mRNA delivery system

Abstract

To provide nanoparticles suitable for mRNA delivery. [Solution] Nanoparticles for mRNA delivery comprising a cationic polymer and an anionic polysaccharide, wherein the cationic polymer is chitosan or a salt thereof. A composition comprising the nanoparticles and mRNA.

Description

Technical Field

[0001] The present invention relates to an mRNA delivery system.

Background Art

[0002] Clinical gene therapy has been performed using viral vectors. However, there remains a concern about pathogenicity, and particularly in gene therapy for congenital genetic diseases, long-term pathogenicity has been a problem (Non-Patent Documents 1 and 2). The solution is the development of non-viral vectors without pathogenicity. PEG-lipid nanoparticles encapsulating mRNA with a vaccine against novel coronavirus have attracted attention (Non-Patent Document 3).

[0003] However, since PEG-lipid nanoparticles have poor cell affinity, the development of an active gene delivery system is desired for targeting cells with specific genetic diseases.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

[0005] The present invention aims to provide nanoparticles suitable for mRNA delivery.

[0006] Furthermore, the present invention also aims to provide a pharmaceutical product comprising nanoparticles suitable for mRNA delivery and mRNA. [Means for solving the problem]

[0007] The inventors have previously developed a plasmid DNA (pDNA) delivery system using polysaccharide nanoparticles and have succeeded in obtaining effective antitumor effects by performing suicide gene therapy on cancer cells in animal experiments (J Gene Med 2013; 15:83-92; Biomaterials 33(2012)7251-7260; J Gene Med. 2017;19:e2968). Therefore, we investigated an mRNA delivery system using polysaccharide nanoparticles (chitosan / hyaluronic acid nanoparticles, chitosan / chondroitin sulfate nanoparticles) that were effective in the pDNA delivery system. As a result, we succeeded in finding polysaccharide nanoparticles suitable for mRNA delivery, and completed the present invention.

[0008] The gist of this invention is as follows: (1) Nanoparticles for mRNA delivery comprising a cationic polymer and an anionic polysaccharide, wherein the cationic polymer is chitosan or a salt thereof. (2) Nanoparticles of chitosan as described in (1), wherein the molecular weight of the chitosan is 1k to 50k. (3) The nanoparticles according to (1), wherein the anionic polysaccharide is hyaluronic acid or a salt thereof and / or chondroitin sulfate or a salt thereof. (4) Nanoparticles of hyaluronic acid as described in (3), wherein the molecular weight of the hyaluronic acid is 10k to 4000k. (5) The nanoparticles described in (3), wherein the molecular weight of chondroitin sulfate is 1k to 50k. (6) The nanoparticles described in (3), wherein the degree of sulfation of chondroitin sulfate is 0.3 to 2. (7) Nanoparticles according to (1), wherein the ratio of the amino group of chitosan to the negative charge of the anionic polysaccharide is 1:0.2 to 8. (8) A composition comprising the nanoparticles described in (1) and mRNA. (9) The composition according to (8), wherein the nanoparticles described in (1) form a complex with mRNA. (10) The composition according to (8) or (9), wherein the ratio of phosphate groups of mRNA to amino groups of chitosan is 1:4 to 16. (11) The composition according to (10), wherein the ratio of the phosphate group of mRNA, the amino group of chitosan, and the negative charge of the anionic polysaccharide is 1:4 to 16:4 to 32. (12) The composition according to (9), wherein the particle size of the nanoparticles forming a complex with mRNA is 30 to 500 nm. (13) The composition according to (8) used to prevent and / or treat at least one disease selected from infectious diseases, cancer, monogenic disorders, cardiovascular diseases, neurological diseases and ocular diseases. [Effects of the Invention]

[0009] The present invention provides nanoparticles suitable for mRNA delivery. [Brief explanation of the drawing]

