Magnetic RNA nanoparticles for mRNA delivery for expression of therapeutic protein

Abstract

The present invention relates to magnetic RNA nanoparticles for mRNA delivery for the expression of therapeutic proteins in cells. The features and advantages of the present invention are summarized as follows: (a) The present invention provides magnetic RNA nanoparticles for mRNA delivery for the expression of a therapeutic protein, and a method for synthesizing same. (b) The magnetic RNA nanoparticles for delivering mRNA for the expression of therapeutic proteins according to the present invention can express a therapeutic protein by delivering mRNA produced in vitro into cells. (c) According to the present invention, it is possible to obtain magnetic nanoparticles for mRNA delivery, which are uniformly dispersed and homogenous in size, avoiding aggregation during synthesis. (d) Compared to RNA nanoparticles without a magnetic nanoparticle core, the magnetic RNA nanoparticles for mRNA delivery according to the present invention exhibit remarkably high RNA delivery efficiency. (e) The magnetic nanoparticles for mRNA delivery according to the present invention have a remarkably high intracellular protein expression rate compared to lipofectamine.

Description

Magnetic RNA nanoparticles for mRNA delivery for therapeutic protein expression

[0001] The present invention relates to magnetic RNA nanoparticles for mRNA delivery for intracellular therapeutic protein expression.

[0002]

[0003] mRNA is a ribonucleic acid molecule that encodes a protein. 1 The presence of mRNA is essential for various genes to be expressed and become functional proteins. Therefore, active research is underway to utilize mRNA in various fields, such as vaccines. 2-5 However, when delivering mRNA itself, which is a single-stranded nucleic acid, into a living body, it is very important to ensure that the mRNA is delivered without being degraded through an appropriate delivery system due to RNA-degrading enzymes present in the body. 6 .

[0004] The conventional delivery system is VLP 7-8 , exosome 9 , LNP 10-14 , micelle 15 These delivery systems utilize a method of loading mRNA into particles, but have the limitation that not all mRNA can be loaded into each particle. Furthermore, problems such as side effects resulting from the accumulation of particle components in the body are arising. Therefore, the development of a method that addresses both safety and delivery efficiency in delivering mRNA into the body is necessary.

[0005]

[0006] Numerous papers and patents are referenced and cited throughout this specification. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety to provide a clearer understanding of the state of the art and the scope of the present invention.

[0007]

[0008] The present inventors sought to develop a method for delivering mRNA to express therapeutic proteins within cells. As a result, they developed a nanoparticle comprising a magnetic nanoparticle, T7 RNA polymerase, and an RNA shell. They confirmed that the nanoparticle was capable of expressing proteins within living cells, thereby completing the present invention.

[0009] The purpose of the present invention is to provide a method for manufacturing magnetic nanoparticles having an RNA shell for intracellular protein expression.

[0010] Another object of the present invention is to provide magnetic nanoparticles having an RNA shell for intracellular protein expression.

[0011] Another object of the present invention is to provide an isolated host cell comprising the magnetic nanoparticles.

[0012] Another object of the present invention is to provide a method for introducing the magnetic nanoparticles into isolated host cells.

[0013] Another object of the present invention is to provide a method for producing a target protein, comprising a step of introducing magnetic nanoparticles into isolated host cells.

[0014]

[0015] In order to achieve the above-described object, the present invention provides a method for producing magnetic nanoparticles having a self-assembled mRNA shell, comprising: (i) a step of surface-modifying magnetic nanoparticles with T7 RNA polymerase; and (ii) a step of performing rolling circle transcription (RCT) using a circular DNA template strand encoding a target protein to be expressed and the magnetic nanoparticles surface-modified with T7 RNA polymerase.

[0016] According to one embodiment of the present invention, the magnetic nanoparticles may be any one selected from ZnFe2O4, CoFe2O4, NiFe2O4, MnFe2O4, MgFe2O4, LiFe5O8, Fe3O4, Fe2O3, FePt, and CoPt.

[0017] According to one embodiment of the present invention, the T7 RNA polymerase may be linked to the surface of the magnetic nanoparticle by covalent bonding or electrostatic bonding.

[0018] According to one embodiment of the present invention, the covalent bond between the T7 RNA polymerase and the magnetic nanoparticles may be formed by an EDC-NHS coupling reaction.

[0019] According to one embodiment of the present invention, the magnetic nanoparticles may be PEGylated using PEG having a thiol group and a carboxyl group at each of the two terminal ends.

[0020] According to one embodiment of the present invention, the pegylation may be performed on magnetic nanoparticles coated with APTES ((3-Aminopropyl)triethoxysilane) to expose amine functional groups.

[0021] According to one embodiment of the present invention, the APTES-coated magnetic nanoparticles may be surface-modified by coating the surface of the magnetic nanoparticles with an unsaturated fatty acid and then modifying the surface with APTES.

[0022] According to one embodiment of the present invention, the unsaturated fatty acid may be oleic acid.

[0023] According to one embodiment of the present invention, the circular DNA template strand may include a promoter region base sequence for T7 RNA polymerase and a ribosome binding base sequence.

[0024] According to one embodiment of the present invention, the circular DNA template strand may sequentially include a promoter region base sequence for T7 RNA polymerase, a ribosome binding base sequence, and a base sequence containing genetic information for expression of a target protein to be expressed.

[0025] According to one embodiment of the present invention, the circular DNA template strand can be used at a concentration of 0.5 to 2.5 nM.

[0026] According to one embodiment of the present invention, the RCT can be performed while stirring at 400 to 700 rpm.

[0027] In order to achieve the other objects described above, the present invention provides magnetic nanoparticles having a self-assembled mRNA shell manufactured by the above method.

