mRNA cap analogs and their uses

Abstract

One aspect relates to a compound of formula 1, a cap analog containing the compound, an mRNA 5'-capped with the cap analog, a method for producing an mRNA using the cap analog, a use of the cap analog in producing an mRNA, and a pharmaceutical composition for expressing a desired peptide or protein, the pharmaceutical composition containing an mRNA 5'-capped with the cap analog.

Description

[Technical Field]

[0001] The present invention relates to cap analogs and methods for expressing proteins using the same, and more specifically to mRNA cap analogs, 5'-capped mRNAs containing the same, methods for producing the same, pharmaceutical compositions for expressing a desired peptide or protein containing the 5'-capped mRNA, and methods for producing a desired peptide or protein using the 5'-capped mRNA. [Background technology]

[0002] RNA therapeutics have recently emerged as a new paradigm for the treatment of infectious diseases and cancer. In particular, the development of mRNA vaccines using in vitro transcription has emerged as the most promising approach to combating RNA virus infections, overcoming the limitations of traditional vaccines such as attenuated, inactivated, and synthetic antigen vaccines. RNA virus infections are relatively highly transmissible and undergo rapid mutation due to their unstable structure, necessitating the rapid development of safe vaccines. mRNA vaccines address these infectious diseases and can shorten the vaccine development process, as the development period for mRNA vaccine candidates is approximately one month. Furthermore, once introduced into the body, mRNA is degraded within 24 hours. Unlike DNA vaccines, mRNA does not enter the nucleus but is translated into proteins by ribosomes in the cytoplasm, eliminating the risk of genetic mutations and offering excellent safety.

[0003] The following components are required for in vitro synthesis of mRNA molecules: a linearized DNA template, RNA polymerase (typically T7 RNA polymerase), four ribonucleoside triphosphates (NTPs) A, G, C, and U (or mΨ, 1-methylpseudouridine), and a cap analog. In some cases, poly(A) polymerase can be used to polyadenylate 200–250 adenosines at the 3' end. Alternatively, a capping enzyme can be used instead of a cap analog to synthesize the 5' capping structure at the 5' end. Compared to other components that are relatively well-optimized, the 5' capping structure, which has a complex structure and plays a crucial role in the protein translation of mRNA molecules, is the most expensive component during in vitro synthesis, and research into its efficient synthesis is actively underway.

[0004] After transcription, mRNA undergoes a maturation process to efficiently translate the coding protein. During this process, 7-methylguanosine ( 7m The process in which the 5'-5'-triphosphate chain is linked to the 5'-cap structure ( 7m The 5' capping structure can protect the 5' end of mRNA from biodegradation by 5' exonucleases, affecting the transport of mRNA from the nucleus to the cytoplasm. In particular, the 5' capping structure is recognized by eukaryotic translation initiation factor 4E (eIF4E) to form a translation initiation complex, thereby playing an important role in protein expression.

[0005] For such capped mRNA to be clinically applicable, it must have a structure in which the 2' hydroxyl group of the first nucleotide linked to 7-methylguanosine via a 5'-triphosphate chain is methylated (Cap1 mRNA). When the unmethylated Cap0 structure is used, MDA5 (an intracellular sensor protein) recognizes the Cap0 structure and induces an immune inflammatory response when exogenous mRNA with the Cap0 structure is introduced into the body, thereby impairing the binding of the mRNA to eIF4E and inhibiting protein expression. In contrast, exogenous Cap1 mRNA with a methylated 2' OH is not recognized by MDA5 when introduced into the body, and therefore does not induce an immune response. Therefore, it can induce relatively high protein expression, making it suitable for clinical use.

[0006] Commercialized mRNA vaccines based on the aforementioned Cap1 mRNA structure include Moderna's mRNA-1273 and Pfizer / BioNTech's BNT162b2. Both mRNA vaccines were developed to combat the infectious disease caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), an RNA virus that has caused a global pandemic. To reduce production costs, the BNT162b2 mRNA vaccine employs a co-transcriptional capping method, which involves simultaneously introducing a chemically synthesized cap analog during in vitro mRNA synthesis, resulting in the cap analog forming the 5' end of the mRNA, rather than the conventional capping method that uses a capping enzyme.

