

[0173] RNA extraction from liver tissue or liver cells was performed according to the manufacturer's protocol for the Easy-BLUE total RNA extraction kit (iNtRON Biotechnology, 17061). cDNA synthesis was performed according to the manufacturer's protocol for the PrimeScript RT reagent kit (TAKARA, RR036A). 1,000 ng of RNA served as a template for cDNA synthesis, and the synthesis was performed at 37°C for 15 min and 95°C for 1 min. RT-qPCR was performed using QuantStudio 3 (ThermoFisher Scientific) according to the manufacturer's protocol, and TB green (TAKARA, RR420) was used as the reaction reagent according to the manufacturer's protocol.

[0174] The primer sequences used are as follows.

[0175] FR18s rRNAGTA ACC CGT TGA ACC CCA TTCCA TCC AAT CGG TAG TAG CGPou5f1ACA TCG CCA ATC AGC TTG GAGA ACC ATA CTC GAA CCA CAT CCSox2ACA GAT GCA ACC GAT GCA CCTGG AGT TGT ACT GCA GGG CGKlf4GCA CAC CTG CGA ACT CAC ACCCG TCC CAG TCA CAG TGG TAAc-MycACC ACC AGC AGC GAC TCT GATGC CTC TTC TCC ACA GAC ACC

[0176] As a result, it was confirmed that Yamanaka factor was effectively expressed in the experimental group compared to the control group (Fig. 1b).

[0177]

[0178] 1-3. Confirmation of OSKM protein expression

[0179] After 72 hours of negative administration, the livers of control and experimental mice (n = 3 per group) were removed, fixed in 4% paraformaldehyde (Sigma) for 12 hours, embedded in paraffin, and subjected to immunohistochemical analysis.

[0180] For immunohistochemical analysis, 4 μm-thick paraffin sections were treated with 0.3% H2O2 (in methanol) to block endogenous peroxidase activity. The slides were then immersed in tris-EDTA buffer (pH 9.0) for antigen retrieval and treated in a water bath at 95°C for 20 min. Subsequently, the slides were washed with TBST (0.1% Triton X-100 in tris-buffered saline) for 10 min and blocked with 10% normal goat serum in TBST for 1 h. The slides were incubated with primary antibody (OCT4 antibody (BD, 611203, 1:400 dilution)) overnight at 4°C or for 1 h at room temperature. After washing three times with TBST, the slides were treated with HRP-conjugated secondary antibody (Jackson Immunoresearch, 111-035-003 / 115-035-003) in TBST (1:10000 dilution) for 2 hours at room temperature. Next, 3,3'-diaminobenzidine (DAB) solution (Vector Laboratories, SK-4100) was reacted for 2–6 minutes. Nuclear staining was performed using hematoxylin (Sigma, GHS3). The slides were mounted with MOWIOL mounting solution (Sigma), and the samples were observed using a light microscope (Leica DM500) or an automated multimodal tissue analysis system (PerkinElmer, Vectra).

[0181] As a result, it was confirmed that OCT4 protein was effectively expressed in the experimental group compared to the control group (Fig. 1c).

[0182] In addition, after 24 (D1), 48 (D2), and 72 (D3) hours of drinking water administration, the livers of the control and experimental mice were extracted, homogenized, and proteins were extracted using RIPA buffer (supplemented with 1% protease inhibitor (Roche's cOmplete product) and 0.1% sodium orthovanadate (T&I's BSO-9100 product)). After quantifying the proteins, 10-20 μg of proteins were loaded onto SDS-PAGE, and the expression of OCT4 protein was confirmed by western blot. The antibody information used in the western blot is as follows: Primary antibody: OCT4 (BD, 611004, 1:1000 dilution), beta-actin (Santa Cruz, sc-47778, 1:1000) / Secondary antibody: HRP-conjugated secondary antibody (Jackson Immunoresearch, 111-035-003 / 115-035-003) in TBST (1:10000 dilution)

[0183] As a result, the OCT4 protein gradually increased from 48 hours (D2), and a distinct band of OCT4 protein was confirmed at 72 hours (D3) (Fig. 1d).

[0184]

[0185] 1-4. Confirmation of expression of liver cell function markers

[0186] After 72 hours of negative administration, liver cells were isolated from control and experimental mice (n = 3 per group) using a two-step collagenase perfusion method, and RNA was extracted. cDNA was synthesized using this as a template, and quantitative real-time PCR was performed to determine the expression levels of Alb, Arg1, AldoB, Cyp3a11, Pepck, and Ttr genes, which are markers of liver cell function, in the experimental group.

[0187] The primer sequences used are as follows.

[0188] FRAlbGCG CAG ATG ACA GGG CGG AAGTG CCG TAG CAT GCG GGA GGArg1CAT TGG CTT GCG AGA CGT AGA CGCT GAA GGT CTC TTC CAT CAC CAldoBTGT CTG GAG GTA TGA GTG AGGCTG GGT TGC CTT CTT GTT TGCCyp3a11CCT GGG TGC TCC TAG CAA TCGGA GAG GCG TTT GAC CAT CAPepckGGC GAT GAC ATT GCC TGG ATG ATGT CTT CAC TGA GGT GCC AGG ATtrCAC CAA ATC GTA CTG GAA GAC AGTC GTT GGC TGT GAA AAC CAC

[0189] As a result, liver cells in the experimental group of mice showed an overall decrease in liver cell-specific gene expression due to reprogramming factor expression (Fig. 1e). This confirmed the possibility of partial dedifferentiation of liver cells by reprogramming factor expression.

[0190]

[0191] Additionally, after 72 hours of negative administration, livers were removed from control and experimental mice (n = 3 per group), and OCT (Optimal cutting temperature) blocks were prepared, and HNF4A protein was stained through immunofluorescence staining.

