Composition for delivering genetic material having improved long-term storage stability

Abstract

The present invention relates to a composition for delivering genetic material having improved long-term storage stability. The composition comprises an ionic liquid including a choline cation and an anion, and lipid nanoparticles including an ionizable lipid, a polyethylene glycol (PEG)-lipid, a phospholipid, and a sterol lipid, thereby enabling intracellular delivery of genetic material while effectively suppressing degradation of genetic material (nucleic acid) during long-term storage and maintaining long-term stability.

Description

Composition for delivering genetic material with improved long-term storage stability

[0001] The present invention relates to a composition for delivering genetic material with improved long-term storage stability. More specifically, the invention relates to a composition and a method for preparing the same that can deliver genetic material into a cell by including an ionic liquid containing choline cations and anions, and lipid nanoparticles containing ionizable lipid, PEG (polyethylene glycol)-lipid, phospholipid, and sterol lipid, thereby inhibiting the degradation of genetic material (nucleic acid) during long-term storage and maintaining long-term stability.

[0002] Nucleic acid-based gene therapies and vaccines are attracting attention as next-generation medical technologies with high potential for the prevention and treatment of diseases. In particular, nucleic acids such as mRNA and siRNA are being actively researched as therapeutic agents for various infectious and intractable diseases because they can directly regulate the expression of target proteins. However, there is a fundamental limitation in that stable delivery and storage are difficult because nucleic acids are easily degraded by enzymes such as nucleases or hydrolysis reactions in in vivo and in vivo environments.

[0003] To address these issues, lipid nanoparticles (LNPs) have been developed as carriers to protect nucleic acids and enhance intracellular delivery efficiency. Lipid nanoparticles can protect nucleic acids from the external environment by encapsulating them internally, and their efficacy and safety have been proven through their application in mRNA-based COVID-19 vaccines.

[0004] However, it has been pointed out that lipid nanoparticles still lack sufficient long-term storage stability. In particular, since refrigeration or freezing at low temperatures is often required, this leads to increased costs and logistical constraints during transportation and storage. These limitations act as major factors hindering the rapid and stable supply of vaccines and gene therapies in the event of a large-scale infectious disease outbreak, such as a pandemic.

[0005] Therefore, there is a need to develop a composition capable of delivering genetic material into cells, inhibiting the degradation of genetic material (nucleic acid) during long-term storage, and maintaining long-term stability, as well as a method for manufacturing the same.

[0006] The present invention aims to provide a composition for delivering genetic material and a method for manufacturing the same, which can deliver genetic material into a cell, inhibit the degradation of genetic material (nucleic acid) during long-term storage, and maintain long-term stability.

[0007] In addition, the present invention aims to provide a composition having high genetic material (drug) loading efficiency and an average particle size suitable for use as a vaccine delivery vehicle, and a method for manufacturing the same.

[0008] The present invention is capable of various modifications and may have various embodiments, and specific embodiments are to be illustrated and described in detail. However, this is not intended to limit the invention to specific embodiments, and it should be understood that the invention includes all modifications, equivalents, and substitutions that fall within the spirit and scope of the invention.

[0009] The terms used in this application are used merely to describe specific embodiments and are not intended to limit the invention. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this application, terms such as "comprising" or "having" are intended to specify the presence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0010] Where in this specification, when a quantity, concentration, or other value or parameter is given as an enumeration of a range, a preferred range, a preferred upper limit, and a preferred lower limit, it should be understood that any pair of any upper range limit or preferred value and any lower range limit or preferred value are specifically disclosed, regardless of whether the range is disclosed separately.

[0011] Where a range of numerical values ​​is mentioned in this specification, unless otherwise stated, the range and the scope of the parent invention within that range are not intended to be limited to the specific value mentioned when defining the range.

[0012] Furthermore, to prevent clutter caused by overlapping content, redundant details have been omitted below. In other words, the content of the invention is not limited solely to the following description, and should be interpreted in accordance with the overall context of the invention.

[0013] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the present invention pertains. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this application. Specific details for the implementation of the above invention are described below.

[0014] composition for delivering genetic material

[0015] The present invention provides a composition for delivering genetic material with improved long-term storage stability.

[0016] FIG. 1 shows a schematic diagram of lipid nanoparticles (mRNA-LNP) loaded with genetic material according to the prior art and a composition for delivering genetic material (CDHP-mRNA-LNP) according to one embodiment of the present invention.

[0017] Referring to FIG. 1, specifically, the present invention provides a composition for transferring genetic material comprising an ionic liquid and lipid nanoparticles, wherein the lipid nanoparticles comprise ionizable lipid, PEG (polyethylene glycol) lipid, phospholipid, and sterol lipid, and the ionic liquid comprises choline cations and anions.

[0018] Ionic liquids are attracting attention in the field of biomolecular protection and stabilization due to their high ionic conductivity, compositional diversity, and stabilizing effects at the molecular level. While some studies have reported that ionic liquids can enhance the structural stability of nucleic acids and inhibit their degradation, attempts to apply them to lipid nanoparticle-based genetic material carriers to improve long-term storage stability are still limited.

[0019] Accordingly, the present invention aims to provide genetic material-carrying lipid nanoparticles that enable intracellular delivery of genetic material by introducing an ionic liquid, while simultaneously effectively inhibiting the degradation of genetic material (nucleic acid) during long-term storage and maintaining long-term stability. Through this, the invention aims to improve the storage stability issues of existing lipid nanoparticle delivery systems and contribute to the development of more practical gene therapies and / or vaccines.

[0020] The above ionic liquid may include choline dihydrogen phosphate, choline chloride, or both.

[0021] The above ionic liquid may be supported within the lipid nanoparticles.

[0022] Specifically, the loading ratio of the ionic liquid supported on the lipid nanoparticles may be 5 to 15 weight%, preferably 10 weight%, based on the total weight of the ionic liquid used in the preparation of the composition.

[0023] It is preferable that the above ionic liquid is not present outside the lipid nanoparticles. In the present invention, by having the ionic liquid present inside the lipid nanoparticles, the hydrolysis of genetic material can be effectively inhibited, and toxicity during the process of delivering genetic material into the cell can be reduced compared to when the ionic liquid is present outside.

[0024] The above composition may contain 1 to 2,000 mM of the ionic liquid, preferably 100 to 2,000 mM, and more preferably 400 to 2,000 mM. If the ionic liquid is less than 1 mM, it is undesirable because the effects of inhibiting genetic material degradation and improving long-term stability due to the inclusion of the ionic liquid are not sufficiently expressed. If it exceeds 2,000 mM, it is undesirable because the efficiency of intracellular delivery of genetic material may decrease as the average particle size increases, and it may deviate from the particle size range suitable for the intended vaccine delivery vehicle.

[0025] The above composition may contain a total of 1 to 15 mM of the ionized lipid, the PEG-lipid, the phospholipid, and the sterol lipid. That is, the total lipid concentration of the composition may be 1 to 15 mM. The total lipid concentration of the composition may preferably be 2 to 14 mM, more preferably 3 to 13 mM. In addition, to form particles with an average particle size (e.g., 100 to 150 nm) suitable for a vaccine delivery vehicle, the total lipid concentration may be, for example, 3 to 6.5 mM. If the total lipid concentration is less than 1 mM, the encapsulation efficiency of the genetic material decreases, which is undesirable; and if it exceeds 15 mM, the intracellular delivery efficiency of the genetic material may decrease as the average particle size increases, and it may fall outside the range suitable for the intended vaccine delivery vehicle, which is also undesirable.

