C(5)-halogenated pyrimidine ribonucleotide-bound mRNA and method for increasing protein expression using same

Abstract

The present invention relates to mRNA to which C (5)-halogenated pyrimidine ribonucleotide is bound and a technique for increasing protein expression using same. Various C(5) halogenated pyrimidine (cytidine / uridine) ribonucleotides were synthesized and single- and double-base modified rNTPs were bound to cap-dependent and cap-independent mRNA and then examined for transcription and translation efficiency. As a result, the translation efficiency of fluorinated and chlorinated modified mRNA was 4-5 times higher than that of unmodified mRNA in the case of single base modification, and the combination of pseudouridine and 5-fluorocytidine was significantly increased by about 10 times or more compared to unmodified RNTP in the case of double base modification. Therefore, the present invention can be advantageously utilized in a treatment method requiring modified mRNA.

Description

C(5)-halogenated pyrimidine ribonucleotide-bound mRNA and method for increasing protein expression using the same

[0001] The present invention relates to mRNA bound to C(5)-halogenated pyrimidine ribonucleotide and a technique for increasing protein expression using the same.

[0002]

[0003] mRNA vaccines are playing a pivotal role in treating the COVID-19 pandemic, which is spreading globally. The main components of mRNA vaccines are modified nucleosides (pseudouridine, ψ) and methyl nucleosides (m1ψ). These mRNAs possess numerous advantages, including the ability to evade the innate immune response, efficiently express antigens, and avoid harmful intrachromosomal integration, a serious side effect of DNA-based vaccines. Therefore, many research institutions have been conducting preclinical and clinical studies on the therapeutic applications of mRNA, demonstrating therapeutic efficacy using modified mRNA platforms.

[0004] Foreign mRNA introduced into cells is recognized by various pattern recognition receptors (PRRs) in the endosome or cytosol, which induce the activation of the type I interferon (IFN-I) pathway and cause an excessive immune response that inhibits the expression of antigen proteins. However, mRNA introduced with chemically modified nucleosides such as pseudouridine and 1-methylpseudouridine is known to increase the efficiency of protein expression by reducing the efficiency of type I interferon activity by avoiding the activation of TLR7, TLR8, and other innate immune sensors. Modified nucleosides are analogs other than the existing A, U, G, and C, and mRNA introduced with these modified nucleic acids can bring about slightly different secondary structure modifications even with the same sequence, and it is known that it avoids excessive immunity by not being recognized by RNA pattern recognition receptors, or it also brings about different efficiency in terms of RNA structural stability.

[0005] Through research, it was demonstrated that the immunogenicity of in vitro-transcribed mRNA was reduced by suppressing the toll-like receptor (TLR)-mediated immunogenicity of mRNA by modifying the bases N6-methyladenosine (m6A), pseudouridine (ψ), 5-methylcytidine (m5C), 2-thioridine (s2U), and 5-methyluridine (m5U) in a single or double manner, and the stability and high translational capacity of mRNA were demonstrated by substituting uridine with pseudouridine. These two research results were important works that changed the perception and reality of mRNA and contributed to the recent development of modified mRNA as a therapeutic agent. In addition, many researchers have reported the effects of modified mRNA in various therapeutic fields using m5C / ψ or m5C / s2U modified mRNA for therapeutic applications of mRNA. Therefore, it is important to develop new and diverse modified mRNAs for utilization in various therapeutic fields. However, although the present inventors screened various commercial purine and pyrimidine modified mRNAs for protein expression, most of them did not show efficient transcription and translation efficiencies.

[0006] Accordingly, the present inventors have tried to find a modified nucleotide that can increase protein expression efficiency, and as a result, they focused on the C(5) modification of pyrimidine, which has high biostability and bioavailability among pyrimidine modifications, and can produce more stable mRNA. Accordingly, they confirmed that introducing a modified nucleotide in which a halogen is substituted at the 5' position of the cytidine and uridine pyrimidine bases into the mRNA structure can avoid excessive immune response and increase the expression efficiency of the antigen protein. In addition, unlike the usual decrease in transcription efficiency when using modified nucleotides, they confirmed that the transcription efficiency was not decreased when using a modified nucleotide in which a halogen is substituted at the 5' position of the cytidine and uridine pyrimidine bases, and thus completed the present invention.

[0007]

[0008] The problem to be solved by the present invention is to provide a method for increasing protein expression efficiency using an mRNA structure to which a C(5)-halogenated pyrimidine ribonucleotide is bound.

[0009]

[0010] In order to solve the above technical problem, in one embodiment of the present invention, an mRNA structure is provided in which a modified nucleotide is introduced in which the 5th carbon of the pyrimidine base of cytidine or uridine is substituted with a halogen.

[0011] In another embodiment, a composition for RNA synthesis comprising the modified nucleotide is provided.

[0012] In addition, in an embodiment of the present invention, a method for increasing protein expression efficiency using mRNA into which the modified nucleotide has been introduced is provided.

[0013] In the present invention, the composition for RNA synthesis may be characterized by further comprising pseudouridine or methylpseudouridine.

[0014] In the present invention, the method may be characterized in that protein expression efficiency increases through cap-dependent translation.

[0015] In the present invention, the method may be characterized by further comprising pseudouridine or methylpseudouridine, and in particular, may be characterized by comprising 5-Fluoro cytidine triphosphate (5-F rCTP) represented by chemical formula 1 and pseudouridine, and in this case, the protein may be characterized by being an antigen protein.

[0016] The above modified nucleotide may be selected from the group consisting of the following chemical formulas 1 to 4.