[0192] For OCT blocks, livers were removed from mice, washed in PBS, and fixed in 4% PFA at 4°C for 12 hours. The fixed liver tissues were immersed in 30% sucrose in PBS solution at 4°C overnight to induce dehydration. The prepared liver tissues were cryopreserved in Tissue-Tek optimal cutting temperature (OCT) compound (Sakura Finetek). For immunofluorescence analysis, 8-μm-thick frozen sections were incubated with primary antibody (HNF4a antibody (Abcam, ab41898, 1:400)) in 4% bovine serum albumin (BSA) in TBST overnight at 4°C, followed by incubation with fluorochrome-conjugated secondary antibody (Invitrogen, Alexa Fluor 488 / 594 / 647 goat IgG, host species: Mouse, Rabbit) for 2 hours at room temperature. Nuclear staining was performed using 4',6-Diamidino-2-phenylindole (DAPI). Slides were mounted with a cover glass using MOWIOL mounting solution, and samples were observed using a fluorescence microscope (Olympus BX53 or Leica THUNDER Imager).

[0193] HNF4A is a transcription factor essential for maintaining liver cell function and homeostasis. The decreased expression of HNF4A, along with the decreased expression of liver cell-specific markers identified in Examples 1-4, suggests that expression of reprogramming factors can induce partial dedifferentiation of liver cells (Fig. 1f).

[0194]

[0195] 1-5. Checking glycogen storage in liver cells

[0196] After 48 (D2) and 96 (D4) hours of negative administration, livers were removed from control and experimental mice (n = 2 per group), fixed, and embedded in paraffin. The intracellular glycogen storage was confirmed through Periodic acid-Schiff staining.

[0197] In the same manner as in Example 1-3, paraffin blocks were prepared from liver tissue, and 5 μm-thick sections were prepared. PAS staining was performed using the Periodic Acid Schiff Stain kit (ScyTek Laboratories, PAS-2-IFU) according to the manufacturer's protocol. After staining, slides were mounted using Canada balsam (Sigma) and observed using an optical microscope (Leica DM500) or an automated multimodal tissue analysis system (PerkinElmer, Vectra).

[0198] As a result, it was confirmed that the glycogen storage capacity, an essential function of the liver, was reduced by the expression of reprogramming factors (Fig. 1g).

[0199]

[0200] 1-6. Confirmation of liver cell division capacity

[0201] After 72 hours of negative administration, livers were removed from control and experimental mice (n = 3 per group), and OCT (Optimal Cutting Temperature) blocks were prepared. Immunofluorescence staining was performed for Ki-67 protein and bromodeoxyuridine (BrdU), respectively. The antibodies used were Ki-67 antibody (Abcam, ab16667, 1:400) and BrdU antibody (Novus Biologicals, NBP2-14890, 1:200).

[0202] Pulse-and-chase experiments were performed for BrdU staining. BrdU (150 mg / kg, dissolved in 0.9% NaCl) was administered intraperitoneally 3 h before euthanasia of mice (i.e., after 69 h of doxycycline administration).

[0203] As a result, it was confirmed that the division ability of liver cells increased in the experimental group of mice in which the reprogramming factor was expressed (Fig. 1h, Fig. 1i).

[0204]

[0205] 1-7. Confirmation of changes in liver cell ploidy

[0206] After 72 hours of oral administration, liver cells were isolated from control and experimental mice using a two-step collagenase perfusion method and fixed in 75% ethanol in PBS for 15 minutes at room temperature. The fixed liver cells were treated with 1% RNase A in PBS for 30 minutes at room temperature to remove RNA contamination, and the RNase-treated liver cells were stained with PI solution (1 mg / ml in distilled water). The ploidy of the liver cells was then evaluated using a BD celesta flow cytometer.

[0207] As a result, 2N and 4N increased in the experimental group mice compared to the control group mice, confirming that liver cell-specific drainage decreased after reprogramming (Fig. 1j).

[0208]

[0209] 1-8. Confirmation of liver cell dedifferentiation through expression of liver cell-specific reprogramming factors.

[0210] Liver cells were isolated from the transgenic mice of Example 1-1 using a two-step collagenase perfusion method. The isolated liver cells were stained with trypan blue, and the number of viable cells was measured using a hemacytometer. 1x10 were then seeded onto a collagen-coated 60 pi culture dish. 6 Cells were seeded and cultured in Dulbecco's modified Eagle's medium (DMEM, supplemented with 10% FBS and 1% penicillin / streptomycin). The culture medium was changed 3 hours after seeding to stabilize the liver cells, and then every 24 hours. To induce the expression of reprogramming factors, 0.5-1 μg / ml doxycycline was dissolved in the culture medium and treated. For the control group, ultra pure water (UPW, Beiosesang W2006), a solvent for doxycycline, was dissolved in the culture medium and treated. After in vitro culture for a total of 72 hours, RNA was extracted from the cells of the experimental and control groups, cDNA was synthesized using this as a template, and quantitative real-time PCR was performed.

[0211] The primer sequences used are as follows.

[0212] FRFahACG ACT GGA GCG CAC GAG ACAGG GCT GGC TGT GGC AGA GAHnf4aAAA TGT GCA GGT GTT GAC CACTC ACG CTC CTC CTG AAG AACyp3a11CCT GGG TGC TCC TAG CAA TCGGA GAG GCG TTT GAC CAT CACyp2e1TGG TCC TGC ATG GCT ACA AGCGG GCC TCA TTA CCC TGT TT

[0213] As a result, the expression of liver cell-specific markers Fah, Hnf4a, Cyp3a11, and Cyp2e1 was all reduced (Fig. 1k). This suggests that the phenomenon of liver cell dedifferentiation through in vivo reprogramming is reproduced through the expression of liver cell-specific reprogramming factors.

[0214]

[0215] [Example 2] Confirmation of the liver regeneration effect through bioreprogramming

[0216] After observing the phenomenon of liver cell dedifferentiation according to bio-reprogramming in Example 1, bio-reprogramming was induced in an acetaminophen-induced liver injury model (AILI) to determine whether enhanced liver regeneration could be induced.

[0217] Acetaminophen (APAP, Sigma A7085) was dissolved in normal saline at 55°C at 20 mg / ml and administered intraperitoneally to the transgenic mice of Example 1 at a dose of 300 mg / kg or 500 mg / kg at 36°C. APAP administration was performed after a 12-hour fast, and food was provided immediately after APAP administration. Doxycycline was administered via drinking water immediately after APAP administration in the same manner as in Example 1, and was administered for up to 3 days (Fig. 2a).