[0026] By controlling the above ionic liquid concentration and total lipid concentration, the average size and uniformity of the formed composition (particles) can be controlled.

[0027] Among the components of lipid nanoparticles according to one example, the ionized lipid is an ionizable compound having properties similar to lipids, and can play a role in encapsulating the genetic material within the lipid nanoparticles with high efficiency through electrostatic interaction with the genetic material.

[0028] The above ionized lipids are 1-octylnonyl ester (SM-102), (6Z, 9Z, 28Z, 31Z)-hephtatriaconta 6,9,28,31-tetraene-19-yl4-(dimethylamino)butanoate (DLin-MC3-DMA), 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP), [(4-hydroxybutyl)azandyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-CDAP), 1,2-Dilinoleoyl-3-Dimethylaminopropane (DLin-DAP), 1,2-Dilinoleyloxy-N,N-Dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-Dimethylaminomethyl-[1,3]-Dioxolane (DLin-K-DMA), 2,2-Dilinoleyl-4-(2-Dimethylaminoethyl)-[1,3]-Dioxolane (DLin-KC2-DMA), 1,2-Dioleoyl-3-Dimethylammonium Propane (DODAP), N,N-Dimethyl-(2,3-Dioleyloxy)Propyamine (DODMA), Dioctadecylamidoglycyl Spermine (DOGS), Spermine Cholesteryl Carbamate (GL-67), Bis-Guanidinium-Spermidine-Cholesterol (BGTC), 3β-(N(N',N'-dimethylaminoethane)-carbamoyl) cholesterol (DC-Chol), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydecyl)amino)ethyl)(2-hydroxydecyl)amino)ethyl)piperazine-1-yl)ethylazandiyl)dododecane-2-ol (C12-200), Nt-butyl-N'-tetradecylamino-propionamidine (diC14-amidine), dimethyldioctadecylammonium bromide (DDAB), N(1,2-dimyristyl oxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N,N-dioleylN,N-dimethylammonium chloride (DODAC), dioleyl Oxypropyl-3-dimethylhydroxyethylammonium bromide (DORIE), N-(1-(2,3-dioleyl oxyl)propyl)-N-2-(sperminecarboxamide)ethyl)-N,It may include one or more selected from the group consisting of N-dimethylammonium trifluoroacetate (DOSPA), N-(1-(2,3-dioleyl oxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), aminopropyl-dimethyl-bis(dodecyloxy)-propane aluminum bromide (GAP-DLRIE), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dipalmityl-3-trimethylammonium propane (DPTAP), 1,2-distearyl-3-trimethylammonium propane (DSTAP), and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), and preferably may include 1-octylnonyl ester (SM-102).

[0029] In this specification, "PEG(polyethyleneglycol)-lipid," "lipid-PEG," "PEG-lipid," or "lipid-PEG" refers to a form in which lipid and PEG are conjugated, meaning a lipid in which a polyethylene glycol (PEG) polymer, which is a hydrophilic polymer, is bonded to one end. The PEG-lipid contributes to the particle stability of lipid nanoparticles and serves to prevent aggregation between nanoparticles.

[0030] The above PEG-lipids may include one or more selected from the group consisting of poly(ethylene glycol)-dimyristoyl glycerol (PEG-DMG), poly(ethylene glycol)-disteroyl glycerol (PEG-DSG), poly(ethylene glycol)-dipalmitoyl glycerol (PEG-DPG), poly(ethylene glycol)-dimyristoylphosphatidylethanolamine (PEG-DMPE), poly(ethylene glycol)-disteroylphosphatidylethanolamine (PEG-DSPE), poly(ethylene glycol)-dipalmitoylphosphatidylethanolamine (PEG-DPPE), methoxypoly(ethylene glycol)-N,N-ditetradecylacetamide (ALC-0159, mPEG-DTA), and poly(ethylene glycol)-ceramide (PEG-CER), and preferably, may include poly(ethylene glycol)-dimyristoyl glycerol (PEG-DMG).

[0031] The molecular weight of PEG in the above PEG-lipid may be 1,000 to 5,000 daltons, 1,500 to 5,000 daltons, 2,000 to 5,000 daltons, 1,000 to 3,000 daltons, 1,500 to 3,000 daltons, 2,000 to 3,000 daltons, 1,000 to 2,600 daltons, 1,500 to 2,600 daltons, 2,000 to 2,600 daltons, 1,000 to 2,500 daltons, 1,500 to 2,500 daltons, or 2,000 to 2,500 daltons.

[0032] According to one example, the phospholipid among the components of the lipid nanoparticle plays a role in protecting the core formed by the interaction of ionized lipids and genetic material (mRNA) within the lipid nanoparticle by surrounding it, and can facilitate cell membrane passage and endosomal escape during intracellular drug delivery by binding to the phospholipid bilayer of the target cell.

[0033] The above phosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylethanolamine (DSPE), It may include one or more selected from the group consisting of phosphatidylethanolamine (PE), dipalmitoylphosphatidylethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS), and 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine], and preferably may include distearoylphosphatidylcholine (DSPC).

[0034] According to one example, among the components of lipid nanoparticles, sterol lipids provide structural rigidity to the lipid filling within the lipid nanoparticles and can play a role in improving the stability of the nanoparticles by being dispersed in the core and surface of the nanoparticles.

[0035] The above sterol lipids may include one or more selected from the group consisting of cholesterol and beta-sitosterol, and preferably may include cholesterol.

[0036] According to one example, the lipid nanoparticles may include 1-octylnonyl ester (SM-102), poly(ethylene glycol)-dimyristoyl glycerol (PEG-DMG), distearoylphosphatidylcholine (DSPC), and cholesterol.

[0037] The lipid nanoparticles may comprise 30 to 60 mol% of the ionized lipid, 0.5 to 5 mol% of the PEG-lipid, 5 to 20 mol% of the phosphoric lipid, and 20 to 50 mol% of the sterol lipid, based on 100 mol% of the total lipid nanoparticles.

[0038] Specifically, the lipid nanoparticles may contain 30 to 60 mol%, preferably 40 to 57 mol%, 45 to 55 mol%, 47 to 53 mol%, and more preferably 48 to 52 mol% of the ionized lipid, based on 100 mol% of the total lipid nanoparticles.

[0039] In addition, the lipid nanoparticles may contain 0.5 to 5 mol%, preferably 0.8 to 3.5 mol%, 0.9 to 2.5 mol%, 1 to 2 mol%, 1.1 to 1.8 mol%, and more preferably 1.2 to 1.7 mol% of the PEG-lipid based on 100 mol% of the total lipid nanoparticles.

[0040] In addition, the lipid nanoparticles may contain 5 to 20 mol%, preferably 7.5 to 17 mol%, 8 to 15 mol%, 8.5 to 13 mol%, and more preferably 9 to 12 mol% of the phosphorus lipid, based on 100 mol% of the total lipid nanoparticles.

[0041] In addition, the lipid nanoparticles may contain 20 to 50 mol% of the sterol lipid, preferably 25 to 48 mol%, 30 to 45 mol%, 33 to 43 mol%, 34 to 41 mol%, and more preferably 35 to 40 mol%, based on 100 mol% of the total lipid nanoparticles.