[0007] The cap analog used in BNT162b2 is a third generation cap analog, Cleancap® AG (3'OMe), developed by Trilink (US Patent 10,913,768). 7m G (3’OMe) pppA (2’OMe)This trimeric cap analog has a pG structure and can be incorporated during mRNA synthesis to synthesize the Cap1 mRNA structure without the use of enzymes. Because the trimeric cap analog contains one or more bases compared to GTP, it exhibits stronger hydrogen bonding strength during base pairing with the DNA template strand for transcription initiation. Therefore, unlike the previous dimeric second-generation cap analogs (U.S. Patent 7,074,596), which competitively base pair with guanosine triphosphate (GTP) on the DNA template, the trimeric cap analog preferentially base pairs with GTP, allowing for reduced cap analog input during in vitro mRNA synthesis and, as a result, reduced mRNA impurities with pppG 5' ends.

[0008] BNT162b2, which incorporates a trimer cap analog, faced a temporary shortage of raw material due to overwhelming demand for the global COVID-19 pandemic. Furthermore, the cap analog remains the most expensive component in mass-producing mRNA vaccines. Rapid and cost-effective production and supply of cap analogs is essential for the production of mRNA vaccines to effectively combat highly contagious RNA virus infections.

[0009] The currently commercially available manufacturing process for trimer cap analogs can be broadly divided into six steps as shown below (US Patent 10,913,768).

[0010] Reaction Scheme 1 [ka]

[0011] In the reaction formula 1, G represents guanosine, 7mG means 7-methylguanosine, N means any one of adenosine, cytidine, guanosine, and uridine, p means -P(=O)O2-, Me means methyl, and Im means imidazolide. Furthermore, when viewed in detail, pN, which is a reactant used in the final manufacturing process, (2’OMe) If the step of synthesizing pN is included, a long process of at least 10 steps must be carried out.

[0012] In particular, when Trilink's cap analogs are mass-produced, the main factor that increases production costs and synthesis time in the entire manufacturing process is the purification step using ion exchange chromatography (mainly DEAE resin) that must be performed between manufacturing steps. After purifying the intermediates at each manufacturing step, a large amount of buffer solution (mainly triethylammonium bicarbonate (TEAB) buffer) used in column purification must be distilled in order to proceed to the next step in organic solvent. In the entire manufacturing process, pG (3’OMe) , ppG (3’OMe) ,pp 7m G (3’Ome) , pN (2’OMe) pN, 7m G (3’OMe) pppN (2’OMe) Since pN must be purified at each stage of production, a total of five ion exchange column purifications are required. Such lengthy process, including column purification and buffer distillation, increases the time and cost required for the production of cap analogs, making them less economical. Summary of the Invention [Problem to be solved by the invention]

[0013] One aspect is to provide a cap analog that can increase the efficiency of in vitro synthesis of 5'-capped mRNA molecules, not only increasing the efficiency of protein expression from capped mRNA, but also enabling economical production by reducing the number of synthesis and purification steps of the cap analog itself.

[0014] Another embodiment is to provide a 5'-capped mRNA as the cap analog.

[0015] Yet another aspect is to provide a method for producing mRNA using the cap analog.

[0016] Yet another aspect is to provide a composition or kit for producing 5'-capped mRNA, comprising the cap analog.

[0017] Yet another aspect is to provide a pharmaceutical composition for expressing a peptide or protein of interest, comprising said cap analog.

[0018] Yet another embodiment is to provide a cell containing a 5'-capped mRNA as the cap analog.