[0218]

[0219] 2-1. Tissue necrosis 2 days after induction of liver damage

[0220] Forty-eight hours after administration of APAP (300 mg / kg), the mouse livers were removed, fixed, and embedded. Hematoxylin (H9627, Sigma Aldrich) and eosin (000E0614, Samchun Chemicals) staining was performed according to the manufacturer's protocol, and the degree of tissue necrosis was confirmed (n=4 per group).

[0221] In addition, 48 hours after APAP administration, the livers of the mice were removed, proteins were extracted, and western blot was performed using OCT4 antibody or Cleaved Caspase-3 (Asp175) antibody (Cell signaling technology, #9661, 1:1000 dilution) to confirm the expression of OCT4 and cleaved caspase-3, thereby confirming cell death after AILI due to bio-reprogramming.

[0222] Finally, 48 hours after APAP administration, the mouse livers were removed, fixed, and embedded. TdT-mediated dUTP Nick End Labeling (TUNEL) staining was performed using an in situ apoptosis detection kit (RnD Systems, 4810-30-K) according to the manufacturer's protocol to determine the degree of tissue necrosis. To evaluate apoptosis after AILI, tissues where apoptosis occurred were stained with 3,3'-Diaminobenzidine (DAB), and nuclei were stained with hematoxylin (Sigma, GHS3). The samples were observed under a light microscope (Leica DM500).

[0223] As a result, we were able to confirm the effect of reducing liver damage according to bioreprogramming in the acetaminophen-induced liver damage model (Fig. 2b), and we were also able to confirm through western blot and TUNEL staining that both apoptotic cell death and necrotic cell death were also reduced (Fig. 2c, Fig. 2d).

[0224]

[0225] 2-2. Tissue necrosis over time after induction of moderate / severe liver damage

[0226] In order to verify whether the reduction in liver damage by bio-reprogramming confirmed in Example 2-1 was due to an increase in liver regeneration ability or a reduction in liver damage itself, two doses of AILI models were used to determine the degree of liver damage over time (Fig. 2e).

[0227] After inducing moderate AILI (300 mg / kg, 24 hours, D1) and severe AILI (500 mg / kg, 96 hours, D4) by APAP administration, the mouse livers were removed, fixed, embedded, and H&E stained to determine the degree of tissue necrosis (n=3 per group).

[0228] As a result, it was confirmed that the degree of tissue necrosis was high in sever AILI (Fig. 2f).

[0229]

[0230] 2-3. Evaluation of liver damage and liver regeneration over time after induction of moderate / severe liver damage

[0231] After APAP administration at 300 mg / kg or 500 mg / kg, mice were bled and livers were removed at 48 and 96 hours, respectively. Submandibular blood collection was performed using a 21G syringe needle to puncture the facial vein 1-2 mm deep, and the resulting blood was collected in tubes and refrigerated. After bled and liver extraction were completed, the mouse blood was stored at room temperature without shaking, and the serum was separated by centrifugation at 3000 g for 10 minutes, aliquoted, and stored at -80°C. The aliquoted serum was used to analyze AST / ALT levels using a serum analyzer (Fujifilm, Dri-chem). Meanwhile, the mouse livers were fixed and embedded after extraction, and Ki-67 protein was stained using immunohistochemistry (n = 4-6 per group).

[0232] As a result, there was no significant difference in the AST / ALT levels at 48 hours (Fig. 2g), but the AST / ALT levels at 96 hours were significantly reduced in the mice in which bio-reprogramming was induced together with AILI (Fig. 2h), confirming that although AILI was at the same level, liver regeneration was effectively performed by the bio-reprogramming factor. In addition, it was confirmed that the expression of Ki-67 protein, a liver regeneration marker, increased in the mice in which bio-reprogramming was induced together with AILI (Fig. 2i), confirming that the present invention has the potential to be a regenerative medicine drug for bio-reprogramming.

[0233] For reference, the 96-hour time point corresponds to the point at which liver regeneration is completed, and since Ki-67 positive cells at the 96-hour time point are a phenotype that indicates that regeneration is not completely completed, the observation of fewer Ki-67 positive cells in the APAP+Dox group compared to the APAP only group means that liver regeneration occurred more quickly through bioreprogramming (Fig. 2j).

[0234]

[0235] [Example 3] Confirmation of the mechanism for inducing liver progenitor cells through bioreprogramming.

[0236] Typically, liver regeneration after acute liver injury involves the interaction and participation of most cells that make up the liver. It is well known that liver cells near the site of necrosis or apoptosis due to liver injury undergo dedifferentiation, increasing the expression of fetal / oncogenic genes and enabling them to function as hepatic progenitor cells (Ben-Moshe et al., Cell stem cell, 2022 / DOI: 10.1016 / j.stem.2022.04.008). After observing the dedifferentiation of liver cells and enhanced liver regenerative capacity through in vivo reprogramming, we aimed to investigate the correlation between the endogenous liver regeneration mechanism inherent in liver tissue and the dedifferentiation of liver cells through exogenous in vivo reprogramming.

[0237] To this end, in the AILI mouse model of Example 2, the livers of the mice were extracted at 24, 48, 72, and 96 hours after administration of 300 mg / kg of APAP, fixed, and OCT blocks were prepared, and SOX9 and HNF4A were stained using immunofluorescence staining. The antibodies used were HNF4a antibody (Abcam, ab41898, 1:400) and SOX9 antibody (Merck, ab5535, 1:400), respectively.

[0238] SOX9 is a representative marker of hepatic progenitor cells induced after liver damage. It is rarely expressed in liver cells while maintaining liver homeostasis, but is observed to be expressed together with HNF4A, a liver cell marker, during liver damage (Pu et al., Nature Genetics volume 55, pages 651-664 (2023)).

[0239] Compared to control mice in which liver damage was not induced, SOX9 / HNF4A immunofluorescence staining of the liver 48 hours after AILI confirmed a significant increase in SOX9-expressing cells around the hepatic vein (Fig. 3a). In addition, when SOX9+ liver cells in the hepatic vein and periportal vein were quantified through immunofluorescence staining at 24, 48, 72, and 96 hours after AILI, it was confirmed that there was almost no induction of hepatic progenitor cells around the portal vein, whereas there was a significant increase in the induction of hepatic progenitor cells around the hepatic vein at 24 and 48 hours (Fig. 3b).