[0042] According to one embodiment of the present invention, the composition of the present invention comprises an ionic liquid and lipid nanoparticles, wherein the ionic liquid comprises choline dihydrogen phosphate or choline chloride, and the lipid nanoparticles may comprise 1-octylnonyl ester (SM-102), poly(ethylene glycol)-dimyristoyl glycerol (PEG-DMG), distearoylphosphatidylcholine (DSPC), and cholesterol.

[0043] According to one embodiment of the present invention, the composition of the present invention comprises an ionic liquid and lipid nanoparticles, wherein the ionic liquid is supported on the lipid nanoparticles, and the ionic liquid comprises choline dihydrogen phosphate or choline chloride, and the lipid nanoparticles may comprise 1-octylnonyl ester (SM-102), poly(ethylene glycol)-dimyristoyl glycerol (PEG-DMG), distearoylphosphatidylcholine (DSPC), and cholesterol.

[0044] According to one embodiment of the present invention, the composition of the present invention comprises choline dihydrogen phosphate and lipid nanoparticles, and the lipid nanoparticles may comprise 1-octylnonyl ester (SM-102), poly(ethylene glycol)-dimyristoyl glycerol (PEG-DMG), distearoylphosphatidylcholine (DSPC), and cholesterol. Specifically, the composition may comprise 1 to 2,000 mM of choline dihydrogen phosphate, for example, one or more of 1, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,500, and 2,000 mM, or a range between two of these values. In addition, the total lipid concentration of the above composition may be 1 to 15 mM, for example, one or more of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 mM, or a range between two of these values. In addition, the lipid nanoparticles may comprise 30 to 60 mol% of 1-octylnonyl ester (SM-102), 0.5 to 5 mol% of poly(ethylene glycol)-dimyristoyl glycerol (PEG-DMG), 5 to 20 mol% of distearoylphosphatidylcholine (DSPC), and 20 to 50 mol% of cholesterol, based on 100 mol% of total lipid nanoparticles.

[0045] In a specific aspect, the composition of the present invention comprises 2,000 mM of choline dihydrogen phosphate and lipid nanoparticles, wherein the lipid nanoparticles comprise, based on 100 mol% of total lipid nanoparticles, 30 to 60 mol% of 1-octylnonyl ester (SM-102), 0.5 to 5 mol% of poly(ethylene glycol)-dimyristoyl glycerol (PEG-DMG), 5 to 20 mol% of distearoylphosphatidylcholine (DSPC), and 20 to 50 mol% of cholesterol, and the total lipid concentration of the composition may be 3.125 mM.

[0046] According to one embodiment of the present invention, the composition of the present invention comprises choline chloride and lipid nanoparticles, and the lipid nanoparticles may comprise 1-octylnonyl ester (SM-102), poly(ethylene glycol)-dimyristoyl glycerol (PEG-DMG), distearoylphosphatidylcholine (DSPC), and cholesterol. Specifically, the composition may comprise 1 to 2,000 mM of choline chloride, for example, one or more of 1, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,500, and 2,000 mM, or a range between two of these values. In addition, the total lipid concentration of the above composition may be 1 to 15 mM, for example, one or more of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 mM, or a range between two of these values. In addition, the lipid nanoparticles may comprise 30 to 60 mol% of 1-octylnonyl ester (SM-102), 0.5 to 5 mol% of poly(ethylene glycol)-dimyristoyl glycerol (PEG-DMG), 5 to 20 mol% of distearoylphosphatidylcholine (DSPC), and 20 to 50 mol% of cholesterol, based on 100 mol% of total lipid nanoparticles.

[0047] In a specific aspect, the composition of the present invention comprises 2,000 mM of choline chloride and lipid nanoparticles, wherein the lipid nanoparticles comprise, based on 100 mol% of total lipid nanoparticles, 30 to 60 mol% of 1-octylnonyl ester (SM-102), 0.5 to 5 mol% of poly(ethylene glycol)-dimyristoyl glycerol (PEG-DMG), 5 to 20 mol% of distearoylphosphatidylcholine (DSPC), and 20 to 50 mol% of cholesterol, and the total lipid concentration of the composition may be 3.125 mM.

[0048] The above genetic material delivery composition may have an average particle size of 50 to 200 nm, preferably 60 to 190 nm, 70 to 180 nm, 80 to 170 nm, 90 to 160 nm, 95 to 155 nm, and more preferably 100 to 150 nm. The above genetic material delivery composition has an average particle size within the above range, making it easy to introduce into cells and allowing for high efficiency in delivering genetic material to target organs and / or cells, so it can be utilized as a vaccine delivery vehicle.

[0049] According to one embodiment of the present invention, the composition of the present invention comprises an ionic liquid and lipid nanoparticles, wherein the ionic liquid comprises choline dihydrogen phosphate or choline chloride, and the lipid nanoparticles comprise 1-octylnonyl ester (SM-102), poly(ethylene glycol)-dimyristoyl glycerol (PEG-DMG), distearoylphosphatidylcholine (DSPC), and cholesterol, and the composition may have an average particle size of 50 to 500 nm. Here, the particle size may be one or more of 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, and 500 nm, or a range between two of these values.

[0050] The above composition further comprises a genetic material, and the genetic material may be supported on the lipid nanoparticles. Specifically, the above composition comprises an ionic liquid, lipid nanoparticles, and a genetic material, and the ionic liquid and the genetic material may be supported within the lipid nanoparticles.

[0051] The above genetic material may be nucleic acid and may include, for example, one or more selected from the group consisting of messenger ribonucleic acid (mRNA), small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, transfer ribonucleic acid (tRNA), and antisense oligodeoxynucleotide (AS-ODN).

[0052] In some aspects, where the genetic material is nucleic acid, the nucleic acid may be in a naked form, which means nucleic acid not bound to a protein, lipid, or other protective molecule. Additionally, the nucleic acid may be associated with one or more lipids, proteins, carbohydrates, and / or other organic compounds (e.g., squalene).

[0053] The above composition contains genetic material between 0.001 and 100 mg / mL, for example, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 20, 30, It may contain genetic material in a range between two of the following values: 40, 50, 60, 70, 80, 90, and 100 mg / mL. The genetic material may be nucleic acid, preferably messenger ribonucleic acid (mRNA) or ribonucleic acid (RNA) (e.g., yeast RNA). In a specific aspect, the genetic material is mRNA, and the composition may contain mRNA in an amount of 0.01 to 10 mg / mL, 0.05 to 1 mg / mL, 0.07 to 0.5 mg / mL, e.g., 0.1 mg / mL. In a specific aspect, the genetic material is RNA (e.g., yeast RNA), and the composition may contain RNA in an amount of 0.01 to 10 mg / mL, 0.05 to 1 mg / mL, 0.07 to 0.5 mg / mL, e.g., 0.1 mg / mL.

[0054] The above composition may have a pH of 5.0 to 9.0, and the pH may be, for example, one or more of 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0, or be a range between any two of these values. there is.

[0055] In this specification, "encapsulation" also means "encapsulation" and refers to encapsulating a delivery substance to efficiently incorporate it into the body, and genetic material encapsulation efficiency (encapsulation efficiency, EE) refers to the content of genetic material encapsulated within lipid nanoparticles relative to the total genetic material content used in the manufacture, and ionic liquid encapsulation efficiency refers to the content of ionic liquid encapsulated within lipid nanoparticles relative to the total ionic liquid content used in the manufacture.