[0019] Yet another embodiment is to provide a cell comprising, as said cap analog, a protein or peptide translated from a 5'-capped mRNA. [Means for solving the problem]

[0020] One embodiment provides a compound of Formula 1, or a pharmaceutically acceptable salt thereof: [ka]

[0021] where n=0, 1 or 2; Y1, Y2, and Y3 are each independently O or S; R1 is C 1~6 alkyl or CH2Ph, R2 and R3 are each independently H or a sulfonyl-containing group, provided that at least one of R2 and R3 is a sulfonyl-containing group, said sulfonyl-containing group being independently selected from the group consisting of mesyl, esyl, triflyl, tresyl, tosyl, brosyl, nosyl, and dansyl; R4 and R5 are each independently OH or methoxy; R6 is OH or a mononucleotide or oligonucleotide having 1 to 7 bases, Z and Z' are each independently a natural, modified or unnatural nucleoside base.

[0022] Another embodiment provides a cap analog, which is a compound of Formula 1.

[0023] Yet another embodiment provides a 5'-capped mRNA as the cap analog.

[0024] Yet another aspect provides a method for producing a 5'-capped mRNA, comprising including the cap analog during mRNA synthesis.

[0025] Yet another aspect provides a composition or kit for producing 5'-capped mRNA, comprising the cap analog.

[0026] Yet another aspect provides a pharmaceutical composition for expressing a desired peptide or protein, comprising a 5'-capped mRNA as the cap analog and a pharmaceutically acceptable carrier.

[0027] Yet another embodiment provides a cell comprising a 5'-capped mRNA as the cap analog.

[0028] Yet another embodiment provides a cell comprising, as the cap analog, a protein or peptide translated from a 5'-capped mRNA. [Effects of the Invention]

[0029] In one embodiment, cap analogs, such as TriLink's trimer cap analogs, can be used during mRNA synthesis to increase the efficiency of in vitro synthesis of 5'-capped mRNA molecules and the efficiency of peptide or protein expression from the capped mRNA. In particular, they can further increase initial protein expression after transfection in a cap-dependent translation manner, making them particularly useful for expressing genes where initial protein expression is important (e.g., the expression of the nsp1 portion of self-amplifying RNAs). Furthermore, the cap analogs can be synthesized economically in terms of time and cost by reducing the number of synthesis steps and purification steps. This, along with the increased efficiency of peptide or protein expression using the capped mRNA, can contribute to the overall economical synthesis of 5'-capped mRNA.

[0030] Therefore, similar to currently commercially available trimer cap analogs, this cap analog increases the efficiency of in vitro synthesis of 5'-capped mRNA molecules, significantly increases the peptide or protein expression efficiency of capped mRNA compared to conventional trimer cap analogs, and can be produced more economically.

[0031] As such, the cap analogs have excellent advantages in terms of efficacy and production costs, and mRNAs containing the cap analogs disclosed herein can be very useful in treating or preventing diseases in mammals, including humans. [Brief explanation of the drawings]

[0032] [Figure 1]This is a graph showing the relative in vitro transcription (IVT) yield (%) of compound (8) of Example 8 compared to 7mG(3'OMe)pppA(2'OMe)pG after measuring the in vitro transcription (IVT) yield of capped luciferase mRNA using compound (8) of Example 8 according to one embodiment of the present invention or 7mG(3'OMe)pppA(2'OMe)pG as a cap analog. [Figure 2] This graph shows the results of quantifying the expression level of translated luciferase protein after transfection of HEK293T cells with luciferase mRNA produced by in vitro transcription (IVT) using compound (8) of Example 8 or 7mG(3'OMe)pppA(2'OMe)pG as a cap analog (mean ± standard deviation (n=3), ***: P<0.05, NS: not significant). [Figure 3] 1 is a graph showing the results of calculating the AUC (area under the curve) value of the total luciferase protein expression level from 1 hour to 48 hours after transfection with compound (8) of Example 8 or luciferase mRNA capped with 7mG(3'OMe)pppA(2'OMe)pG according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention will be described in further detail below.