[0028] According to one embodiment of the present invention, the average diameter of the magnetic nanoparticles may be 100 to 600 nm.

[0029] According to one embodiment of the present invention, the polydispersity index (PDI) of the magnetic nanoparticles may be 0.01 to 0.4.

[0030] In order to achieve another object mentioned above, the present invention provides a magnetic nanoparticle having a self-assembled mRNA shell comprising the following composition:

[0031] (i) magnetic nanoparticles;

[0032] (ii) APTES ((3-Aminopropyl)triethoxysilane) layer;

[0033] (iii) PEGylation layer;

[0034] (iv) T7 RNA polymerase layer; and

[0035] (v) mRNA envelope layer of the gene encoding the target protein.

[0036] In order to achieve another object mentioned above, the present invention provides an isolated host cell containing the magnetic nanoparticle.

[0037] In order to achieve another object mentioned above, the present invention provides a method for producing a target protein, comprising a step of introducing the magnetic nanoparticles into a separated host cell.

[0038] According to one embodiment of the present invention, the magnetic nanoparticles may be introduced into isolated host cells using magnetofection.

[0039]

[0040] The features and advantages of the present invention are summarized as follows:

[0041] (a) The present invention provides magnetic RNA nanoparticles and a synthesis method for mRNA delivery for therapeutic protein expression.

[0042] (b) The magnetic RNA nanoparticle for mRNA delivery for expression of a therapeutic protein of the present invention can express a therapeutic protein by delivering mRNA produced in a test tube into a cell.

[0043] (c) According to the present invention, magnetic nanoparticles for mRNA delivery can be obtained that are not synthesized in a clump, are uniformly dispersed, and have a homogeneous size.

[0044] (d) The magnetic nanoparticles for mRNA delivery according to the present invention have significantly higher RNA delivery efficiency than RNA nanoparticles without a magnetic nanoparticle core.

[0045] (e) The magnetic nanoparticles for mRNA delivery according to the present invention have a significantly higher intracellular protein expression rate compared to lipofectamine.

[0046]

[0047] Figures 1a and 1b illustrate a method for synthesizing magnetic RNA nanoparticles for mRNA delivery for therapeutic protein expression and a schematic diagram for delivering the nanoparticles to cells to express the therapeutic protein.

[0048] Figures 2a and 2b show the characteristics of magnetic nanoparticles according to the method of attaching T7 polymerase to the surface of the magnetic nanoparticles. Figure 2a shows a bright-field image of the magnetic nanoparticles according to the attachment method. Figure 2b shows the size distribution of the magnetic nanoparticles according to the attachment method using dynamic light scattering. Figures 2c and 2f show the attachment of T7 RNA polymerase by modifying the surface of the magnetic nanoparticles. Figures 2c, 2d, 2e, and 2f show TEM images according to the step of modifying the surface of the magnetic nanoparticles. Figure 2g shows the Zeta potential representing the surface potential of each magnetic nanoparticle.

[0049] Figures 3a to 3d illustrate magnetic nanoparticles having an RNA shell. Figures 3a, 3b, and 3c illustrate TEM images of RNA nanoparticles, magnetic nanoparticles having an RNA shell under no stirring conditions, and magnetic nanoparticles having an RNA shell under stirring conditions, respectively. Figure 3d illustrates an SEM image of magnetic nanoparticles having an RNA shell under stirring conditions. Figure 3e illustrates the size distribution of magnetic nanoparticles having an RNA shell under each stirring condition for optimizing the stirring conditions.

[0050] Figures 4a to 4f show protein expression of nanoparticles delivered into cells. Figures 4a and 4d are confocal fluorescence microscopy images after nanoparticle treatment in HEK-293 cells and MDA-MB-231 cells. Figure 4b is a graph showing the difference in protein expression rates between magnetic RNA nanoparticles for mRNA delivery and lipofectamine 3000 for therapeutic protein expression in HEK-293 cells. Figure 4e is a graph showing the difference in protein expression rates between nanoparticle delivery using magnetic transfection and general RNA nanoparticles in MDA-MB-231 cells. Figures 4c and 4f are graphs showing the cytotoxicity results according to the concentration of nanoparticles in each cell.

[0051] Figures 5a and 5b illustrate protein expression and protein activity of nanoparticles delivered into cells. Figure 5a is a confocal fluorescence microscopy image showing whether protein expression and activity were observed when therapeutic protein mRNA and target sequence gRNA were delivered via nanoparticles to red fluorescent HEK-293 cells. Figure 5b is a graph showing the degree of knockout of red fluorescent protein (TurboFP602) by quantifying the confocal fluorescence microscopy image.

[0052]

[0053] The present inventors sought to develop a method for delivering therapeutic protein mRNA for intracellular protein expression. As a result, they developed an mRNA delivery vehicle with an RNA shell containing magnetic nanoparticles, and confirmed that the protein was expressed within cells.

[0054] The magnetic RNA nanoparticle composition for mRNA delivery for therapeutic protein expression of the present invention comprises a nanoparticle comprising a magnetic nanoparticle, a T7 RNA polymerase bound to the surface of the magnetic nanoparticle, and an mRNA shell.

[0055] The term “magnetic nanoparticle” as used herein refers to nano-sized spherical particles composed of iron oxide. The magnetic nanoparticles have the advantage of being easy to manufacture and being able to control the desired particle size.

[0056] In this specification, “mRNA shell” means an mRNA shell that is created by self-assembly of a circular DNA template strand by T7 RNA polymerase, and the mRNA shell can be modified depending on the circular DNA template strand.

[0057]

[0058] Hereinafter, the present invention will be described in detail.