[0056] According to one example, the loading efficiency can be calculated by a commonly used method, for example, the dielectric material loading efficiency may be measured through RiboGreen analysis and a UV / Vis spectrophotometer.

[0057] A composition for delivering genetic material according to one example can contain genetic material, particularly mRNA or RNA, with high loading efficiency. Lipid nanoparticles, previously known as genetic material carriers, contained genetic material at a low rate, which limited their use as compositions for delivering genetic material. In contrast, a composition for delivering genetic material according to one example can contain genetic material with high loading efficiency, specifically with a loading efficiency of 90% or more.

[0058] The above composition can stably store the genetic material at a temperature greater than 0°C, greater than 4°C, greater than 15°C, greater than 25°C, greater than 30°C, or greater than 35°C for one or more of 1 day, 7 days, 14 days, 30 days, 60 days, 90 days, or 120 days, or for a period between any two of these values. Preferably, the above composition can stably store the genetic material at 4°C for 14 days or more.

[0059] The above composition may include a pharmaceutically acceptable carrier and may be formulated according to conventional methods into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as topical preparations, suppositories, and sterile injectable solutions.

[0060] The above-mentioned pharmaceutically acceptable carriers include, but are not limited to, those commonly used in the art, such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Additionally, the pharmaceutical composition of the present invention comprises fillers, extenders, binders, wetting agents, disintegrants, diluents or excipients such as surfactants, and other pharmaceutically acceptable additives.

[0061] When the above composition is formulated into an oral solid dosage form, it includes tablets, pills, powders, granules, capsules, etc., and such solid dosage forms may include at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., and may include, but are not limited to, lubricants such as magnesium stearate, talc, etc.

[0062] When the above composition is formulated into an oral liquid form, it includes suspensions, liquid formulations, emulsions, syrups, etc., and includes diluents such as water and liquid paraffin, humectants, sweeteners, flavorings, preservatives, etc., but is not limited thereto.

[0063] When the above composition is formulated for parenteral administration, it includes sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspensions include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Witepsol, macrogol, Tween 61, cacao oil, laurin oil, glycerogelatin, etc. may be used as bases for suppositories, but are not limited thereto.

[0064] Method for preparing a composition for transferring genetic material

[0065] The present invention provides a method for preparing a composition for delivering genetic material, comprising: (a) preparing an organic phase solution containing ionizable lipid, PEG (polyethylene glycol) lipid, phospholipid, and sterol lipid; (b) preparing an aqueous phase solution containing an ionic liquid; and (c) preparing a composition for delivering genetic material containing an ionic liquid and lipid nanoparticles by controlling the flow rates of each of the organic phase solution and the aqueous solution; wherein the lipid nanoparticles contain the ionizable lipid, the PEG lipid, the phospholipid, and the sterol lipid, and the ionic liquid contains choline dihydrogen phosphate, choline chloride, or both.

[0066] Specifically, the aforementioned genetic material delivery composition can be prepared by the method for preparing the genetic material delivery composition described above.

[0067] Step (a) above may be a step of preparing an oil phase by dissolving ionizable lipid, PEG (polyethylene glycol)-lipid, phospholipid, and sterol lipid in an organic compound, and the organic compound may be anhydrous ethanol.

[0068] In addition, the ionized lipid : PEG-lipid : phosphoric lipid : sterol lipid may be included in a molar ratio of 30 to 60 : 0.5 to 5 : 5 to 20 : 20 to 50, preferably 48 to 52 : 1.2 to 1.7 : 9 to 12 : 35 to 40, and more preferably 50 : 1.5 : 10 : 38.5.

[0069] In this specification, the molar ratio refers to the mole ratio.

[0070] According to one embodiment of the present invention, step (a) may be a step of preparing an oil phase solution by dissolving 1-octylnonyl ester (SM-102), poly(ethylene glycol)-dimyristoyl glycerol (PEG-DMG), distearoylphosphatidylcholine (DSPC) and cholesterol in an organic compound, and the organic compound may be anhydrous ethanol.

[0071] In a specific aspect, step (a) may be a step of preparing an oil phase by dissolving 1-octylnonyl ester (SM-102), poly(ethylene glycol)-dimyristoyl glycerol (PEG-DMG), distearoylphosphatidylcholine (DSPC), and cholesterol in an organic compound in a molar ratio of 30 to 60 : 0.5 to 5 : 5 to 20 : 20 to 50, and the organic compound may be anhydrous ethanol. The molar ratio may preferably be 48 to 52 : 1.2 to 1.7 : 9 to 12 : 35 to 40, more preferably 50 : 1.5 : 10 : 38.5.

[0072] According to one embodiment of the present invention, step (b) may be a step of adding an ionic liquid to an aqueous phase solution and adjusting the pH as needed to prepare an aqueous solution containing the ionic liquid.

[0073] Here, the pH adjustment may be adjusting the pH of the aqueous solution to 2.5 to 6.0, and the pH may be, for example, one or more of 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0, or a range between any two of these values. By adjusting the pH of the above aqueous solution to 2.5 to 6.0, protonation of ionized lipids is induced, and accordingly, the protonated ionized lipids effectively interact with genetic material (e.g., mRNA), thereby facilitating the formation of nanoparticles.

[0074] In a specific aspect, the ionic liquid is choline dihydrogen phosphate, and step (b) can prepare an aqueous solution containing the ionic liquid by adding 1 to 5,000 mM, e.g., 500 mM, 1,000 mM, or 2,000 mM of choline dihydrogen phosphate to an aqueous solution and then adjusting the pH to pH 2.5 to pH 6.0, e.g., pH 4.0.

[0075] In a specific aspect, the ionic liquid is choline chloride, and step (b) can prepare an aqueous solution containing the ionic liquid by adding 1 to 2,000 mM of choline chloride, e.g., 500 mM, 1,000 mM, or 2,000 mM to an aqueous solution, and then adjusting the pH to pH 2.5 to pH 6.0, e.g., pH 4.0.

[0076] In step (b) above, the aqueous phase may additionally include genetic material.

[0077] According to one embodiment of the present invention, step (b) may be a step of adding an ionic liquid and a dielectric material to an aqueous phase solution and adjusting the pH as needed to prepare an aqueous solution containing the ionic liquid and the dielectric material.

[0078] More specifically, step (b) may be a step of preparing an aqueous solution containing an ionic liquid and a genetic material by dissolving an ionic liquid and a genetic material of 1 to 2,000 mM in an aqueous solution, and the ionic liquid may be, for example, one or more of 1, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,500, and 2,000 mM, or a concentration in a range between two of these values.

[0079] The genetic material may be nucleic acid and may include, for example, one or more selected from the group consisting of messenger ribonucleic acid (mRNA), small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, transfer ribonucleic acid (tRNA), and antisense oligodeoxynucleotide (AS-ODN). Preferably, the genetic material may include messenger ribonucleic acid (mRNA) or ribonucleic acid (RNA) (e.g., yeast RNA).

[0080] In some aspects, where the genetic material is nucleic acid, the nucleic acid may be in a naked form, which means nucleic acid not bound to a protein, lipid, or other protective molecule. Additionally, the nucleic acid may be associated with one or more lipids, proteins, carbohydrates, and / or other organic compounds (e.g., squalene).