[0010] [Figure 1]Comparison of gene expression activity in HuH7 cells using polysaccharide nanoparticles containing pDNA and mRNA encoding luciferase. Molecular weight of chitosan is 46.4k. (-): pDNA / chitosan complex (P:N=1:5) or mRNA / chitosan complex (P:N=1:5), HA: pDNA / chitosan / HA complex or mRNA / chitosan / HA complex (P:N:(-)=1:5:16), CS: pDNA / chitosan / CS complex or mRNA / chitosan / CS complex (P:N:(-)=1:5:16), naked: pDNA or mRNA. P:N:(-) represents P: phosphate group of the gene, N: amino group of chitosan, and (-): negative charge of the anionic polysaccharide. [pDNA]= [mRNA]= 3.0 μg / mL. Transfection time = 4 hours, post-transfection time = 20 hours. The results are mean ± standard deviation (n=4). [Figure 2] (Upper panel) Dependence of mRNA / chitosan complex gene expression activity in HuH7 cells on chitosan molecular weight (14.7k, 15.2k, 46.4k) and composition ratio (P:N=1:5, 1:10, 1:15). P:N is where P is the phosphate group of the gene and N is the amino group of chitosan. [mRNA] = 1.5 μg / mL. Transfection time = 4 hours, post-transfection time = 20 hours. Results are mean ± standard deviation (n=3-4). (Lower panel) Dependence of mRNA / chitosan complex gene expression activity in HuH7 cells on composition ratio (P:N=1:X, X = 4, 6, 8, 10, 12, 16). P:N is where P is the phosphate group of the gene and N is the amino group of chitosan. Chitosan molecular weight is 14.7k. [mRNA] = 1.5 μg / mL. Transfection time = 4 hours, post-transfection time = 20 hours. Results are mean ± standard deviation (n=2-3). [Figure 3]Comparison of gene expression activity in HuH7 cells using mRNA-containing polysaccharide nanoparticles. The molecular weight of chitosan is 14.7kJ. The x-axis represents: (-): mRNA / chitosan complex (P:N=1:10), CS40: mRNA / chitosan / CS40 complex (P:N:(-)=1:10:16 and 1:10:32), HA: mRNA / chitosan / HA complex (P:N:(-)=1:10:16 and 1:10:32). P:N:(-) represents P: phosphate group of the gene, N: amino group of chitosan, and (-): negative charge of the anionic polysaccharide. [mRNA] = 1.5 μg / mL. Transfection time = 4 hours, post-transfection time = 20 hours. Results are mean ± standard deviation (n=4). [Figure 4] Molecular weight dependence of chondroitin sulfate on gene expression activity of mRNA / chitosan / CS complex (P:N:(-)=1:5:16) in HuH7 cells. Molecular weight of chitosan is 14.7k. The x-axis represents (-): mRNA / chitosan complex (P:N=1:10). CS10, CS22, and CS40 are mRNA / chitosan / CS complexes (P:N:(-)=1:10:16). Molecular weights of chondroitin sulfate are CS10: 10k, CS22: 22k, and CS40: 40k. [mRNA]=1.5 μg / mL and 3.0 μg / mL. P:N:(-) represents P: phosphate group of the gene, N: amino group of chitosan, and (-): negative charge of the anionic polysaccharide. Transfection time = 4 hours, post-transfection time = 20 hours. The results are mean ± standard deviation (n=2-3). [Figure 5]Comparison of gene expression activity in HuH7 cells using polysaccharide nanoparticles containing pDNA and mRNA encoding luciferase. The molecular weight of chitosan is 14.7k. The horizontal axis represents: (-): pDNA / chitosan complex (P:N=1:10) or mRNA / chitosan complex (P:N=1:10), HA: pDNA / chitosan / HA complex or mRNA / chitosan / HA complex (P:N:(-)=1:10:16), CS: pDNA / chitosan / CS complex or mRNA / chitosan / CS complex (P:N:(-)=1:10:16), naked: pDNA or mRNA. [pDNA]=[mRNA]=3.0 μg / mL. P:N:(-) represents P: phosphate group of the gene, N: amino group of chitosan, and (-): negative charge of the anionic polysaccharide. Transfection time = 4 hours, post-transfection time = 20 hours. The results are mean ± standard deviation (n=4). [Figure 6] This study compared gene expression activity in HuH7 cells using polysaccharide nanoparticles containing luciferase-encoding pDNA and mRNA, and also compared gene expression activity with chitosan molecular weights of 46.4k and 14.7k. The vertical axis represents relative expression activity, with mRNA gene expression activity set to 100. The horizontal axis represents: (-): pDNA / chitosan complex or mRNA / chitosan complex, HA: pDNA / chitosan / HA complex or mRNA / chitosan / HA complex, CS: pDNA / chitosan / CS complex or mRNA / chitosan / CS complex. [pDNA]=[mRNA]=3.0 μg / mL. P:N:(-) represents P: phosphate group of the gene, N: amino group of chitosan, (-): negative charge of the anionic polysaccharide. Transfection time = 4 hours, post-transfection time = 20 hours. Results are mean ± standard deviation (n=4). [Figure 7]Comparison of gene expression activities in HepG2 cells using polysaccharide nanoparticles containing pDNA encoding luciferase and mRNA, and comparison of gene expression activities with chitosan having molecular weights of 46.4k and 14.7k. The vertical axis represents gene expression activity (upper figure) and relative expression activity when the gene expression activity with mRNA is set to 100 (lower figure). The horizontal axis represents (-): pDNA / chitosan complex or mRNA / chitosan complex, HA: pDNA / chitosan / HA complex or mRNA / chitosan / HA complex, CS40: pDNA / chitosan / CS40 complex or mRNA / chitosan / CS40 complex. P:N:(-) represents P: phosphate group of the gene, N: amino group of chitosan, (-): negative charge of the anionic polysaccharide. [pDNA] = [mRNA] = 3.0 μg / mL. Transfection time = 4 hours, post-transfection time = 20 hours. Results are mean ± standard deviation (n = 4). [Figure 8] Comparison of gene expression activity (upper figure) and intracellular uptake amount (lower figure) in HuH7 cells using polysaccharide nanoparticles containing mRNA. The molecular weight of chitosan is 14.7k. The horizontal axis represents (-): mRNA / chitosan complex, HA: mRNA / chitosan / HA complex, CS: mRNA / chitosan / CS40 complex. P:N:(-) represents P: phosphate group of the gene, N: amino group of chitosan, (-): negative charge of the anionic polysaccharide. [mRNA] = 1.5 μg / mL. Transfection time = 4 hours, post-transfection time = 24 hours. Results are mean ± standard deviation (n = 4). *: p < 0.05, ND: no significant difference.