[0034] Unless otherwise defined, all technical terms used herein are used in the sense commonly understood by those of ordinary skill in the art in the field related to the present invention. Furthermore, while preferred methods and samples are described herein, similar or equivalent methods and samples are also included within the scope of the present invention. Furthermore, numerical values described herein are considered to include the meaning of "about" even if not explicitly stated. The contents of all publications cited as references in this specification are incorporated herein in their entirety by reference.

[0035] Definition of Terms As used herein, the following terms have the meanings defined below unless otherwise specified.

[0036] As used herein, the term "complementary" or "complementarity," in the context of a complex between a cap analog and a DNA template, refers to standard Watson / Crick base-pairing rules. For example, the sequence "5'-AGTC-3'" is complementary to the sequence "3'-TCAG-5'." The complementarity need not be perfect; the duplex may contain mismatched base pairs, degenerate nucleotides, or unmatched nucleotides. One of ordinary skill in the art can experimentally determine duplex stability by considering numerous variables, including, for example, the length of the oligonucleotide, the base composition of the oligonucleotide and its sequence, the occurrence of mismatched base pairs, ionic strength, hybridization buffer components, and reaction conditions.

[0037] Complementarity can be "complete" or "total" when all nucleotide bases of the two nucleic acid strands are matched by recognized base-pairing rules, "partial" when only a portion of the nucleotide bases of the cap analog and the DNA target are matched by recognized base-pairing rules, or "absent" when none of the nucleotide bases of the two nucleic acid strands are matched by recognized base-pairing rules.

[0038] The term "nucleoside" as used herein includes all naturally occurring nucleosides. The base rings most frequently found in naturally occurring nucleosides are purine and pyrimidine rings. Naturally occurring purine rings include, for example, adenine, guanine, and N6-methyladenine. Naturally occurring pyrimidine rings include, for example, cytosine, thymine, 5-methylcytosine, and uracil. Naturally occurring nucleosides include, but are not limited to, ribo-, 2'-O-methyl-, or 2'-deoxyribo-derivatives of, for example, adenosine, guanosine, cytidine, thymidine, uridine, inosine, 7-methylguanosine, or uridine.

[0039] The terms "nucleoside analog," "modified nucleoside," or "nucleoside derivative" include synthetic nucleosides as described herein. Nucleoside derivatives also include nucleosides with modified base and / or sugar moieties, with or without protecting groups, such as 2'-deoxy-2'-fluorouridine and 5-fluorouridine. The compounds and methods provided herein encompass not only the above base rings and their synthetic analogs, but also non-natural heterocyclic substituted base sugars and acyclic substituted base sugars. Other nucleoside derivatives that can be utilized in the present invention include, for example, LNA nucleosides, halogen-substituted purines (e.g., 6-fluoropurine), halogen-substituted pyrimidines, N6-ethyladenine, N4-(alkyl)-cytosine, 5-ethylcytosine, and the like (U.S. Patent No. 6,762,298).

[0040] The term "modified nucleoside base" refers to a base contained in the "modified nucleoside," such as a halogen-substituted purine (e.g., 6-fluoropurine), a halogen-substituted pyrimidine, N6-ethyladenine, N4-(alkyl)-cytosine, 5-ethylcytosine, and the like.

[0041] The term “C 1―n"Alkyl" means a linear or branched saturated hydrocarbon radical chain of 1 to n carbons. Specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, isopentyl, and n-hexyl.

[0042] In the process of synthesizing a substance, the term "in situ method" means that the obtained product is subjected to the next step of reaction in the original vessel without any additional purification process.

[0043] One embodiment relates to a compound of Formula 1, or a pharmaceutically acceptable salt thereof: chemical formula 1 [ka]

[0044] where n=0, 1 or 2; Y1, Y2, and Y3 are each independently O or S; R1 is C 1~6 alkyl or CH2Ph, R2 and R3 are each independently H or a sulfonyl-containing group, provided that at least one of R2 and R3 is a sulfonyl-containing group, said sulfonyl-containing group being independently selected from the group consisting of mesyl, esyl, triflyl, tresyl, tosyl, brosyl, nosyl, and dansyl; R4 and R5 are each independently OH or methoxy; R6 is OH or a mononucleotide or oligonucleotide having 1 to 7 bases, Z and Z' are each independently a natural, modified or unnatural nucleoside base.