[0081] Step (b) above may be a step of preparing an aqueous phase by dissolving 0.001 to 100 mg / mL of genetic material in an aqueous solution, and the genetic material is, for example, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, The concentration may be one or more of 8.0, 9.0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 mg / mL, or a range between two of these values. The genetic material may be nucleic acid, preferably may include messenger ribonucleic acid (mRNA) or ribonucleic acid (RNA) (e.g., yeast RNA). In a specific aspect, the genetic material is mRNA, and the mRNA may be dissolved at 0.01 to 10 mg / mL, 0.05 to 1 mg / mL, 0.07 to 0.5 mg / mL, e.g., 0.1 mg / mL. In certain aspects, the genetic material is RNA (e.g., yeast RNA), and the RNA can be dissolved in an amount of 0.01 to 10 mg / mL, 0.05 to 1 mg / mL, 0.07 to 0.5 mg / mL, e.g., 0.1 mg / mL.

[0082] In a specific aspect, step (b) may be a step of preparing an aqueous solution containing an ionic liquid and a genetic material by dissolving 1 to 2,000 mM of an ionic liquid in an aqueous solution, adjusting the pH to pH 2.5 to pH 6.0, e.g., pH 4.0, and then adding 0.001 to 100 mg / mL of genetic material, wherein the ionic liquid is choline dihydrogen phosphate or choline chloride, and the genetic material is messenger ribonucleic acid (mRNA) or ribonucleic acid (RNA). The ionic liquid may be 1 to 2,000 mM, e.g., 500 mM, 1,000 mM, or 2,000 mM, and the genetic material may be 0.001 to 100 mg / mL, e.g., 0.1 mg / mL.

[0083] In step (c), the flow rate ratio of the oil phase to the aqueous phase may be 1:1 to 1:5, preferably 1:2 to 1:4, and more preferably 1:3. It is desirable for the flow rate ratio to be within the above range so as to ensure the formation stability and uniformity of the lipid nanoparticles.

[0084] The above manufacturing method can be performed based on microfluidic technology.

[0085] Methods for storing genetic material

[0086] The present invention provides a method for storing genetic material, comprising the step of storing genetic material using a composition for delivering genetic material, wherein the composition for delivering genetic material comprises an ionic liquid and lipid nanoparticles, wherein the lipid nanoparticles comprise ionizable lipid, PEG (polyethylene glycol) lipid, phospholipid, and sterol lipid, and the ionic liquid comprises choline dihydrogen phosphate, choline chloride, or all of these.

[0087] In a specific aspect, the method comprises the step of storing genetic material using a composition for delivering genetic material, wherein the composition for delivering genetic material comprises an ionic liquid, lipid nanoparticles, and genetic material, wherein the ionic liquid and the genetic material are supported on the lipid nanoparticles, wherein the lipid nanoparticles comprise ionizable lipid, PEG (polyethylene glycol) lipid, phospholipid, and sterol lipid, and wherein the ionic liquid may comprise choline dihydrogen phosphate, choline chloride, or all of these.

[0088] The above method may include a method of storing the genetic material in the above composition at a temperature greater than 0°C, greater than 4°C, greater than 15°C, greater than 25°C, greater than 30°C, or greater than 35°C for one or more of 1 day, 7 days, 14 days, 30 days, 60 days, 90 days, or 120 days, or for a period between any two of these values.

[0089] In some aspects, after storage, the genetic material may be preserved at a level of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% based on biological function and efficacy, or at a level between any two of these values.

[0090] Considering the ease of storage and transport, even if biological function and efficacy are preserved at 50% or less (e.g., about 45%, about 40%, about 35%, about 30%, about 25%, etc.), the composition described herein is considered to provide substantial value.

[0091] Genetic material transfer method

[0092] The present invention provides a method for delivering genetic material, comprising the step of administering a genetic material delivery composition to a subject in an effective amount, wherein the genetic material delivery composition comprises an ionic liquid and lipid nanoparticles, wherein the lipid nanoparticles comprise ionizable lipid, PEG (polyethylene glycol)-lipid, phospholipid, and sterol lipid, and the ionic liquid comprises choline dihydrogen phosphate, choline chloride, or all of these.

[0093] The term "administration" above means introducing a specific substance to a subject by an appropriate method, and the route of administration of the composition may be through any general route as long as it can reach the target tissue. Additionally, the composition of the present invention may be administered by any device capable of transporting the active substance (genetic material) to the target tissue.

[0094] The above "subject" refers to all animals including humans (patients), monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, guinea pigs, etc., and specifically may refer to mammals and may also refer to biological samples.

[0095] The above "effective amount" may be a "therapeutically effective amount." As used herein, a "therapeutically effective amount" means an amount sufficient to provide any improvement or benefit to the subject. In other words, a "therapeutically effective" amount means an amount that provides the effect of alleviating, relieving, or reducing one or more clinical symptoms in the subject. A person skilled in the art will understand that the therapeutic effect does not necessarily have to be complete or curative, and that it is sufficient if some benefit is provided to the subject. In certain aspects, the above therapeutically effective amount may not be curative.

[0096] The above composition may be stored for 1 day, 7 days, 14 days, 30 days, 60 days, 90 days, 120 days or more at a temperature above 0°C (e.g., 4°C, 25°C, 15 to 30°C, or 0 to 8°C) before being administered to a subject.

[0097] The above composition may be administered intravenously, intradermally, intra-arterially, intragraftally, intraperitoneally, intralesionally, intracranially, intraspinally, intraarachnoidally, intra-articularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, locally, intratumorally, intramuscularly, subcutaneously, subconjunctivally, intravesically, mucosa, intrapericardially, intraumbilically, intraocularly, orally, inhaled, injected, infused, continuous infusion, administered via catheter, lavage, in a cream formulation, a lipid composition (e.g., liposomes), or a combination thereof.

[0098] The composition for delivering genetic material according to the present invention can deliver genetic material into a cell, inhibit the degradation of genetic material during long-term storage, and improve the long-term stability of lipid nanoparticles.

[0099] In addition, the above composition can maintain the biological function of genetic material for a long period when stored for a long period.

[0100] In addition, the above composition has a high genetic material (drug) loading efficiency and can have an average particle size suitable as a vaccine delivery vehicle.

[0101] According to the method for preparing a composition for delivering genetic material according to the present invention, it is possible to produce a composition that enables intracellular delivery of genetic material, suppresses degradation of genetic material during long-term storage, maintains the biological function of genetic material for a long period, and improves the long-term stability of lipid nanoparticles.

[0102] In addition, the above manufacturing method can adjust the average particle size of the composition to a range suitable for a vaccine delivery vehicle.

[0103] FIG. 1 shows a schematic diagram of a lipid nanoparticle (mRNA-LNP) carrying genetic material according to the prior art and a composition for delivering genetic material (CDHP-mRNA-LNP) according to one embodiment of the present invention.

[0104] Figure 2 is a schematic diagram showing the process of periodic monitoring experiments for evaluating the stability of nucleic acids within lipid nanoparticles.

[0105] Figure 3 is a schematic diagram showing the intracellular transfusion process for evaluating the function of nucleic acids within lipid nanoparticles.

[0106] Figure 4 shows the results of evaluating nucleic acid preservation ability in a genetic material delivery composition (CDHP-mRNA-LNP), lipid nanoparticles (mRNA-LNP, control), an ionic liquid mixture (Naked mRNA+CDHP), and Naked mRNA.

[0107] Figure 5 shows the results of the evaluation of nucleic acid function in the genetic material delivery composition (CDHP LNP) of Example 1.