[0017] [Chemical Formula 1]

[0018]

[0019] [Chemical Formula 2]

[0020]

[0021] [Chemical Formula 3]

[0022]

[0023] [Chemical Formula 4]

[0024]

[0025]

[0026] By synthesizing various C(5) halogenated pyrimidine (cytidine / uridine) ribonucleotides and incorporating single- and double-base modified rNTPs into cap-dependent and cap-independent mRNAs, we confirmed the transcription and translation efficiency. As a result, fluorinated and chlorinated modified mRNAs showed a translation efficiency 4-5 times higher than that of unmodified mRNA in the case of single-base modification, and in the case of double-base modification, the combination of pseudouridine and 5-fluorocytidine was confirmed to increase the translation efficiency by about 10 times or more compared to unmodified rNTPs, so it can be usefully utilized in therapeutic methods that depend on modified mRNA.

[0027]

[0028] Figure 1 is a schematic diagram showing the structure of purchased 5-fluorocytidine (1a) and 5-fluorouridine (2a) ribonucleosides.

[0029] Figure 2 is a schematic diagram showing the C(5)-halogenation (Cl, Br, and I) synthesis of cytidine and uridine ribonucleosides.

[0030] Figure 3 is a diagram showing the process of synthesizing C(5)-halogenated cytidine ribonucleoside 5`-O-triphosphate using a one-pot synthesis method.

[0031] Figure 4 is a diagram showing the process of synthesizing C(5)-halogenated uridine ribonucleoside 5`-O-triphosphate using a one-pot synthesis method.

[0032] Figure 5 is a schematic diagram briefly showing an mRNA to which a C(5)-halogenated pyrimidine ribonucleotide of the present invention is bound and an experimental method for increasing protein expression using the same.

[0033] Figure 6 shows the results of in vitro transcription (cap-dependent) of C(5)-halogenated pyrimidine-linked mRNA: (a) Luciferase mRNA transcription band. Lane 1: Ladder, Lane 2: Luciferase template, Lane 3: Luciferase mRNA (all native), Lane 4: Luciferase mRNA (with Pseudo-rUTP), Lane 5: Luciferase mRNA (with 5-fluoro-rCTP), Lane 6: Luciferase mRNA (with 5-fluoro-rUTP), Lane 7: Luciferase mRNA (with 5-chloro-rCTP), Lane 8: Luciferase mRNA (with 5-chloro-rUTP), Lane 9: Luciferase mRNA (with 5-bromo-rCTP), Lane 10: Luciferase mRNA (with 5-bromo-rUTP), Lane 11: Luciferase mRNA (with 5-iodo-rCTP), Lane 12: Luciferase mRNA (with 5-iodo-rUTP), Lane 13: Luciferase mRNA (containing pseudo-rUTP and 5-Fluoro-rCTP), Lane 14: Luciferase mRNA (containing pseudo-rUTP and 5-chloro-rCTP), Lane 15: Luciferase mRNA (containing 5-fluoro-rCTP and 5-chloro-rUTP), Lane 16: Luciferase mRNA (containing 5-fluoro-rUTP and 5-chloro-rUTP), Lane 17: Luciferase mRNA (containing 5-fluoro-rCTP and 5-fluoro-rUTP), Lane 18: Luciferase mRNA (containing 5-chloro-rCTP and 5-chloro-rUTP). b) Bar graph of transcribed yield. This graph is plotted to show the yield of transcribed mRNA containing C(5) halogenated pyrimidine nucleotides based on the concentration of transcribed mRNA (ng / μl), and for the bar graph, the nanodrop assay was performed in triplicate and the average value was obtained.

[0034] Figure 7: (a) GFP mRNA transcription (cap-dependent). Lane 1: Ladder, Lane 2: GFPTemplate, Lane 3: GFPmRNA (all native), Lane 4: GFPmRNA (with Pseudo-rUTP), Lane 5: GFPmRNA (with 5-fluoro-rCTP), Lane 6: GFPmRNA (with 5-fluoro-rUTP), Lane 7: GFPmRNA (with 5-chloro-rCTP), Lane 8: GFPmRNA (with 5-chloro-rUTP), Lane 9: GFPmRNA (with 5-bromo-rCTP), Lane 10: GFPmRNA (with 5-bromo-rUTP), Lane 11: GFPmRNA (with 5-iodo-rCTP), Lane 12: GFPmRNA (with 5-Iodo-rUTP), Lane 13: GFPmRNA (with Pseudo-rUTP and Lane 14: GFP mRNA (including PseudorUTP and 5-Chloro-rCTP), Lane 15: GFP mRNA (including 5-Fluoro-rCTP and 5-Chloro-rUTP), Lane 16: GFP mRNA (including 5-Fluoro-rUTP and 5-Chloro-rCTP), Lane 17: GFP mRNA (including 5-Fluoro-rCTP and 5-Chloro-rUTP), Lane 18: GFP mRNA (including 5-Fluoro-rCTP and 5-Chloro-rUTP). (b) Bar graph of transcribed yield. This graph is plotted to indicate the yield of transcribed mRNA containing C(5) halogenated pyrimidine nucleotides based on the concentration of transcribed mRNA (ng / μl). For the bar graph, the nanodrop assay was performed in triplicate and the average value was obtained.

[0035] Figure 8: (a) Translation yield of luciferase-capped mRNA (error bars represent standard deviations of three samples); (b) Ranking of rNTP combinations by translation yield (luminescence values ​​here are the average of three samples).

[0036] Figure 9 is a graph showing the translation rate of IRES GFP mRNA.

[0037] Figures 10 to 12 show the relative expression efficiency of TLR 7, IFN alpha, and RIG-1 by intracellular introduction of mRNA into which modified nucleotides according to the present invention have been introduced for immunogenicity confirmation, respectively.

