Nucleic acid delivery system using lipid nanoparticles

Abstract

The present invention relates to: a nucleic acid delivery complex comprising lipid nanoparticles; a composition comprising the nucleic acid delivery complex for delivering nucleic acids; and a nucleic acid delivery method comprising a step of treating, with the nucleic acid delivery complex, an object to which a nucleic acid is delivered.

Description

Nucleic acid delivery system using lipid nanoparticles

[0001] [Cross-reference with related applications]

[0002] This application claims the benefit of priority from Korean Patent Application No. 10-2023-0117624, filed September 5, 2023, the entire contents of which are incorporated herein by reference.

[0003] The present invention relates to a nucleic acid delivery complex comprising a lipid nanoparticle, a nucleic acid delivery composition comprising the nucleic acid delivery complex, and a nucleic acid delivery method comprising a step of treating the nucleic acid delivery complex to a nucleic acid delivery target.

[0004]

[0005] Lipid nanoparticles (LNPs) are nanometer-sized particles (nm, 10-9 m) composed of lipids. Lipid nanoparticles can encapsulate small molecular drugs, peptides, proteins, messenger RNA (mRNA), etc., and they have the characteristic of enabling endocytosis and cytosolic release by controlling the particle size and chemical / physical properties of the particles. Due to their unique composition and size-dependent properties, lipid nanoparticles can encapsulate lipophilic and hydrophilic drugs, and they have the advantages of being non-biologically toxic, not requiring organic solvents, and easy control of drug release and drug targeting. In addition, it has advantages such as increased drug stability, improved bioavailability, protection and controlled release of drugs sensitive to external environment (light, moisture, etc.), wide range of application (skin, oral, intravenous injection, etc.), and ease of mass production, so it greatly contributes to increasing the delivery efficiency of substances with low stability such as nucleic acids.

[0006] In particular, lipid nanoparticles (LNPs) have been used to deliver various nucleic acids. However, these nucleic acids have low stability, making it difficult to deliver them intact into cells. Therefore, LNPs capable of delivering these nucleic acids to specific sites in the body or within bacteria are needed, highlighting the need for improved LNP formulations that can be used for nucleic acid delivery.

[0007] Accordingly, the inventors of the present invention completed the present invention by identifying an LNP capable of delivering nucleic acid specifically to bacteria.

[0008]

[0009] One object of the present invention is to provide a nucleic acid delivery complex.

[0010] Another object of the present invention is to provide a composition for nucleic acid delivery comprising the nucleic acid delivery complex.

[0011] Another object of the present invention is to provide a nucleic acid delivery method comprising a step of treating the nucleic acid delivery complex with a nucleic acid delivery target.

[0012] In order to achieve the above object, one aspect of the present invention provides a nucleic acid delivery complex comprising: a lipid nanoparticle comprising 5 to 20 parts by weight of a helper lipid, 20 to 40 parts by weight of an ionizable lipid, 10 to 30 parts by weight of a cationic lipid, 0.5 to 3 parts by weight of a polyethylene glycol lipid (Lipid-anchored PEG), and 30 to 50 parts by weight of cholesterol; and a nucleic acid entrapped inside the lipid nanoparticle.

[0013] Another aspect of the present invention provides a composition for nucleic acid delivery comprising the nucleic acid delivery complex.

[0014] Another aspect of the present invention provides a nucleic acid delivery method using the nucleic acid delivery complex.

[0015] The nucleic acid delivery complex of the present invention can efficiently deliver a nucleic acid to be delivered into a cell, and thus has the advantage of being useful as a tool for regulating or editing gene expression. Furthermore, the nucleic acid delivery complex of the present invention enhances nucleic acid delivery efficiency by incorporating a cationic lipid at a certain content and identifying a type of antibiotic that can be used as a membrane permeability enhancer for LNPs. Furthermore, the LNP of the present invention is capable of delivering nucleic acids to Gram-negative bacteria, and thus can be utilized for nucleic acid delivery to Gram-negative bacteria.

[0016] The effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the following description.

[0017]

[0018] Figure 1 is a schematic diagram schematically showing the structure of the nucleic acid delivery complex of the present invention.

[0019] Figure 2 is a result showing the configuration of the nucleic acid delivery complex of the present invention.

[0020] Figure 3 shows a schematic diagram of an experiment for measuring the nucleic acid delivery efficiency of the nucleic acid delivery complex of the present invention.

[0021] Figure 4 shows the results of measuring the number of colonies transformed by nucleic acid delivery in the nucleic acid delivery complex library of the present invention.

[0022] Figure 5 shows the types and composition ratios of lipids constituting the nucleic acid delivery complexes LNP 470 and LNP 496 of the present invention in a circular graph.

[0023] Figure 6 shows the results of observing the nucleic acid delivery complexes LNP 470 and LNP 496 of the present invention using a cryo microscope.

[0024] Figure 7 shows the results of colony count measurement according to the presence or absence of cationic lipid in the nucleic acid delivery complex of the present invention.

[0025] Figure 8 shows the results of measuring the number of colonies according to the specific gravity of the ionizable lipid and cationic lipid included in the nucleic acid delivery complex of the present invention.

[0026] Figure 9 shows the results of measuring the number of colonies when the nucleic acid delivery complex of the present invention was treated with polymyxin B and polymyxin E.

[0027] Figure 10 shows the results of measuring the number of colonies according to the type of membrane permeability enhancer.

[0028] Figure 11 shows the results of measuring the number of colonies according to the treatment concentration of polymyxin B.

[0029] Figure 12 shows the results of measuring the number of colonies according to the treatment concentration of PA108 and AMP2.

[0030] Figure 13 shows the results of confirming the nucleic acid delivery effect in Escherichia pergusoni and Escherichia marmotae isolated from sepsis patients. In the graph of Figure 13, 11, 23, and 25 represent Escherichia pergusoni, and 17 represents Escherichia marmotae.

