mRNA delivery system for enhancing protein expression and use thereof

Abstract

Provided in the present invention are an mRNA delivery system, and specifically provided are a pharmaceutical composition containing mRNA and a potentiating protein, and a preparation thereof and the use thereof. The delivery system of the present invention comprises mRNA and a potentiating protein. The application of the delivery system enables more efficient delivery of mRNA into a target cell and enhances the expression thereof in the target cell.

Description

mRNA delivery systems that enhance protein expression and their applications Technical Field

[0001] This invention relates to a drug delivery system, and more particularly to a pharmaceutical composition containing mRNA and an enhancing protein, its formulation, and its application. Background Technology

[0002] Messenger RNA (mRNA) plays a crucial role in modern biotechnology and medicine. As a key intermediate molecule in the transmission of genetic information, it guides protein synthesis, thereby regulating numerous biological processes and cellular functions. In several key applications, such as gene therapy, vaccine development, and protein replacement therapy, efficient in vivo expression of mRNA is a core element in achieving desired therapeutic effects. However, researchers have long faced numerous challenges, the most prominent being how to improve the expression efficiency and stability of mRNA in vivo or in vitro.

[0003] In past research and practice, various methods have been explored to improve mRNA expression. For example, structural modifications to mRNA have been employed, including but not limited to optimizing its 5' cap structure, 3' poly(A) tail, and adjusting the nucleotide sequence of the coding region. These measures have improved mRNA stability and translation efficiency to some extent. Simultaneously, in terms of delivery systems, various carriers, such as lipid nanoparticles, have been developed to more effectively deliver mRNA into target cells, thereby promoting its expression.

[0004] Despite the progress made, many limitations still exist. For example, in some complex physiological environments or disease models, even with advanced delivery vectors, the expression level of mRNA is still insufficient to produce significant therapeutic effects, or its expression duration is short enough to maintain long-term biological effects.

[0005] Therefore, improvements to mRNA delivery systems are still needed to deliver mRNA more effectively into target cells and promote its expression. Summary of the Invention

[0006] (a) Technical problems to be solved

[0007] To address the shortcomings of existing technologies, this invention provides an mRNA delivery system that can more effectively deliver mRNA into target cells and enhance its expression within those cells.

[0008] (II) Technical Solution

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] In one aspect, the present invention provides a composition for delivering mRNA, the composition comprising mRNA and an enhancer protein.

[0011] In some embodiments, the enhancing protein is a protein that can improve mRNA translation levels, and the enhancing protein is selected from human eIF4E (uniprot accession number P06730), mouse eIF4E (uniprot accession number P63073), human CBP80 (uniprot accession number Q09161), human CBP20 (uniprot accession number P52298), human eIF4A (uniprot accession number P60842), human eIF4G (uniprot accession number Q04637), human eIF4B (uniprot accession number P23588), and eIF3A (uniprot accession number P23588). One or more of the following: prot login number Q14152, eIF1 (uniprot login number P41567), eIF1AX (uniprot login number P47813), human PABPC1 (uniprot login number A0A087WTT1), human LARP1 (uniprot login number Q6PKG0), human LARP4 (uniprot login number Q71RC2), human RACK1 (uniprot login number P63244), human mTOR (uniprot login number P42345), and human MAPK1 (uniprot login number P28482).

[0012] In some embodiments, the synergistic protein is selected from eIF4E and / or CBP80.

[0013] In some embodiments, the synergistic protein is selected from human eIF4E, mouse eIF4E, and / or human CBP80.

[0014] In one aspect, the present invention provides a pharmaceutical formulation for delivering mRNA, said pharmaceutical formulation comprising the above-described composition.

[0015] In some embodiments, the pharmaceutical formulation is selected from lipid nanoparticles (LNP), cationic lipid complexes (lipoplex, LPX), lipid polymers, polymer nanoparticles, exosomes, inorganic nanoparticles, or biological microvesicles.

[0016] In some embodiments, the pharmaceutical formulation is selected from lipid nanoparticles (LNPs).

[0017] In one aspect, the present invention provides a lipid nanoparticle composition comprising mRNA, the aforementioned synergistic protein, and lipid nanoparticles; said lipid nanoparticles include ionizable lipids, phospholipids, structural lipids, and PEG lipids. Optionally, the mRNA and synergistic protein are partially or completely encapsulated by the lipid nanoparticles.

[0018] In some embodiments, the molar ratio of the mRNA to the aforementioned synergist protein is (1-50):(1-50).

[0019] In some embodiments, the molar ratio of the mRNA to the aforementioned synergist protein is (1-30):(1-30).

[0020] In some embodiments, the molar ratio of the mRNA to the aforementioned synergist protein is (1-25):(1-25).

[0021] In some embodiments, the molar ratio of the mRNA to the aforementioned synergist protein is (1-20):(1-20).

[0022] In some embodiments, the molar ratio of the mRNA to the aforementioned enhancing protein is (1-10):(1-10).

[0023] In some embodiments, the molar ratio of the mRNA to the aforementioned synergist protein is (1-5):(1-5).

[0024] In some implementations, the molar ratio of the mRNA to the aforementioned synergist protein is approximately 1:1.

[0025] In some embodiments, the molar ratio of the sum of the ionizable lipids, the phospholipids and the structural lipids, and the PEG lipids is (35-65):(35-65):(0.5-5).

[0026] In some embodiments, the molar ratio of the sum of the ionizable lipids, the phospholipids and the structural lipids, and the PEG lipids is (40-50):(35-65):(1-3).

[0027] In some embodiments, the molar ratio of the sum of the ionizable lipids, the phospholipids and the structural lipids, and the PEG lipids is (50-65):(35-65):(1-3).

[0028] In some embodiments, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is (40-50):(5-15):(30-50):(1-3).

[0029] In some embodiments, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is (40-50):(10-15):(35-45):(1.5-2.5).

[0030] In some embodiments, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is (50-65):(5-15):(30-50):(1-3).

[0031] In some embodiments, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is (50-65):(10-15):(35-45):(1.5-2.5).

[0032] In some embodiments, the content of ionizable lipids is 35 mol%-65 mol%, the content of phospholipids and the sum of the structural lipids is 35 mol%-65 mol%, and the content of PEG lipids is 0.5 mol%-5 mol%, where mol% is calculated based on the total number of moles of lipids in the lipid nanoparticle composition.

[0033] In some embodiments, the content of ionizable lipids is 40 mol%-50 mol%, preferably 45 mol%-49 mol%, and more preferably 48 mol%-49 mol%.

[0034] In some embodiments, the content of ionizable lipids is 50 mol%-65 mol%, preferably 51 mol%-55 mol%, and more preferably 52 mol%-53 mol%.

[0035] In some embodiments, the sum of phospholipids and the structural lipids is 35 mol%-65 mol%, preferably 35 mol%-55 mol%, and more preferably 45 mol%-50 mol%.

[0036] In some implementations, the phospholipid content is 5 mol%-15 mol%, preferably 10 mol%-15 mol.

[0037] In some implementations, the structured lipid content is 30 mol%-50 mol%, preferably 35 mol%-45 mol%.

[0038] In some implementations, the PEG lipid content is 1 mol%-5 mol%, preferably 1.5 mol%-2.5 mol%.

[0039] In some embodiments, the N / P ratio of the ionizable lipid to mRNA is 3.5-5.5, preferably 5.2.