[0038]

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0040] The present invention relates to mRNA having various halogens introduced at the C(5) position of a pyrimidine base. C(5)-halogenated (F, Cl, Br, I) pyrimidine ribonucleoside triphosphates were synthesized and incorporated into mRNA during an in vitro transcription process. As a result, it was confirmed that, except for iodine (I), small and electronegative C(5)-halogenated pyrimidines exhibit higher in-vitro transcription reaction efficiency (F > Cl > Br). Among C(5)-halogenated pyrimidine ribonucleotides, it was confirmed that mRNAs incorporated with C(5)-halogenated cytidines (5-F rCTP and 5-Cl rCTP) exhibited more prominent protein expression than C(5)-halogenated uridine-modified mRNA and unmodified mRNA. In particular, in the case of mRNA introduced with fluorine (5-F rCTP) and chlorine (5-Cl rCTP), it was confirmed that protein expression was about 4-5 times higher efficiency than unmodified mRNA, similar to pseudouridine (ψ). When pseudouridine (ψ) and fluorocytidine (5-F rCTP) were simultaneously introduced into mRNA and double-linked, the protein expression efficiency was confirmed to be excellent, increasing up to 10-fold. In addition, it was confirmed that the efficiency of cap-dependent protein expression was much higher in mRNA linked to C(5)-halogenated pyrimidine ribonucleotides than in cap-independent (IRES) expression. Therefore, these results can be usefully utilized in the field of development of modified mRNA-based therapeutics.

[0041] The present invention provides an mRNA structure in which a modified nucleotide is introduced using a modified nucleotide in which a halogen is bonded to the 5th carbon atom of the pyrimidine base of cytidine and uridine, i.e., the modified CTP, the modified UTP, and ATP (Adenosine triphosphate) and GTP (Guanosine triphosphate). Compounds in which a halogen is bonded to the 5th carbon atom of the pyrimidine base of cytidine and uridine have been utilized for antiviral purposes, but have not been utilized to increase protein expression efficiency by introducing them into an mRNA structure.

[0042] In addition, a composition for RNA synthesis is provided, which includes a modified nucleotide in which the 5th carbon of the pyrimidine base of cytidine or uridine is substituted with a halogen.

[0043] In the present invention, the composition for RNA synthesis may be characterized by further comprising pseudouridine or methylpseudouridine.

[0044] In addition, a method for increasing protein expression efficiency using an mRNA structure into which the modified nucleotide is introduced is provided.

[0045] In the present invention, the method may be characterized in that protein expression efficiency increases through cap-dependent translation.

[0046] In the present invention, the method may be characterized by further comprising pseudouridine or methylpseudouridine, and in particular, may be characterized by comprising 5-Fluoro cytidine triphosphate (5-F rCTP) represented by chemical formula 1 and pseudouridine, and in this case, the protein may be characterized by being an antigen protein.

[0047] In the present invention, a method for increasing protein expression efficiency using an mRNA structure comprises an mRNA structure including ATP (Adenosine triphosphate), GTP (Guanosin triphosphate), CTP (Cytidine TriPhosphate) and UTP (Uridine TriPhosphate), and at least one ribonucleotide among the CTP and UTP may have a halogen functional group bonded to the 5th carbon of a pyrimidine base, and may be characterized in that it is particularly substituted with F or Cl (chemical formulae 1 to 4).

[0048] In the present invention, the halogen may be one or more halogens selected from the group consisting of F (Fluorine), Cl (chlorine), Br (Bromine), and I (Iodine), and more preferably one or more halogens selected from the group consisting of F (Fluorine), Cl (chlorine), and Br (Bromine). In the present invention, the bonding of a halogen (F, Cl, Br, and I) functional group to the 5th carbon of one or more ribonucleotide pyrimidines among the CTP and UTP is shown in the synthetic process and NMR data in Example 1 below, and the specific chemical formulas are shown as Chemical Formulas 1 to 8 (see Table 3).

[0049] The ribonucleotide of the above CTP is a pyrimidine with an F (fluorine) functional group attached to the 5th carbon, and is represented by the following chemical formula 1.

[0050] [Chemical Formula 1]

[0051]

[0052] Another ribonucleotide of the above CTP is one in which a Cl (chlorine) functional group is bonded to the 5th carbon of a pyrimidine, and is represented by the following chemical formula 3.

[0053] [Chemical Formula 3]

[0054]

[0055] The method for increasing the protein expression efficiency is to increase the level of protein expression through cap-dependent translation, and in the present invention, it was confirmed that the cap-dependent protein expression efficiency is much higher in mRNA to which C(5)-halogenated pyrimidine ribonucleotides are bound than in cap-independent (IRES) expression.

[0056]

[0057] <Example 1> Synthesis of C(5)-halogenated pyrimidine ribonucleosides and 5'-O-triphosphates of these ribonucleosides

[0058] In the present invention, halogen derivatives (F, Cl, Br, I) were incorporated at the C(5) position of uridine and cytidine ribonucleosides. Although several methods for the C(5) halogenation of pyrimidine nucleosides have been previously reported, in the present invention, halogen derivatives other than fluoro derivatives were also synthesized from available cytidine and uridine ribonucleosides. These derivatives were synthesized using 1H-NMR and ESI mass spectrometry. RNA transcription was performed using T7 RNA polymerase by attaching triphosphates to the C(5) halogen derivatives of pyrimidine ribonucleosides. Pyrimidine triphosphates with C(5) halogens were synthesized using the Ludwig method, which does not require 2' and 3' hydroxyl protection of the nucleoside. It was observed that the time interval required for triphosphate synthesis varied depending on the halogen attached to the C(5) position of pyrimidine ribonucleosides. We found that triphosphate synthesis was substrate-dependent, and optimized the reaction conditions by extending the reaction time from 2 to 3 hours after pyrophosphate addition for all nucleotides to obtain good yields for each nucleotide. Each triphosphate compound was then purified by HPLC and confirmed using 31P-NMR.