[0031] Figure 14 is a schematic diagram showing the process of bacterial death according to CHRISPR delivery, and shows the result of a decrease in the number of bacterial colonies as the nucleic acid delivery vehicle of the present invention delivers CHRISPR nucleic acid.

[0032] Figure 15 shows the results of increasing the survival rate of mice and Galleria meronella infected with bacteria as the nucleic acid delivery system of the present invention delivers CHRISPR nucleic acid.

[0033] Figure 16 shows the results of comparing the nucleic acid delivery efficiency of the nucleic acid delivery complex of the present invention by bacterial type.

[0034] Figures 17a and 17b show the results of confirming the nucleic acid delivery effect of the nucleic acid delivery complex of the present invention in human cells.

[0035] Figure 18 shows the cytotoxicity results of the nucleic acid delivery complex of the present invention in human cells.

[0036]

[0037] Hereinafter, the present invention will be described in detail.

[0038]

[0039] 1. Nucleic acid delivery complex of the present invention

[0040] One aspect of the present invention provides a nucleic acid delivery complex.

[0041] The nucleic acid delivery complex comprises a lipid nanoparticle and a nucleic acid entrapped within the lipid nanoparticle.

[0042] The lipid nanoparticles described above are nanometer-sized particles composed of lipids, and have the characteristic of being capable of endocytosis by controlling the particle size or chemical / physical properties. Accordingly, the lipid nanoparticles can deliver nucleic acids captured within the particles into cells.

[0043] The lipid nanoparticles may include helper lipid, ionizable lipid, cationic lipid, lipid-anchored PEG, and cholesterol.

[0044] The helper lipid may be one that surrounds the nucleic acid to maintain the structure of the lipid nanoparticle, and for example, the helper lipid may be dipalmitoylphosphatidylcholine (DOPC), N,N-dimyristylamide of 2-hydroxyacetic acid (ALC-0159), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-diphytanoyl-sn-glycero-3-phosphocholine (4ME), 1,2-distearoylphosphatidylcholine (DSPC). and, specifically, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0045] The helper lipid may be included in the lipid nanoparticles in an amount range consisting of a lower limit selected from the group consisting of 5, 6, 7, 8, and 9 parts by weight and an upper limit selected from the group consisting of 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 parts by weight, based on the total weight of the lipid nanoparticles. For example, the helper lipid may be included in the lipid nanoparticles in an amount of 5 to 20 parts by weight, and specifically, in an amount of 5 to 18 parts by weight, 6 to 16 parts by weight, 7 to 14 parts by weight, 8 to 12 parts by weight, and more specifically, in an amount of 10 parts by weight.

[0046] The above ionized lipid may play a role in promoting the formation of a nucleic acid structure and improving the delivery efficiency after being introduced into cells, and for example, the above ionized lipid may be SM-102 (9-Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate), BP-26127 (7-[(2-Hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]heptyl 2-octyldecanoate), MC3 (4-(dimethylamino)-butanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl ester), L319 (Di((Z)-Non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate), 306Oi10(tetrakis(8-methylnonyl) 3,3',3'',3'''-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate), C13-113-3(1-[2-[2-[bis(2-hydroxytridecyl)amino]ethyl-methylamino]ethylamino]tridecan-2-ol), C13-113-4(1-[2-[2-[bis(2-hydroxytridecyl)amino]ethyl-methylamino]ethyl-(2-hydroxytridecyl)amino]tridecan-2-ol), DODMA (1,2-dioleyloxy-3-dimethylaminopropane), C12-200(1,1'-[[2-[4-[2-[[2-[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1-piperazinyl]ethyl]imino]bis-2-dodecanol),C13-112-3(1-[2-[2-[2-[bis(2-hydroxytridecyl)amino]ethoxy]ethoxy]ethylamino]tridecan-2-ol), C13-112-4(14,23-bis(2-hydroxytridecyl)-17,20-dioxa-14,23-diazahexatriacontane-12,25-diol) There may be etc., specifically, DODMA (1,2-dioleyloxy-3-dimethylaminopropane), ALC-0315 ([(4-Hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate)) or Lipid 215(3-[4,4-Bis(octyloxy)-1-oxobutoxy]-2-[[[[3-(diethylamino)propoxy]carbonyl]oxy]methyl]propyl (9Z,12Z)-9,12-octadecadienoate) may be

[0047] The ionized lipid may be included in the lipid nanoparticles in a content range consisting of a lower limit selected from the group consisting of 20, 21, 22, 23, 24, 25, 26, 27, 28, and 29 parts by weight and an upper limit selected from the group consisting of 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40 parts by weight, based on the total weight of the lipid nanoparticles. For example, the ionized lipid may be included in the lipid nanoparticles in a content of 20 to 40 parts by weight, and specifically, in a content of 22 to 38 parts by weight, 24 to 36 parts by weight, 26 to 34 parts by weight, or 28 to 32 parts by weight, and more specifically, in a content of 30 parts by weight.

[0048] The cationic lipid may play a role in increasing the delivery efficiency of nucleic acids by suppressing the aggregation of the nanoparticle shell and enabling a stable dispersion state, and may include, for example, DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), TAP14 (2,3-di(tetradecanoyloxy)propyl-trimethylazanium), etc., and specifically, may be DOTAP (1,2-dioleoyl-3-trimethylammonium-propan), TAP16 (1,2-dipalmitoyl-3-trimethylammonium-propane), or TAP18 (1,2-stearoyl-3-trimethylammonium-propane).

[0049] The cationic lipid may be included in the lipid nanoparticles in a content range consisting of a lower limit selected from the group consisting of 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 parts by weight and an upper limit selected from the group consisting of 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 parts by weight, based on the total weight of the lipid nanoparticles. For example, the cationic lipid may be included in the lipid nanoparticles in a content of 10 to 30 parts by weight, and specifically, in a content of 12 to 28 parts by weight, 14 to 26 parts by weight, 16 to 24 parts by weight, or 18 to 22 parts by weight, and more specifically, in a content of 20 parts by weight.