[0040] In some embodiments, the ionizable lipid is selected from 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA; "XTC2"), and 2,2-dilinoleyl-4-(3-dimethylaminopropyl). -[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), 2,2-dilinyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinyl-4-N-methylpepiazino-[1,3]-dioxolane (DLi n-K-MPZ), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleyl... 1,2-Dilinoleothio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleoyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleoyloxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), and 1,2-dilinoleoyl-3-trimethylaminopropane chloride (DLin-TAP).Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleyloxy-N,N-dimethylaminopropane ( DODMA), 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2 ...',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), 3-(N',N'-dioleoyl)-carbamoyl)cholesterol (DC-Chol), 3-(N',N'-dioleoyl)-carbamoyl)-cholesterol (DC-Chol), 3-(N',N'-dioleoyl)-carbamoyl)-carbamoyl) Myristyloxypropyl-3-yl)-N,N-dimethyl-N-carboxyethylammonium bromide (DMRIE), 2,3-dioleoxy-N-[2(spermine-formylamino)ethyl]-N,N-dimethyl-1-propanetrimonium trifluoroacetate (DOSPA), bis(octadecylaminoglycyl)spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-β-oxybut-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-β-oxy) The following are included in the list of one or more of the following: 3'-oxaproloxy)-3-dimethyl-1-(cis,cis-9',1-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleoyloxybenzylamine (DMOBA), 1,2-N,N'-dioleoylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilinoleoylcarbamoyl-3-dimethylaminopropane (DLincarbDAP), MC3, SM-102, and ALC-0315.

[0041] In some embodiments, the ionizable lipid is a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof:

[0042] (I).

[0043] In some embodiments, R1 is selected from H, vinyl, and C1 to C3 alkyl. In one specific embodiment, R1 is H; in one specific embodiment, R1 is vinyl; in one specific embodiment, R1 is C1 to C3 alkyl; for example, R1 is C1 alkyl, C2 alkyl, or C3 alkyl.

[0044] In some embodiments, R2, R3, and R4 are each independently a C7-C15 straight-chain alkyl group; in some preferred embodiments, R2, R3, and R4 are each independently selected from C8-C12 straight-chain alkyl groups. For example, R2, R3, and R4 are each independently a C7, C8, C9, C10, C11, C12, C13, C14, or C15 straight-chain alkyl group. In some preferred embodiments, R1 is an ethyl group; in some preferred embodiments, R1 is H. In some preferred embodiments, R2 is a C8 or C11 straight-chain alkyl group. In some preferred embodiments, R3 and R4 are both C8 straight-chain alkyl groups.

[0045] In some implementations, X1 and Y1 are different, X2 and Y2 are different, X1 and X2 are each independently selected from C=O or O, and Y1 and Y2 are each independently selected from C=O or O; in some preferred implementations, X1 and X2 are both C=O, and Y1 and Y2 are both O.

[0046] In some implementation schemes, m and n are each independently selected from 5, 6, 7, and 8.

[0047] In some implementations, o is selected from 2, 3, 4, 5, and 6; in some preferred implementations, o is 3 or 6.

[0048] In some embodiments, the ionizable lipid compound is selected from one of the following structures:

[0049] Compound 1

[0050] ,

[0051] Compound 2

[0052] ,

[0053] Compound 3

[0054] ,

[0055] Compound 4

[0056] ,

[0057] Compound 5

[0058] ,

[0059] Compound 6

[0060] ,

[0061] Compound 7

[0062] or

[0063] Compound 8

[0064] .

[0065] In some embodiments, the ionizable lipids provided in this application are compounds of formula IIA, or pharmaceutically acceptable salts thereof, or stereoisomers thereof:

[0066] .

[0067] In some implementation schemes, R 1a and R 1b Independently selected from -CH3. In one specific embodiment, R 1a For -CH3; in a specific embodiment, R 1b It is -CH3.

[0068] In some implementations, R2 and R3 are each independently represented by H.

[0069] In some embodiments, R4 is selected from C8, C9, C10, C11, or C12 straight-chain alkyl groups. In some preferred embodiments, R4 is selected from C10 or C11 straight-chain alkyl groups. For example, R4 is a C8 straight-chain alkyl group, a C9 straight-chain alkyl group, a C10 straight-chain alkyl group, a C11 straight-chain alkyl group, or a C12 straight-chain alkyl group.

[0070] In some embodiments, R5 and R6 are independently selected from C6, C7, and C8 straight-chain alkyl groups; for example, R5 and R6 are independently C6, C7, or C8 straight-chain alkyl groups. In some preferred embodiments, R5 and R6 are independently selected from C6 or C8 straight-chain alkyl groups. For example, R5 and R6 are independently C8 or C6 straight-chain alkyl groups.

[0071] In some embodiments, R5 and R6 are both C8 straight-chain alkyl; in some embodiments, R5 is a C6 straight-chain alkyl and R6 is a C8 straight-chain alkyl; in some embodiments, R5 is a C8 straight-chain alkyl and R6 is a C6 straight-chain alkyl.

[0072] In some implementations, X1 and X2 are both C=0, and Y1 and Y2 are both 0; in some implementations, X1 is 0, Y1 is C=0, X2 is C=0, and Y2 is 0; in some implementations, X1 is C=0, Y1 is 0, X2 is 0, and Y2 is C=0. In some implementations, X1 and X2 are both 0, and Y1 and Y2 are both C=0.

[0073] In some implementation schemes, o and p are each independently selected from 4, 5, 6, 7, 8, and 9.

[0074] In some implementations, q is selected from 2, 3, 4 or 5; in some preferred implementations, q is 2, 3 or 4.

[0075] In some embodiments, the ionizable lipid compound is selected from one of the following structures or a pharmaceutically acceptable salt thereof or a stereoisomer thereof:

[0076] Compound 9

[0077] ,

[0078] Compound 10

[0079] ,

[0080] Compound 11

[0081] ,

[0082] Compound 12

[0083] ,

[0084] Compound 13

[0085] or

[0086] Compound 14

[0087] .

[0088] In some embodiments, the ionizable lipid is a compound of formula (III), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof:

[0089] Formula (III)

[0090] R1 is ;

[0091] R2 and R3 are C6-C8 straight-chain alkyl groups;

[0092] n is selected from 2, 3, 4, 5, 6;

[0093] o is selected from 1, 2, 3, 4, 5, 6, 7, 8;

[0094] p is selected from 5, 6, 7, 8, 9.

[0095] In some implementations, R2 and R3 are both C8 straight-chain alkyl groups.

[0096] In some implementations, n is 2 or 6.

[0097] In some implementations, o is 5.

[0098] In some implementations, p is 7.

[0099] In some embodiments, the ionizable lipid compound is selected from one of the following structures:

[0100] Compound 15 or

[0101] Compound 16 .

[0102] In some embodiments, the phospholipid is selected from one or more of the following compounds: 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-distearateoyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-diundecanoyl-sn-glycerol-3-phosphate choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), and 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0Diether PC). 1-Oleoyl-2-cholesterolylhemisuccinoyl-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16LysoPC), 1,2-dilinolenoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicyl-sn-glycerol-3-phosphate choline, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphate choline), 1,2- Dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-diphydanoyl-sn-glycerol-3-phosphate ethanolamine (ME16.0PE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenooyl-sn-glycerol-3-phosphate ethanolamine), 1,2-dioleoyl-sn-glycerol-3-phosphate sodium salt (DOPG), dioleoylphosphatidylserine (DOPS), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearate-phosphatidyl-ethanolamine (DSPE), dipalmitoyl... Neutral lipids include sphingolipids, bis(myristoyl)phosphatidylethanolamine (DPPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine (LPE). These neutral lipids can be synthetic or of natural origin.