[0240] Meanwhile, after 72 hours of drinking water administration to the mice of Example 1, liver cells from the control and experimental groups were isolated by two-step collagenase perfusion, RNA was extracted, and cDNA was synthesized using the RNA as a template. The expression of liver progenitor cell-specific markers was confirmed through qPCR. In addition, the expression of EpCAM and HNF4A was confirmed through immunofluorescence staining using EpCAM antibody (Invitrogen, 14-9326-82, 1:400 dilution) and HNF4A antibody, and SOX9 was stained and SOX9+ liver cells in the hepatic vein and periportal vein were quantified.

[0241] The primer information used at this time is as follows.

[0242] FRSox9AGA TAA GTT CCC CGT GTG CATGA CGT GTG GCT TGT TCT TGCd44CTC CAG ACA ACC ACC AGG ATATC CGT TCT GAA ACC ACG TCLy6aGAC CCT GGA GGC ACA CAG CCCAT GTG GGA ACA TTG CAG GAC CCCCd133CTG CGA TAG CAT CAG ACC AAG CCTT TTG ACG AGG CTC TCC AGA TCEpcamGAG TCC GAA GAA CCG ACA AGG AGAT GTG AAC GCC TCT TGA AGC G

[0243] As a result, similar to the pattern of hepatic progenitor cell induction observed after AILI, the expression of hepatic progenitor cell-specific markers in liver cells after in vivo reprogramming was significantly increased (Fig. 3c), and representative hepatic progenitor cell markers, EpCAM (Yoon et al., Hepatology, 2011 / DOI 10.1002 / hep.24122) and HNF4A, were co-expressed, confirming that hepatic progenitor cells were induced by in vivo reprogramming factors (Fig. 3c, Fig. 3d).

[0244] Finally, we confirmed the expression of SOX9 through immunohistochemical staining, and found that unlike the hepatic progenitor cells induced specifically around the hepatic vein after AILI, the hepatic progenitor cells induced after in vivo reprogramming were not location-specific (Fig. 3e).

[0245] In summary, we confirmed that in vivo reprogramming induces hepatic progenitor cells, which exhibits patterns similar to intrinsic liver regeneration mechanisms. However, unlike intrinsic liver regeneration mechanisms, the induction of hepatic progenitor cells is non-location-specific, providing insight into the accelerated liver regeneration mechanism following acute liver injury by in vivo reprogramming.

[0246] It is generally known that the induction of hepatic progenitor cells after liver injury occurs through interactions with other cells present in the niche surrounding liver cells. In particular, it has been recently revealed that Kupffer cells, liver-specific macrophages, secrete the cytokine IL-6 after liver injury and induce the dedifferentiation of hepatocytes surrounding the damaged area (Li et al., Cell stem cell, Volume 30, Issue 3, pages 283-299 (2023)). Therefore, we aimed to determine whether the dedifferentiation of hepatocytes by in vivo reprogramming is mediated by an immune response (Fig. 4a).

[0247] ROSA26-rtTA mice were produced by mating the transgenic mice of Example 1 with C57BL6 / J wild-type mice to obtain heterozygous mice, and then mating the heterozygous mice with each other. 8-week-old ROSA26-rtTA mice or the transgenic mice of Example 1 were administered regular drinking water without doxycycline to the control group, and doxycycline (0.15 mg / ml) was administered drinking water to the experimental group (n = 3 per group).

[0248] After 72 hours of drinking, liver cells were isolated from the control and experimental groups of ROSA26-rtTA mice or the transgenic mice of Example 1 using a two-step collagenase perfusion method, and proteins were extracted. The expression levels of HNF4A, SOX9, pSTAT3, and PCNA were then confirmed through western blotting. The antibodies used are as follows: HNF4A (Cell signaling technology, #3113, 1:1000 dilution), pSTAT3 (Cell signaling technology, #9145, 1:1000 dilution), and PCNA (Santa Cruz, sc-56, 1:1000 dilution).

[0249] As a result of the experiment, it was confirmed that in the case of the transgenic mouse of Example 1, phosphorylation of STAT3 was significantly increased. STAT3 is a signaling factor known to be important for the dedifferentiation of hepatocytes during the liver regeneration process after liver damage (Li et al., Cell stem cell, Volume 30, Issue 3 pages 283-299 (2023), and it was confirmed that the phosphorylation of the corresponding protein increased by the expression of reprogramming factors. In addition, it was confirmed that the expression of Sox9, a liver progenitor cell marker, increased, the expression of HNF4A, a hepatocyte marker, decreased, and the expression of PCNA, a cell division marker, increased by the expression of reprogramming factors. On the other hand, in the case of ROSA26-rtTA mice, when hepatocytes were isolated after 72 hours of doxycycline administration and Western blotting was performed, it was confirmed that there was no significant increase in STAT3 phosphorylation or SOX9 expression, indicating that the effect of inducing liver progenitor cells by cell reprogramming is not related to the activation of rtTA protein (Fig. 4b).

[0250] In addition, the livers of the transgenic mice of Example 1 were extracted from the APAP administration group (300 mg / kg ip injection) or the doxycycline administration group (n = 3 per group) at 0, 24, 48, 72, and 96 hours from the time of administration, and the protein was used to confirm the expression levels of pSTAT3, SOX9, and PCNA through western blotting. Vinculin (Santa Cruz, sc-73614, 1:1000) was used as a control. Through this, it was confirmed that the phosphorylation of STAT3 preceded the induction of liver progenitor cells after AILI, and it was confirmed that the regeneration of the liver occurred rapidly at 48 and 72 hours after AILI through the change in the expression level of PCNA, a cell division marker. In addition, it was confirmed that the induction of liver progenitor cells occurred through a signaling pathway similar to AILI through the expression levels of SOX9 and pSTAT3 proteins after bioreprogramming, and in particular, it was confirmed that the induction of phosphorylation of STAT3 preceded the increase in SOX9 expression (Fig. 4c).