Mode for Carrying Out the Invention

[0011] Hereinafter, the present invention will be described in detail.

[0012] The present invention provides nanoparticles for mRNA delivery, comprising a cationic polymer and an anionic polysaccharide, wherein the cationic polymer is chitosan or a salt thereof.

[0013] The nanoparticles of the present invention can be used as a non-viral drug delivery system (DDS) for delivering nucleic acid drugs. In addition, they can be used as carriers for introducing genes and nucleic acids into cells in gene editing and molecular biology research.

[0014] The molecular weight of chitosan is preferably 1k to 50k, more preferably 10k to 40k, and even more preferably 10k to 15k. The molecular weight of chitosan can be measured by methods such as the viscosity method and high performance liquid chromatography.

[0015] Chitosan is a type of polysaccharide and is obtained by deacetylating chitin obtained from the exoskeletons of crustaceans such as crabs and shrimps. The degree of deacetylation of chitosan is preferably 50% to 100%, more preferably 80% to 100%, and even more preferably 90% to 99%. The degree of deacetylation of chitosan can be measured by methods such as the colloidal titration method, infrared absorption spectroscopy method, hydrolysis method, and enzymatic decomposition method.

[0016] Examples of chitosan salts include hydrochloride, acetate, and lactate.

[0017] The anionic polysaccharide is preferably hyaluronic acid or its salt and / or chondroitin sulfate or its salt.

[0018] Hyaluronic acid is a type of linear glycosaminoglycan, in which N-acetylglucosamine and D-glucuronic acid are linked linearly. Hyaluronic acid can be isolated from chicken combs, and there are also those produced by lactic acid bacteria. The molecular weight of hyaluronic acid is preferably 10k to 4000k, more preferably 100k to 2000k, and even more preferably 400k to 1500k. The molecular weight of hyaluronic acid can be measured by methods such as the viscosity method and high performance liquid chromatography.

[0019] Examples of hyaluronic acid salts include sodium salt, potassium salt, and ammonium salt.

[0020] Chondroitin sulfate is a type of linear glycosaminoglycan, with a structure in which sulfate is bonded to a repeating sugar chain of N-acetylgalactosamine and D-glucuronic acid disaccharides. Chondroitin sulfate can be isolated from shark and bovine cartilage. The molecular weight of chondroitin sulfate is often 1k to 50k, preferably 10 to 45k, and more preferably 20k to 40k. The molecular weight of chondroitin sulfate can be measured by viscosity spectroscopy, high-performance liquid chromatography, polyacrylamide gel electrophoresis, multi-angle light scattering, etc.