[0045] The pharmaceutically acceptable salt refers to a salt commonly used in the pharmaceutical field, specifically, a base addition salt.The salts include, for example, monovalent metal salts, divalent metal salts, amine salts, or amino acid salts.The monovalent metal salts include Na salts, Li salts, or K salts, the divalent metal salts include Ca salts, Zn salts, or Mg salts, the amine salts include trimethylamine salts, triethylamine salts, ammonium salts, pyridine salts, or picoline salts, and the amino acid salts include, but are not limited to, arginine salts, lysine salts, or histidine salts.

[0046] The pharmaceutically acceptable salt may be in the form of a salt that allows the compound of Chemical Formula 1 to be in a stable, electrically neutral form in aqueous liquid.

[0047] The compound of formula 1, or a pharmaceutically acceptable salt thereof, may be, for example, a compound of formula 1a: chemical formula 1a [ka]

[0048] where: n=0, 1 or 2; X1, X2, X3, and X4 are each independently absent, a monovalent metal ion, a divalent metal ion, or a combination thereof, wherein X1, X2, X3, and X4 are selected such that the compound of Formula 1 is electroneutral; Y1, Y2, and Y3 are each independently O or S; R1 is C 1~6 alkyl or CH2Ph, R2 and R3 are each independently H or a sulfonyl-containing group, provided that at least one of R2 and R3 is a sulfonyl-containing group, said sulfonyl-containing group being independently selected from the group consisting of mesyl, esyl, triflyl, tresyl, tosyl, brosyl, nosyl, and dansyl; R4 and R5 are each independently OH or methoxy; R6 is OH or a mononucleotide or oligonucleotide having 1 to 7 bases, Z and Z' are each independently a natural, modified or unnatural nucleoside base.

[0049] In one embodiment, the monovalent metal ion is Na + , Li + and K. + and the divalent metal ion is selected from the group consisting of Mg 2+ , Zn 2+ and Ca 2+ The compound is selected from the group consisting of:

[0050] In one embodiment, the sulfonyl-containing group is a mesyl, esyl, or tosyl group.

[0051] In one embodiment, the C 1―6 Alkyl is methyl, ethyl, propyl, isopropyl or t-butyl.

[0052] In one embodiment, in Formula 1, Z and Z' are each independently a natural purine or pyrimidine base moiety, or a modified form thereof. More specifically, Z and Z' are each independently selected from the group consisting of guanine, adenine, cytosine, thymine, uracil, and modified forms thereof. In one embodiment, Z and Z' are adenine and guanine, respectively.

[0053] In one embodiment, in Formula 1, R6 is OH, a mononucleotide, or an oligonucleotide having 2 to 5 bases.

[0054] In one embodiment, the compound having the structure of Formula 1a is In chemical formula 1a, n=0, 1 or 2; X1, X2, X3, and X4 are each independently absent or Na; Y1, Y2, and Y3 are each independently O or S; R1 is C 1~6 is alkyl, R2 and R3 are each independently H or a sulfonyl-containing group, with the proviso that at least one of R2 and R3 is a sulfonyl-containing group, said sulfonyl-containing groups being each independently selected from the group consisting of mesyl, esyl, and tosyl; R4 and R5 are each independently OH or methoxy; R6 is OH or a mononucleotide or oligonucleotide having 1 to 3 bases, Z is a compound that is a natural, modified or unnatural nucleoside base.

[0055] In one embodiment, R4 is methoxy and R5 is OH.

[0056] In one embodiment, the compound of Formula 1 is a compound selected from the group consisting of the compounds listed in Table 1 below: [Table 1]

[0057] In the abbreviated compound names, the G means guanosine, 7m G means 7-methylguanosine, A means adenosine, p means -P(=O)(OH)O-, Es means esyl, Ms means mesyl, Ts means tosyl, and Me means methyl.