[0103] In some implementations, the phospholipid is DSPC.

[0104] In some embodiments, the structural lipid is selected from one or more of cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, tomatidine, tomatine, ursolic acid, and α-tocopherol.

[0105] In some implementations, the structural lipid is cholesterol.

[0106] In some implementations, the structural lipids include one or more of cholesterol and corticosteroids such as prednisolone, dexamethasone, and hydrocortisone.

[0107] In some implementations, the structural lipid is cholesterol.

[0108] In some embodiments, the PEG lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, or PEG-modified dialkylglycerol.

[0109] In some embodiments, the PEG lipid is DMG-PEG2000, DMPE-PEG2000, or DSPE-PEG2000.

[0110] In some embodiments, the lipid nanoparticles have a diameter of about 30 nm to about 200 nm.

[0111] In some embodiments, the lipid nanoparticles have a diameter of about 70 nm to about 150 nm.

[0112] In some embodiments, the lipid nanoparticles have a diameter of about 90 nm to about 110 nm.

[0113] In one aspect, the present invention provides a pharmaceutical composition comprising the above-described lipid nanoparticle composition and a pharmaceutically acceptable excipient or auxiliary ingredient.

[0114] In one aspect, the present invention provides a method for delivering a therapeutic and / or preventive agent to mammalian cells, the method comprising administering the above-described lipid nanoparticles or the above-described pharmaceutical composition to a subject, the administration comprising contacting cells with the nanoparticle composition or the pharmaceutical composition to deliver the therapeutic and / or preventive agent to the cells.

[0115] In some embodiments, the mammalian cell is located in a mammal.

[0116] In some implementations, the mammal is a human.

[0117] In some embodiments, the lipid nanoparticles are administered via intravenous, intramuscular, intradermal, subcutaneous, intranasal, or inhalation.

[0118] In one aspect, the present invention provides a method for generating a target polypeptide in mammalian cells, the method comprising contacting the cells with the aforementioned lipid nanoparticles or the aforementioned pharmaceutical composition to deliver a therapeutic and / or preventive agent to the cells, wherein the therapeutic and / or preventive agent is mRNA encoding the target polypeptide, thereby enabling the mRNA to be translated in the cells to generate the target polypeptide.

[0119] In some embodiments, the mammalian cell is located in a mammal.

[0120] In some implementations, the mammal is a human.

[0121] In some embodiments, the lipid nanoparticles or pharmaceutical composition are administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

[0122] In one aspect, the present invention provides a method for treating and / or inhibiting a disease or symptom in a mammal, the method comprising administering to the mammal a therapeutically effective amount of the lipid nanoparticles or the pharmaceutical composition thereof.

[0123] In some implementations, the disease or condition is characterized by dysfunctional or abnormal protein or peptide activity.

[0124] In some implementations, the disease or condition is selected from infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases, renal vascular diseases, or metabolic diseases.

[0125] In some implementations, the mammal is a human.

[0126] In some embodiments, the lipid nanoparticles or pharmaceutical composition are administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

[0127] In one aspect, the present invention provides a method for specifically delivering a therapeutic and / or preventive agent to a mammalian organ, the method comprising administering the lipid nanoparticles or the pharmaceutical composition to the mammal, the administration comprising contacting the mammalian organ with the nanoparticles, thereby delivering the therapeutic and / or preventive agent to the organ.

[0128] In some implementations, the mammal is a human.

[0129] In some embodiments, the lipid nanoparticles or pharmaceutical composition are administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

[0130] In some embodiments, the mammal is pretreated 24 hours or less prior to the contact or administration step.

[0131] In some embodiments, the mammal is pretreated approximately one hour prior to the contact or administration step.

[0132] (III) Beneficial Effects

[0133] This invention provides an mRNA delivery system that, compared with existing technologies, has the following advantages:

[0134] The delivery system of the present invention includes mRNA and enhancer protein. By applying the above delivery system, mRNA can be delivered more effectively into target cells, thereby enhancing its expression in target cells. Attached Figure Description

[0135] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0136] Figure 1. Detection of the effect of mRNA / eIF4E nanoparticles on luciferase expression at the cellular level;

[0137] Figure 2. Detection of the effect of mRNA / eIF4E nanoparticles on luciferase expression at the animal level;

[0138] Figure 3. Effects of different enhancing proteins and mRNA encapsulation on mRNA expression;

[0139] Figure 4. Effects of different enhancing proteins and mRNA encapsulation on mRNA spleen expression distribution;

[0140] Figure 5. Effects of different enhancing proteins and mRNA encapsulation on mRNA gastric expression distribution;

[0141] Figure 6. Effects of different enhancing proteins and mRNA encapsulation on the distribution of mRNA intestinal expression. Detailed Implementation

[0142] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0143] The technical solutions in the embodiments of the present invention are intended to solve the above-mentioned technical problems, and the overall approach is as follows:

[0144] This invention provides an mRNA delivery system comprising mRNA and an enhancer protein. By applying the above delivery system, mRNA can be delivered more effectively to target cells, thereby enhancing its expression within the target cells.

[0145] definition:

[0146] The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the terms referenced have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as understood by those skilled in the art.

[0147] The references to "an embodiment" or "an implementation" in the specification indicate that the described embodiment may include a specific aspect, feature, structure, part, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, part, or characteristic. Furthermore, such phrases may, but do not necessarily, refer to the same embodiment mentioned in other parts of the specification. Moreover, when a specific aspect, feature, structure, part, or characteristic is described in connection with an embodiment, whether explicitly stated or not, it is within the knowledge of those skilled in the art to influence or relate such aspect, feature, structure, part, or characteristic with other embodiments.

[0148] Unless the context explicitly indicates otherwise, the singular forms “a,” “an,” and “the” include plural indicators. Thus, for example, a reference to “a compound” includes multiple such compounds, such that a compound X includes multiple compounds X. It should also be noted that claims can be drafted to exclude any optional elements. Thus, this statement is intended to serve as a priori basis for the use of exclusive terms such as “unique,” ​​“only,” etc., in conjunction with any element described herein, and / or for the reference to or “negation” of claim elements.

[0149] The term “and / or” means any one of the matters, any combination of the matters, or all the matters relating to the term. The phrase “one or more” is readily understood by those skilled in the art, especially when read in the context in which it is used. For example, one or more substituents on a benzene ring refers to one to five, or one to four substituents (e.g., if the benzene ring is disubstituted).

[0150] The term "about" can refer to a variation of ±5%, ±10%, ±20%, or ±25% of a specified value. For example, "about 50%" can have a variation of 45% to 55% in some embodiments. For integer ranges, the term "about" can include one or two integers greater than and / or less than the integers cited at each end of the range. Unless otherwise specified herein, the term "about" is intended to include values ​​near the cited range that are equivalent to functional aspects of a single ingredient, composition, or embodiment, such as weight percentage. The term "about" can also modify the endpoints of the cited range as discussed above in this paragraph.