[0059] One aspect of the present invention relates to a method for producing magnetic nanoparticles having a self-assembled mRNA shell, comprising the steps of: (i) surface-modifying magnetic nanoparticles with T7 RNA polymerase; and (ii) performing rolling circle transcription (RCT) using a circular DNA template strand encoding a target protein to be expressed and the magnetic nanoparticles surface-modified with T7 RNA polymerase.

[0060]

[0061] First, step (i) is a step of surface-modifying magnetic nanoparticles with T7 RNA polymerase.

[0062] The magnetic nanoparticles may be any one selected from ZnFe2O4, CoFe2O4, NiFe2O4, MnFe2O4, MgFe2O4, LiFe5O8, Fe3O4, Fe2O3, FePt, and CoPt, and preferably ZnFe2O4. In the case of the ZnFe2O4, it is known that the magnetic efficiency is the best clinically. 16-20 .

[0063] The above magnetic nanoparticles may be particles having an average diameter of 1 to 50 nm, preferably 2 to 20 nm.

[0064] The T7 RNA polymerase may be covalently or electrostatically linked to the surface of the magnetic nanoparticle, and preferably may be covalently linked. However, when the T7 RNA polymerase and the magnetic nanoparticle are linked electrostatically, the particle size may become excessively large or the particles may clump together (Figs. 2a and 2b).

[0065] In one specific example, the covalent bond between the T7 RNA polymerase and the magnetic nanoparticles may be formed by an EDC-NHS coupling reaction. When the T7 RNA polymerase and the magnetic nanoparticles are covalently bonded by an EDC-NHS coupling reaction, the nanoparticles are synthesized with a constant size and their dispersibility increases (Figs. 2a and 2b).

[0066] At this time, the magnetic nanoparticles before being bound to the T7 RNA polymerase may be PEGylated using PEG having a thiol group and a carboxyl group at each end. The PEGylation may be performed on magnetic nanoparticles coated with APTES ((3-Aminopropyl)triethoxysilane) so that the amine functional group is exposed. In addition, the APTES-coated magnetic nanoparticles may be surface-modified with APTES after coating the surface of the magnetic nanoparticles with an unsaturated fatty acid. In addition, the unsaturated fatty acid may be oleic acid. Forming an APTES layer on the surface of the magnetic nanoparticles in the above manner increases the surface area of ​​the magnetic nanoparticles, thereby exposing more functional groups during surface modification. In addition, by forming a PEGylated layer thereafter, a carboxyl functional group capable of performing surface modification through EDC-NHS coupling can be created on the surface of the magnetic nanoparticles.

[0067] The magnetic nanoparticles of the present invention, surface-modified as described above, do not separate the polymerase bound to the surface. The polymerase bound to the surface continuously transcribes RNA, the transcribed RNA self-assembles, and the RNA located inside is protected from the outside, thereby reducing the risk of modification. In other words, when using magnetic nanoparticles surface-modified as described above, not only does the RNA transcription efficiency increase, but the stability of the transcribed RNA also increases. In addition, when magnetic nanoparticles are used as cores, intracellular delivery can be achieved with higher efficiency through magnetofection.

[0068]

[0069] Next, step (ii) is a step of performing RCT using a circular DNA template strand encoding a target protein to be expressed and magnetic nanoparticles surface-modified with the T7 RNA polymerase.

[0070] The present invention performs RCT using a magnetic nanoparticle core coupled with RNA polymerase, thereby overcoming limitations of conventional RCT, such as significant loss during the washing process, relatively low protein expression, and difficulty in controlling protein expression due to large variations in the amount depending on the absorption rate of cells even when the same amount is processed.

[0071] The above circular DNA template strand may include a promoter region base sequence for T7 RNA polymerase and a ribosome binding base sequence.

[0072] Specifically, the circular DNA template strand may be a circular double-stranded plasmid DNA that sequentially includes a promoter region base sequence, which is a binding site where T7 RNA polymerase initiates polymerization during transcription, a ribosomal binding site (RBS) base sequence, which is a ribosome binding site of the produced mRNA, and a base sequence containing genetic information for expression of the target protein to be expressed.

[0073] In one specific example, the promoter region base sequence for the T7 RNA polymerase may be the base sequence represented by SEQ ID NO: 1, and the ribosome binding base sequence may be the base sequence represented by SEQ ID NO: 2.

[0074] The above circular DNA template strand is RCT'd using T7 RNA polymerase bound to the surface of the magnetic nanoparticle, and self-assembles to form an mRNA shell of the magnetic nanoparticle, and the magnetic nanoparticle having the mRNA shell enters the cell and expresses the target protein.

[0075] In one specific embodiment, the circular DNA template strand can be used at a concentration of 0.5 to 2.5 nM, preferably 0.5 to 1.5 nM, more preferably 0.8 to 1.2 nM.

[0076] In one specific example, the RCT may be performed while stirring at 400 to 700 rpm, preferably 600 to 700 rpm, and more preferably 640 to 660 rpm. When the RCT is performed under stirring conditions of less than 400 rpm or more than 700 rpm, the size of the magnetic nanoparticles produced becomes excessively large, and agglomeration occurs (Fig. 3e).

[0077] The magnetic nanoparticles having a self-assembled mRNA shell of the present invention manufactured by the above method have a relatively uniform size distribution with an average diameter of 100 to 600 nm, specifically 150 to 500 nm, and more specifically 150 to 300 nm, and are synthesized by distributing them as individual nanoparticles without clumping together. If the size of the nanoparticles is less than the lower limit, the nanoparticles may be easily decomposed, and if it exceeds the upper limit, the intracellular delivery efficiency may decrease.

[0078] In addition, the magnetic nanoparticles of the present invention have a polydispersity index (PDI) of very low values, such as 0.01 to 0.4, specifically 0.1 to 0.35, and more specifically 0.2 to 0.35.