[0251] Meanwhile, the livers of the transgenic mice of Example 1 were removed 48 hours and 72 hours after administration from the APAP administration group (300 mg / kg ip injection) or the doxycycline administration group (n = 3 per group), and the F4 / 80 protein was stained through immunofluorescence staining using F4 / 80 antibody (Cell signaling technology, #70076S, 1:400 dilution). F4 / 80 protein is a macrophage detection marker, and macrophages are representative immune cells that infiltrate the liver through an immune response. As a result of the experiment, it was confirmed that infiltration of immune cells expressing F4 / 80 occurred around the hepatic vein after AILI, whereas no significant infiltration pattern was observed in the bio-reprogramming group. It was confirmed that the effect of inducing hepatic progenitor cells by cell reprogramming was caused by signaling similar to the immune response that occurs after liver damage (Fig. 4d).

[0252] In conclusion, we confirmed that the induction of hepatic progenitor cells by in vivo reprogramming is dependent on the expression of reprogramming factors and is mediated by signaling pathways similar to the liver's inherent regenerative mechanisms. However, unlike the AILI model, immune cell infiltration did not occur after in vivo reprogramming, confirming that STAT3 phosphorylation in liver cells occurs in an immune-independent manner, leading to the induction of hepatic progenitor cells.

[0253]

[0254] [Example 4] Development of a liver tissue-specific mRNA delivery system based on lipid nanoparticles (C12-SPM-GAL LNP)

[0255] We developed a liver tissue-specific lipid nanoparticle (LNP) mRNA delivery system and confirmed its mRNA delivery activity and cytotoxicity in in vitro cells.

[0256] C12-SPM-GAL LNP, a liver tissue-specific lipid nanoparticle, was prepared by mixing C12-SPM, dioleoylphosphatidylethanolamine (DOPE), cholesterol, polyethylene glycol (PEG)-linked ceramide (PEG-ceramide), and galactosyl ceramide.

[0257] Specifically, C12-SPM-GAL LNPs were prepared as follows. C12-SPM (synthesized by Medigen), DOPE (#850725, Avanti Polar Lipids), cholesterol (C8667 Sigma-Aldrich), C16-PEG2000 ceramide (#880180, Avanti Polar Lipids), C16 Galactosyl(α) ceramide (#860431, Avanti Polar Lipids), which are lipid components of C12-SPM-GAL LNP, were dissolved in ethanol at concentrations of 50 mg / ml, 10 mg / ml, 10 mg / ml, 5 mg / ml, and 2.5 mg / ml, respectively. The mass of C12-SPM used was set to 5 times the mass of the mRNA to be formulated, and C12-SPM:DOPE:cholesterol:C16-PEG2000 ceramide:C16 Galactosyl(α) ceramide = An organic phase was prepared by adding ethanol in a molar ratio of 20:26.5:50.5:1.5:1.5.

[0258] The aqueous phase, in which mRNA was dissolved in 10 mM sodium citrate (pH 4.0) at a concentration of 0.0238 mg / ml so as to have a mass ratio of 5:1 with C12-SPM, and the organic phase prepared above were mixed at a volume ratio of 3:1 using a microfluidic mixing device (Ignite, Precision NanoSystems) to prepare a formulation (the expected mRNA concentration in the total volume upon formulation completion was set to 0.017857 mg / ml). After stabilization at room temperature for 15 min, dialysis was performed in phosphate buffered saline (PBS) at 4 °C for 16 h using a 3.5 kDa MWCO cassette (66330, Thermo Fisher Scientific), and the resulting mixture was concentrated to the desired concentration using an ultracentrifugal filter (UFC901024, Sigma-Aldrich) before use (Fig. 5a).

[0259] After preparing C12-SPM-GAL LNPs loaded with luciferase mRNA (L-7202, TriLink BioTechnologies), mouse embryo-derived fibroblasts (Y. et al., Three-dimensional brain-like microenvironments facilitate the direct reprogramming of fibroblasts into therapeutic neurons. Nat Biomed Eng2, 522-539, 2018) were treated for 24 hours, and the mRNA delivery efficiency was compared with that of lipofectamine (Invitrogen), an in vitro cell gene delivery reagent, through luciferase luminescence analysis. Mouse embryo-derived fibroblasts were seeded at 5X10 in a 24-well plate. 4Cells were seeded at a cell density of 10 cells per well, and 24 hours later, lipofectamine and C12-SPM-GAL mRNA-LNPs were diluted in Opti-MEM (31985070, Gibco) at a volume of 10% of the cell culture medium at a concentration of 0.5 μg per well based on mRNA concentration, and transfection was performed by evenly dropping them onto the cell culture medium. The control group (no treatment, NT), the control group treated with luciferase mRNA loaded onto lipofectamine, and the experimental group in which luciferase mRNA was loaded onto C12-SPM-GAL LNP were compared.

[0260] After 24 hours of treating the culture medium with Lipofectamine and C12-SPM-GAL LNP, the cells were broken up with cell lysis buffer (E1531, Promega). The cell lysate was treated with D-luciferin (E8110, Promega), a substrate of luciferase, and the luminescence signal was measured to confirm the degree to which luciferase mRNA was delivered into the cells and expressed as protein. As a result, compared to the untreated control group and lipofectamine, it was confirmed that when luciferase mRNA was delivered using C12-SPM-GAL LNP, the efficiency of mRNA delivery and expression was significantly higher (Fig. 5b).

[0261] Meanwhile, mouse-derived fibroblasts were treated with the control group (no treatment, NT), lipid nanoparticles containing DLin-MC3-DMA (Med Chem Express), an ionizable lipid for RNA delivery approved by the FDA, as a core lipid, and C12-SPM-GAL lipid nanoparticles developed in the present invention at the same concentrations as in Fig. 5b, and after 24 hours, the cells were replaced with a cell culture medium containing a 6-fold dilution of a 10 mg / ml MTT (methylthiazolyldiphenyl-tetrazolium bromide) (Sigma Aldrich) solution and incubated for 4 hours. After removing the cell culture medium, DMSO was treated in an amount equal to half of the cell culture medium volume to dissolve MTT formazan crystals and the 560 nm absorbance of the solution was measured to evaluate cytotoxicity.