[0021] The degree of sulfation of chondroitin sulfate is preferably 0.3 to 2, more preferably 0.5 to 1.5, and more preferably 1.0 to 1.3. The degree of sulfation of chondroitin sulfate can be measured by ion chromatography or elemental analysis.

[0022] Examples of chondroitin sulfate salts include sodium salts, potassium salts, and ammonium salts.

[0023] The ratio of the amino group of chitosan to the negative charge of the anionic polysaccharide is preferably 1:0.2 to 8, more preferably 1:0.5 to 5, and more preferably 1:0.8 to 2.5.

[0024] The nanoparticles of the present invention can be produced by mixing a chitosan solution (for example, a chitosan concentration of 1 to 20% by mass) with an anionic polysaccharide solution (for example, an anionic polysaccharide concentration of 1 to 35% by mass).

[0025] The nanoparticles of the present invention can form complexes with mRNA. The present invention also provides a composition comprising the above-mentioned nanoparticles and mRNA. Although mRNA molecules are large and polar and cannot pass through the lipid bilayer of cells, the formation of a complex between mRNA and nanoparticles makes it possible to transport mRNA into the cytoplasm. The nanoparticles can fuse with the cell membrane and release the encapsulated mRNA into the cytoplasm. The mRNA content in the composition is often 1 to 30% by mass, preferably 2 to 25% by mass, and more preferably 5 to 20% by mass.

[0026] mRNA can be either protein-coding RNA or non-protein-coding RNA, but when used as mRNA pharmaceuticals or mRNA vaccines, it is preferable that it be RNA that codes for the target protein.

[0027] The nucleotides constituting mRNA may be of the natural type or modified nucleotides, but it is preferable that they contain at least one modified nucleotide. Introducing modified nucleotides improves the stability of mRNA and suppresses adverse reactions to exogenous RNA. Modified nucleotides are preferably those in which the base portion has been modified, such as pyrimidine nucleotides with a substitution at position 5, or pseudouridines which may have a substitution at position 1. Specifically, examples include 5-methylcytidine, 5-methoxyuridine, 5-methyluridine, pseudouridine, and 1-alkylpseudridine.

[0028] When used as mRNA pharmaceuticals or mRNA vaccines, mRNA should preferably contain a cap structure (5' Cap), a 5' untranslated region (5'-UTR), a target protein translation region, a 3' untranslated region (3'-UTR), and a poly(A) tail. The sequence of the target protein translation region should preferably be optimized for codons.

[0029] mRNA can be produced by in vitro transcription (IVT) from template DNA. Kits containing the reagents necessary for synthesizing RNA by IVT are commercially available, and mRNA can be produced from template DNA using these kits.

[0030] The target protein encoded by the mRNA should be selected according to the purpose.

[0031] For the prevention and / or treatment of infectious diseases, target proteins encoded by mRNA often encode antigen proteins that induce host immunity against the antigen. Examples include the spike protein of SARS-CoV-2 (COVID-19 vaccine), the hemagglutinin (HA) protein of the influenza virus (influenza vaccine), the G protein of the rabies virus (rabies vaccine), the outer membrane protein of the Zika virus (Zika virus vaccine), the Env protein or Gag protein of HIV (HIV vaccine), and the glycoprotein B of CMV (cytomegalovirus vaccine).

[0032] Examples of target proteins encoded by mRNA to prevent and / or treat cancer include tumor-specific antigens (e.g., NY-ESO-1, MAGE-A3, MAGE-A4, WT1, etc.) and immune checkpoint inhibitor molecules (e.g., PD-L1, etc.).

[0033] Examples of mRNA-encoded target proteins for preventing and / or treating monogenic disorders include ornithine transcarbamylase (OTC) (ornithine transcarbamylase deficiency), propionyl-CoA carboxylase alpha / beta (propionic acidemia), fully functional CFTR (cystic fibrosis), glucose-6-phosphatase (G6Pase) (glycogen storage disease type I), and glycogen debranching enzyme (AGL) (glycogen storage disease type III).

[0034] Examples of mRNA-encoded target proteins for preventing and / or treating cardiovascular disease include VEGF-A.