[0050] In particular, the nucleic acid delivery complex of the present invention may further enhance nucleic acid delivery efficiency by including a cationic lipid. In a specific embodiment of the present invention, the nucleic acid delivery efficiency was compared depending on the presence or absence of the cationic lipid. As a result, it was confirmed that when lipid nanoparticles including the cationic lipid DOTAP were used, the number of cell colonies delivered with the nucleic acid was high.

[0051] In addition, the nucleic acid delivery complex of the present invention may have a mixing ratio of ionizable lipid and cationic lipid of 45:10 to 15:40, specifically 40:15 to 20:35. When the mixing ratio is within the above range, the zeta potential, which is the electrical potential between the cell surface and the surrounding cell culture medium, is high at +20 mV or more, so that aggregation does not occur and a stable dispersed state is exhibited, thereby improving the nucleic acid delivery efficiency.

[0052] The polyethylene glycol lipid may prevent the nanoparticle shell from being decomposed and improve distribution in the body, for example, Maleimide-PEG2000(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000]), ALC-0159(Methoxypolyethyleneglycoloxy(2000)-N,N-ditetradecylacetamide), C16-PEG2000(N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]}), DOPE-PEG1000-Amine(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000]), DOPE-PEG2000-Amine(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]), DSPE-PEG-Biotin(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)]), DSPE-PEG-Sugar5000, There may be DSPE-PEG-Sugar10000, etc., specifically, C14-PEG(MW 2000)(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]), DSPE-PEG-mannose(MW 5000)(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(polyethylene glycol)-Mannose-5000) or C18-PEG(MW 2000)( 1,It may be 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]).

[0053] The polyethylene glycol lipid may be included in the lipid nanoparticles in a content range consisting of a lower limit selected from the group consisting of 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, and 1.4 parts by weight and an upper limit selected from the group consisting of 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, and 3.0 parts by weight, based on the total weight of the lipid nanoparticles. For example, the polyethylene glycol lipid may be included in the lipid nanoparticles in a content of 0.5 to 3.0 parts by weight, and specifically, in a content of 0.6 to 2.8 parts by weight, 0.8 to 2.6 parts by weight, 1.0 to 2.4 parts by weight, or 1.1 to 2.2 parts by weight, and more specifically, in a content of 1.5 parts by weight.

[0054] The above cholesterol is an organic substance of the steroid series with hydrophobic properties, and may contribute to maintaining the structure of lipid nanoparticles by surrounding nucleic acids together with the helper lipid.

[0055] The cholesterol may be included in the lipid nanoparticles in a content range consisting of a lower limit selected from the group consisting of 30, 31, 32, 33, 34, 35, and 36 parts by weight and an upper limit selected from the group consisting of 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50 parts by weight, based on the total weight of the lipid nanoparticles. For example, the cholesterol may be included in the lipid nanoparticles in a content of 0.5 to 3.0 parts by weight, and specifically, in a content of 30 to 50 parts by weight, 31 to 48 parts by weight, 32 to 46 parts by weight, 34 to 44 parts by weight, or 36 to 42 parts by weight, and more specifically, in a content of 38.5 parts by weight.

[0056] Accordingly, the lipid nanoparticle of the present invention may comprise 5 to 20 parts by weight of helper lipid, 20 to 40 parts by weight of ionizable lipid, 10 to 30 parts by weight of cationic lipid, 0.5 to 3 parts by weight of polyethylene glycol lipid (Lipid-anchored PEG), and 35 to 47 parts by weight of cholesterol.

[0057] In one specific example of the present invention, the lipid nanoparticle of the present invention may contain 30 parts by weight of the ionized lipid, 25 parts by weight of the cationic lipid, 10 parts by weight of the helper lipid, 2 parts by weight of the polyethylene glycol lipid, and 38.5 parts by weight of the cholesterol.

[0058] In one specific example of the present invention, the lipid nanoparticle of the present invention may contain 30 parts by weight of the ionized lipid, 25 parts by weight of the cationic lipid, 10 parts by weight of the helper lipid, 2 parts by weight of the polyethylene glycol lipid, and 33 parts by weight of the cholesterol.

[0059] The lipid nanoparticles of the present invention may have a zeta potential, which is the electrical potential difference between the cell surface and the surrounding culture medium, of +17 mV or more. When the zeta potential is as described above, aggregation of the nanoparticle shell can be suppressed and a stable dispersion state can be maintained. In one embodiment of the present invention, the lipid nanoparticles of the present invention have a zeta potential of 17.1 mV or more or 20.5 mV or more.

[0060] The lipid nanoparticles of the present invention may have an encapsulation efficiency (EE %) of 70% or more. The encapsulation efficiency is a value indicating the percentage of drug successfully captured in micelles or nanoparticles. When the lipid nanoparticles of the present invention have the encapsulation efficiency as described above, the lipid nanoparticles of the present invention can effectively encapsulate and transport the drug. In one embodiment of the present invention, the lipid nanoparticles of the present invention have an encapsulation efficiency of 76% or 71%.

[0061] The structure of the lipid nanoparticle of the present invention may be schematically illustrated as in Fig. 1. Referring to Fig. 1, the lipid nanoparticle includes an outer membrane and an inner membrane, and each lipid particle includes a hydrophilic head portion and a hydrophobic tail portion, so that the hydrophilic head portions of the helper lipid, ionizable lipid, cationic lipid, and polyethylene glycol lipid may interact and be positioned toward the outer surface of the outer membrane, and the hydrophobic tail portions of the helper lipid, ionizable lipid, and polyethylene glycol lipid and the cholesterol may be positioned toward the inner membrane of the outer membrane. In addition, the targeting portion may be in a form in which it interacts and is bound to the hydrophilic portion of the polyethylene glycol lipid. In addition, the lipid nanoparticle may include the helper lipid, the ionized lipid, the cationic lipid, and the cholesterol in the inner membrane, and the hydrophilic head portions of the helper lipid, the ionized lipid, and the cationic lipid may interact to form the shape of the inner membrane surrounding the nucleic acid inside the lipid nanoparticle, and the hydrophobic tail portions of the helper lipid, the ionized lipid, and the cationic lipid and the cholesterol may be positioned so as to face the outside of the inner membrane. The nucleic acid may be included in the inner membrane of the lipid nanoparticle. In the lipid nanoparticle having the above structure, the helper lipid and the cholesterol may stabilize the structure of the lipid nanoparticle and maintain the structure, and the cationic lipid may suppress the aggregation of the membrane of the lipid nanoparticle to induce a stable membrane structure and lipid distribution.