[0058] The compound of Chemical Formula 1 can be prepared, for example, by the methods illustrated in the Examples. Those skilled in the art can prepare the compound of Chemical Formula 1 by appropriately modifying the methods illustrated in the Examples below, and by changing the reaction conditions, reaction order, and reactants.

[0059] A specific example of the compound of Formula 1 can be prepared by the method shown in Reaction Scheme 2a below. Reaction Scheme 2a [ka]

[0060] Specific details of exemplary preparation methods of the above reaction schemes are described in the following Examples.

[0061] The above reaction scheme 2a can be briefly represented by the following abbreviations: [ka]

[0062] wherein G means guanosine, 7m G means 7-methylguanosine, A means adenosine, p means -P(=O)(OH)O-, TBS means tertiary butyldimethylsilyl, Me means methyl, Im means imidazolide, and Ms means mesyl.

[0063] The above preparation method can produce the compound of Formula 1 through four steps. Therefore, the compound of Formula 1 can be produced more economically with fewer steps than conventional known methods for producing cap analogs.

[0064] The compound of Formula 1 according to one embodiment can be used as a cap analog, and thus, 5'-capped mRNA can be prepared using the cap analog, which is the compound of Formula 1.

[0065] Accordingly, one particular embodiment provides a cap analog that is a compound of Formula 1 above.

[0066] In addition, one particular embodiment provides a method for producing 5'-capped mRNA, which comprises including a cap analog, which is a compound of Formula 1, during mRNA synthesis.

[0067] In addition, one particular embodiment provides a composition or kit for producing 5'-capped mRNA, which comprises a cap analog that is the compound of Chemical Formula 1.

[0068] In addition, one particular embodiment provides a use of the cap analog, which is the compound of Chemical Formula 1, for synthesizing mRNA.

[0069] In addition, one particular embodiment provides a 5'-capped mRNA using the compound of Formula 1 as a cap analog.

[0070] The method for producing 5'-capped mRNA using the cap analog of Formula 1 can be carried out by any method known in the art. In one embodiment, 5'-capped mRNA can be produced by a co-transcriptional capping method, which is an mRNA synthesis method in which a chemically synthesized cap analog is simultaneously introduced during in vitro mRNA synthesis, and the cap analog constitutes the 5' end. Specifically, the method can include introducing the cap analog of Formula 1 into a mixture containing RNA polymerase under conditions that allow transcription of a polynucleotide template by the RNA polymerase, and incubating the mixture for a time sufficient to allow transcription of the template.

[0071] The cap analog of Chemical Formula 1 according to one embodiment can increase the transcription efficiency of mRNA in vitro compared to the efficiency of initiation using standard GTP, ATP, CTP, or UTP, and subsequently increase the protein expression efficiency of the capped mRNA transcribed during the translation process. The increased transcription efficiency can increase mRNA synthesis by, for example, about 10%, about 20%, about 40%, about 60%, about 80%, about 90%, about 100%, about 150%, about 200%, or about 500% compared to mRNA synthesis using conventional methods.

[0072] The cap analogue, which is the compound of Formula 1 according to the above embodiment, is a conventional trimeric cap analogue ( 7m G (3’OMe) pppA (2’OMe) pG), the protein expression level of the 5'-capped mRNA molecule can be significantly increased. More specifically, in the compound of Formula 1, the 3'-OH residue of the mRNA molecule is monosulfonylated with a cap analog, and the mRNA molecule is 5'-capped with a conventional Trilink trinucleotide 3'-methoxy cap analog ( 7m G (3’OMe) pppA (2’OMe) After transfection, mRNA molecules 5'-capped with pG can significantly increase early protein expression, thereby significantly increasing the overall protein expression level (see Experimental Examples).

[0073] The cap analogue, which is the compound of Formula 1 according to one embodiment, has the advantage that it can be synthesized economically in terms of time and cost by reducing the number of synthesis steps and column purification steps compared to the conventional Trilink trimer cap analogue.