[0035] Examples of mRNA-encoded target proteins for preventing and / or treating neurological diseases include TH, AADC, GCH, APOE4, PQBP1, SOD1, and FUS.

[0036] Examples of target proteins encoded by ocular disease mRNA include RPE65, CRB1, and RPGRIP1.

[0037] Furthermore, by incorporating mRNA and guide RNA encoding genome editing enzymes such as CRISPR / Cas9, cytidine base editor (CBE), adenine base editor (ABE), and Prime editor, it becomes possible to repair gene mutations in these single-gene disorders. In some cases, donor DNA (repair template) may also be included.

[0038] In the composition of the present invention, the ratio of phosphate groups of mRNA to amino groups of chitosan is preferably 1:4 to 16, more preferably 1:5 to 12, and more preferably 1:5 to 10. Furthermore, the ratio of phosphate groups of mRNA, amino groups of chitosan, and negative charges of anionic polysaccharides is preferably 1:4 to 16:4 to 32, more preferably 1:4 to 12:6 to 24, and more preferably 1:5 to 10:8 to 16.

[0039] In the composition of the present invention, the nanoparticles preferably form a complex with mRNA. The nanoparticles forming the complex with mRNA preferably have a particle size of 30 to 500 nm, more preferably 50 to 300 nm, and more preferably 90 to 250 nm. Furthermore, the nanoparticles forming the complex with mRNA preferably have a zeta potential of -10 to -60 mV, more preferably -20 to -50 mV, and more preferably -30 to -40 mV. The particle size and zeta potential of the nanoparticles can be measured by dynamic light scattering.

[0040] The mRNA-nanoparticle complex can be prepared by mixing an mRNA solution (e.g., mRNA concentration of 0.5-5% by mass), a chitosan solution (e.g., chitosan concentration of 1-20% by mass), and an anionic polysaccharide solution (e.g., anionic polysaccharide concentration of 1-35% by mass). It is preferable to mix the mRNA solution and the anionic polysaccharide solution first, and then add the chitosan solution. These solutions should be prepared using PBS(-) adjusted to pH 6.5, and the complex should be prepared by mixing each solution in a 1.5 mL plastic tube by pipetting.

[0041] The compositions of the present invention can be used to prevent and / or treat diseases or symptoms involving the expression of target genes, such as infectious diseases, cancer, monogenic diseases, cardiovascular diseases, neurological diseases, and ocular diseases.

[0042] Examples of infectious diseases include the novel coronavirus, AIDS, and hepatitis viruses.

[0043] Examples of cancers include melanoma, leukemia, prostate cancer, and brain tumors.

[0044] Examples of single-gene disorders include hemophilia, thrombosis, congenital immune disorders, and thalassemia.

[0045] Examples of cardiovascular diseases include hypercholesterolemia and amyloidosis.

[0046] Examples of neurological diseases include Parkinson's disease, Alzheimer's disease, and ALS.

[0047] Examples of eye diseases include retinitis pigmentosa and age-related macular degeneration.

[0048] In the present invention, "treatment" means recovery, remission, alleviation, and / or delay of the worsening of a disease or symptoms in a patient who has developed a disease or is exhibiting symptoms.

[0049] In this invention, "prevention" means reducing the incidence rate of a disease or symptom. Prevention includes reducing the risk of progression of a disease or symptom, or reducing the severity of such diseases or symptoms, and also includes preventing recurrence.

[0050] When the composition of the present invention is used as a pharmaceutical for the prevention and / or treatment of the above-mentioned diseases or symptoms, the mRNA-nanoparticle complex can be administered orally or parenterally to human or animal subjects by means of injection into veins, subcutaneous tissue, muscles, or the abdominal cavity, inhalation into the nasal cavity, oral cavity, or lungs, direct administration into lesional tissue or nearby blood vessels, or by supporting it on a porous material such as a gel, sponge, or nonwoven fabric and implanting it. The dosage will vary depending on the target subject, target disease or symptoms, and route of administration, but for example, the single dose of the active ingredient (mRNA) is usually about 0.1 mg to 1 mg / kg body weight, preferably about 0.2 mg to 0.5 mg / kg body weight, administered orally, intramuscularly, subcutaneously, into lesional tissue, or intravenously (preferably continuously or every other day).