[0062] In one specific embodiment of the present invention, the lipid nanoparticle of the present invention may be one in which the ionizable lipid is DODMA, ALC-0315 or Lipid 215, the cationic lipid is DOTAP, TAP16 or TAP18, the helper lipid is DOPE, and the polyethylene glycol lipid is C14-PEG (MW 2000), DSPE-PEG-mannose (MW 5000) or C18-PEG (MW 2000).

[0063] The nucleic acid delivery complex comprises a nucleic acid entrapped inside the lipid nanoparticle and can deliver the nucleic acid into a cell.

[0064] The nucleic acid is a polymer composed of units called nucleotides, and is responsible for the function of assisting in the storage, transmission, and expression of genetic information, and may include, for example, DNA, plasmid DNA, siRNA, mRNA, sRNA, CRISPR, gRNA, etc. The nucleic acid has low stability, making it difficult to deliver it intact into cells. However, by utilizing the nucleic acid delivery complex of the present invention, the nucleic acid is protected by lipid nanoparticles, thereby increasing the stability, and thus the efficiency of nucleic acid delivery can be increased.

[0065] In addition, the lipid nanoparticle may further comprise a targeting moiety. The targeting moiety may serve to specifically bind to a cell to which the nucleic acid is to be delivered, thereby localizing the lipid nanoparticle and the nucleic acid captured therein to the cell, thereby increasing the delivery efficiency of the nucleic acid. The targeting moiety may include biotin, amine, sugar, etc., but is not limited as long as it can specifically bind to the cell to which the nucleic acid is to be delivered.

[0066] The above lipid nanoparticles can be produced through conventional methods used to produce lipid nanoparticles, for example, ultrasonication, high-pressure homogenization, ultrafiltration, solvent emulsification / evaporation, microemulsion, etc.

[0067] In particular, the nucleic acid delivery complex may be capable of delivering nucleic acids into the cell of a Gram-negative bacterium.

[0068] The above Gram-negative bacteria are a type of prokaryotic cell, and may have an outer membrane composed of lipopolysaccharides, lipoproteins, and other complex polymers instead of a cell wall with a relatively small amount of peptidoglycan compared to Gram-positive bacteria. The Gram-negative bacteria have the characteristic of having a thinner peptidoglycan layer in their cell wall and the presence of an outer membrane compared to Gram-positive bacteria. Representative Gram-negative bacteria may include Klebsiella penumoniae, Salmonella spp., Pseudomonas aeruginosa, Haemophilus influenzae, Enterobacter spp., Yersinia pest, etc. Specifically, the Gram-negative bacteria may be Escherichia coli, Escherichia fergusonii, Shigella sonnei, Klebsiella pneumoniae, or Citrobacter freundii.

[0069] In a specific embodiment of the present invention, nucleic acids were delivered to Gram-negative bacteria, including Escherichia coli, Escherichia pergusoni, Shigella sonnei, and Citrobacter freundii, using the nucleic acid delivery system of the present invention. As a result, it was confirmed that nucleic acid delivery using the nucleic acid delivery system of the present invention is possible for Gram-negative bacteria such as E. coli, Escherichia pergusoni, Shigella sonnei, Klebsiella pneumoniae, and Citrobacter freundii.

[0070]

[0071] 2. Application of the nucleic acid delivery complex of the present invention

[0072] Another aspect of the present invention provides a composition for nucleic acid delivery comprising the nucleic acid delivery complex.

[0073] The above nucleic acid delivery complex is the same as that described in the table of contents of '1. Nucleic acid delivery complex of the present invention' and therefore will not be described again.

[0074] The above composition for nucleic acid delivery may further include a membrane permeability enhancer for lipid nanoparticles.

[0075] The above membrane permeability enhancer helps the lipid nanoparticles to enter the cell membrane. Specifically, it may increase the membrane permeability of the cell membrane of the cell that is the target of nucleic acid delivery, thereby facilitating the lipid nanoparticles to pass through the cell membrane and enter the cell. Increasing the membrane permeability may mean destroying the cell membrane of the cell to create a pore in the cell membrane. However, any lipid nanoparticle may be used without limitation as long as it helps the lipid nanoparticles to enter the cell membrane. More specifically, the membrane permeability enhancer may have an activity that binds to the outer cell membrane of bacteria and destroys the cell membrane. For example, the membrane permeability enhancer may be an antimicrobial agent such as polymyxin-B, polymyxin-E, antimicrobial peptides 2 (AMP2), and PA108 (2-(4-chlorophenyl)-6-methyl-4-nitrophenol).

[0076] The polymyxin B or polymyxin E may be included in the composition in an amount range consisting of a lower limit selected from the group consisting of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9 μg / ml and an upper limit selected from the group consisting of 2.5, 2.6, 2.7, 2.8, 2.9, and 3.0 μg / ml, based on the total amount of the nucleic acid delivery complex. For example, the polymyxin B or polymyxin E may be included in the composition in an amount of 0.5 to 3.0 μg / ml, 0.6 to 2.7 μg / ml, 0.7 to 2.5 μg / ml, or 0.9 to 2.1 μg / ml, specifically, 1.5 μg / ml, based on the total amount of the nucleic acid delivery complex.