[0074] Specifically, when preparing the compound of Formula 1, an optimized starting material (e.g., G (2’TBS) By selecting the starting material (G), the conventional two-step process of converting guanosine to guanosine monophosphate and then to diphosphate can be shortened to a one-step process of immediately converting guanosine to diphosphate, and the next step is 7-methylation of guanosine in situ, thereby shortening the crystallization / purification process. (2’TBS) ) is mesylated at the 3'OH and 5'OH, and then 7-methylguanosine diphosphate (pp 7m 7-methylguanosine diphosphate (pp 7m The synthesis steps up to G) can be shortened by two steps compared to the conventional Trilink trimer cap analogue production method (the method shown in Reaction Scheme 1 below). 7mG (3’OMe) pppN (2’OMe) The method for producing pN requires a total of five ion exchange column purifications in the following reaction scheme 1. (3’OMe) , ppG (3’OMe) ,pp 7m G (3’Ome) , pN (2’OMe) pN, 7m G (3’OMe) pppN (2’OMe) pN) (see US Pat. No. 10,913,768), the cap analog containing the compound of Formula 1 can be prepared by the following reaction scheme 2 (purification target: pp 7m G (3’OR) , pN (2’OMe) pN, 7m G (3’OR) pppN (2’OMe) pN), and can be prepared through a total of three ion exchange column purifications. Therefore, the capped analog of the compound of Formula 1 according to one embodiment can be prepared using a significantly fewer number of purification steps and a shorter synthesis process than conventional capped analogs.

[0075] Reaction Scheme 1 [ka]

[0076] Reaction Scheme 2 [ka]

[0077] In the reaction formulas 1 and 2, G represents guanosine; 7m G represents 7-methylguanosine, N represents any one of adenosine, cytidine, guanosine, and uridine, p represents -P(=O)O2-, TBS represents tertiary butyldimethylsilyl, Me represents methyl, Im represents imidazolide, and R represents a substituent selected from the group consisting of tosyl, brosyl, nosyl, esyl, mesyl, triflyl, tresyl, and dansyl.

[0078] Therefore, the capped analog of the compound of Formula 1 according to the above embodiment has the advantage of further reducing the manufacturing time and cost of the capped analog itself.

[0079] In one embodiment, a method for producing 5'-capped mRNA can include adding at least one modified NTP to a transcription reaction. The modification of the at least one modified NTP does not substantially impair RNA polymerase-mediated synthesis of mRNA. The modified NTP can include, for example, one or more modified nucleoside bases, one or more modified sugars, or one or more modified 5'-triphosphates. Such modified NTPs are incorporated onto the 3' end of cap analogs, which do not block transcription and support additional primer extension. The modified group of the modified NTP can be a detectable label or a detectable marker. Thus, after transcription, the produced mRNA containing a detectable label or marker can be identified by size, mass, color, and / or affinity capture. In one embodiment, the detectable label or marker is a fluorescent dye, and the affinity capture label is biotin.

[0080] In one embodiment, one or more components of the transcription reaction (e.g., cap analog and / or NTP) can be labeled with a detectable label or marker. Thus, after transcription, mRNA molecules can be identified by, for example, size, mass, affinity capture, or color. For example, the detectable label is a fluorescent dye and the affinity capture label is biotin.

[0081] The kit or composition for producing 5'-capped mRNA may contain all transcription reagents for normal mRNA synthesis (e.g., FLuc mRNA). More specifically, the kit may contain one or more reagents selected from the group consisting of a cap analog, a container labeled for transcription, a manual for mRNA synthesis, one or more unmodified NTPs, one or more modified NTPs (e.g., methylpseudouridine-5'-triphosphate), RNA polymerase, other enzymes, a reaction buffer, magnesium, and a DNA template.

[0082] 5'-capped mRNA produced using a cap analog according to one embodiment contains the cap analog within its structure, and can be administered to a living organism and used to express a protein in the organism.