[0108] Figure 6 shows the results of the nucleic acid function evaluation in the genetic material delivery composition (CC LNP) of Example 2.

[0109] Figure 7 shows the results of confirming antigen expression 48 hours after injecting the genetic material delivery composition (RT-mRNA-LNP) of Example 2, which had been stored at room temperature for 7 days, into a mouse model.

[0110] Examples are provided to aid in understanding the present invention. The following examples are provided merely to facilitate a better understanding of the invention, and the scope of the invention is not limited by these examples.

[0111] <Example>

[0112] Example 1: Preparation of compositions for genetic material delivery (CDHP-LNP, CDHP-mRNA-LNP)

[0113] The composition for delivering genetic material according to the present invention was prepared using a microfluidic-based Nanoassemblr™ Ignite system. The total flow rate was set to 12 mL / min, and the flow rate ratio of the aqueous phase to the organic phase was set to 3:1.

[0114] The above oil phase was prepared by dissolving ionized lipids (1-octylnonyl ester, SM-102), phospholipids (distearoylphosphatidylcholine, DSPC), cholesterol, and PEG-lipids (DMG-PEG 2000) in anhydrous ethanol in a molar ratio of 50:10:38.5:1.5.

[0115] The above aqueous phase was prepared by dissolving a nucleic acid (yeast RNA or mRNA) at a concentration of 0.1 mg / mL with a nitrogen-phosphorus (N / P) ratio of 6 in an ionic liquid (choline dihydrogen phosphate, CDHP) solution (pH 4.0) with a concentration of 0.5 M to 2.0 M.

[0116] The prepared genetic material delivery composition was dialyzed against Dulbecco's Phosphate Buffer Saline (DPBS) solution for 1 hour and 30 minutes using a 50 kDa Amicon filter to remove residual solvent and impurities.

[0117] Example 2: Preparation of a composition for genetic material delivery (CC-LNP)

[0118] A genetic material transfer composition (CC-LNP) was prepared in the same manner as in Example 1, except that choline chloride (CC) was used instead of choline dihydrogen phosphate (CDHP) as the ionic liquid.

[0119] Comparative Example 1: Preparation of lipid nanoparticles (mRNA-LNP)

[0120] Lipid nanoparticles were prepared in the same manner as in Example 1, except that a 0.1 M citric acid buffer solution with a pH of 4.0 was used instead of an ionic liquid solution (pH 4.0).

[0121] <Experimental Example>

[0122] Experimental Example 1: Evaluation of Physical Properties and Nucleic Acid Loading Efficiency of a Composition for Genetic Material Delivery

[0123] The particle size, polydispersity index (PDI), and zeta potential of the genetic material delivery composition prepared according to the example were measured using Dynamic Light Scattering (DLS, Malvern Zetasizer) equipment. In addition, the Quant-iT RiboGreen quantification kit (Invitrogen) was used to quantify the ratio and concentration of nucleic acids loaded onto lipid nanoparticles. RiboGreen emits fluorescence upon binding to RNA; while free RNA in solution is directly detected, RNA encapsulated within lipid nanoparticles is not effectively detected until the particles are dissolved with a surfactant. Accordingly, the fluorescence intensity of the sample was measured before and after treatment with Triton X-100, and the encapsulation efficiency (EE) was calculated using the difference. All samples were diluted to the same concentration and dispensed into a black 96-well plate for analysis, and fluorescence intensity was measured using a microplate reader (Hidex) under conditions of excitation 485 nm and emission 520 nm.

[0124] (1) Analysis of physical properties according to the concentration of ionic liquid when total lipid concentration is 12.5 mM

[0125] In the composition for genetic material delivery prepared according to Example 1 and the lipid nanoparticles prepared according to Comparative Example 1, under conditions where the total lipid concentration of ionized lipids, PEG-lipids, phospholipids, and sterol lipids is 12.5 mM, the physical properties according to the concentration of the ionic liquid (CDHP) are summarized in Table 1 below.

[0126] Comparative Example 1 (LNP) Example 1 (CDHP-LNP) 0.5 M CDHP 1 M CDHP 2 M CDHP Size (d. nm) 89.69±0.70 30 80.80±0.169 290.75±0.88 05 165.4±1.83 4 PDI 0.133±0.015 0.130±0.009 0.132±0.012 0.066±0.010 Zeta potential (mV) -0.164 -1.92 -1.40 -0.77 0 EE (%) 98.27 98.14 98.42 97.87

[0127] According to Table 1, lipid nanoparticles prepared according to Comparative Example 1 and a composition (CDHP-LNP) containing 0.5 to 1 M CDHP formed uniform particles with a size of about 80 to 90 nm and a low PDI (0.11 to 0.13). On the other hand, the composition (CDHP-LNP) containing 2.0 M CDHP showed a significantly increased particle size of about 165.4 nm, but a very uniform distribution with a PDI of 0.066. This suggests that the high concentration of ionic liquid influenced the expansion of the particle's internal structure.

[0128] (2) Analysis of physical properties according to the concentration of ionic liquid when the total lipid concentration is 3.125 mM

[0129] In the composition for genetic material delivery prepared according to Example 1 and the lipid nanoparticles prepared according to Comparative Example 1, under conditions where the total lipid concentration of ionized lipids, PEG-lipids, phospholipids, and sterol lipids is 3.125 mM, the physical properties according to the concentration of the ionic liquid (CDHP) are summarized in Table 2 below.

[0130] Comparative Example 1 (LNP) Example 1 (CDHP-LNP) 1 M CDHP 2 M CDHP Size (d. nm) 10 1.7±2.8 9 2 11 0.0±0.4 9 3 3 13 2.1±2.3 8 6 PDI 0.1 20±0.0 2 4 0.1 7 2±0.0 5 0.0 99±0.0 2 7 Zeta potential (mV) -0.8 9 7 -1.1 1 -1.5 7 EE (%) 9 2.5 3 9 2.6 3 9 4.4 9

[0131] According to Table 2, when prepared with a total lipid concentration lowered to 3.125 mM, the particle size was 132.1 ± 2.386 nm under 2 M CDHP conditions, which is a significant decrease compared to the 12.5 mM total lipid concentration condition (165.4 nm). Furthermore, the PDI remained at a low level of 0.099, and the encapsulation efficiency (EE) was also excellent at 94.49%. This demonstrates that the problem of increased particle size that occurs when using high-concentration ionic liquids can be effectively controlled by adjusting the total lipid concentration.

[0132] (3) Analysis of physical characteristics according to changes in total lipid concentration under 2M CDHP conditions

[0133] In the composition for genetic material delivery prepared according to Example 1, the physical properties according to the total lipid concentration under the condition that the ionic liquid (CDHP) is 2.0 M are summarized in Table 3 below.

[0134] Example 1 (CDHP-LNP, 2 M CDHP)3.125mM6.25mM12.5mMSize (d.nm)132.1±2.386153.7±0.2082165.4±1.834PDI0.099±0.0270.077±0.0130.066±0.010Zeta potential (mV)-3.54-1.18-0.770EE (%)94.4996.4697.87

[0135] Table 3 shows the results of stepwise adjustment of total lipid concentration to secure a particle size in the low 100 nm range suitable for a vaccine delivery vehicle while containing 2M CDHP. According to Table 3, as the total lipid concentration was reduced from 12.5 mM to 6.25 mM to 3.125 mM, the particle size showed a sequential decreasing trend from 165.4 nm to 153.7 nm to 132.1 nm. In particular, under the 3.125 mM condition, the smallest particle size (132.1 nm) was secured while maintaining a high nucleic acid loading efficiency of over 94%.