[0079] Magnetic nanoparticles according to the present invention exhibit increased intracellular permeability, resulting in significantly higher RNA delivery efficiency compared to RNA nanoparticles lacking a magnetic nanoparticle core. Furthermore, when magnetic nanoparticles according to the present invention are administered into cells to deliver RNA, the expression rate of the target protein is significantly higher compared to conventional cationic lipid particles (LNPs).

[0080]

[0081] Another aspect of the present invention relates to magnetic nanoparticles having self-assembled mRNA shells manufactured by the above method.

[0082] Another aspect of the present invention relates to magnetic nanoparticles having a self-assembled mRNA shell comprising the following composition:

[0083] (i) magnetic nanoparticles;

[0084] (ii) APTES ((3-Aminopropyl)triethoxysilane) layer;

[0085] (iii) PEGylation layer;

[0086] (iv) T7 RNA polymerase layer; and

[0087] (v) mRNA envelope layer of the gene encoding the target protein.

[0088] Since the “magnetic nanoparticle having a self-assembled mRNA shell” of the present invention and the “manufacturing method” thereof have already been described above, their description is omitted to avoid excessive duplication.

[0089] The average diameter of the magnetic nanoparticles is 400 to 800 nm, specifically 500 to 700 nm, and more specifically 550 to 650 nm, and has a relatively uniform size distribution. If the size of the magnetic nanoparticles is not uniform, it may be difficult to control the mRNA concentration within the nanoparticles.

[0090] The polydispersity index (PDI) of the above magnetic nanoparticles may be 0.01 to 0.4, specifically 0.1 to 0.35, and more specifically 0.2 to 0.35.

[0091] Magnetic nanoparticles according to the present invention exhibit increased intracellular permeability, resulting in significantly higher RNA delivery efficiency compared to RNA nanoparticles lacking a magnetic nanoparticle core. Furthermore, when magnetic nanoparticles according to the present invention are administered into cells to deliver RNA, the expression rate of the target protein is significantly higher compared to delivery using conventional cationic lipid nanoparticles (LNPs).

[0092]

[0093] Another aspect of the present invention relates to an isolated host cell comprising the magnetic nanoparticles.

[0094] Since the “magnetic nanoparticle having a self-assembled mRNA shell” of the present invention has already been described above, its description is omitted to avoid excessive duplication.

[0095] By introducing the magnetic nanoparticles into isolated host cells, isolated host cells containing magnetic nanoparticles can be produced, and the isolated cells can express a target protein by the mRNA shell on the surface of the introduced magnetic nanoparticles.

[0096] The above-mentioned isolated host cells may be somatic cells or stem cells, and may be appropriately selected by those skilled in the art according to the purpose, and there is no particular limitation on their type. In the present invention, the intracellular introduction of the magnetic nanoparticles may be performed by those skilled in the art by appropriately selecting a known transformation method, but magnetofection is preferably used.

[0097] As used herein, “magnetofection” refers to a method of delivering RNA into cells (transfection) by a magnetic field acting on the RNA shell on the surface of magnetic nanoparticles. Specifically, it is a method in which magnetic nanoparticles having the mRNA shell of the present invention are concentrated in target cells under the influence of an external magnetic field, and then genetic material (nucleic acid) is absorbed through endocytosis and / or pinocytosis. This method has the advantage of maintaining the structure of the cell membrane intact, unlike other physical transfection methods such as electroporation or gene guns that damage the cell membrane. In addition, the high cell permeability of magnetic nanoparticles provides the advantages of high genetic material (nucleic acid) delivery efficiency and high biocompatibility.

[0098] In a specific embodiment of the present invention, magnetic nanoparticles having an RNA shell containing mRNA of the target protein Cas9 and guide RNA (gRNA) for a red fluorescent protein target gene were manufactured, and these were introduced into human kidney cells by autotransfection, and then the expression and activity of the Cas9 protein were confirmed (Fig. 5b).

[0099]

[0100] Another aspect of the present invention relates to a method for producing a target protein, comprising the step of introducing the magnetic nanoparticles into an isolated host cell.

[0101] Since the “magnetic nanoparticles having self-assembled mRNA shells” and “isolated host cells” of the present invention have already been described above, their descriptions are omitted to avoid excessive duplication.

[0102] The above magnetic nanoparticles may be introduced into isolated host cells using magnetofection.

[0103]

[0104] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0105]

[0106] Example

[0107] Experimental materials and methods

[0108] Preparation of materials

[0109] RPMI1640 medium, DMEM medium, and antibiotics (penicillin-streptomycin; 10,000 IU / mL sodium penicillin and 10,000 mg / mL streptomycin sulfate in 0.83% saline) were purchased from Welgene Incorporation (Daegu, South Korea). Fetal bovine serum (FBS) was purchased from Hyclone (Utah, USA). Trypsin (Trypsin-EDTA solution, 1×) and phosphate-buffered saline (PBS; pH 7.4, 10 mM) were purchased from Thermofisher (Massachusetts, USA). PEG with thiol and carboxyl groups at both ends was purchased from Creative PEGWork (North Carolina, USA). pCMV-T7-SpCas9-P2A-EGFP was purchased from Addgene (Massachusetts, USA). T7 RNA polymerase (50,000 units / mL) and ribonucleic acid solution (25 mM each) were purchased from New England BioLabs Incorporation (Massachusetts, USA). The sequence forming the circular template for generating guide RNA was manufactured by Bioneer (Daejeon, South Korea).