[0262] As a result, it was confirmed that the C12-SPM-GAL lipid nanoparticles developed in the present invention had no cytotoxicity at all (Fig. 5c).

[0263]

[0264] [Example 5] mRNA delivery activity and toxicity of a liver-specific lipid nanoparticle (LNP) mRNA delivery system in mouse liver tissue

[0265] In Example 4, luciferase mRNA was encapsulated in C12-SPM-GAL (GAL), a liver tissue-specific lipid nanoparticle-based mRNA delivery system developed, and DLin-MC3-DMA-based lipid nanoparticle (MC3), a positive control, and diluted in PBS to enable injection of 100 μl at a dose of 0.3 mg / kg based on mRNA mass into mice (C57BL / 6, Orient Bio, n=3~4) via intravenous injection (retroorbital sinus injection). As a negative control, the same volume of PBS was intravenously injected.

[0266] At 6, 12, 24, 48, and 96 hours after intravenous injection, luciferin (D-luciferin, P1043, Promega) dissolved in normal saline at 15 mg / ml was injected intraperitoneally in a volume of 200 μl at a dose of 150 mg / kg, and 10 minutes later, the luminescence signal was measured using an in vivo imaging system (IVIS, PerkinElmer) to compare the expression of luciferase mRNA in the body by time point.

[0267] As a result, when mRNA was injected using C12-SPM-GAL lipid nanoparticles, a significantly higher level of luciferase expression in liver tissue was confirmed compared to the group that was injected using DLin-MC3-DMA-based lipid nanoparticles, which was the positive control. In particular, when mRNA was injected using C12-SPM-GAL lipid nanoparticles, a transient expression pattern was observed with maximum expression 6 hours after injection and a rapid decrease within 24 hours. This suggests that not only can the toxicity of Oct4, Sox2, Klf4, and c-Myc genes be controlled for long-term expression through transient expression induction, but also that the expression pattern can be precisely controlled when multiple administrations are performed (Fig. 6a).

[0268] Meanwhile, when luciferase mRNA was encapsulated in C12-SPM-GAL (GAL) lipid nanoparticles and DLin-MC3-DMA lipid nanoparticles, respectively, and 0.3 mg / kg was intravenously injected into mice, the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) contents were analyzed 24 hours later. As a result, it was confirmed that there was no significant increase in values ​​compared to the control group injected with PBS (Figs. 6b, 6c), and thus, it was confirmed that C12-SPM-GAL lipid nanoparticles are safe mRNA delivery vehicles that do not show liver toxicity when intravenously injected.

[0269]

[0270] [Example 6] Synthesis of reprogramming factor mRNA and validation of lipid nanoparticle-encapsulated formulations

[0271] Four mRNAs encoding the retrograde reprogramming factors Oct4, Sox2, Klf4, and c-Myc (collectively OSKM) were synthesized from plasmid DNA via in vitro transcription (IVT).

[0272] IVT was performed by linearizing plasmid DNA encoding four types of factors (custom-made by Vectorbuilder) with NotI restriction enzyme (R001S, New England Biolabs) and purifying DNA through DNA purification (K3037, Bioneer), followed by mRNA in vitro transcription (MEGAscript T7 Transcription Kit, AM1333, Invitrogen). In the mRNA transcription process, 5' capping was performed during the transcription process through co-transcriptional capping, and 3'-0-Me-m was used as the 5' cap.7 G(5')ppp(5')ARCA cap analog (S1411S, New England Biolabs) was used. In addition, to alleviate the immunogenicity of mRNA in the body, nucleoside-modified mRNA was synthesized by replacing UTP with N1-Methylpseudouridine-5'-Triphosphate (N-1081, TriLink BioTechnologies) during mRNA synthesis.

[0273]

[0274] Sequence of pmRVac-mc-Myc plasmid DNA vector

[0275]

[0276]

[0277] DNA Length: 3520 bp

[0278]

[0279] Sequence of pmRVac-mKlf4 plasmid DNA vector

[0280]

[0281]

[0282] DNA Length: 3625 bp

[0283]

[0284] Sequence of pmRVac-mOct4 plasmid DNA vector

[0285]

[0286]

[0287] DNA Length: 3259 bp

[0288]

[0289] Sequence of pmRVac-mSox2 plasmid DNA vector

[0290]

[0291]

[0292] DNA Length: 3148 bp

[0293]

[0294] It was confirmed through 1% agarose gel electrophoresis that four types of mRNAs matching the size of the sequence were synthesized (Fig. 7a).

[0295] Four types of C12-SPM-GAL LNPs (OSKM mRNA LNPs) encapsulating mRNAs encoding four different types of dedifferentiation reprogramming factors were produced and characterized. The process for producing C12-SPM-GAL LNPs for each mRNA was the same as in Example 4.

[0296] To measure mRNA encapsulation efficiency, a ribogreen assay (R11490, Invitrogen) was performed. RiboGreen dye binds to RNA and emits fluorescence at 480–520 nm, but cannot penetrate the interior of lipid nanoparticles. Therefore, the mRNA encapsulation efficiency within lipid nanoparticles can be quantified by comparing the case where the manufactured lipid nanoparticles were treated with RiboGreen dye and the case where the lipid nanoparticles were broken by treating them with 2% Triton X-100 (Sigma Aldrich) and then treated with RiboGreen dye. When the encapsulation efficiency was quantified, it was confirmed that all showed a high encapsulation efficiency of over 85%. When the particle size and PDI (polydispersity index) were measured using dynamic light scattering (DLS) equipment, it was confirmed that uniform nanoparticles were formed with a size of less than 100 nm and a PDI of less than 0.2 (Fig. 7b).

[0297] The four mRNA-LNPs, Oct4, Sox2, Klf4, and c-Myc, were treated with the same amounts as in Example 4 for 24 hours, and then the proteins were isolated and subjected to western blotting to confirm the intracellular protein expression of each mRNA. In this case, the primary antibodies used were Oct4 (sc-5279, Santa Cruz Biotechnology), Sox2 (sc-365823, Santa Cruz Biotechnology), Klf4 (AF3158, R&D systems), and c-Myc (AF3696, R&D systems), and the secondary antibodies used were anti-mouse HRP-linked IgG (7076P2, Cell Signaling Technology) and anti-rabbit HRP-linked IgG (7074P2, Cell Signaling Technology).