[0051] The mRNA-nanoparticle complex may be formulated with a pharmaceutically acceptable carrier and administered to a subject. Examples of carriers include water and physiological saline. Other additives such as buffers, pH adjusters, osmotic regulators, protective agents, stabilizers, and solubilizers may also be added. The composition of the present invention may be sterilized.

[0052] The composition of the present invention is preferably prepared in dosage forms of drug units that are suitable for the dosage of the active ingredient. Examples of such dosage forms of drug units include injections (ampoules), intravenous infusions, nasal sprays, oral preparations (such as capsules), and patch preparations (such as transdermal patches), and it is preferable that each dosage form of drug unit contains typically 0.1 mg to 1.0 mg / kg of the active ingredient (mRNA). [Examples]

[0053] The present invention will be described in more detail below with reference to examples. [Example 1] 1. Introduction In this example, we investigated mRNA delivery systems using polysaccharide nanoparticles (chitosan / hyaluronic acid nanoparticles, chitosan / chondroitin sulfate nanoparticles). As a result, we successfully identified polysaccharide nanoparticles suitable for mRNA delivery to hepatocytes.

[0054] 2. Materials and experimental methods (1) Cell seeding into 48-well plates Cells at 90% confluence (HuH7 or HepG2 cells, RIKEN BioResource Research Center (BRC)) were harvested by trypsin treatment and suspended in serum-containing medium. Cells were stained with trypan blue, and the number of viable cells was measured using a hemocytometer, yielding 4.0 × 10⁶ cells. 4 The required cell quantity to achieve the desired cells / well was calculated. Serum-containing medium was added to each well of a 48-well plate (code 3830-48, IWAKI) to a total volume of 250 μL. The calculated amount of cells was seeded and cultured at 37 °C and 5% CO2 for 24 hours to allow the cells to adhere.

[0055] (2) Solution preparation of composite materials [Preparation of Chitosan Solution] The chitosan used (Yaizu Suisan) is shown in Table 1. The chitosan solution (DDA: 82.4%) was dissolved in PBS(-) prepared to pH 6.5. This was shaken overnight in an EYELA CUTE MIXER CM-1000 and filtered and sterilized using a 0.22 μm filter (MILLEX GV, MILLIPORE) to obtain a chitosan solution (pH 6.5).

[0056] [Table 1]

[0057] [Preparation of anionic polysaccharide solution] The anionic polysaccharides used are shown in Table 2. The anionic polysaccharides were dissolved in PBS(-) prepared at pH 6.5. HA: Hyaluronic acid (Seikagaku Corporation), CS: Chondroitin sulfate (Seikagaku Corporation).

[0058] [Table 2]

[0059] (3) Fabrication of the complex The mixture ratio (charge ratio of the phosphate group (P) of the gene, the amino group (N) of chitosan, and the negative charge (-) of the anionic polysaccharide) was set to either P:N:(-) = 1:5:16 or P:N:(-) = 1:10:16. The gene was either pDNA (pGl4.51 vector, Promega) or mRNA (Cleancap FLuc mRNA, Trilink Biotechnologies).

[0060] The gene / chitosan / anionic polysaccharide ternary complex was prepared in DMEM / 10 mM MOPS (code 23415-54, nacalai tesque) solution (pH 6.5). Two 1.5 mL tubes, A and B, were prepared. 35 μL of MOPS solution, 3 μL of pDNA or mRNA solution, and 12 μL of anionic polysaccharide solution were added to tube A and pipetted thoroughly. Meanwhile, 47 μL of MOPS solution and 3 μL of chitosan solution were added to tube B and pipetted thoroughly. Then, the solution from B was added to A and pipetted, and the mixture was allowed to stand for 15 minutes. At this point, the final concentration of pDNA or mRNA was 30 μg / mL.

[0061] The gene / chitosan binary complex was prepared in the same way as the ternary complex, using DMEM / 10 mM MOPS solution (pH 6.5). Two tubes, A and B, were prepared. 47 μL of MOPS solution and 3 μL of pDNA or mRNA solution were added to tube A and thoroughly pipetted. Meanwhile, 47 μL of MOPS solution and 3 μL of chitosan solution were added to tube B and similarly thoroughly pipetted. Then, the solution from B was added to A and pipetted, and the mixture was allowed to stand for 15 minutes. At this point, the final concentration of pDNA or mRNA was 30 μg / mL.