[0110] sgRNA template (SEQ ID NO: 3): Phosphate-5'-AAAAGCACCGACTCGGTGCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTATAGCTCTAAAAC-3'

[0111] TurboFP602 target protospacer T7 promoter template (SEQ ID NO: 4): Phosphate-5'-CGTAGGGCTTGCCTTCGCCCTAATACGACTCACTATAG-3'

[0112] CRISPR primer-TurboFP602 (SEQ ID NO: 5): 5'-CTTTTCTATAGTGAGTCGTATTAGGGCGAAGGCAAGCCCTACGGTTTT-3'

[0113] PLK1 target protospacer T7 promoter template (SEQ ID NO: 6): Phosphate-5'-TTAATGTATCGAGGGCTCCATAATACGACTCACTATAG-3'

[0114] CRISPR primer-PLK1 (SEQ ID NO: 7): 5'-CTTTTCTATAGTGAGTCGTATTATGGAGCCCTCGATACATTAAGTTTT-3'

[0115] All chemicals used in this study were purchased as reagent grade.

[0116]

[0117] Preparation of magnetic nanoparticles (ZnFe2O4)

[0118] The following method was used to synthesize magnetic nanoparticles of 10 nm size. 21 . 1,2-Hexadecanediol (final concentration 10 mM), oleic acid (final concentration 6 mM), and oleylamine (final concentration 6 mM) are placed in a three-necked round flask, and then 2 mM Fe (acac) 3 (Iron (III) acetylacetonate) and 1 mM ZnCl 2 are added to the flask, followed by 40 mL of Trioctylamine. Then, the water inside is heated to 130 °C and vaporized for 30-90 minutes. The mixture is heated at 10 °C per minute to 200 °C and stirred at 300 rpm using a magnetic stirrer. After reaching 200 °C, heat to 300 °C and react for 1 hour, then naturally cool to 30 °C. Wash the reactant with ethanol, collect the magnetic nanoparticles using a centrifuge, and disperse the magnetic nanoparticles using toluene.

[0119]

[0120] Preparation of functionalized magnetic nanoparticles

[0121] To create amine functional groups on the surface of magnetic nanoparticles, magnetic nanoparticles coated with oleic acid were synthesized as magnetic nanoparticles coated with (3-Aminopropyl)triethoxysilane (APTES). 0.8% (v / v%) APTES and 1 μL of Tetramethylammonium hydroxide (TMAOH) were added to 6 mg of magnetic nanoparticles dissolved in 5 mL of toluene, and the reaction was carried out at room temperature with stirring at 400 rpm for 72 hours. Afterwards, the mixture was washed three times with a magnet and ethanol, dispersed in ethanol, and stored.

[0122] To carry out PEGylation, PEG (SH-PEG-COOH) with a thiol group and a carboxyl group at each end was used. 6 mg of magnetic nanoparticles were reacted with 3 mM 4-(N-Maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (Sulfo-SMCC) using a rotator stirrer at 80 rpm for 30 min, and then 3 mM SH-PEG-COOH was added and reacted under the same conditions. The magnetic nanoparticles with PEG attached to the surface were washed three times using a magnet and nuclease-free water (NFW) and then stored in NFW.

[0123]

[0124] Surface modification of magnetic nanoparticles using T7 polymerase

[0125] An EDC-NHS coupling reaction was used to modify the surface of PEGylated magnetic nanoparticles using T7 RNA polymerase. 1 mM each of N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-Hydroxysuccinimide (NHS) were added to 0.5 mL of PEG-labeled magnetic nanoparticle solution and reacted at room temperature for 30 min. 5,000 units of T7 RNA polymerase were then added to the mixture and reacted at 4°C for more than 12 h.

[0126]

[0127] Synthesis of magnetic nanoparticles with RNA shells

[0128] Rolling circle transcription (RCT) technology was used to synthesize RNA nanoparticles. The polymerase buffer consisted of 80 mM Tris-HCl, 12 mM MgCl2, 2 mM DTT, and 4 mM spermidine. Magnetic nanoparticles with polymerase buffer, 1 mM each rNTP, 1 nM pCMV-T7-SpCas9-P2A-EGFP, 1 nM sgRNA circular template, and 100 units of T7 polymerase attached were mixed and then subjected to RCT reaction at 37°C and 650 rpm with stirring for more than 20 h to produce magnetic nanoparticles with RNA shells (average diameter: 201.01 nm, polydispersity index (PDI): 0.29).

[0129]

[0130] dynamic light scattering

[0131] The size of each nanoparticle was measured using dynamic light scattering. Measurements were performed using an ELS-Z (Otsuka electronics, Osaka, Japan). Each nanoparticle was diluted in the solvent in which it was dispersed and measured, and dynamic light scattering measurement data were obtained from 150 accumulations. Each data set was repeated three times.

[0132]

[0133] Surface potential measurement

[0134] The surface potential of each synthesized nanoparticle was measured using an ELS-Z (Otsuka electronics, Osaka, Japan). Each nanoparticle was diluted to 10% in the solvent in which it was dispersed and measured. Surface potential data were obtained by repeating 10 accumulations of data three times to obtain a single data set.

[0135]

[0136] Transmission electron microscopy

[0137] Standard electron microscopy grids with a coated carbon film (200 mesh) and Lacey carbon grids (200 mesh) were purchased from TED PELLA Incorporation (California, USA). For transmission electron microscopy, 10 μL of each nanoparticle solution was placed on a standard electron microscopy grid with a coated carbon film (200 mesh) and a Lacey carbon grid (200 mesh). All samples were dried after removing all moisture. Images were obtained at 80.0 kV using a JEM-2100F (JEOL, Tokyo, Japan).