[0059]

[0060] <1-1> Reagents

[0061] All reagents for halogenation and triphosphate synthesis were purchased commercially and used without further purification. H-NMR and 31P NMR spectra were analyzed using an NMR spectrometer (Bruker AV 400, 162 MHz) using DMSO-d6 and D2O as solvents. UV-Vis spectra of these compounds were analyzed using a Cary Series UV-Vis spectrophotometer (Agilent Technology) and a 1-cm path-length quart cuvette. The absorbance change was measured immediately after irradiating the cuvette solution with UV light. The luciferase plasmid was constructed using VectorBuilder (Vector ID: VB220810-1198vwd). All oligonucleotides used were purchased from Cosmo Genetec, and the luciferase protein substrate for the 'Luciferase Assay System' was purchased from Promega. 96-well polystyrene-treated cell culture plates were purchased from Corning, and 60 mL cell culture flasks were purchased from SPL Life Sciences. 2X TOPsimple™ DyeMIX-Tenuto PCR master-mix, T7 RNA polymerase, and RNase inhibitor were purchased from Enzynomics, and 3´-O-Me-m7G(5')ppp(5')G RNA Cap Structure Analog (ARCA) and DNase I were purchased from New England Biolabs (NEB). The RNA purification kit 'RNA Clean and Concentrator' was purchased from Zymo Research. Cellulose, ethanol, fetal bovine serum, pen / strip, and trypsin were purchased from Sigma-Aldrich. Lipofectamine™ MessengerMAX™ transfection reagent, Opti-MEM, DMEM, and Heracell 240i CO2 incubator were purchased from Thermo Fisher.The Colibri+ LB 916 microvolume spectrophotometer and the Centro LB 960 Microplate Luminometer were purchased from Berthold Technologies.

[0062]

[0063] <1-2> Process and reaction of each halogenation

[0064] The reaction of cytidine and uridine is also the structure of commercially available 5-F-cytidine / uridine (Fig. 1). 5-Chlorocytidine and uridine (1b and 2b) were synthesized. Specifically, to synthesize cytidine 1b, 0.300 g (1.2 mM) of cytidine (1) was dissolved in 0.5 M HCl and DMF (6 ml), stirred at room temperature for 1 h, then the temperature was lowered to 0 °C. A 0.342 g, 1.984 mM m-CPBA solution in DMF was added over 20 min, and the temperature was then raised to RT (15–25 °C). After stirring for 6 hours, the reaction mixture was extracted with EtOAc and water, and the progress of the reaction was checked by TLC. The aqueous layer was collected and dried using a rota-evaporator, and a slurry was prepared and subjected to silica column chromatography using 0-5% methanol and DCM to obtain the compound as a white solid powder with a yield of 65% and 0.385 g.

[0065] 1H NMR (400 MHz, DMSO-d6): δ 8.32 (s, 1H), 7.84 (s,1H), 7.22 (s, 1H), 5.717 (d, 1H,J= 3.2Hz), 5.386 (d, 1H, J= 4.8Hz) 5.236 (t, 1H, J= 4.8Hz), 5.498 (d, 1H, J= 5.6Hz), 3973-3850 (m, 2H), 3.844 (t,1H,J= 2.8Hz), 3.726- 3.689 (dq, 1H), 3.596-3.554 (dq, 1H), 3.341 (s, 8H), 2.506 (t, 4H,J= 1.6Hz). MS (ESI, LRMS, m / z): calculated for C9H 12 ClN3O5 ([m]): m / z 277.66; Found: [M-2] 275.6.

[0066] In addition, to synthesize uridine 2b, 0.300 g (1.2 mM) of uridine (2) was dissolved in 0.5 M HCl, DMF (6 ml), stirred at room temperature for 1 h, then lowered to 0 °C, and 0.339.7 g, 1.968 mM m-CPBA solution with DMF was added for 20 min, and then the temperature was increased to RT. After stirring for 6 h, the reaction mixture was extracted with EtOAc and water, and the aqueous layer was collected and dried using a rota-evaporator, and TLC was checked to determine the progress of the reaction. In addition, a slurry was prepared and silica column chromatography was performed using 0-5% methanol and DCM to obtain 93% of the compound, 0.594 g of white solid powder.

[0067] 1H NMR (400 MHz, DMSO-d6): δ 11.865 (s, 1H), 8.415 (s, 1H), 5.464 (d, 1H, J= 4.4Hz), 5.441 (d, 1H, J= 5.3Hz), 5.290 (t, 1H, J= 4.7Hz), 5.085 (d, 1H, J= 5.4Hz), 4.045 (dd, 1H, J=4.8Hz, J= 4.8Hz), 3.998 (dd, 1H, J=5Hz, J=5Hz), 3.867 (t, 1H, J= 2.3Hz), 3.721-3.672 (dq, 1H), 3.606-3.558 (dq, 1H), 3.336 (s, 22H), 2.506 (t, 18H, J=1.6Hz).MS (ESI, LRMS, m / z): calculated for C9H 11 ClN2O6 ([m]): m / z 278.03; Found: [M-2] 276.6.

[0068] 5-Bromocytidine and uridine (1c and 2c) were synthesized. Specifically, to synthesize cytidine 1c, 0.300 g (1.2 mM) of cytidine (1) and 0.285 g (1.6 mM) of N-bromosuccinimide (NBS) were placed in a 200 ml RB flask, dissolved in anhydrous DMF (5 ml), and stirred overnight at room temperature in a nitrogen atmosphere. After the reaction was completed, the solvent was removed using an evaporator, and a slurry was prepared, which was then subjected to column chromatography using 0-5% methanol and DCM to obtain 0.379 g of a white solid powder (yield: 90%).