[0298] As a result, compared to the control group (no treatment, NT) that was not treated at all, it was confirmed that all four types of proteins were effectively overexpressed by treatment with four types of mRNA-LNPs (Oct4, Sox2, Klf4, and c-Myc) produced from mouse-derived fibroblasts (Fig. 7c).

[0299] In addition, after treating mouse-derived fibroblasts with the four mRNA-LNPs of Oct4, Sox2, Klf4, and c-Myc for 24 hours, immunofluorescence staining was performed using Oct4 (sc-5279, Santa Cruz Biotechnology), Sox2 (sc-365823, Santa Cruz Biotechnology), and DAPI (A2412, TCI America). As a result, it was confirmed that Oct4 and Sox2 were expressed as proteins within the cells (Fig. 7d).

[0300] From the above results, it was confirmed that mRNA encoding the dedifferentiation reprogramming factors Oct4, Sox2, Klf4, and c-Myc can be synthesized and produced as a uniform lipid nanoparticle formulation, and that this can be delivered into cells in vitro to effectively induce protein expression.

[0301]

[0302] [Example 7] Verification of in vivo liver cell reprogramming based on OSKM mRNA-LNP

[0303] As a control, PBS, C12-SPM-GAL LNPs encapsulating luciferase (Luc) mRNA, which has no function in liver tissue, and four types of C12-SPM-GAL LNPs encapsulating Oct4, Sox2, Klf4, and c-Myc (OSKM) mRNA, respectively, were prepared in the same manner as in Example 4, and these were administered intravenously to normal C57BL / 6 mice at a total of 0.3 mg / kg or 0.6 mg / kg based on the mass of the mRNAs encapsulated in the same amount of Oct4, Sox2, Klf4, and c-Myc mRNA-LNPs, or as a control, 100 μl intravenous injection of Luc mRNA-LNPs at 0.3 mg / kg or 0.6 mg / kg was performed (n=4~5). Based on the previous results showing maximum expression 6 hours after intravenous injection of LNP, analysis was conducted by sacrificing mice 24 hours (30 h) and 48 hours (54 h) after the time of maximum expression (6 h) (Fig. 8a).

[0304] First, liver tissue was collected from the sacrificed mouse, RNA was extracted from the liver tissue using the same method as in Example 1-2, and cDNA was synthesized. Then, Oct4 (Pou5f1), Sox2, Klf4, and c-Myc (Myc) mRNA uptake levels were confirmed through quantitative real-time PCR analysis.

[0305] As a result, it was confirmed that four types of mRNA were present in the liver tissue compared to the control group injected with PBS even after 30 and 54 hours of intravenous injection (Fig. 8b).

[0306] PBS, Luc mRNA-LNP, and OSKM mRNA-LNP were intravenously injected at concentrations of 0.3 mg / kg and 0.6 mg / kg, respectively. After 54 hours, mouse liver tissues were collected and stained with hematoxylin (H9627, Sigma Aldrich) and eosin (000E0614, Samchun Chemicals), and Masson's trichrome (MT) (B8563, Sigma Aldrich) according to the manufacturer's protocol.

[0307] As a result, it was confirmed that no necrosis or fibrosis occurred in liver tissue in all samples (Fig. 8c).

[0308] Liver tissue was collected 30 hours after intravenous injection of OSKM mRNA-LNP, and quantitative real-time PCR for Sox9 and Ki-67 was performed.

[0309] As a result, compared to the control group injected with PBS, it was confirmed that the expression of Sox9, a liver progenitor-like cell (LPLC) marker, and Ki-67, a cell proliferation marker, significantly increased in the experimental group injected with OSKM mRNA-LNP (Fig. 8d), and in particular, it was confirmed that cells expressing Sox9 and Ki-67 increased around the portal vein of the liver (Fig. 8e).

[0310] Liver tissue was pulverized 30 hours after intravenous injection of OSKM mRNA-LNP, protein was extracted, and Western blot analysis was performed. The antibody used was phospho-histone H3 (Ser10) (Cell signaling technology, #9701, 1:1000 dilution).

[0311] As a result, compared to the control group injected with PBS, it was confirmed that the experimental group injected with OSKM mRNA-LNP showed an increase in Sox9 expression, along with an increase in histone H3 phosphorylation (pHH3) and PCNA protein expression (Fig. 8f).

[0312] In summary, the above results show that intravenous injection of OSKM mRNA-LNP effectively dedifferentiates liver cells, increasing the proliferative liver progenitor-like cell population.

[0313]

[0314] [Example 8] Effect of OSKM mRNA-LNP on promoting liver regeneration in a mouse model of liver damage

[0315] Acetaminophen (APAP) was intraperitoneally injected into mice at a concentration of 400 mg / kg to induce liver damage. Since liver tissue regeneration begins 24 hours after acetaminophen-induced liver injury (AILI), OSKM mRNA encapsulated in C12-SPM-GAL LNPs was intravenously injected into mice 18 hours after APAP administration, 6 hours before the peak expression time of OSKM at 24 h, when liver tissue regeneration begins. Controls included a control group that was not induced with AILI (Cont, Normal), a control group that was not treated after AILI (Mock), and a control group injected with luciferase mRNA-LNP (Luc) (n=14). The mouse strains and drug administration methods used in this experiment were the same as in Example 7. Liver tissues were harvested 48 and 96 hours after APAP injection and analyzed (Fig. 9a).

[0316] One day after intraperitoneal injection of APAP, whole liver photographs and H&E staining of liver tissue sections showed that necrosis occurred in the tissues around the central vein due to AILI induction (Fig. 9b).

[0317] The survival rate of mice injected with OSKM mRNA-LNP was significantly increased compared to the untreated control group (Mock) or the control group injected with luciferase mRNA-LNP (Luc) after AILI induction (Fig. 9c).

[0318] H&E staining of liver tissue 96 hours after intraperitoneal injection of APAP confirmed that the necrotic area (dotted line) around the central vein was significantly reduced in the liver tissue of mice injected with OSKM mRNA-LNP compared to the liver tissue of mice injected with luciferase mRNA-LNP (Fig. 9d).