[0151] As will be understood by those skilled in the art, all numerical values ​​(including those representing amounts of components, properties such as molecular weight, reaction conditions, etc.) are approximate and should be understood to be optionally modified by the term "about" in all cases. These values ​​may vary depending on the desired properties sought to be obtained by those skilled in the art using the teachings of this specification. It should also be understood that such values ​​are fixedly included in the variations that are necessarily caused by the standard deviation present in their respective test measurements.

[0152] Each operation and step can be described as a series of separate, alternating operations in a manner that aids in understanding the implementation scheme. However, the order of description should not be interpreted as implying that these operations are sequential.

[0153] The specification may use the terms "one implementation" or "some implementations," each referring to one or more identical or different implementations. Furthermore, the terms "comprising," "including," and "having" are synonymous when used in connection with implementations.

[0154] As those skilled in the art will understand, for any and all purposes, particularly in providing written description, all ranges cited herein also encompass any and all possible subranges and combinations thereof, as well as the individual values ​​constituting the ranges, particularly integer values. A cited range (e.g., weight percentage or carbide group) includes each specific value, integer, decimal, or entity within the range. Any listed range can be readily considered adequately descriptive and capable of being decomposed into at least equal halves, tertiaries, quarters, quintiles, or tenths of the same range. As a non-limiting example, each range discussed herein can be readily decomposed into the lower third, middle third, and upper third, etc. Also as those skilled in the art will understand, all language, such as “up to,” “at least,” “greater than,” “less than,” “exceeding,” “or more,” etc., includes cited numerical values, and such terms refer to ranges that can subsequently be decomposed into subranges as discussed above. In the same manner, all ratios cited herein also include all subratios falling within a wider range. Therefore, the specific values ​​cited for radicals, substituents, and ranges are merely illustrative; they do not exclude other defined values ​​or other values ​​within the defined ranges of radicals and substituents.

[0155] Those skilled in the art will also readily understand that when members are grouped together in a common manner, such as in a Markush group, the invention covers not only the entire group listed as a whole, but also each member of that group and all possible subgroups of the main group. Furthermore, for all purposes, the invention covers not only the main group, but also the main group lacking one or more members. Therefore, the invention contemplates the explicit exclusion of any one or more members of the cited group. Thus, this condition can be applied to any of the disclosed categories or embodiments, thereby excluding any one or more of the cited elements, classes of things, or embodiments from said category or embodiment, for example, from limitations used for explicit negation.

[0156] The term "effective amount" refers to an amount that effectively treats a disease, ailment, and / or condition, or produces the cited effect. For example, an effective amount could be an amount that effectively reduces the progression or severity of a disease or symptom that is being treated. The determination of a therapeutically effective amount is entirely within the capabilities of those skilled in the art, particularly in light of the disclosure provided herein. The term "effective amount" is intended to include the amount of a compound described herein or a combination of compounds described herein that, for example, effectively treat or prevent a disease or ailment of the host, or treat the symptoms of said disease or ailment. Thus, "effective amount" generally means an amount that provides the desired effect.

[0157] The term "treatment" includes (i) preventing the occurrence of a disease, pathology, or medical condition (e.g., prevention); (ii) suppressing a disease, pathology, or medical condition, or halting its development; (iii) alleviating a disease, pathology, or medical condition; and / or (iv) reducing symptoms associated with said disease, pathology, or medical condition. Therefore, the term "treatment" can be extended to prevention and can include preventing, reducing, stopping, or reversing the progression or severity of a condition or symptom being treated. Thus, the term "treatment" can include (as appropriate) medical, therapeutic, and / or preventative application.

[0158] The term "inhibition" refers to slowing down, stopping, or reversing the growth or progression of a disease, infection, condition, or cell group. Inhibition can be, for example, greater than about 20%, 40%, 60%, 80%, 90%, 95%, or 99% compared to growth or progression occurring in the absence of treatment or exposure.

[0159] The term "contact" refers to establishing a physical connection between two or more entities. For example, contacting a mammalian cell with a nanoparticle composition means that the mammalian cell and the nanoparticles share a physical connection. Methods for contacting cells with external entities in vivo and in vitro are well known in the field of biology. For example, nanoparticle compositions can be contacted with mammalian cells placed within mammals via various routes of administration (e.g., intravenous, intramuscular, intradermal, and subcutaneous), and can involve various amounts of the nanoparticle composition. Furthermore, the nanoparticle composition can contact more than one mammalian cell.

[0160] The term "specific delivery" or "specific transport" refers to the delivery of more (e.g., at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times) of therapeutic and / or preventative drugs to a target tissue (such as mammalian liver) compared to non-target tissues. The level of nanoparticle delivery to a specific tissue can be measured by comparing the weight of proteins produced in the tissue to the weight of the tissue, comparing the amount of therapeutic and / or preventative drugs in the tissue to the weight of the tissue, comparing the weight of proteins produced in the tissue to the total weight of proteins in the tissue, or comparing the amount of therapeutic and / or preventative drugs in the tissue to the total amount of therapeutic and / or preventative drugs in the tissue.

[0161] The term "encapsulation ratio" refers to the amount of therapeutic and / or preventive agents that form part of the nanoparticle composition, relative to the total amount of therapeutic or preventive agents used in the preparation of the nanoparticle composition. For example, if 97 mg of therapeutic and / or preventive agents are encapsulated in the nanoparticle composition out of a total of 100 mg initially provided to the composition, the encapsulation ratio can be 97%. The term "encapsulation" can refer to complete, substantial, or partial encapsulation, closure, surrounding, or sealing.

[0162] The term “expression” of a nucleic acid sequence refers to the translation of mRNA into a polypeptide or protein and / or post-translational modifications of the polypeptide or protein.

[0163] The term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in a cell culture, in a petri dish, etc., rather than events that occur within a living organism (e.g., an animal, plant, or microorganism).

[0164] The term "in vivo" refers to events that occur within an organism, such as an animal, plant, or microorganism or its cells or tissues.

[0165] The term "ex vivo" refers to an event that occurs outside of an organism (such as an animal, plant, or microorganism, or its cells or tissues). Ex vivo events can occur in environments with minimal alteration from the natural (e.g., internal) environment.

[0166] For “polymer nanoparticles”, polymer nanoparticles suitable for use as adjuvants in this invention include poly(α-hydroxy acid), polyhydroxybutyric acid, polylactone (including polycaprolactone), polydioxanone, polyvalerone, polyorthoester, polyanhydride, polycyanoacrylate, tyrosine-derived polycarbonate, polyvinylpyrrolidone or polyester-amide, and combinations thereof.

[0167] For “inorganic nanoparticles”, such as gold nanorods or silica-based nanoparticles (e.g., mesoporous silica nanoparticles (MSN)).

[0168] Regarding "exosomes," exosomes can also be used as carriers of the mRNA or drug delivery media described in this invention. For the review, see Ha et al., July 2016, Acta Pharmaceutica Sinica B, Vol. 6, No. 4, pp. 287-296; https: / / doi.org / 10.1016 / j.apsb.2016.02.001.

[0169] The term "nanoparticle composition" refers to a composition comprising one or more lipids. Nanoparticle compositions typically have particle sizes on the order of micrometers or smaller and may include a lipid bilayer. Nanoparticle compositions include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipid complexes. For example, a nanoparticle composition may be a liposome having a lipid bilayer with a diameter of 500 nm or smaller.