[0062] (4) Transfection After confirming cell adhesion, the serum-containing medium was removed, and 225 μL of DMEM / 10 mM MOPS FBS(+) medium (pH 6.5) was added. Then, 25 μL of the prepared complex was added to each cell, and the cells were incubated at 37 °C and 5% CO2 for 4 hours.

[0063] (5) Post-transfection After transfection, the culture medium was removed, washed once with serum-containing medium, and then 250 μL of serum-containing medium was added again, and the cells were incubated for 20 hours. This time was defined as the post-transfection time.

[0064] (6) Cell collection The culture medium was removed from each sample and washed three times with PBS(-). Then, 100 μL of Cell Culture Lysis Reagent (code E153A, Promega) was added, and the cells were lysed by shaking at room temperature for 15 minutes. The cell lysates were collected in tubes.

[0065] (7) Luciferase assay The cell solution was centrifuged at 4 °C at 12,000 rpm (13,800 × g) for 5 minutes. After centrifugation, 20 μL of the supernatant and 100 μL of luciferin solution (code E151A, Promega) were placed in a tube and gently pipetted. The relative light units (RLU) were measured using a luminometer (Gene Light GL-200A, Microtec-Nichion).

[0066] (8) Protein assay The protein assay was performed using the DC protein assay kit (BIO-RAD). Absorbance at 690 nm was measured using a microplate reader (Multiskan FC, 51119050, Thermo). Protein concentration was quantified from the absorbance of each sample using a calibration curve of BSA solution.

[0067] (9) Calculation of gene expression activity The RLU obtained by the luciferase assay was corrected for the protein concentration of the cell solution obtained by the protein assay to calculate RLU / mg protein. This value was defined as gene expression activity.

[0068] 3. Results and Discussion 3.1 mRNA expression activity under pDNA-optimized conditions (HuH7 cells) Figure 1 shows the results of gene expression activity in a complex prepared using chitosan (molecular weight 46.4k), which has been conventionally used for pDNA delivery, with a ternary complex composition ratio of P:N:(-) = 1:5:16. The pDNA results showed a significant increase in expression activity for both binary and ternary complexes compared to the control group (naked) administered only pDNA. In particular, the CS ternary complex showed higher values ​​than the HA ternary complex. On the other hand, the mRNA results showed lower expression activity values ​​than the pDNA complexes under all conditions.

[0069] 3.2 Molecular weight dependence of chitosan To improve mRNA expression activity, we investigated the molecular weight dependence of chitosan. The results of gene expression activity with mRNA / chitosan complexes are shown in Figure 2.

[0070] Evaluation of gene expression activity in mRNA / chitosan complexes showed that using low molecular weight chitosan resulted in the highest transgene expression activity at a P:N = 1:10 ratio.

[0071] 3.3 Dependence of the ternary complex on its composition ratio We investigated the effect of the ternary complex composition ratio to improve mRNA expression activity.

[0072] The gene expression activity of ternary complexes containing hyaluronic acid (HA) and chondroitin sulfate (CS40) was evaluated at charge ratios of P:N:(-)=1:10:16 and P:N:(-)=1:10:32. The results are shown in Figure 3. The complex containing chondroitin sulfate showed higher gene expression activity than the complex containing hyaluronic acid. Furthermore, the complex prepared with the charge ratio P:N:(-)=1:10:16 showed higher gene expression activity than the complex with P:N:(-)=1:10:32.

[0073] 3.4 Molecular weight dependence of chondroitin sulfate Of the three molecular weights of chondroitin sulfate, the highest expression activity was obtained with CS40 (Figure 4).

[0074] 3.5 Gene expression activity under optimized conditions (HuH7 cells) Transfection was performed in HuH7 cells using a complex prepared with chitosan at a molecular weight of 14.7k and a composition ratio of P:N:(-) = 1:10:16. The results of gene expression activity are shown in Figure 5. Furthermore, the relative gene expression activity, with the gene expression activity when transfected with pDNA or mRNA only (naked) set to 100, is shown in Figure 6.