[0319] In addition, immunohistochemical staining of cleaved caspase-3 in liver tissues 96 hours after intraperitoneal injection of APAP showed that the expression level of cleaved caspase-3 around the central vein was significantly increased in the untreated control group (Mock) and the control group (Luc) injected with luciferase mRNA-LNP after AILI induction, whereas it was similar to that in normal mice (Cont) in mice injected with OSKM mRNA-LNP, indicating that liver cell apoptosis was significantly alleviated (Fig. 9e). Western blot analysis of cleaved caspase-3 expression by isolating total protein from liver tissues 96 hours after intraperitoneal injection of APAP also confirmed that cleaved caspase-3 was reduced to a similar level as that in normal mice in mice injected with OSKM mRNA-LNP at a concentration of 0.3 mg / kg (Fig. 9f).

[0320] To confirm whether the liver regeneration promotion effect as described above was due to the dedifferentiation reprogramming of liver cells, liver tissues were collected 48 hours after intraperitoneal injection of APAP and immunofluorescence staining was performed. As a result, it was confirmed that the liver progenitor-like cell (LPLC) marker Sox9 and the cell proliferation marker Ki-67 both showed significantly higher expression levels in the liver tissues of mice injected with OSKM mRNA-LNP compared to mice injected with luciferase mRNA-LNP (Fig. 9g). In addition, when liver tissues were collected 48 hours and 96 hours after intraperitoneal injection of APAP and western blot analysis was performed, the western blot results 48 hours after intraperitoneal injection of APAP showed that the expression level of the LPLC marker Sox9 was significantly increased in mice treated with OSKM mRNA-LNP at both concentrations of 0.3 mg / kg and 0.6 mg / kg, and the western blot results 96 hours after intraperitoneal injection of APAP showed that the cell cycle marker histone H3 phosphorylation (pHH3) was increased in mice injected with OSKM mRNA-LNP.

[0321]

[0322] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0323]

[0324] National Research and Development Project 1 that supported this invention

[0325] [Project ID] 9991008836

[0326] [Assignment Number] 00432867 (RS-2024-00432867)

[0327] [Ministry Name] Ministry of Science and ICT

[0328] [Name of Project Management (Specialist) Institution] National Research Foundation of Korea

[0329] [Research Project Name] Artificial Stem Cell-Based Regenerative Therapy Technology Development Project

[0330] [Research Project Name] Development of Organ-Specific ABC-Like Cell Formation and Control Technology through In Vivo Reprogramming

[0331] [Name of the project performing organization] Seoul National University Industry-Academic Cooperation Foundation

[0332] [Research Period] May 1, 2024 - December 31, 2029

[0333]

[0334] National Research and Development Project 2 that supported this invention

[0335] [Project ID] 9991008834

[0336] [Assignment Number] 00432653 (RS-2024-00432653)

[0337] [Ministry Name] Ministry of Science and ICT

[0338] [Name of Project Management (Specialist) Institution] National Research Foundation of Korea

[0339] [Research Project Name] Artificial Stem Cell-Based Regenerative Therapy Technology Development Project

[0340] [Research Project Name] Treatment of Fatty Liver Disease Based on Liver Tissue-Specific Lipid Nanoparticles

[0341] Development of artificial blastocyst-mediated regeneration technology for

[0342] [Name of the project performing organization] Yonsei University

[0343] [Research Period] May 1, 2024 - December 31, 2029

Claims

1. A lipid nanoparticle comprising an ionized lipid comprising one or more tertiary amine structures, a helper phospholipid, cholesterol or a cholesterol analogue, a polyethylene glycol-linked lipid, and galactosyl ceramide.

2. A lipid nanoparticle according to claim 1, characterized in that the lipid nanoparticle is for mRNA delivery.

3. A lipid nanoparticle according to claim 2, characterized in that the lipid nanoparticle is for liver-specific delivery of mRNA.

4. A lipid nanoparticle according to claim 1, characterized in that it comprises an ionized lipid having one or more tertiary amine structures: a helper phospholipid: cholesterol or a cholesterol analogue: a lipid linked to polyethylene glycol: galactosyl ceramide in a molar ratio of 20 to 50: 10 to 30: 30 to 60: 0.5 to 2: 0.5 to 2. 5.mRNA; and An mRNA-lipid nanoparticle complex comprising a lipid nanoparticle according to any one of claims 1 to 4.

6. A method for producing an mRNA-lipid nanoparticle complex of claim 5, comprising the following steps: (a) a step of preparing an organic phase lipid nanoparticle by dissolving an ionized lipid containing one or more tertiary amine structures, a helper phospholipid, cholesterol or a cholesterol analogue, a polyethylene glycol-linked lipid, and galactosyl ceramide in alcohol; and (b) A step of mixing the organic lipid nanoparticles with mRNA in an aqueous phase to encapsulate mRNA in the lipid nanoparticles.

7. In paragraph 6, after step (b), (c) A manufacturing method further comprising a step of dialyzing and / or filtering the lipid nanoparticles encapsulated with the mRNA.

8. A manufacturing method in claim 6, wherein in step (b), the organic phase containing the lipid nanoparticles and the aqueous phase containing mRNA are mixed in a volume ratio of 1:1.5 to 5.

9. A manufacturing method in claim 6, wherein in step (b), the ionized lipid containing one or more tertiary amine structures and mRNA are mixed at a mass ratio of 5 to 20:

1.

10. A pharmaceutical composition for preventing or treating liver disease, comprising a cell reprogramming factor mRNA and a lipid nanoparticle of any one of claims 1 to 4.

11. A pharmaceutical composition for preventing or treating liver disease, characterized in that in claim 10, the cell reprogramming factors are Oct4, Sox2, Klf4 and C-Myc.

12. A pharmaceutical composition for preventing or treating liver disease, characterized in that in claim 10, the liver disease is selected from the group consisting of acute liver damage, chronic liver damage, hepatitis, cirrhosis, liver failure, cirrhosis, and liver cancer.

13. A pharmaceutical composition for preventing or treating liver disease, characterized in that the composition in claim 10 is administered once or multiple times, and when administered multiple times, is administered at intervals of 6 to 48 hours.

Images