[0170] The term "lipid component" refers to a component of a nanoparticle composition comprising one or more lipids. For example, a lipid component may include one or more cationic / ionizable lipids, PEGylated lipids, structured lipids, or other lipids such as phospholipids.

[0171] The terms “ionizable lipid” or “cationic lipid” refer to any of the many lipid species that carry a net positive charge at a selected pH, such as physiological pH (e.g., pH about 7.0).

[0172] The term "PEG lipid" or "PEGylated lipid" refers to lipids containing polyethylene glycol components, such as PEG-conjugated lipids, PEG-modified lipids, or PEG-modified lipids.

[0173] The term "phospholipid" refers to a lipid comprising a phosphate ester moiety and one or more carbon chains, such as unsaturated fatty acid chains. Phospholipids may contain one or more (e.g., double or triple bonds) bonds (e.g., one or more unsaturated bonds). Certain phospholipids can facilitate fusion with membranes. For example, cationic phospholipids can interact with one or more negatively charged phospholipids in a membrane (e.g., a cell membrane or intracellular membrane). Phospholipid fusion with a membrane can allow one or more elements of a lipid-containing composition to cross the membrane, thereby allowing, for example, the delivery of one or more elements into the cell.

[0174] The nanoparticle compositions of the present invention may also comprise salts of one or more compounds. The salts may be pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" refers to a derivative of the disclosed compound wherein the parent compound is modified by converting an existing acid or base portion into its salt form (e.g., by reacting the free base with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; bases or organic salts of acidic residues such as carboxylic acids, etc. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, disaccharide, dodecyl sulfate, ethanesulfonate, fumarate, glucoheponicate, glucoheponicate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, palmitate, pectate, 3-phenylpropionate, phosphate, picrate, neopentyl ester, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, etc.

[0175] Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. Pharmaceutically acceptable salts of this application include, for example, conventional non-toxic salts of parent compounds formed from non-toxic inorganic or organic acids. Pharmaceutically acceptable salts of this application can be synthesized by conventional chemical methods from parent compounds containing a basic or acidic moiety. Typically, these salts can be prepared by reacting the free acidic or basic form of these compounds with a stoichiometric amount of a suitable base or acid in water, in an organic solvent, or in a mixture of both. Non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are typically preferred.

[0176] The term "polydispersion index" is a ratio that describes the uniformity of the particle size distribution of a system. Smaller values, such as less than 0.3, indicate a narrow particle size distribution.

[0177] The term "method of administration" may include intravenous, intramuscular, intradermal, subcutaneous, or other methods of delivering the composition to a subject. Any method of administration may be chosen for targeted delivery (e.g., specific delivery) to a particular area or system of the body.

[0178] The term "patient" refers to a subject who may seek or need treatment, needs treatment, is receiving treatment, will receive treatment, or is a subject for whom a trained professional is to provide care for a particular disease. The term "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms that, to a reasonable extent of medical judgment, are suitable for contact with tissues in humans and animals without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0179] The term "pharmaceuticalally acceptable excipient or adjuvant" refers to a diluent, excipient, or carrier administered co-administered with a therapeutic agent, and which, to the extent of reasonable medical judgment, is suitable for contact with human and / or other animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, sterile liquids such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is an exemplary carrier when the pharmaceutical composition is administered intravenously. Physiological saline and aqueous solutions of glucose and glycerol can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. The compositions may also contain small amounts of wetting agents, emulsifiers, or pH buffers as needed. Oral formulations may contain standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. Specifically, excipients include, but are not limited to, anti-adhesion agents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (pigments), modifiers, emulsifiers, fillers (diluents), film-forming agents or coatings, flavorings, fragrances, flow enhancers, lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water for hydration. More specifically, excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, dicalcium hydrogen phosphate, calcium stearate, croscarmellose sodium, croscarmellose polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethyl cellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, phenylparaben, retinyl palmitate, shellac, silica, sodium carboxymethyl cellulose, sodium citrate, sodium glycolate starch, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (α-tocopherol), vitamin C, and xylitol.

[0180] The pharmaceutical compositions of the present invention can act systemically and / or locally. For this purpose, they can be administered via suitable routes, such as by injection (e.g., intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular injection, including infusion) or transdermal administration; or by oral, sublingual, nasal, transmucosal, topical, ophthalmic formulation, or inhalation administration. For these routes of administration, suitable dosage forms can be used to administer the pharmaceutical compositions of the present invention. These dosage forms include, but are not limited to, tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, and syrups.

[0181] The term "single unit dose" refers to the dose of any therapeutic agent administered in a single dose / once / through a single route / at a single point of contact, i.e., a single application.

[0182] The term "fractionated dose" refers to dividing a single unit dose or the total daily dose into two or more doses.

[0183] The term "total daily dose" is a given or prescribed amount over 24 hours. It can be administered as a single unit dose.

[0184] In the context of nanoparticle compositions, “particle size” or “average particle size” refers to the average diameter of the nanoparticle composition.

[0185] The terms "subject" or "patient" refer to any organism to which the composition according to this application may be administered, for example, for experimental, diagnostic, preventive, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals, such as mice, rats, rabbits, non-human primates, and humans) and / or plants.

[0186] The term "target cell" refers to any one or more target cells. These cells can be found in vitro, in vivo, in situ, or in the tissues or organs of an organism. The organism can be an animal, preferably a mammal, more preferably a human, and most preferably a patient.

[0187] The term "target tissue" refers to any one or more target tissue types in which therapeutic and / or prophylactic delivery will result in the desired biological and / or pharmacological effect. Examples of target tissues include specific tissues, organs, and their systems or groups. In a particular application, target tissues may be the kidney (e.g., intracoronary or intrafemoral) or blood vessels (e.g., via intratumoral injection), the kidney, lung, spleen, or vascular endothelium. "Non-target tissue" refers to any one or more tissue types in which the expression of encoded proteins does not result in the desired biological and / or pharmacological effect. In a particular application, non-target tissues may include the liver and spleen.

[0188] The terms "therapeutic agent" or "preventive agent" refer to any pharmaceutical agent that, when administered to a subject, has a therapeutic, diagnostic, and / or preventive effect and / or induces the desired biological and / or pharmacological action. Therapeutic agents are also known as "active agents" or "active components." Such substances include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutic agents, small molecule drugs, proteins, and nucleic acids.

[0189] The term "transfection" refers to the introduction of a species (e.g., RNA) into cells. Transfection can be performed, for example, in vitro, outside the body, or in vivo.

[0190] The term "mol%" is calculated based on the total number of moles of lipids in the lipid nanoparticle composition, that is, the percentage of lipid components in the lipid nanoparticle composition relative to the total lipids present in the lipid nanoparticle composition.

[0191] The term total lipids refers to all lipids in a lipid nanoparticle composition. Specifically, total lipids can consist of ionizable lipids, phospholipids, structural lipids, and PEG lipids.

[0192] The term "N:P ratio" or "N / P ratio" refers to the molar ratio of ionizable nitrogen atoms to phosphate groups in the RNA in a lipid nanoparticle composition.

[0193] To better understand the above technical solutions, the following will provide a detailed explanation of the technical solutions in conjunction with the accompanying drawings and specific implementation methods.

[0194] Example 1: Sample preparation of mRNA / enhancing protein

[0195] 1.1 mRNA preparation

[0196] (1) Plasmid linearization

[0197] The plasmid encoding firefly luciferase was linearized with restriction endonuclease SapⅠ. The reaction system is shown in Table 1. The plasmid containing the target gene was digested at 37℃ for 3 h.