[0069] 1H NMR (400 MHz, DMSO-d6): δ 8.398 (s, 1H), 7.850 (s, 1H), 7.004 (s, 1H), 5.714 (d, 1H, J=3.2Hz), 5.388 (d, 1H, J=4.8Hz), 5.239 (t, 1H, J=4.8Hz), 4.996 (d, 1H, J=5.2Hz), 4.108 (d, 1H, J=5.2Hz), 3.953 (dd, 2H, J=5.2Hz, J=3.6Hz)3.845 (t, 1H, J=2.4Hz), 3.729-3.688 (dq, 1H), 3.591-3.543 (dq, 1H), 3.339 (s, 20H), 3.171 (d, 2H, J=5.2Hz), 2.506 (t, 10H, J=2Hz).MS (ESI, LRMS, m / z): calculated for C9H 12 BrN3O5 ([m]): m / z 321.00; Found: [M-2] 319.6.

[0070] In addition, to synthesize uridine 2c, 0.300 g (1.2 mM) of uridine (2) and 0.285 g (1.6 mM) of N-Bromosuccinimide (NBS) were added to a 200 ml RB flask, dissolved in anhydrous DMF (5 ml), and stirred overnight at room temperature in a nitrogen atmosphere. After the reaction was completed, the solvent was removed using an evaporator, and a slurry was prepared, which was then subjected to column chromatography using 0-5% methanol and DCM to obtain the product as a final product in the form of a white solid powder (70%), 0.2956 g.

[0071] 1H NMR (400 MHz, DMSO-d6): δ 11.827 (s, 1H), 8.490 (s, 1H), 5.730 (d, 1H, J=4.4Hz), 5.440 (s, 1H), 5.290 (s, 1H), 5.082 (s, 1H), 4.040 (d, 1H, , J=4Hz), 3.988 (t, 1H, J=4.4Hz), 3.868 (t, 1H, , J=2.4Hz), 3.694 (d, 1H, , J=12Hz), 3.579 (d, 1H, , J= 12Hz), 3.341 (s, 6H), 2.506 (t, 3H, , J= 2Hz).MS (ESI, LRMS, m / z): calculated for C9H 11 BrN2O6 ([m]): m / z 321.98; Found: [M-1] 320.6.

[0072] 5-Iodocytidine and iodouradine (1d and 2d) were synthesized. Specifically, to synthesize iodocytidine 1d, 0.300 g (1.2 mM) of cytidine (1) and 0.5399 g (2.4 mM) of N-Iodosuccinimide were placed together in a 200 ml RB flask and dissolved in anhydrous DMF (6 ml). After stirring at 60 °C for 8 hours in a nitrogen atmosphere, the reaction was confirmed by TLC, and the solvent was removed using a rota-evaporator to make a slurry, which was then subjected to column chromatography to obtain 0.4074 g of powder (87%).

[0073] 1H NMR (400 MHz, DMSO-d6): δ 8.412 (s, 1H), 7.811 (s, 1H), 6.616 (s, 1H), 5.707 (d, 1H, J= 3.6Hz), 5.369 (d, 1H, J= 4.8Hz), 5.224 (t, 1H, J= 4.8Hz), 4.989 (d, 1H, J= 5.2Hz), 3.941 (dd, 2H, J= 5.6Hz, J= 4.4Hz), 3.840 (t, 1H, J= 2.4Hz), 3.718-3.680 (dq, 1H), 3.580-3.538 (dq, 1H), 3.341 (s, 19H), 2.506 (t, 6H, J= 1.6Hz).MS (ESI, LRMS, m / z): calculated for C9H 12 lN3O5([m]): m / z 368.98; Found: [M-1] 367.6.

[0074] In addition, to synthesize iodide uridine 2d, 0.300 g (1.2 mM) of uridine (2) and 0.5399 g (2.4 mM) of N-Iodosuccinimide were placed together in a 200 ml RB flask and dissolved in anhydrous DMF (6 ml). After stirring for 8 hours at 60 °C in a nitrogen environment, the reaction was confirmed by TLC, and the solvent was removed using a rota-evaporator to make a slurry, which was then subjected to column chromatography. Finally, 73% powder, 0.3426 g was obtained.

[0075] 1H NMR (400 MHz, DMSO-d6): δ 11.690 (s, 1H), 8.482 (s, 1H), 5.722 (d, 1H, J=4.4Hz), 5.422 (d, 1H, J=5.6Hz), 5.265 (t, 1H, J=4.8Hz), 5.076 (d, 1H, J=5.6Hz), 4.034 (dd, 1H,J=4.8Hz, J=4.8Hz), 3.979 (dd, 1H, J=4.8Hz, J=4.8Hz), 3.863(t, 1H, , J=2Hz), 3.699-3.657 (dq, 1H), 3.592-3.551 (dq, 1H), 3.337 (s, 15H), 2.506 (t, 3H, , J= 1.6Hz).MS (ESI, LRMS, m / z): calculated for C9H 11 IN2O6([m]): m / z 369.97; Found: [M-1] 368.5.

[0076]

[0077] <1-3> Preparation of 5'-O-triphosphates of C(5)-halogen-substituted pyrimidine ribonucleosides

[0078] To prepare 5'-O-triphosphates of C(5)-halogen-substituted pyrimidine ribonucleosides, C(5)-halogenated cytidine ribonucleoside 5'-O-triphosphate was synthesized by a one-pot synthesis method (Fig. 3). In addition, C(5)-halogenated uridine ribonucleoside 5'-O-triphosphate was synthesized by a one-pot synthesis method (Fig. 4). Various time intervals and HPLC retention times of C(5)-halogenated cytidine ribonucleosides after pyrophosphate addition are shown in Table 1, and the time intervals and HPLC retention times of C(5)-halogenated uridine nucleosides after pyrophosphate addition are shown in Table 2.