[0138]

[0139] cell culture

[0140] Breast cancer cell lines (MDA-MB-231) and renal epithelial cell lines (HEK-293) were obtained from the Korea Cell Line Bank (Seoul, Korea). The red fluorescent protein-expressing renal epithelial cell line (Red fluorescence HEK-293) was obtained from Innoprot (Bizkaia, Spain). The breast cancer cell line was cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin. The renal epithelial cell line was cultured in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin. The red fluorescent protein-expressing renal epithelial cell line (Red fluorescence HEK-293) was cultured in DMEM medium supplemented with 10% FBS, 1% penicillin-streptomycin, and 5 μg / mL puromycin. Cells were cultured in TC-grade petri dishes (Falcon, New York, USA) under conditions of 37°C and 5% CO2.

[0141]

[0142] Fluorescence microscopy analysis to confirm expression

[0143] To confirm the expression of fluorescent proteins within cells, fluorescence images were analyzed using a confocal microscope. A Stellaris 5 (Leica, Wetzlar, Germany) confocal microscope was used. Cells were prepared in 24-well and 96-well plates, respectively, and cell nuclei were stained with Hoechst 33342. Cell nuclei were observed using a 405-nm wavelength laser, and fluorescent protein expression was confirmed using a 488-nm wavelength laser.

[0144]

[0145] Cytotoxicity assay

[0146] To determine the cytotoxicity of nanoparticles, PrestoBlue Cell Viability Reagent (Invitrogen, California, USA) was used for analysis. Cells were prepared in 96-well plates and analyzed 24 hours after nanoparticle treatment. Cytotoxicity analysis was performed by observing absorbance at a wavelength of 600 nm.

[0147]

[0148] Production of sgRNA circular templates

[0149] In this experiment, a circular sgRNA template was produced for RNA synthesis using rolling circle transcription technology. Specifically, the TurboFP602 target protospacer T7 promoter template (SEQ ID NO: 4) and CRISPR primer-TurboFP602 (SEQ ID NO: 5), and the PLK1 target protospacer T7 promoter template (SEQ ID NO: 6) and CRISPR primer-PLK1 (SEQ ID NO: 7) were each mixed at a concentration of 100 μM and reacted at 4°C for more than 12 hours to synthesize double-stranded DNA. Afterwards, 1 μM of the sgRNA template (SEQ ID NO: 3), 10 μM each of the double-stranded DNAs, 1X ligation buffer, and 4 units of T4 ligase were added and reacted at 20°C for 12 hours to synthesize. The sgRNA circular template in this experiment is incorporated into the RNA shell, including the guide RNA sequence capable of cleaving the target protein, TurboFP602.

[0150]

[0151] Results and Discussion

[0152] Binding and characterization of T7 RNA polymerase with magnetic nanoparticles

[0153] Zinc iron magnetic nanoparticles (ZnFe2O4), which are highly magnetic nanoparticles, were synthesized. The magnetic nanoparticles were synthesized to have a size of 10 nm, and their size and morphology were measured using a transmission electron microscope (TEM). To surface-modify the magnetic nanoparticles with T7 polymerase, amine functional groups were exposed on the surface using APTES. PEGylated magnetic nanoparticles were then synthesized using PEG, resulting in magnetic nanoparticles capable of binding to T7 polymerase. Magnetic nanoparticles and T7 polymerase were coupled to produce T7 polymerase-labeled magnetic nanoparticles. To optimize the attachment of T7 polymerase to the surface of the magnetic nanoparticles, the EDC-NHS coupling reaction and the electrostatic method were compared. Figure 2a shows brightfield images of nanoparticle dispersions according to each coupling method, and Figure 2b shows the size distributions of surface-modified magnetic nanoparticles using each coupling method. In the case of the EDC-NHS coupling reaction method, the nanoparticles were synthesized with a consistent size and were well dispersed, resulting in a dark brown color, which is the color of magnetic nanoparticles. On the other hand, in the case of surface modification through simple electrostatic bonding, the bonding was non-specific, resulting in particles that were too large in size or particles clumping together, which resulted in the particles settling (Figures 2a and 2b).

[0154] The size and shape of each magnetic nanoparticle were measured by TEM. Figures 2c, 2d, 2e, and 2f show TEM images of each magnetic nanoparticle. It can be confirmed that the layer around the magnetic nanoparticles becomes thicker as the surface of each magnetic nanoparticle is modified (Figures 2c, 2d, 2e, and 2f). Figure 2g shows the Zeta potential of each nanoparticle. In the case of magnetic nanoparticles (MNP), the surface potential is negative because the surface is composed of oleic acid. When the surface was modified with APTES (MNP-APTES), the potential was positive because the amine functional groups were exposed on the surface. After PEGylation (PEGylated MNP), the carboxyl functional groups formed the surface, so it was negative. After surface modification with T7 polymerase (T7-MNP), the zeta potential was confirmed to shift toward positive.

[0155]

[0156] Fabrication and characterization of magnetic nanoparticles with RNA shells

[0157] Magnetic nanoparticles with self-assembled mRNA shells containing magnetic nanoparticles as cores were prepared by rolling circle transcription (RCT) using magnetic nanoparticles surface-modified with T7 polymerase and a circular DNA plasmid (pCMV-T7-SpCas9-P2A-EGFP) in which the T7 RNA promoter, ribosome binding site (RBS), and Cas9 sequences are sequentially linked.

[0158] The mRNA nanoparticles with the mRNA shells fabricated above were compared with mRNA nanoparticles fabricated without magnetic nanoparticles. Figures 3a and 3b show TEM images of each nanoparticle. While the mRNA nanoparticles without magnetic nanoparticles do not show a magnetic nanoparticle core (Figure 3a), the mRNA nanoparticles fabricated by attaching T7 polymerase to the magnetic nanoparticles can be confirmed to have a magnetic nanoparticle core (white arrow) (Figure 3b).