[0198] Table 1. Plasmid linearization restriction enzyme digestion system

[0199]

[0200] Take 2 μL of the enzyme digestion product and perform 1% agarose gel electrophoresis to check the linearization of the plasmid. Purify the linearized plasmid using a PCR product recovery kit (Comway Century).

[0201] (2) In vitro transcription and purification

[0202] The linearized recombinant plasmid obtained in step (1) was used as a template for in vitro transcription using a high-yield T7 RNA transcription kit. The high-yield T7 RNA transcription kit, product name: High Yield T7 RNA Synthesis Kit, Shanghai Zhaowei Technology Development Co., Ltd., product catalog number: ON-040; 5× Reaction Buffer, 100mM ATP Solution, 100mM CTP Solution, 100mM GTP Solution, Enzyme mix, DNase I, Ammonium Acetate Stop Solution, and Lithium Chloride (LiCl) Precipitation Solution are all components of the high-yield T7 RNA transcription kit. 100mM ΨUTP Solution (pseudouridine triphosphate), full name: N1-Me-pUTP, 100mM. Each component was added according to the following system (Table 2) (taking a 20μL reaction system as an example), mixed well, and reacted at 37℃ for 3h.

[0203] Table 2. In vitro transcription system

[0204]

[0205] Among them, CleanCap AG is m7G(5')ppp(5')(2'-OMeA)pG.

[0206] After transcription, 1 μL of DNase I was added, and the mixture was incubated at 37°C for 15 min. Then, 15 μL of Ammonium Acetate Stop Solution was added and mixed well. Next, 1 / 3 volume of 7.5 M Lithium Chloride (LiCl) Precipitation Solution was added (to a final concentration of 2.5 M), and the mixture was incubated at -20°C for 30 min. The mixture was centrifuged at 12000 g for 15 min, and the RNA precipitate was discarded. 1 mL of 70% ethanol was added to wash the RNA, and the mixture was centrifuged at 12000 g for 5 min, discarding the supernatant. After air-drying, 50 µL of RNase-free water was added to dissolve the precipitate, and mRNA quantification was performed using a UV spectrophotometer to obtain capped in vitro transcribed mRNA.

[0207] 1.2 Lipid nanoparticle (LNP) encapsulation

[0208] The luciferase mRNA stock solution obtained in step 1.1 was dispersed in 20 mM acetic acid solution (pH 5.0) to obtain an RNA solution with an mRNA concentration of 200 µg / mL. The solution was mixed with enhancing protein (human eIF4E, mouse eIF4E, or human CBP80) at a molar ratio of 1:1. The mixture was further mixed with compound 9 at a molar ratio of cholesterol:DSPC:DMG-PEG2000 of 50:38.5:10:1.5 to form a lipid mixture. The flow rates of the aqueous and oil phases were controlled using a T-junction method to mix the mRNA and lipid mixture. The syringe pump was started to mix the mRNA solution with the lipid mixture to form LNPs. The mixture was then diluted 2-fold with diluent, concentrated by centrifugation through ultrafiltration, and subjected to a 5-fold solution replacement. The resulting solution was adjusted to pH 7.0–8.0 with Tris aqueous solution to obtain an LNP-encapsulated mRNA solution. LNPs are lipid nanoparticles.

[0209] The prepared encapsulated samples are shown in Table 1:

[0210] Table 1. Prepared Encapsulated Samples

[0211]

[0212] Example 2: Enhancement of mRNA / eIF4E on mRNA expression

[0213] 2.1 Detection of the effect of mRNA / eIF4E nanoparticles on luciferase expression at the cellular level

[0214] Cell lines from different species were seeded into 96-well plates. After 24 hours, 50 ng of sample-loaded material was added to each well (as shown in Figure 1). After 24 hours, the culture medium was removed, and luciferase activity was quantitatively analyzed using a luciferase detection system (Promega, E1501). GraphPad Prism 8 software was used to perform multiple t-tests for statistical analysis of the significance between groups. p<0.001).

[0215] As shown in Figure 1, regardless of whether it was human, monkey, or mouse cells, the luciferase activity expressed in the samples co-encapsulated with human eIF4E and mRNA was significantly higher than that in the samples containing only mRNA. This indicates that human eIF4E can enhance the expression of mRNA in cells.

[0216] 2.2 Animal-level detection of the effect of mRNA / eIF4E nanoparticles on luciferase expression

[0217] The LNP-loaded mRNA sample prepared in Example 1.2 was diluted with PBS buffer to obtain an injection solution with a concentration of 50 μg / ml. BALB / c female mice weighing approximately 20g were administered the solution via tail vein injection using an insulin syringe, with each mouse receiving 200 μl. Six hours after injection, in vivo animal imaging was performed using Perkinelmer's IVIS to detect the in vivo expression of luciferase. The substrate was D-luciferin sodium salt (GOLDBIO, LUCNA-1G), prepared with physiological saline to a concentration of 15 mg / ml, sterilized by filtration through a 0.22 μm filter, aliquoted, and stored at -20°C protected from light. Before imaging, each mouse weighing approximately 20g was intraperitoneally injected with 200 μl of the substrate solution for 10-20 minutes. The mice were then anesthetized with isoflurane gas and placed on the imaging plate for in vivo fluorescence detection.

[0218] GraphPad Prism 8 software was used to perform multiple t-tests for statistical analysis to determine the significance between groups. (p<0.01). The results are shown in Figure 2. Similar to the results at the cellular level, the luciferase activity expressed in mice by samples co-encapsulated with human eIF4E and mRNA was significantly higher than that of samples containing only mRNA.

[0219] Example 3: Effects of different enhancing proteins and mRNA encapsulation on mRNA expression

[0220] This invention further investigates the effects of different species of eIF4E (human and mouse) and different enhancing proteins (eIF4E and CBP80) on luciferase mRNA expression at the cellular level. Using a similar experimental method to Example 2.1, human 293T cells and mouse BHK21 cells were seeded in 96-well plates. After 24 hours, 50 ng of the loaded sample was added to each well (as shown in Figure 1). After 24 hours, the culture medium was removed, and luciferase activity was quantitatively analyzed using a luciferase detection system (Promega, E1501). GraphPad Prism 8 software was used to perform multiple t-tests for statistical analysis of the significance between groups (…). p<0.001).

[0221] As shown in Figure 3, the luciferase activity of samples co-encapsulated with these three enhancing proteins and mRNA in 293T cells and BHK21 cells was significantly higher than that of samples containing only mRNA. This indicates that eIF4E and CBP80 can enhance the expression of mRNA in cells.

[0222] Example 4: Effects of different enhancing proteins and mRNA encapsulation on in vivo mRNA expression and distribution

[0223] The drug administration to mice was performed using a method similar to that in Example 2.2. BALB / c female mice weighing approximately 20g were administered insulin via tail vein injection, with each mouse receiving 200μl. Six hours after injection, the substrate, D-luciferin sodium salt (GOLDBIO, LUCNA-1G), was prepared with physiological saline to a concentration of 15 mg / ml, sterilized by filtration through a 0.22μm filter, aliquoted, and stored at -20°C protected from light. Before imaging, each mouse weighing approximately 20g was intraperitoneally injected with 200μl of the substrate solution, allowing it to act for 10-20 minutes. Tissues from the mice (heart, liver, spleen, lung, kidney, stomach, and intestine) were then subjected to fluorescence imaging using PerkinElmer IVIS to quantitatively detect the expression of luciferase in each tissue.