[0077] The antimicrobial peptide 2 may be included in the composition in an amount range consisting of a lower limit selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, and 9 μg / ml and an upper limit selected from the group consisting of 100, 110, 120, 130, 140, and 150 μg / ml, based on the total amount of the nucleic acid delivery complex. For example, the antimicrobial peptide 2 may be included in the composition in an amount of 5 to 150 μg / ml, 6 to 130 μg / ml, 7 to 120 μg / ml, or 9 to 110 μg / ml, specifically, 10 or 100 μg / ml, based on the total amount of the nucleic acid delivery complex.

[0078] The PA108 may be included in the composition in an amount range consisting of a lower limit selected from the group consisting of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9 μg / ml and an upper limit selected from the group consisting of 100, 110, 120, 130, 140, and 150 μg / ml, based on the total amount of the nucleic acid delivery complex. For example, the PA108 may be included in the composition in an amount of 0.5 to 150 μg / ml, 0.6 to 130 μg / ml, 0.7 to 120 μg / ml, or 0.9 to 110 μg / ml, specifically, 1.0, 10, or 100 μg / ml, based on the total amount of the nucleic acid delivery complex.

[0079] The composition described above can be used to deliver nucleic acids into cells to induce cell transformation. Specifically, the nucleic acids in the composition can be delivered into cells, allowing the recipient cells to accept and incorporate the external nucleic acid genetic material, thereby expressing its genetic information. For example, CRISPR nucleic acids can be delivered to induce the death of the target cell.

[0080] In one embodiment of the present invention, a nucleic acid delivery complex comprising the lipid nanoparticles having plasmid DNA comprising CRISPR-Cas13a and gRNA encapsulated therein is used.

[0081] In a specific embodiment of the present invention, plasmid DNA containing a green fluorescent gene (GFP) and an ampicillin resistance gene (AmpR) was captured within the lipid nanoparticles, thereby delivering the nucleic acid into Escherichia coli. As a result, the DNA was delivered into the E. coli, transformation occurred, and the E. coli emitted green fluorescence. Furthermore, the E. coli was able to survive even in a medium containing ampicillin. Thus, the nucleic acid delivery complex or composition of the present invention can be used to engineer cells to express desired genetic properties.

[0082]

[0083] Another aspect of the present invention provides a nucleic acid delivery method comprising the step of treating the nucleic acid delivery complex to a nucleic acid delivery target cell.

[0084] The above nucleic acid delivery target cell may refer to a target cell to which the genetic information of the nucleic acid is to be expressed.

[0085] In the step of treating the nucleic acid delivery complex to a nucleic acid delivery target cell, a membrane permeability enhancer of the lipid nanoparticle may be treated together.

[0086] Accordingly, the nucleic acid delivery method may include a step of treating a nucleic acid delivery target cell with the nucleic acid delivery complex and the membrane permeability enhancer of the lipid nanoparticle.

[0087] The meaning and content of the above membrane permeability enhancer are the same as those described above, so they will not be described again.

[0088]

[0089] Hereinafter, the present invention will be described in detail through examples and experimental examples.

[0090] However, the following examples and experimental examples are only intended to illustrate the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

[0091]

[0092] [Example 1]

[0093] Nucleic acid delivery complex of the present invention

[0094] [1-1] Construction of nucleic acid delivery complex of the present invention and production of library

[0095] The lipid nanoparticles (LNP) of the present invention are composed of a helper lipid, cholesterol, an ionizable lipid, a cationic lipid, a lipid-anchored PEG, and a targeting moiety, and have a structure in which the lipid nanoparticles surround a nucleic acid to be delivered (Fig. 1). As shown in Fig. 2, DOPC, ALC-0159, POPC, 4ME, dioleophyl-phosphatidylethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) as helper lipids at a ratio of 5 to 20%, ALC-0315, SM-102, BP-26127, MC3, L319, 306Oi10, Lipid 215, C13-113-3, C13-113-4, DODMA, DODMA C12-200, C13-112-3 or C13-112-4 as ionizable lipids at a ratio of 20 to 40%, and DOTAP, DOTMA, TAP14, TAP16 or TAP18 as cationic lipids at a ratio of 10 to 30%. Lipid nanoparticles (LNPs) containing 0.5 to 3% of C14-PEG-2000, C18-PEG-2000, Maleimide-PEG2000, ALC-0159, C16-PEG2000, DOPE-PEG1000-Amine, DOPE-PEG2000-Amine, DSPE-PEG-Biotin, DSPE-PEG-Sugar5000, or DSPE-PEG-Sugar10000 as a lipid-anchored PEG, and cholesterol as the remainder of the lipid combination, were prepared. Into these lipid nanoparticles, plasmid DNA (pAD123::INR7, pKLC21::IMP, pKLC21::16s, or pKLC21) was included as the nucleic acid to be delivered, thereby preparing a library of 511 nucleic acid delivery complexes.

[0096] [1-2] Selection of the nucleic acid delivery complex of the present invention

[0097] For the library of Example 1-2, in order to select the LNP with the best nucleic acid delivery efficiency, it was measured whether the LNP could deliver the plasmid DNA (pAD123::INR7) of SEQ ID NO: 1 to E. coli (BW25113). Specifically, the pAD123::INR7 contains the green fluorescent gene (GFP) of SEQ ID NO: 2 and the ampicillin resistance gene (AmpR) of SEQ ID NO: 3. Therefore, when the plasmid DNA as above is delivered into E. coli, transformation occurs, green fluorescence is emitted, and the characteristic of survival even in a medium containing ampicillin was utilized (Fig. 3). Therefore, after culturing the 511 LNP libraries in an ampicillin-containing medium, the nucleic acid delivery efficiency of the LNP library was evaluated by measuring the number of colonies of cells that had survived the delivery of the nucleic acids. At this time, for LNPs 1 to 460, antibiotics were not treated to improve the membrane permeability of LNPs, and for LNPs 461 to 511, polymyxin-B or polymyxin-E was treated with 1.5 μg / ml of the antibiotics described above.

[0098] As a result, as illustrated in Fig. 4, more than 1,000 colonies were observed in specific LNPs such as LNP 470 and LNP 496, confirming high nucleic acid transfer efficiency. In particular, it was confirmed that when the membrane permeability was improved by treating with the above antibiotics, the nucleic acid transfer efficiency was improved.