[0136] (4) Analysis of physical properties according to changes in PEG-lipid content during the preparation of 2M CDHP-LNP

[0137] In the composition for genetic material delivery prepared according to Example 1, when the ionic liquid (CDHP) is 2.0 M and the total lipid concentration is 12.5 mM, the physical properties according to the PEG-lipid content are summarized in Table 4 below.

[0138] Example 1 (CDHP-LNP, 2 M CDHP, Total lipid concentration 12.5 mM) PEG-lipid 0.5 mol% PEG-lipid 1.5 mol% PEG-lipid 3 mol% PEG-lipid 4 mol% PEG-lipid 5 mol% Size (d. nm) 19 3.3±2.9 14 16 5.4±1.8 34 15 7.9±1.1 02 16 4.4±1.2 58 18 1.2±1.7 47 PDI 0.1 72±0.0 21 0.0 66±0.0 10 0.1 31±0.0 14 0.1 18±0.0 14 0.1 09±0.0 23 Zeta potential (mV) -0.9 55 -0.7 70 -0.7 68 0.3 45 0.3 31 (%)97.397.8794.7391.1499.46

[0139] According to Table 4, as the ratio of PEG-lipid content increased, it was observed that the size of some particles decreased (about 157.9 nm), but the noticeable size reduction effect was not as significant as that of controlling the total lipid concentration.

[0140] Experimental Example 2: Confirmation of Ionic Liquid Loading in Lipid Nanoparticles

[0141] In the genetic material delivery composition prepared according to the example, it was confirmed whether the ionic liquid was supported on the lipid nanoparticles. Specifically, lipids were removed from the lipid nanoparticles using the Matyash lipid extraction method, and the internal ionic liquid (CDHP) and genetic material (RNA) were separated; subsequently, the amount of ionic liquid actually supported within the particles was quantified using the same ion chromatography analysis method.

[0142] Experimental Example 3: Evaluation of the nucleic acid preservation ability of a composition for genetic material delivery

[0143] Agarose gel electrophoresis was performed to qualitatively evaluate the mRNA protective performance of ionic liquid-supported lipid nanoparticles. In this experiment, 996 nucleotide-long EGFP mRNA (5-moUTP) was used as the model mRNA.

[0144] Figure 2 is a schematic diagram illustrating the process of periodic monitoring experiments for evaluating the stability of nucleic acids within lipid nanoparticles. According to Figure 2, the nucleic acid preservation ability of the genetic material delivery composition was evaluated as follows.

[0145] Comparative Example 1 (control LNP) and Example 1 (1M CDHP LNP and 2M CDHP LNP) loaded with EGFP mRNA were stored in a refrigerated environment (4°C), and the preservation of internal nucleic acids was evaluated by degrading the lipid nanoparticles at regular intervals. After collecting each sample, the LNP was degraded by diluting it 15-fold with 1% Triton X-100 solution and reacting it for 5 minutes. The RNA exposed after LNP degradation was mixed with a gel loading dye (Thermo Scientific) and then injected into a 1% agarose gel containing a nucleic acid-specific fluorescent dye (SYBR gold, Invitrogen). Electrophoresis was performed for 30 minutes under conditions of 50 V or 100 V. After electrophoresis, the fluorescence signal was measured using a gel image analysis device (Azure c600). The acquired fluorescence images were quantified using ImageJ. To correct for deviations in fluorescence intensity between gels, an equal amount of DNA ladder was injected as an internal standard, and relative fluorescence values ​​were calculated based on this.

[0146] Figure 4 shows the results of evaluating nucleic acid preservation ability in a genetic material delivery composition (CDHP-mRNA-LNP), lipid nanoparticles (mRNA-LNP, control), an ionic liquid mixture (Naked mRNA+CDHP), and Naked mRNA. Specifically, the mRNA preservation ability was evaluated by storing samples for a long period in a refrigerated environment (4℃) and periodically performing agarose gel electrophoresis. Based on the mRNA concentration of each sample measured using the Quant-iT RiboGreen quantification kit, 0.02 μg of all LNP and Triton X-100-treated LNP samples were injected during gel electrophoresis.

[0147] According to Figure 4, the fluorescence intensity of mRNA stored in Comparative Example 1 (control LNP) for 2 weeks decreased by approximately 82.5% compared to the initial level, whereas no significant decrease was observed in the fluorescence intensity of mRNA stored in Example 1 (LNP loaded with 1M CDHP and 2M CDHP). In addition, in the final analysis performed after about 3 months, it was confirmed that the mRNA stored in Comparative Example 1 (control LNP) was completely degraded, whereas the mRNA stored in Example 1 (2M CDHP LNP) was confirmed to maintain its intact form without degradation. This demonstrates that lipid nanoparticles loaded with ionic liquids have superior nucleic acid preservation capabilities compared to control lipid nanoparticles.

[0148] Experimental Example 4: Evaluation of the Functionality of Nucleic Acids in a Composition for Genetic Material Delivery

[0149] Figure 3 is a schematic diagram illustrating the intracellular transfection process for evaluating the function of nucleic acids within lipid nanoparticles. Referring to Figure 3, CDHP LNP loaded with EGFP mRNA (Example 1) was transfected into HeLa cells to evaluate the functionality of the internal nucleic acids.

[0150] Specifically, the genetic material delivery composition of Example 1 or Example 2 (CDHP LNP or CC LNP loaded with EGFP mRNA) stored in a refrigerated environment (4℃) was applied to cells and cultured in a CO2 incubator for 24 hours. After culture, the fluorescence expression of EGFP protein within the cells was confirmed using confocal microscopy. The mRNA concentration of each sample was measured using the Quant-iT RiboGreen quantification kit, and based on the quantification results, all LNP samples were injected at an equal dose of 0.4 μg during transfection. The EGFP mRNA delivered into the cells was translated in the cytoplasm and expressed as EGFP protein, and the fluorescence signal of the expressed EGFP protein demonstrated that the mRNA performed its translation function normally even in an ionic liquid-loaded lipid nanoparticle environment.

[0151] Figure 5 shows the results of the nucleic acid function evaluation in the genetic material delivery composition (CDHP LNP) of Example 1, and Figure 6 shows the results of the nucleic acid function evaluation in the genetic material delivery composition (CC LNP) of Example 2.

[0152] According to Figure 5, it was confirmed that RNA stored within lipid nanoparticles loaded with CDHP ionic liquid functions normally.

[0153] According to Figure 6, it was confirmed that mRNA stored in lipid nanoparticles loaded with a choline-based ionic liquid with only the anion changed also functions normally. This demonstrates that mRNA in lipid nanoparticles loaded with a choline-based ionic liquid containing choline as a cation maintains functionality within cells, regardless of the type of anion.

[0154] Experimental Example 5: Confirmation of antigen expression of a genetic material delivery composition stored at room temperature for a certain period

[0155] Figure 7 shows the results of confirming antigen expression 48 hours after injecting the genetic material delivery composition (RT-mRNA-LNP) of Example 2, which had been stored at room temperature for 7 days, into a mouse model. Here, the mRNA used in the preparation of the composition of Example 2 was SARS CoV-2 Spike mRNA with a length of 4,285 nucleotides.