[0159] In addition, when using magnetic nanoparticles as cores, the synthesis was carried out under stirring conditions to ensure that each nanoparticle was well dispersed. Figure 3c shows a TEM image of magnetic nanoparticles manufactured under stirring conditions, and Figure 3d shows a SEM (Scanning electron microscope) image of magnetic nanoparticles manufactured under stirring conditions.

[0160] Additionally, the range for optimizing stirring conditions and attaching T7 polymerase to the surface of magnetic nanoparticles was established. Figure 3e shows the results showing the size distribution of magnetic nanoparticles according to changes in stirring speed, measured using dynamic light scattering.

[0161] After attaching T7 polymerase to the surface of magnetic nanoparticles, when the RCT reaction was performed at 650 rpm, it was confirmed that not only were the smallest-sized nanoparticles synthesized, but they were also well-dispersed. At 0 rpm and 850 rpm, it was confirmed that the nanoparticles were not only large but also clumped (Fig. 3e). In addition, it was confirmed that the magnetic nanoparticles with mRNA shells manufactured under the optimized stirring conditions of 400 to 700 rpm, more specifically, 450 to 650 rpm, were not synthesized in a clump but were distributed as individual nanoparticles (Figs. 3c, 3d, and 3e).

[0162]

[0163] Confirmation of intracellular delivery and protein expression of magnetic nanoparticles with RNA shells

[0164] HEK-293 and MDA-MB-231 were seeded at 5 × 10 in 24-well plates, respectively. 4 cells / well and 3×10 4 Cells were seeded per well and incubated at 37°C in a cell culture incubator. After 24 hours, each cell was treated with magnetic nanoparticles (M-RNPs) having an RNA shell containing a plasmid including the spCas9 gene and the EGFP gene (treatment concentration: 15% (v / v %)) or lipofectamine 3000 (DNA concentration: 500 ng, lipofectamine concentration: 1.5 uL) and incubated at 37°C. After 24 hours, the protein expression of the mRNA delivered into each cell was confirmed using confocal fluorescence microscopy analysis. Figures 4a and 4d are fluorescence images obtained through confocal fluorescence microscopy analysis, Figures 4b and 4e are graphs comparing the amount of protein expression, and Figures 4c and 4f are graphs obtained through a cytotoxicity test.

[0165] Specifically, the protein expression levels were compared when the target protein was delivered to HEK-293 cells using magnetic nanoparticles with an RNA shell and when delivered using Lipofectamine 3000. As a result, it was confirmed that when a plasmid containing the spCas9 gene and the EGFP gene was delivered to HEK-293 cells using the magnetic nanoparticles with an RNA shell of the present invention (M-RNP), the expression rate of the fluorescent protein was higher than when delivered using Lipofectamine 3000 (Figs. 4a and 4b).

[0166] In addition, the protein expression levels were compared when MDA-MB-231 cells were treated with RNA nanoparticles (RNP) and when magnetic nanoparticles with RNA shells were introduced via magnetofection (M-RNP Magnetofection). Magnetofection was performed for 4 hours using a neodymium magnet. As a result, it was confirmed that the protein expression level in MDA-MB-231 cells was very low when only RNA nanoparticles were delivered, whereas a significantly high protein expression rate was observed when magnetic nanoparticles with RNA shells were introduced via magnetofection (Figs. 4d and 4e).

[0167] Additionally, cytotoxicity tests were conducted on each cell at various concentrations to determine the optimal concentration for nanoparticle delivery. In each cell, cytotoxicity was confirmed to be unaffected when nanoparticles were administered to cells at concentrations up to 15% (Figures 4c and 4f).

[0168]

[0169] Expression of therapeutic proteins and confirmation of protein activity

[0170] Magnetic nanoparticles with an RNA shell containing mRNA of Cas9, a therapeutic protein, and guide RNA (gRNA) for a red fluorescent protein (TurboFP602) target gene were fabricated. Red fluorescent HEK-293 cells were seeded in a 24-well plate at a density of 5 × 10 4Cells were seeded per well and incubated at 37°C in a cell culture incubator. After 24 hours, magnetic nanoparticles with RNA shells were treated at 15% (v / v%) and incubated at 37°C. After 24 hours, the protein expression and activity of the delivered mRNA were confirmed using confocal fluorescence microscopy. Figure 5a is a fluorescence image obtained using confocal fluorescence microscopy, in which Hoechst 33342 (blue) represents the cell nucleus, EGFP (green) represents the Cas9 protein labeled with green fluorescent protein, and TurboFP602 (red) represents the red fluorescent protein. Figure 5b is a graph confirming the expression and activity of the therapeutic protein Cas9 protein. In Figure 5b, red represents red fluorescent protein, yellow represents Cas9 protein labeled with red fluorescent protein and green fluorescent protein, and green represents Cas9 protein labeled with green fluorescent protein.

[0171] As a result, it was confirmed that the mRNA of the therapeutic protein was properly expressed in the cell, showing green fluorescence through green fluorescent protein, and that red fluorescent protein decreased (Fig. 5a). At this time, when nanoparticles were produced through RCT by mixing the plasmid containing the spCas9 gene and the EGFP gene, and the circular DNA template strand of the guide RNA at ratios of 1:1, 1:2, 1:4, and 1:8, respectively, it was confirmed that the highest protein activity was shown at ratios of 1:1 and 1:2 (Fig. 5b).