[0099] Accordingly, the specific lipid types and composition ratios of the LNP 470 and LNP 496 are summarized in Fig. 5, Table 1, and Table 2, and the LNP 470 and LNP 496 were selected as the LNPs of the present invention to be used in subsequent experiments.

[0100] Composition of LNP470 Type Content (mol%) Ionized lipid DODMA 30 Cationic lipid DOTAP 25 Cholesterol Cholesterol 33 Helper lipid DOPE 10 PEG-lipid C14-PEG 20 00 2

[0101] Composition of LNP496 Type Content (mol%) Ionized lipid DODMA 30 Cationic lipid DOTAP 25 Cholesterol Cholesterol 33 Helper lipid DOPE 10 PEG-lipid DSPE-PEG-Mannose 2

[0102] Furthermore, the properties of LNP 470 and LNP 496 selected as described above were analyzed. Specifically, the average diameter (Z-Avg), weight average molecular weight (polydispersity index, PdI), zeta potential, and encapsulation efficiency (Encapsulation efficiency, EE %) were measured. As a result, as shown in Table 3 below, the average diameters of LNP 470 and LNP 496 were 110.9 nm and 134 nm, respectively, the polydispersity index (PdI) values ​​were 0.165 and 0.18, respectively, the zeta-potential values ​​were 20.5 mV and 17.1 mV, respectively, and the encapsulation efficiency (EE %) values ​​were 76% and 71%, respectively.

[0103] (n=5)LNP 470LNP 496Z-Avg. (d.nm)134110.9PdI0.1650.18Zeta-potential (mV)20.517.1EE (%)7671

[0104] Furthermore, the actual morphology of the lipid nanoparticles of LNP 470 and LNP 496 was observed through cryo-electron microscopy (cryo-EM, cryo-EM). As a result, as shown in Fig. 6, particle shapes characteristic of lipid nanoparticles were observed in LNP 470 and LNP 496.

[0105] [1-3] Confirmation of the effect of cationic lipids in the nucleic acid delivery complex of the present invention

[0106] To compare the change in nucleic acid delivery efficiency according to the addition of cationic lipids among the above LNP combinations, the number of E. coli colonies with and without cationic lipids was measured using the same method as in Example 1-2. At this time, polymyxin B was treated equally for all LNP combinations, and 25 mol% of DOTAP was used as the cationic lipid.

[0107] As a result, as shown in Fig. 7, in the case of LNP 470 and LNP 496 of the present invention including cationic lipids, the number of colonies was measured to be about 400 or more compared to the case without cationic lipids, confirming that the nucleic acid delivery ability was significantly superior.

[0108] [1-4] Specific proportions of ionizable lipids and cationic lipids in the nucleic acid delivery complex of the present invention

[0109] In order to confirm the change in nucleic acid delivery efficiency according to the mixing ratio of ionizable lipid and cationic lipid in the LNP of the present invention, lipid nanoparticles were manufactured by using DODMA as the ionizable lipid and DOTAP as the cationic lipid, and adjusting the ratio of DODMA:DOTAP to various ratios ranging from 55:0 to 20:35. The nucleic acid delivery efficiency was evaluated by combining the ionizable lipid and cationic lipid at various ratios and measuring the number of E. coli colonies in the same manner as in Example 1-2.

[0110] As a result, as shown in Fig. 8, when the ratio of ionized lipids and cationic lipids was 45:10 to 25:30, the number of colonies was more than 100, and the zeta potential, which is the electric potential difference between the cell surface and the surrounding culture medium, was high at +20 mV or more, indicating that no aggregation occurred and a stable dispersion state was observed, confirming that the nucleic acid transfer efficiency was high.

[0111]

[0112] [Example 2]

[0113] Confirmation of the type and treatment concentration of the membrane permeability enhancer of the present invention

[0114] In the same manner as in Example 1-2, the nucleic acid delivery complexes containing LNP 470 and / or LNP 496 were treated with various types of membrane permeability enhancers, and then the nucleic acid delivery efficiency was measured. The nucleic acid delivery efficiency was confirmed using polymyxin-E, antimicrobial peptides (AMP), ampicillin, kanamycin, and 2-(4-chlorophenyl)-6-methyl-4-nitrophenol (PA108) as the membrane permeability enhancers.

[0115] Specifically, using LNP 469 of the present invention, various concentrations of the membrane permeability enhancer were treated to Escherichia coli and Escherichia pergusoni isolated from a sepsis patient (corresponding to 23 bacteria in FIG. 13 described below), and then the nucleic acid transfer efficiency was confirmed through the same experimental method as in Example 1-2. In addition, in order to determine the treatment concentration of the membrane permeability enhancer for polymyxin B, various concentrations of polymyxin B were treated to E. coli using LNP 470 and LNP 469, and then the number of E. coli colonies was measured.

[0116] As a result, as shown in Fig. 9, even when treated with polymyxin-E antibiotic as a membrane permeability enhancing agent, more than 1,000 colonies were observed, confirming high nucleic acid transfer efficiency. In addition, as shown in Fig. 10, in the case of E. coli, it was confirmed that nucleic acids were transferred well when AMP2 and PA108 were used among various antibiotics, and these two antibiotics were confirmed to enhance nucleic acid transfer in Escherichia pergusoni. Therefore, it was found that polymyxin B, polymyxin E, and AMP2 and PA108 can be used as membrane permeability enhancing agents of the present invention.

[0117] In addition, as shown in Fig. 11, in the case of polymyxin B, more than 300 colonies were observed at all treatment concentrations of 1 μg / ml, 1.5 μg / ml, and 2 μg / ml, and in particular, in the case of polymyxin B, it was confirmed that the largest number of colonies were produced at 1.5 μg / ml. In addition, as shown in Fig. 12, in the case of PA108, more than 200 colonies were observed at all treatment concentrations of 1 μg / ml, 10 μg / ml, and 100 μg / ml, and in the case of AMP2, it was confirmed that about 200 colonies were observed at all treatment concentrations of 10 μg / ml and 100 μg / ml.