[0079]

[0080]

[0081]

[0082] Specifically, proton sponge (1.2 equiv) and free nucleoside (1 equiv) were placed in a reaction flask with a magnetic stirrer and dried overnight under high vacuum. The mixture was dissolved in trimethyl phosphate (0.3 M) and kept at room temperature for 15 minutes until the compound was completely dissolved, cooled to -15°C, POCl3 (1.2 equiv) was slowly added dropwise until a color change appeared in the reaction slurry and the reaction was continued at the same temperature as above, -15°C, for 1.5 hours, tributyl amine (6 equiv) was slowly added and stirred for 20 minutes, then tributyl ammonium pyrophosphate solution (5.0 equiv) and DMF (0.5 M) were slowly added and stirred for 5 minutes to 3 hours depending on the substrate (ribonucleoside), but the reaction was optimized by increasing the reaction time to more than 2 hours after the addition of pyrophosphate and then stopping the reaction by adding 0.5 M. A 5 M solution (20 vol-equiv) of triethylammonium bicarbonate Et3NH2CO3 (TEAB) was stirred from 0°C to room temperature for 30 minutes, and the resulting solution was lyophilized. The residue was then purified on a reversed-phase (prep pure C18 AQ 100Ao, 5 μm, 250x20) HPLC column (0–20% MeCN as solvent B for 60 minutes, 0.1 M TEEA as solvent A, pH 7.5 for 60 minutes, 100–80%), and the purified compound was lyophilized to provide the triphosphate as a solid. Then, for confirmation, 31P NMR was measured using a Bruker AV-162MHz spectrometer using D2O as solvent, and chemical shifts of δ -23.65 to -23.10 (t, β-P), -11.90 to -11.75 (d, α-P), -11.10 to -10.90 (d, γ-P) were provided in ppm (t=triplet and d= doublet).

[0083]

[0084] <1-4>삼인산염 목록

[0085] 본 발명에서 합성한 삼인산염 목록을 하기 표 3에 나타내었다.

[0086] 번호명칭화학식(NMR data)15-Fluorocytidine triphosphate(5-F rCTP) 31 P NMR wastaken by using a Bruker AV-162 MHz spectrometer with D2O as the solvent and gave chemical shift in ppm around; δ -23.53 to -23.28 (t, β-P), -11.72 to -11.60 (d, α-P), -11.06 to -10.94 (d, γ-P). Where, t=triplet and d= doublet and followed by Electronspray ionization (ESI) mass Spectrometry. Calculated m / z for C9H 15 FN3O 14 P3([M]): m / z 500.98; found ([M-1]): 499.60.25-Fluorouridine triphosphate(5-F rUTP) 31 P NMR was taken by using a Bruker AV-162MHz spectrometer with D2O as the solvent and gave chemical shift in ppm around; δ -23.50 to -23.25 (t, β-P), -11.85 to -11.58 (d, α-P), -11.03to -10.72 (d, γ -P). Where, t=triplet and d= doublet and followed by Electronspray ionization (ESI) mass Spectrometry. Calculated m / z for C9H14 FN2O 15 P3([M]): m / z 501.96; found([M-1]): 500.50.35-Chlorocytidine triphosphate(5-Cl rCTP) 31 P NMR was taken by using a Bruker AV-162MHz spectrometer with D2O as the solvent and gave chemical shift in ppm around; δ -23.56 to -23.31 (t, β-P), -11.98 to -11.68 (d, α-P), -11.06to -10.94 (d, γ -P). Where, t=triplet and d= doublet and followed by Electronsprayionization (ESI) mass Spectrometry. Calculated m / z for C9H 15 ClN3O 14 P3([M]): m / z 516.95; found: 516.50.45-Chlorouridine triphosphate(5-Cl rUTP) 31 P NMR was taken by using a Bruker AV-162MHz spectrometer with D2O as the solvent and gave chemical shift in ppm around; δ -23.57 to -23.33 (t, β-P), -11.85 to -11.73 (d, α-P), -11.06 to -10.59 (d, γ-P). Where, t=triplet and d= doublet and followed by Electronspray ionization (ESI) mass Spectrometry. Calculated m / z for C9H 14 ClN2O 15P3([M]): m / z 517.93; found([M-1]): 516.50.55-Bromocytidine triphosphate(5-Br rCTP) 31 P NMR was taken by using a Bruker AV-162MHz spectrometer with D2O as the solvent and gave chemical shift in ppm around; δ -23.52 to -23.27 (t,β-P), -11.98 to -11.85 (d, α-P), -11.00 to -10.61 (d, γ -P). Where, t=triplet and d= doublet and followed by Electronspray ionization (ESI) mass Spectrometry. Calculated m / z for C9H 15 BrN3O 14 P3([M]): m / z 560.90; found([M-1]): 559.565-Bromouridine triphosphate(5-Br rUTP) 31 P NMR was taken by using a Bruker AV-162MHz spectrometer with D2O as the solvent and gave chemical shift in ppm around; δ -23.54 to -23.30 (t,β-P), -11.89 to -11.56 (d, α-P), -11.06 to -10.74 (d, γ -P). Where, t=triplet and d= doublet and followed by Electronspray ionization (ESI) mass Spectrometry. Calculated m / z for C9H 14 BrN2O 15P3([M]): m / z 561.88; found([M-1]): 560.50.75-Iodocytidine triphosphate(5-I rCTP) 31 P NMR was taken byusing a Bruker AV-162MHz spectrometer with D2O as the solvent and gave chemical shift in ppm around; δ -23.50 to -23.17 (t, β-P), -12.02 to -11.91 (d, α-P), -11.72 to -10.61 (d, γ-P). Where, t=triplet and d= doublet and followed by Electronspray ionization (ESI) mass Spectrometry. Calculated m / z for C9H 15 IN3O 14 P3([M]):m / z 608.88; found([M-1]): 607.40.85-Iodouridine triphosphate(5-I rUTP) 31 P NMR was taken by using a Bruker AV-162MHz spectrometer with D2O as the solvent and gave chemical shift in ppm around; δ -23.54 to -23.29 (t, β-P), -11.93 to -11.80 (d, α-P), -11.72 to -10.61 (d, γ-P). Where, t=triplet and d=doublet. And followed by Electronspray ionization (ESI) mass Spectrometry Calculated m / z for C9H 14 IN2O 15 P3([M]):m / z 609.87; found([M-1]): 608.50.