[0075] The pDNA results showed no significant effect from differences in chitosan molecular weight. On the other hand, the mRNA results showed a significant effect from chitosan molecular weight. Changing the chitosan molecular weight to 14.7k and the composition ratio P:N:(-) = 1:10:16 increased expression activity, achieving a level of expression activity similar to that of pDNA. Therefore, the optimal complex composition for delivering mRNA to cells is a chitosan molecular weight of 14.7k and a complex composition ratio of P:N:(-) = 1:10:16.

[0076] 3.6 Gene expression activity in HepG2 cells The results of gene expression activity are shown in the upper panel of Figure 7, and the relative gene expression activity, with the gene expression activity (naked) when HepG2 cells were transfected with only pDNA or mRNA set to 100, is summarized in the lower panel of Figure 7.

[0077] Under conditions where chitosan had a molecular weight of 46.4k and a composition ratio of P:N:(-) = 1:5:16 (left two columns of Figure 7), mRNA expression activity was lower than that of the pDNA complex.

[0078] Under conditions of chitosan molecular weight 14.7k and P:N:(-) = 1:10:16, a decrease in overall expression activity was observed in pDNA when the molecular weight and composition ratio of chitosan were changed.

[0079] In mRNA, the ternary complex containing chondroitin sulfate (CS40) achieved expression activity comparable to that of pDNA. On the other hand, no improvement in expression activity was observed with the binary complex or the ternary complex containing HA. These results indicate that the mRNA / chitosan / CS ternary complex is suitable for mRNA delivery, and that its optimal complex composition is one with a chitosan molecular weight of 14.7k and a complex composition ratio of P:N:(-) = 1:10:16.

[0080] 3.7 Comparison of gene expression activity and uptake (Huh-7 cells) Figure 8 shows the results of a comparison of gene expression activity and uptake. In terms of gene expression activity, the mRNA / chitosan / CS ternary complex showed higher values ​​than the mRNA / chitosan / HA ternary complex. On the other hand, no difference was observed in intracellular uptake between the two ternary complexes. This suggests that the mRNA / chitosan / CS ternary complex is effective in the intracellular transport mechanism of mRNA.

[0081] 3.8 Measurement of particle size (dynamic light scattering) Table 3 shows the particle size and zeta potential of mRNA / chitosan binary complexes, mRNA / chitosan / CS, or HA ternary complexes (chitosan 14.7k CS40 P:N:(-) = 1:10:16). Values ​​are mean ± SD (n=3).

[0082] [Table 3] [Industrial applicability]

[0083] The nanoparticles of the present invention are suitable for mRNA delivery and can be used for the delivery of nucleic acid drugs. In addition, they can be used as carriers for introducing genes and nucleic acids into cells in gene editing and molecular biology research.

Claims

1. Nanoparticles for mRNA delivery comprising a cationic polymer and an anionic polysaccharide, wherein the cationic polymer is chitosan or a salt thereof.

2. The nanoparticles according to claim 1, wherein the molecular weight of the chitosan is 1k to 50k.

3. The nanoparticle according to claim 1, wherein the anionic polysaccharide is hyaluronic acid or a salt thereof and / or chondroitin sulfate or / a salt thereof.

4. The nanoparticles according to claim 3, wherein the molecular weight of the hyaluronic acid is 10k to 4000k.

5. The nanoparticles according to claim 3, wherein the molecular weight of the chondroitin sulfate is 1k to 50k.

6. The nanoparticle according to claim 3, wherein the degree of sulfation of chondroitin sulfate is 0.3 to 2.

7. The nanoparticles according to claim 1, wherein the ratio of the amino group of chitosan to the negative charge of the anionic polysaccharide is 1:0.2 to 8.

8. A composition comprising the nanoparticles and mRNA described in claim 1.

9. The composition according to claim 8, wherein the nanoparticles according to claim 1 form a complex with mRNA.

10. The composition according to claim 8 or 9, wherein the ratio of phosphate groups of mRNA to amino groups of chitosan is 1:4 to 16.

11. The composition according to claim 10, wherein the ratio of the phosphate group of mRNA, the amino group of chitosan, and the negative charge of the anionic polysaccharide is 1:4 to 16:4 to 32.

12. The composition according to claim 9, wherein the particle size of the nanoparticles forming a complex with mRNA is 30 to 500 nm.

13. The composition according to claim 8, used to prevent and / or treat at least one disease selected from infectious diseases, cancer, monogenic disorders, cardiovascular diseases, neurological diseases, and ocular diseases.

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