[0118]

[0119] [Example 3]

[0120] In vivo application of the nucleic acid delivery complex of the present invention

[0121] After confirming the nucleic acid delivery efficiency of LNP in the in vitro experiments of Examples 1 and 2, the nucleic acid delivery efficiency of LNP of the present invention was also confirmed in vivo.

[0122] [3-1] Isolation and screening of bacteria from sepsis patients

[0123] Using the above LPN 496, the nucleic acid delivery efficiency was confirmed using the same method as in Example 1-2, targeting three strains of Escherichia fergusonii and one strain of Escherichia marmotae isolated from sepsis patients.

[0124] As a result, as shown in FIG. 13, it was confirmed that the number of colonies of the three types of Escherichia pergusoni and Escherichia marmotae was about 100 or more, and it was confirmed that the nucleic acid delivery complex of the present invention can effectively deliver nucleic acids not only to E. coli but also to Escherichia pergusoni and Escherichia marmotae, which are bacteria of the same genus. The Escherichia pergusoni was isolated and selected as a bacterium for the in vivo experiment described below.

[0125] [3-2] Confirmation of the effectiveness of nucleic acid delivery in in vivo animal experiments using Escherichia pergusoni bacteria.

[0126] An experiment was designed to confirm the nucleic acid delivery ability in vivo using the Escherichia pergusoni strains selected as above. Specifically, pKLC21::16s (SEQ ID NO: 5), which contains CRISPER-Cas13a and the gRNA of SEQ ID NO: 4, was used as a plasmid DNA to induce bacterial death following nucleic acid delivery. As a control, pKLC21 plasmid DNA, which does not contain the gRNA for CRISPER-Cas13a function, was used to determine whether there was a difference in the number of bacterial colonies when pKLC21::16s and pKLC21 were delivered into bacteria. If there was a difference in the number of colonies, it was determined that bacterial death was induced (Fig. 14). In addition, the Escherichia pergusoni fungus (pathogen) isolated in Example 3-1 was injected into mice and Galleria mellonella (honey moth larvae), and 5 minutes later, LNP 496 and LNP 470 containing pKLC21::16s and pKLC21 therein were injected, and the survival rates of the mice and Galleria mellonella were measured, thereby confirming whether the nucleic acid delivery complex of the present invention can function significantly in vivo.

[0127] As a result, as shown in the graph of Fig. 14, when pKLC21::16s was delivered, the number of bacterial colonies was significantly reduced compared to when pKLC21 was delivered, indicating that bacterial death was induced as the nucleic acid delivery system of the present invention delivered the CRISPER-Cas13a nucleic acid into the bacteria.

[0128] In addition, as shown in Fig. 15, when lipid nanoparticles not containing the gRNA were administered to mice and Galleria meronella, the survival rates of the two animal models were low, but when lipid nanoparticles containing the gRNA were administered, it was confirmed that the survival rates of the two animal models could be restored. Accordingly, it was found that the lipid nanoparticles of the present invention can deliver CRISPR-Cas / gRNA nucleic acids to bacteria in vivo, thereby reducing pathogens and increasing the survival rate of the gall bladder model.

[0129]

[0130] [Example 4]

[0131] Confirmation of the scope of application of the nucleic acid delivery complex of the present invention

[0132] The range of nucleic acid delivery possible using the nucleic acid delivery complex of the present invention was confirmed in terms of bacteria and human cells.

[0133] [4-1] Aspects of bacteria

[0134] In addition to Escherichia coli and Escherichia pergusoni, for which the nucleic acid delivery ability of the nucleic acid delivery complex of the present invention was confirmed in Examples 2 and 3, the range of bacteria to which the nucleic acid delivery complex of the present invention is applicable was determined. Specifically, the same experiment as in Example 3-2 was conducted with Gram-positive bacteria such as Bacillus subtilis and Staphylococcus aureus, and Gram-negative bacteria such as Shigella sonnei, Klebsiella pneumoniae, and Citrobacter freundii, to confirm the nucleic acid delivery effect.

[0135] As a result, as shown in Fig. 16, while no colony number was measured for Gram-positive bacteria, a high number of bacterial colonies was observed in Gram-negative bacteria such as Shigella sonnei, Klebsiella pneumoniae, and Citrobacter freundii, confirming that the nucleic acid delivery complex of the present invention can deliver nucleic acids into Gram-negative bacteria. Therefore, it was found that nucleic acid delivery into various Gram-negative bacteria such as Escherichia coli, Escherichia pergusoni, Shigella sonnei, Klebsiella pneumoniae, and Citrobacter freundii is possible by utilizing the nucleic acid delivery complex of the present invention.

[0136] [4-2] Aspects of human cells

[0137] The scope of application of the nucleic acid delivery complex of the present invention was expanded by confirming whether the nucleic acid delivery complex of the present invention also has nucleic acid delivery efficacy for human cells. Specifically, Moderna, an mRNA-LNP vaccine, was used as a comparative LNP, and the eGFP gene of SEQ ID NO: 6 was captured within LNP 470 and LNP 496 of the present invention. Using the nucleic acid delivery complex manufactured as described above, the eGFP gene was delivered to HeLa cervical cancer cells and HEK293T human embryonic kidney cells. At this time, an untreated LNP group was used as a control group. The nucleic acid delivery efficiency was evaluated by measuring the fluorescence values ​​of the HeLa cells and the HEK293T cells. At this time, polymyxin B was treated and the fluorescence values ​​were measured when the three types of LNPs were treated as described above.

[0138] As a result, as shown in Figures 17a and 17b, unlike the control group that was not treated with LNP, eGFP was expressed in all of the Moderna, LNP 470, and LNP 496 treatment groups, and it was confirmed that the two types of LNP of the present invention showed higher fluorescence values ​​than the Moderna treatment group. Therefore, it was confirmed that the LNP of the present invention is capable of delivering nucleic acids to human cells.