[0087]

[0088] <Example 2> In vitro transcription analysis of C(5)-halogenated pyrimidine-linked mRNA (cap-dependent mRNA platform)

[0089] To generate modified mRNA, we used an in vitro transcription reaction in which one of the four natural nucleoside triphosphates could be replaced with the corresponding modified nucleoside triphosphate. In the mRNA thus transcribed, single or double bases (uridine and cytidine) were substituted at all positions with C5-halogenated uridine or cytidine. A luciferase template (SI) was prepared from a luciferase plasmid cloned in Escherichia coli, and then the modified luciferase mRNA was prepared through an in vitro transcription reaction. Gel electrophoresis confirmed the appearance of a modified mRNA band (1.7 kb) immediately beneath the template band (Fig. 6a). First, we investigated a cap-dependent in vitro transcription reaction using the luciferase template (Fig. 6b). We confirmed that the transcription yield of mRNA using C5-halogenated uridine and cytidine triphosphate was approximately 10–50% lower than that of the natural nucleotides. The reason for the low yield of modified nucleotides was presumed to be because the active site of T7 RNA polymerase was narrow to recognize the modified nucleoside triphosphate because natural nucleotides were selected as the recognition target of T7 RNA polymerase rather than natural modified nucleotides. In addition, for C(5)-halogenated bases, it was observed that the yield of transcribed mRNA (modified) decreased regularly with size and electronegativity up to brominated nucleotides, and halogen derivatives with smaller size and higher electronegativity showed better transcription yields. Iodinated nucleotides showed higher mRNA transcription yield than brominated nucleotides in the case of single modification of the genetic code, but iodinated nucleotides showed higher transcription yield in the case of double modification. In the case of double modification of the genetic code, the yield of transcribed mRNA was slightly lower than that of single modification and showed an irregular pattern.

[0090]

[0091] <Example 3> In vitro transcription analysis of C(5)-halogenated pyrimidine-linked mRNA (cap-independent mRNA platform)

[0092] Cap-independent mRNA transcription was assayed using C(5) halogenated uridine and cytidine triphosphates containing an IRES (internal ribosome entry site) structure in mRNA. IRES is an RNA element that enables translation initiation in a cap-independent manner. An IRES GFP template was used for cap-independent transcription analysis. All transcription reagents were used similarly to the cap-dependent transcription analysis, but in this case, the ARCA cap was not used. Because the IRES secondary structure of mRNA can affect translation efficiency, the goal was to determine the effect of modified rNTPs on the IRES-containing mRNA and compare it with native rNTPs. Therefore, transcription of all single- and double-modified IRES GFP mRNAs was assayed. Gel analysis revealed that all modified rNTPs were transcribed, with mRNA visible just beneath the template band (Fig. 7a). Interestingly, nanodrop assays for concentration revealed that transcription efficiency of the modified rNTPs was relatively higher when single-modified than when dual-modified (Fig. 7b).

[0093]

[0094] <Example 4> In vitro translation analysis of C(5)-halogenated pyrimidine-linked mRNA (cap-dependent mRNA platform)

[0095] After the incubation period, the medium was removed, and the cells were lysed using 50 μL of 1X lysis buffer provided with the luciferase substrate, and 100 μL of luciferin substrate per sample was taken into a 96-well white plate to perform a luminescence assay, and then each cell lysate sample was added in a volume of 20 μL and mixed by pipetting. Luciferase luminescence was confirmed using a luminometer device as shown in Fig. 8. Fig. 8(a) was ranked based on translation efficiency (Fig. 8(b)). It was confirmed that the dual (pseudouridine (ψ) + C(5)-fluorocytidine and pseudouridine (ψ) + C(5)-chlorocytidine) modified integrated mRNAs of each transcribed mRNA showed significantly improved translation efficiency than the remaining single and dual modified mRNAs containing pseudouridine (ψ) (about 2.5 times more than pseudouridine). Modified mRNAs conjugated with single-stranded 5-Cl rCTP and 5-F rCTP showed approximately 1.1-fold higher translation efficiency than mRNAs conjugated with pseudouridine (ψ). Furthermore, we confirmed that the translation efficiency of mRNAs conjugated with large, low-electronegative C(5)-halogenated (iodocytidine / uridine and bromuridine) ribonucleotides was lower than that of mRNAs conjugated with natural rNTPs.