[0172]

[0173] conclusion

[0174] In this study, magnetic nanoparticles with RNA shells were used to deliver mRNA into cells, enabling the expression of therapeutic proteins. First, T7 polymerase was attached to the surface of the magnetic nanoparticles to deliver RNA using the magnetic nanoparticles as cores. Using this, magnetic nanoparticles with RNA shells were fabricated using a plasmid containing the spCas9 gene and the EGFP gene and a circular DNA template strand containing a guide RNA. When the fabricated magnetic nanoparticles were delivered to renal epithelial cells and breast cancer cells via magnetic transfection, the mRNA of the magnetic nanoparticles resulted in protein expression within the cells. In particular, the protein expression rate (70.60%) when delivered using the RNA shell magnetic nanoparticles of the present invention was significantly higher than the protein expression rate (13.72%) when the target protein was delivered to renal epithelial cells using lipofectamine 3000. In addition, when delivered to breast cancer cells using the magnetic transfection method, the RNA delivery efficiency was significantly higher for magnetic nanoparticles with an RNA shell (72.16%) than for RNA nanoparticles without a magnetic nanoparticle core (3.52%). To confirm the presence of protein activity, nanoparticles containing mRNA of the therapeutic protein Cas9 and guide RNA targeting the red fluorescent protein in a 1:1 or 1:2 ratio were produced in renal epithelial cells expressing red fluorescent protein, and each exhibited a knockout efficacy of approximately 58%. These results indicate that mRNA was delivered into the cell, protein expression occurred, and the protein was expressed in an active state. The magnetic RNA nanoparticles for mRNA delivery for therapeutic protein expression of the present invention can be applied to deliver and express mRNA of various therapeutic proteins.

[0175]

[0176] References

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[0198]

[0199] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. (i) a step of surface-modifying magnetic nanoparticles with T7 RNA polymerase; and (ii) A method for producing magnetic nanoparticles having a self-assembled mRNA shell, comprising: a step of performing rolling circle transcription (RCT) using a circular DNA template strand encoding a target protein to be expressed and magnetic nanoparticles surface-modified with the T7 RNA polymerase; 2. In paragraph 1, A method for producing magnetic nanoparticles having a self-assembled mRNA shell, characterized in that the magnetic nanoparticles are any one selected from among ZnFe2O4, CoFe2O4, NiFe2O4, MnFe2O4, MgFe2O4, LiFe5O8, Fe3O4, Fe2O3, FePt, and CoPt.

3. In paragraph 1, A method for producing a magnetic nanoparticle having a self-assembled mRNA shell, characterized in that the T7 RNA polymerase is linked to the surface of the magnetic nanoparticle by covalent bonding or electrostatic bonding.

4. In paragraph 3, A method for producing magnetic nanoparticles having a self-assembled mRNA shell, characterized in that the covalent bond between the T7 RNA polymerase and the magnetic nanoparticles is formed by an EDC-NHS coupling reaction.

5. In paragraph 4, A method for producing magnetic nanoparticles having a self-assembled mRNA shell, characterized in that the magnetic nanoparticles are PEGylated using PEG having a thiol group and a carboxyl group at each of the two ends.

6. In paragraph 5, A method for producing magnetic nanoparticles having a self-assembled mRNA shell, characterized in that the above-mentioned PEGylation is performed on magnetic nanoparticles coated with APTES ((3-Aminopropyl)triethoxysilane) so that the amine functional group is exposed.

7. In paragraph 6, A method for producing magnetic nanoparticles having a self-assembled mRNA shell, characterized in that the above APTES-coated magnetic nanoparticles are surface-modified by coating the surface of the magnetic nanoparticles with an unsaturated fatty acid and then surface-modifying them with APTES.

8. In paragraph 7, A method for producing magnetic nanoparticles having a self-assembled mRNA shell, characterized in that the unsaturated fatty acid is oleic acid.

9. In paragraph 1, A method for producing a magnetic nanoparticle having a self-assembled mRNA shell, characterized in that the circular DNA template strand includes a promoter region base sequence for T7 RNA polymerase and a ribosome binding base sequence.

10. In paragraph 9, A method for manufacturing a magnetic nanoparticle having a self-assembled mRNA shell, characterized in that the circular DNA template strand sequentially includes a promoter region base sequence for T7 RNA polymerase, a ribosome binding base sequence, and a base sequence containing genetic information for expressing a target protein to be expressed.

11. In paragraph 1, A method for producing magnetic nanoparticles having a self-assembled mRNA shell, characterized in that the above circular DNA template strand is used at a concentration of 0.5 to 2.5 nM.

12. In paragraph 1, A method for producing magnetic nanoparticles having a self-assembled mRNA shell, characterized in that the above RCT is performed while stirring at 400 to 700 rpm.

13. A magnetic nanoparticle having a self-assembled mRNA shell, manufactured by the method according to paragraph 1.

14. In paragraph 13, Magnetic nanoparticles having a self-assembled mRNA shell, characterized in that the average diameter of the magnetic nanoparticles is 100 to 600 nm.

15. In paragraph 13, A magnetic nanoparticle having a self-assembled mRNA shell, characterized in that the polydispersity index (PDI) of the magnetic nanoparticle is 0.01 to 0.

4.

16. Magnetic nanoparticles having a self-assembled mRNA shell comprising the following composition: (i) magnetic nanoparticles; (ii) APTES ((3-Aminopropyl)triethoxysilane) layer; (iii) PEGylation layer; (iv) T7 RNA polymerase layer; and (v) mRNA envelope layer of the gene encoding the target protein.

17. An isolated host cell comprising the magnetic nanoparticle of claim 13 or claim 16.

18. A method for producing a target protein, comprising a step of introducing the magnetic nanoparticle of claim 13 or claim 16 into a separated host cell.

19. In paragraph 18, A method for producing a target protein, characterized in that the magnetic nanoparticles are introduced into isolated host cells using magnetofection.

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