[0139] [4-3] Cytotoxicity in human cells

[0140] In order to confirm the cytotoxicity of the nucleic acid delivery complex of the present invention, the nucleic acid delivery complex was injected into HeLa, a cervical cancer cell, and HEK293T, a human embryonic kidney cell, and the survival rate (%) of each cell was measured.

[0141] As a result, as shown in Fig. 18, compared to the control group that was not treated with the nucleic acid delivery complex of the present invention, the cell viability was generally higher in the case of treatment with LNP 470 and LNP 496 of the present invention, confirming that the LNP of the present invention does not exhibit cytotoxicity. Therefore, it was confirmed that the LNP of the present invention is safe for human cells.

[0142]

[0143] Although representative embodiments of the present invention have been described above as examples, the scope of the present invention is not limited to the specific embodiments described above, and those skilled in the art will be able to make appropriate changes within the scope described in the claims of the present application.

Claims

Lipid nanoparticles comprising 1.5 to 20 parts by weight of a helper lipid, 20 to 40 parts by weight of an ionizable lipid, 10 to 30 parts by weight of a cationic lipid, 0.5 to 3 parts by weight of a polyethylene glycol lipid (Lipid-anchored PEG), and 30 to 50 parts by weight of cholesterol; and Nucleic acid entrapped inside the above lipid nanoparticle; A nucleic acid transfer complex comprising:

2. In claim 1, A nucleic acid delivery complex, wherein the nucleic acid is at least one selected from the group consisting of DNA, plasmid DNA, siRNA, mRNA, sRNA, and CRISPR.

3. In claim 1, A nucleic acid delivery complex, wherein the helper lipid is at least one selected from the group consisting of DOPC, ALC-0159, POPC, 4ME, dioleophyl-phosphatidylethanolamine (DOPE), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

4. In claim 1, A nucleic acid delivery complex into bacteria, wherein the ionized lipid is at least one selected from the group consisting of ALC-0315, SM-102, BP-26127, MC3, L319, 306Oi10, Lipid 215, C13-113-3, C13-113-4, DODMA, DODMA C12-200, C13-112-3, and C13-112-4.

5. In claim 1, A nucleic acid delivery complex, wherein the polyethylene glycol lipid is at least one selected from the group consisting of C14-PEG-2000, C18-PEG-2000, Maleimide-PEG2000, ALC-0159, C16-PEG2000, DOPE-PEG1000-Amine, DOPE-PEG2000-Amine, DSPE-PEG-Biotin, DSPE-PEG-Sugar5000, and DSPE-PEG-Sugar10000.

6. In claim 1, A nucleic acid delivery complex, wherein the helper lipid is included in the lipid nanoparticles in an amount of 5 to 15 parts by weight.

7. In claim 1, A nucleic acid delivery complex, wherein the ionized lipid is included in the lipid nanoparticles in an amount of 25 to 35 parts by weight.

8. In claim 1, A nucleic acid delivery complex, wherein the cationic lipid is included in the lipid nanoparticles in an amount of 15 to 25 parts by weight.

9. In claim 1, A nucleic acid delivery complex, wherein the ratio of ionized lipid and cationic lipid among the above lipid nanoparticles is 50:5 to 20:

35.

10. In claim 1, A nucleic acid delivery complex, wherein the polyethylene glycol lipid is included in the lipid nanoparticles in an amount of 1.0 to 2.0 parts by weight.

11. In claim 1, The nucleic acid transfer complex is a nucleic acid transfer complex that transfers nucleic acid into the cell of a gram-negative bacterium.

12. In claim 12, A nucleic acid delivery complex, wherein the above Gram-negative bacteria is at least one selected from the group consisting of Escherichia coli, Escherichia fergusonii, Shigella sonnei, Klebsiella pneumoniae, and Citrobacter freundii.

13. In claim 1, A nucleic acid delivery complex wherein the lipid nanoparticle further comprises a targeting moiety.

14. In claim 13, A nucleic acid delivery complex wherein the targeting portion specifically binds to bacteria.

15. In claim 13, A nucleic acid delivery complex, wherein the targeting portion is at least one selected from the group consisting of biotin, amine, and sugar.

16. In claim 1, The lipid nanoparticles contain 30 parts by weight of the ionized lipid, 25 parts by weight of the cationic lipid, 10 parts by weight of the helper lipid, 2 parts by weight of the polyethylene glycol lipid, and 33 parts by weight of the cholesterol. The ionizable lipid is DODMA, ALC-0315 or Lipid 215, the cationic lipid is DOTAP, TAP16 or TAP18, the helper lipid is DOPE, and the polyethylene glycol lipid is C14-PEG (MW 2000), DSPE-PEG-mannose (MW 5000) or C18-PEG (MW 2000). Nucleic acid transfer complex.

17. A nucleic acid delivery complex according to any one of claims 1 to 16; and A composition for nucleic acid delivery comprising a membrane permeability enhancer of lipid nanoparticles.

18. In claim 17, A composition for nucleic acid delivery, wherein the membrane permeability enhancer is at least one selected from the group consisting of polymyxin-B, polymyxin-E, antimicrobial peptides 2 (AMP2), and PA108 antimicrobial agent (2-(4-chlorophenyl)-6-methyl-4-nitrophenol).

19. In claim 17, A composition for nucleic acid delivery, wherein the membrane permeability enhancer is included in a content of 0.5 to 2.5 μg / ml based on the total amount of the nucleic acid delivery complex.

20. A nucleic acid delivery method comprising the step of treating a nucleic acid delivery target with a nucleic acid delivery complex according to any one of claims 1 to 16; and a membrane permeability enhancer of a lipid nanoparticle.

21. In claim 20, A method for delivering a nucleic acid, wherein the membrane permeability enhancer is at least one selected from the group consisting of polymyxin-B, polymyxin-E, antimicrobial peptides 2 (AMP2), and PA108 antimicrobial agent (2-(4-chlorophenyl)-6-methyl-4-nitrophenol).

Images