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[0097] <Example 5> In vitro translation analysis of C(5)-halogenated pyrimidine-linked mRNA (cap-independent mRNA platform)

[0098] For cap-independent translation, we used transcribed IRES GFP mRNA lacking a cap analog and containing the EMCV IRES at the 5' end of the mRNA. The effect of mRNA incorporating C(5)-halogenated pyrimidines was investigated. Interestingly, GFP expression analysis using singly and doubly modified rNTPs significantly reduced translation, whereas native mRNA showed significant GFP expression (Fig. 9). IRES modifications induce these results because the IRES secondary structure must be recognized by translation initiation factors. However, the IRES structure incorporating C(5)-halogenated pyrimidines can significantly reduce the translation efficiency of target genes due to the altered secondary structure of the modified IRES, which is different from the native structure. This is because the modified nucleotides cannot form the specific secondary structure required for recognition and translation by the cellular translation machinery.

[0099]

[0100] <Example 6> Confirmation of immunogenicity of C(5)-halogenated pyrimidine-linked mRNA (Experiment to confirm pattern recognition receptor and cytokine production for confirmation of immunogenicity (reverse transcription polymerase chain reaction (RT-PCR)))

[0101] In order to confirm immunogenicity by measuring the relative expression levels of TLR 7, RIG-1 and IFN alpha cytokines, which are pattern recognition receptors of foreign single-stranded mRNA, when mRNA into which modified nucleotides according to the present invention are introduced into cells, THP-1 macrophages, which are immune cells, were used and the relative expression efficiency of TLR 7, RIG-1 and IFN alpha, which are single-stranded RNA pattern recognition receptors, was measured using RT-PCR.

[0102] More specifically, the production of TLR7, a pattern recognition receptor for foreign single-stranded RNA, and RIG-1, and IFN alpha cytokines using THP-1 macrophages was confirmed using the Apoptin mRNA gene with modified nucleotides, and the levels were determined by reverse transcription polymerase chain reaction (RT-PCR). THP-1 monocytes were differentiated into macrophages using 200 ng / ml of Phorbol-12-myristate-13-acetate (PMA). The modified Apoptin mRNA was transduced into macrophages using Lipofectamine MessengerMax (Thermo Fisher Scientific), and after 6 hours of treatment, cells were lysed and each RNA was extracted using the Universal RNA Extraction Kit (Bioneer). After isolation of total RNA, cDNA synthesis was performed using Reverse Transcriptase (Enzynomics) according to the manufacturer's protocol. In the next step, PCR reactions were performed using gene-specific primers for TLR7, IFN alpha, and RIG-1, respectively. The CT (Cycle Threshold value) values ​​for each production amount after PCR were normalized to the CT value of the GAPDH endogenous control to reduce inter-sample variability.

[0103] As a result, when natural mRNA was introduced into cells, the expression efficiency of TLR 7, IFN alpha, and RIG was high, and relatively, as already reported, the expression efficiency was low in the case of mRNA introduced with pseudouridine. In addition, it was confirmed that in the case of mRNA introduced with F-CTP, a modified nucleotide according to the present invention, alone or with pseudouridine and F-CTP in a dual manner, the expression levels of TLR 7, IFN alpha, and RIG-1 were low, similar to the mRNA introduced with pseudouridine. This shows that the increase in the antigen protein expression level of mRNA introduced with F-CTP is due to the effect of reducing excessive immunity (Figs. 10 to 12).

[0104] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single entity may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined manner.

[0105] The scope of the present invention is indicated by the claims set forth below, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

[0106]

[0107] The present invention relates to a method for increasing protein expression efficiency using an mRNA structure to which a C(5)-halogenated pyrimidine ribonucleotide is bound. According to the present invention, by synthesizing various C(5)-halogenated pyrimidine (cytidine / uridine) ribonucleotides and binding single and double base modified rNTPs to cap-dependent and cap-independent mRNAs, and then confirming the transcription and translation efficiency, it was confirmed that fluorinated and chlorinated modified mRNAs had a translation efficiency 4 to 5 times higher than unmodified mRNA in the case of single base modification, and in the case of double base modification, the combination of pseudouridine and 5-fluorocytidine was confirmed to increase the translation efficiency by about 10 times or more compared to unmodified rNTPs, and therefore, it will be useful for therapeutic methods that depend on modified mRNA.

Claims

1. An mRNA structure in which a modified nucleotide is introduced in which the 5th carbon of the pyrimidine base of cytidine or uridine is substituted with a halogen.

2. In paragraph 1, An mRNA structure wherein the modified nucleotide is substituted with a nucleotide selected from the group consisting of F, Cl, Br and I.

3. In paragraph 1, The above modified nucleotide is an mRNA structure represented by a chemical formula selected from the group consisting of the following chemical formulas 1 to 4: [Chemical Formula 1] [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] 4. A composition for RNA synthesis comprising a modified nucleotide in which the 5th carbon of the pyrimidine base of cytidine or uridine is substituted with a halogen.

5. A composition for RNA synthesis, wherein the modified nucleotide in paragraph 4 is represented by a chemical formula selected from the group consisting of the following chemical formulas 1 to 4. [Chemical Formula 1] [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] 6. In paragraph 4, A composition for RNA synthesis, wherein the composition for RNA synthesis further comprises pseudouridine or methylpseudouridine.

7. Method for increasing protein expression efficiency using the mRNA structure of paragraph 1.

8. In paragraph 7, A method for increasing protein expression efficiency, wherein the mRNA structure comprises a compound selected from the group consisting of the following chemical formulas 1 to 4. [Chemical Formula 1] [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] 9. In paragraph 7, The above method is a method for increasing protein expression efficiency, wherein protein expression efficiency is increased by cap-dependent translation.

10. In paragraph 7, A method for increasing protein expression efficiency, wherein the mRNA structure further comprises pseudouridine or methylpseudouridine.

11. In paragraph 10, A method for increasing protein expression efficiency, wherein the mRNA structure comprises a compound represented by the following chemical formula 1 and pseudouridine: [Chemical Formula 1] 12. In paragraph 7, A method for increasing protein expression efficiency, wherein the above protein is an antigen protein.

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