[0224] GraphPad Prism 8 software was used to perform multiple t-tests for statistical analysis to determine the significance between groups. p<0.05; (p<0.001). The results are shown in Figures 4-6. Each enhancing protein altered the expression distribution of mRNA in different tissues, with increased mRNA distribution in the spleen, stomach, and intestine.

[0225] The results above indicate that adding mRNA to the mRNA delivery system can enhance mRNA expression in cells, and that the enhancer protein can alter the expression distribution of mRNA in various tissues, especially increasing the distribution of mRNA in the spleen, stomach, and intestine.

[0226] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composition for delivering mRNA, characterized in that, The composition includes mRNA and enhancer protein.

2. The composition according to claim 1, characterized in that, The enhancing protein is selected from one or more of the following: human eIF4E, mouse eIF4E, human CBP80, human CBP20, human eIF4A, human eIF4G, human eIF4B, eIF3A, eIF1, eIF1AX, human PABPC1, human LARP1, human LARP4, human RACK1, human mTOR, and human MAPK1.

3. The composition according to claim 1, characterized in that, The enhancing protein is selected from eIF4E and / or CBP80.

4. The composition according to claim 1, characterized in that, The enhancing protein is selected from human eIF4E, mouse eIF4E and / or human CBP80.

5. A pharmaceutical formulation for delivering mRNA, characterized in that, The pharmaceutical preparation comprises the composition according to any one of claims 1-4.

6. The pharmaceutical formulation as described in claim 5, characterized in that, The pharmaceutical formulation is selected from lipid nanoparticles, cationic lipid complexes, lipid polymers, polymer nanoparticles, exosomes, inorganic nanoparticles, or biological microvesicles.

7. The pharmaceutical formulation as described in claim 5, characterized in that, The pharmaceutical preparation is selected from lipid nanoparticles.

8. A lipid nanoparticle composition, characterized in that, The lipid nanoparticle composition comprises mRNA, the synergistic protein according to any one of claims 1-4, and lipid nanoparticles; optionally, the mRNA and the synergistic protein according to any one of claims 1-4 are partially or completely encapsulated by the lipid nanoparticles; the lipid nanoparticles include ionizable lipids, phospholipids, structural lipids, and PEG lipids.

9. The lipid nanoparticle composition according to claim 8, characterized in that, The molar ratio of the mRNA to the aforementioned enhancing protein is (1-50):(1-50); preferably, the molar ratio of the mRNA to the aforementioned enhancing protein is (1-30):(1-30); preferably, the molar ratio of the mRNA to the aforementioned enhancing protein is (1-25):(1-25); preferably, the molar ratio of the mRNA to the aforementioned enhancing protein is (1-20):(1-20); preferably, the molar ratio of the mRNA to the aforementioned enhancing protein is (1-10):(1-10); preferably, the molar ratio of the mRNA to the aforementioned enhancing protein is (1-5):(1-5); preferably, the molar ratio of the mRNA to the aforementioned enhancing protein is approximately 1:

1.

10. The lipid nanoparticle composition according to claim 8, characterized in that, The molar ratio of the sum of the ionizable lipids, the phospholipids and the structural lipids, and the PEG lipids is (35-65):(35-65):(0.5-5).

11. The lipid nanoparticle composition according to claim 10, characterized in that, The molar ratio of the sum of the ionizable lipids, the phospholipids and the structural lipids, and the PEG lipids is (40-50):(35-65):(1-3).

12. The lipid nanoparticle composition according to claim 10, characterized in that, The molar ratio of the sum of the ionizable lipids, the phospholipids and the structural lipids, and the PEG lipids is (50-65):(35-65):(1-3).

13. The lipid nanoparticle composition according to claim 10, characterized in that, The molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is (40-50):(5-15):(30-50):(1-3).

14. The lipid nanoparticle composition according to claim 10, characterized in that, The molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is (40-50):(10-15):(35-45):(1.5-2.5).

15. The lipid nanoparticle composition according to claim 10, characterized in that, The molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is (50-65):(5-15):(30-50):(1-3).

16. The lipid nanoparticle composition according to claim 10, characterized in that, The molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is (50-65):(10-15):(35-45):(1.5-2.5).

17. The lipid nanoparticle composition of claim 10, characterized in that, The content of ionizable lipids is 35mol%-65mol%, the content of phospholipids and the sum of the structural lipids is 35mol%-65mol%, and the content of PEG lipids is 0.5mol%-5mol%, where mol% is calculated based on the total number of moles of lipids in the lipid nanoparticle composition.

18. The lipid nanoparticle composition according to claim 10, characterized in that, The content of ionizable lipids is 40 mol%-50 mol, preferably 45 mol%-49 mol, and more preferably 48 mol%-49 mol.

19. The lipid nanoparticle composition according to claim 10, characterized in that, The content of ionizable lipids is 50 mol%-65 mol%, preferably 51 mol%-55 mol%, and more preferably 52 mol%-53 mol%.

20. The lipid nanoparticle composition according to claim 10, characterized in that, The content of the phospholipids and the structural lipids is 35 mol%-65 mol%, preferably 35 mol%-55 mol%, and more preferably 45 mol%-50 mol%.

21. The lipid nanoparticle composition according to claim 10, characterized in that, The phospholipid content is 5 mol%-15 mol, preferably 10 mol%-15 mol.

22. The lipid nanoparticle composition according to claim 10, characterized in that, The structured lipid content is 30 mol%-50 mol%, preferably 35 mol%-45 mol%.

23. The lipid nanoparticle composition according to claim 8, characterized in that, The ionizable lipids are selected from 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA; "XTC2"), and 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane. -Dioxane (DLin-K-C3-DMA), 2,2-dilinyl-4-(4-dimethylaminobutyl)-[1,3]-dioxane (DLin-K-C4-DMA), 2,2-dilinyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinyl-4-N-methylpepiazino-[1,3]-dioxane (DLin-K- MPZ), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleyl-3 1,2-Dilinoleothio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleoyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleoyloxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), and 1,2-dilinoleoyl-3-trimethylaminopropane chloride (DLin-TAP).Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleyloxy-N,N-dimethylaminopropane ( DODMA), 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2 ...',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), 3-(N',N'-dioleoyl)-carbamoyl)cholesterol (DC-Chol), 3-(N',N'-dioleoyl)-carbamoyl)-cholesterol (DC-Chol), 3-(N',N'-dioleoyl)-carbamoyl)-carbamoyl) Myristyloxypropyl-3-yl)-N,N-dimethyl-N-carboxyethylammonium bromide (DMRIE), 2,3-dioleoxy-N-[2(spermine-formylamino)ethyl]-N,N-dimethyl-1-propanetrimonium trifluoroacetate (DOSPA), bis(octadecylaminoglycyl)spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-β-oxybut-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-β-oxy) The following are included in the list of one or more of the following: 3'-oxaproloxy)-3-dimethyl-1-(cis,cis-9',1-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleoyloxybenzylamine (DMOBA), 1,2-N,N'-dioleoylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilinoleoylcarbamoyl-3-dimethylaminopropane (DLincarbDAP), MC3, SM-102, and ALC-0315.