[0156] Specifically, Figure 7 is a histogram of fluorescence intensity distribution using flow cytometry, which is the result of evaluating the level of antigen expression in muscle tissue at the injection site after storing the genetic material delivery composition (CC-mRNA-LNP) of Example 2 at room temperature for 7 days (RT-mRNA-LNP) and then administering it by intramuscular injection at a dose of 5 μg / mouse into the muscle of a mouse.

[0157] At this time, the treatment group of the composition (vehicle) of Example 2 that does not contain mRNA was used as a control, and a commercially available mRNA vaccine was administered for comparison.

[0158] According to Figure 7, when CC-mRNA-LNP was administered compared to the control group, the fluorescence signal was significantly increased, and it was confirmed that antigen expression at a level similar to that of the commercial mRNA vaccine administration group was observed.

[0159]

[0160] The specification omits detailed descriptions of matters that can be sufficiently recognized and inferred by those skilled in the art of the present invention, and various modifications are possible within the scope of not altering the technical concept or essential configurations of the present invention, in addition to the specific examples described in this specification. Accordingly, the present invention may be implemented in a manner different from that specifically described and exemplified in this specification, and this is a matter that can be understood by those skilled in the art.

Claims

1. Contains ionic liquids and lipid nanoparticles, and The above lipid nanoparticles include ionizable lipid, PEG (polyethylene glycol) lipid, phospholipid, and sterol lipid, and The above ionic liquid is a composition for transferring genetic material comprising choline dihydrogen phosphate, choline chloride, or both.

2. In Paragraph 1, A composition in which the above ionic liquid is supported on the above lipid nanoparticles.

3. In Paragraph 2, A composition in which the loading ratio of the ionic liquid supported on the lipid nanoparticles is 5 to 15 weight% based on the total weight of the ionic liquid used in the preparation of the composition.

4. In Paragraph 1, A composition wherein the above composition comprises 1 to 2,000 mM of the ionic liquid.

5. In Paragraph 1, The composition comprises a total of 1 to 15 mM of the ionized lipid, the PEG-lipid, the phosphoric acid lipid, and the sterol lipid.

6. In Paragraph 1, The above ionized lipids are 1-octylnonyl ester (SM-102), (6Z, 9Z, 28Z, 31Z)-hephtatriaconta 6,9,28,31-tetraene-19-yl4-(dimethylamino)butanoate (DLin-MC3-DMA), 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP), [(4-hydroxybutyl)azandyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-CDAP), 1,2-Dilinoleoyl-3-Dimethylaminopropane (DLin-DAP), 1,2-Dilinoleyloxy-N,N-Dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-Dimethylaminomethyl-[1,3]-Dioxolane (DLin-K-DMA), 2,2-Dilinoleyl-4-(2-Dimethylaminoethyl)-[1,3]-Dioxolane (DLin-KC2-DMA), 1,2-Dioleoyl-3-Dimethylammonium Propane (DODAP), N,N-Dimethyl-(2,3-Dioleyloxy)Propyamine (DODMA), Dioctadecylamidoglycyl Spermine (DOGS), Spermine Cholesteryl Carbamate (GL-67), Bis-Guanidinium-Spermidine-Cholesterol (BGTC), 3β-(N(N',N'-dimethylaminoethane)-carbamoyl) cholesterol (DC-Chol), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydecyl)amino)ethyl)(2-hydroxydecyl)amino)ethyl)piperazine-1-yl)ethylazandiyl)dododecane-2-ol (C12-200), Nt-butyl-N'-tetradecylamino-propionamidine (diC14-amidine), dimethyldioctadecylammonium bromide (DDAB), N(1,2-dimyristyl oxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N,N-dioleylN,N-dimethylammonium chloride (DODAC), dioleyl Oxypropyl-3-dimethylhydroxyethylammonium bromide (DORIE), N-(1-(2,3-dioleyl oxyl)propyl)-N-2-(sperminecarboxamide)ethyl)-N,A composition comprising one or more selected from the group consisting of N-dimethylammonium trifluoroacetate (DOSPA), N-(1-(2,3-dioleyl oxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), aminopropyl-dimethyl-bis(dodecyloxy)-propane aluminum bromide (GAP-DLRIE), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dipalmityl-3-trimethylammonium propane (DPTAP), 1,2-distearyl-3-trimethylammonium propane (DSTAP), and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC).

7. In Paragraph 1, A composition in which the above PEG lipid comprises one or more selected from the group consisting of poly(ethylene glycol)-dimyristoyl glycerol (PEG-DMG), poly(ethylene glycol)-disteroyl glycerol (PEG-DSG), poly(ethylene glycol)-dipalmitoyl glycerol (PEG-DPG), poly(ethylene glycol)-dimyristoylphosphatidylethanolamine (PEG-DMPE), poly(ethylene glycol)-disteroylphosphatidylethanolamine (PEG-DSPE), poly(ethylene glycol)-dipalmitoylphosphatidylethanolamine (PEG-DPPE), methoxypoly(ethylene glycol)-N,N-ditetradecylacetamide (ALC-0159, mPEG-DTA), and poly(ethylene glycol)-ceramide (PEG-CER).

8. In Paragraph 1, The above phosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylethanolamine (DSPE), A composition comprising one or more selected from the group consisting of phosphatidylethanolamine (PE), dipalmitoylphosphatidylethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS), and 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine].

9. In Paragraph 1, A composition comprising one or more types selected from the group consisting of cholesterol and beta-sitosterol.

10. In Paragraph 1, A composition comprising, based on 100 mol% of total lipid nanoparticles, 30 to 60 mol% of the ionized lipid, 0.5 to 5 mol% of the PEG-lipid, 5 to 20 mol% of the phosphorus lipid, and 20 to 50 mol% of the sterol lipid.

11. In Paragraph 1, A composition having an average particle size of 50 to 200 nm, wherein the above-mentioned composition for transferring genetic material.

12. In Paragraph 1, The above composition further comprises genetic material, and A composition in which the above genetic material is supported on the above lipid nanoparticles.

13. In Paragraph 1, A composition comprising one or more selected from the group consisting of messenger ribonucleic acid (mRNA), small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, transfer ribonucleic acid (tRNA), and antisense oligodeoxynucleotide (AS-ODN). 14.(a) A step of preparing an organic phase comprising ionizable lipid, PEG (polyethylene glycol)-lipid, phospholipid, and sterol lipid; (b) a step of preparing an aqueous phase containing an ionic liquid; and (c) a step of preparing a composition for genetic material delivery comprising an ionic liquid and lipid nanoparticles by controlling the flow rates of the oil phase and the aqueous phase, respectively; and The lipid nanoparticles comprise the ionized lipid, the PEG-lipid, the phosphoric acid lipid, and the sterol lipid, and A method for preparing a composition for transferring genetic material, wherein the above ionic liquid comprises choline dihydrogen phosphate, choline chloride, or both.

15. In Paragraph 14, A method for manufacturing a composition for transferring genetic material, wherein the flow rate ratio of the oil phase and the water phase is 1:1 to 1:

5.

16. In Paragraph 14, A method for manufacturing a composition for delivering genetic material, wherein the above manufacturing method is performed based on microfluidic technology.

17. In Paragraph 14, A method for manufacturing a composition for delivering genetic material, wherein the above-mentioned aqueous phase further comprises genetic material.

18. A method for storing genetic material, comprising the step of storing genetic material using a composition according to any one of claims 1 to 13.

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