24. The lipid nanoparticle composition according to claim 8, characterized in that, The ionizable lipids are selected from compounds of formula IIA, or pharmaceutically acceptable salts thereof, or stereoisomers thereof. ； R 1a and R 1b Independently selected from -CH3; R2 and R3 are independently selected from H; R4 is selected from C8, C9, C10, C11 or C12 straight-chain alkyl groups; R5 and R6 are independently selected from C6, C7 and C8 straight-chain alkyl groups; X1 and Y1 are different, X2 and Y2 are different, and X1 and X2 are independently selected from C=O or O; Y1 and Y2 are independently selected from C=O or O; o and p are independently selected from 4, 5, 6, 7, 8, and 9; q is selected from 2, 3, 4, or 5; Preferably, R4 is selected from C10 or C11 straight-chain alkyl groups; Preferably, R5 and R6 are independently selected from C6 and C8 straight-chain alkyl groups; more preferably, R5 and R6 are both C8 straight-chain alkyl groups or R5 is a C6 straight-chain alkyl group and R6 is a C8 straight-chain alkyl group. Preferably, q is 2; Preferably, q is 3; Preferably, q is 4; Preferably, X1 and X2 are both C=O, and Y1 and Y2 are both O; Preferably, X1 is 0, Y1 is C=0, and X2 is C=0 and Y2 is 0; Preferably, X1 is C=O, Y1 is O, and X2 is O and Y2 is C=O; Preferably, X1 and X2 are both 0, and Y1 and Y2 are both C=0; More preferably, the ionizable lipid is selected from any one or more of the following compounds: Compound 9 、 Compound 10 、 Compound 11 、 Compound 12 、 Compound 13 or Compound 14 。 25. The lipid nanoparticle composition according to claim 8, characterized in that, The phospholipid is selected from one or more of the following compounds: 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-distearateoyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-diundecanoyl-sn-glycerol-3-phosphate choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), and 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0Diether PC). 1-Oleoyl-2-cholesterolylhemisuccinoyl-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16LysoPC), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicoyl-sn-glycerol-3-phosphate choline, 1,2-bis(docosahexaenooyl-sn-glycerol-3-phosphate choline), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-diphydanoyl-sn-glycerol-3-phosphate ethanolamine (ME16.0PE), 1,2-distearatel-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn- Glyceryl-3-phosphate ethanolamine, 1,2-disarachidanoyl-sn-glyceryl-3-phosphate ethanolamine, 1,2-bis(docosahexaenoyl-sn-glyceryl-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glyceryl-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), dioleoylphosphatidylserine (DOPS), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearate-phosphatidyl - Ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine (LPE).

26. The lipid nanoparticle composition according to claim 8, characterized in that, The phospholipid is selected from DSPC.

27. The lipid nanoparticle composition according to claim 8, characterized in that, The structural lipids are selected from one or more of cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, tomatidine, tomatine, ursolic acid, and α-tocopherol.

28. The lipid nanoparticle composition according to claim 8, characterized in that, The structural lipid in question is cholesterol.

29. The lipid nanoparticle composition according to claim 8, characterized in that, Structural lipids include cholesterol and one or more of corticosteroids such as prednisolone, dexamethasone, and hydrocortisone.

30. The lipid nanoparticle composition according to claim 8, characterized in that, The structural lipid is cholesterol.

31. The lipid nanoparticle composition according to claim 8, characterized in that, The PEG lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, or PEG-modified dialkylglycerol.

32. The lipid nanoparticle composition according to claim 8, characterized in that, The PEG lipid is DMG-PEG2000, DMPE-PEG2000, or DSPE-PEG2000.

33. The lipid nanoparticle composition according to claim 8, characterized in that, The lipid nanoparticles have a diameter of approximately 30 nm to approximately 200 nm.

34. The lipid nanoparticle composition according to claim 8, characterized in that, The lipid nanoparticles have a diameter of approximately 70 nm to approximately 150 nm.

35. The lipid nanoparticle composition according to claim 8, characterized in that, The lipid nanoparticles have a diameter of approximately 90 nm to approximately 110 nm.

36. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the lipid nanoparticle composition according to any one of claims 8-35 and a pharmaceutically acceptable excipient or auxiliary ingredient.

37. A method for delivering a therapeutic agent and / or a preventative agent to mammalian cells, characterized in that, The method includes administering to a subject the lipid nanoparticles of any one of claims 8-35 or the pharmaceutical composition of claim 36, wherein the administration includes contacting cells with the nanoparticle composition or the pharmaceutical composition to deliver a therapeutic agent and / or a preventative agent to the cells.

38. The method as described in claim 37, characterized in that, The mammalian cells mentioned therein are found in mammals.

39. The method as described in claim 37, characterized in that, The mammal mentioned is a human.

40. The method as described in claim 37, characterized in that, The lipid nanoparticles are administered via intravenous, intramuscular, intradermal, subcutaneous, intranasal, or inhalation.

41. A method for producing a target polypeptide in mammalian cells, characterized in that, The method comprises contacting cells with lipid nanoparticles according to any one of claims 8-35 or a pharmaceutical composition according to claim 36 to deliver a therapeutic and / or preventive agent to the cells, wherein the therapeutic and / or preventive agent is mRNA encoding a target polypeptide, thereby enabling the mRNA to be translated in the cells to produce the target polypeptide.

42. The method as described in claim 41, characterized in that, The mammalian cells mentioned therein are found in mammals.

43. The method as described in claim 41, characterized in that, The mammal mentioned is a human.

44. The method as described in claim 41, characterized in that, The lipid nanoparticles or pharmaceutical composition are administered via intravenous, intramuscular, intradermal, subcutaneous, intranasal, or inhalation.

45. A method for treating and / or inhibiting a disease or symptom in a mammal, the method comprising administering to the mammal a therapeutically effective amount of the lipid nanoparticles of any one of claims 8-35 or the pharmaceutical composition of claim 36.

46. ​​The method as described in claim 45, characterized in that, The disease or condition described therein is characterized by dysfunction or abnormal protein or polypeptide activity.

47. The method as described in claim 45, characterized in that, The diseases or conditions mentioned therein are selected from infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases, renal vascular diseases, or metabolic diseases.

48. The method as described in claim 45, characterized in that, The mammal mentioned is a human.

49. The method as described in claim 45, characterized in that, The lipid nanoparticles or pharmaceutical composition are administered via intravenous, intramuscular, intradermal, subcutaneous, intranasal, or inhalation.

50. A method for specifically delivering a therapeutic agent and / or a preventive agent to a mammalian organ, characterized in that, The method comprises administering to a mammal the lipid nanoparticles of any one of claims 8-35 or the pharmaceutical composition of claim 36, the administration comprising contacting a mammalian organ with the nanoparticles, thereby delivering a therapeutic and / or preventative agent to the organ.

51. The method as described in claim 50, characterized in that, The mammal mentioned is a human.

52. The method as described in claim 50, characterized in that, The lipid nanoparticles or pharmaceutical composition are administered via intravenous, intramuscular, intradermal, subcutaneous, intranasal, or inhalation.

53. The method as described in claim 50, characterized in that, The mammals mentioned therein are pretreated 24 hours or less prior to the contact or administration step.

54. The method as described in claim 50, characterized in that, The mammals mentioned therein are pretreated approximately one hour prior to the contact or administration step.

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