[0083] Therefore, yet another aspect provides a method for expressing a desired peptide or protein in a living body, comprising administering to the living body an mRNA containing the cap analog.

[0084] Another aspect provides a pharmaceutical composition for expressing a peptide or protein of interest, comprising mRNA containing the cap analog and a pharmaceutically acceptable carrier.

[0085] In the present application, the term "target peptide or protein" refers to any protein that needs to be expressed in a living body, and in the present application, the mRNA capable of expressing the target peptide or protein is a cap analog according to one embodiment, is 5'-capped, and is capable of expressing the target protein in a living body.

[0086] Depending on the type of the target peptide or protein, the target disease can be treated or prevented in vivo. Therefore, it can be used to treat or prevent any disease that can be treated or prevented by expression of a peptide or protein. Diseases that can be treated or prevented by expression of specific types of peptides or proteins are known, and the pharmaceutical composition can be used to induce expression of the peptide or protein and used to prevent or treat the target disease.

[0087] Another aspect provides a pharmaceutical use of mRNA containing the cap analog for the prevention or treatment of any disease that can be effectively treated by in vivo expression of a peptide or protein.

[0088] The pharmaceutical composition and the treatment or prevention method may be used for gene replacement therapy, genome editing, cancer immunotherapy, or vaccine-based treatment or prevention, etc. In one embodiment, the pharmaceutical composition is an mRNA vaccine.

[0089] The pharmaceutical compositions may be formulated for administration by injection or other suitable route known to those of ordinary skill in the art for treating or preventing a particular condition. Injectable compositions include a pharmaceutically acceptable carrier, such as sterile saline. Injectable compositions may also be formulated as suspensions in lipids or phospholipids, liposomal suspensions, or aqueous emulsions. Methods for formulating the pharmaceutical compositions are well known to those of ordinary skill in the art.

[0090] In one embodiment, the pharmaceutical composition may contain the mRNA containing the cap analog as an active ingredient at a concentration of about 0.01% to 1%, which may vary depending on the frequency, dosage, and method of administration.

[0091] In one embodiment, the pharmaceutical composition can be administered to a mammal, specifically a human, and the dosage will vary depending on the individual's health condition, severity of the disease, weight, age, race, etc., and an appropriate dosage can be determined by a person skilled in the art. In one embodiment, the dosage for a human is in the range of 0.0001 to 100 mg / day, more specifically, in the range of about 0.1 to 50 mg / day.

[0092] The mRNA containing the cap analog according to the above embodiment can be introduced into cells in vivo or in vitro to express proteins or peptides.

[0093] Accordingly, another aspect provides a cell comprising an mRNA comprising a cap analog according to the previous aspect.

[0094] Yet another embodiment provides a cell comprising a protein or peptide translated from an mRNA comprising a cap analog according to the previous embodiment.

[0095] Methods for expressing proteins and peptides in cells in vivo or in vitro using the mRNA are known in the art, and proteins or peptides can be appropriately expressed in cells using such conventional general methods. [Example]

[0096] The following examples are presented to aid in understanding the present invention. The following examples are merely illustrative of the present invention, and it will be apparent to those skilled in the art that various changes and modifications may be made within the scope and spirit of the present invention. It goes without saying that such changes and modifications are also within the scope of the appended claims.

[0097] Explanation of Abbreviations The abbreviations used below are as follows: TBDMS: tertiary butyldimethylsilyl MsCl: methanesulfonyl chloride TEA.3HF: Triethylamine trihydrofluoride THF: tetrahydrofuran ACN: acetonitrile DMS: dimethyl sulfate PW: Pure water DPS: 2,2'-dipyridyl disulfide TEA: Triethylamine PPh3: Triphenylphosphine DMF: dimethylformamide DMTr: 4,4'-dimethoxytrityl DTT: Dithiothreitol

[0098] IVT: in vitro transcription Examples 1 to 3: 7-Methyl-3'-mesyloxy-guanosine 5'-diphosphate imidazolide (Im-pp 7m G (3’OMesyloxy) )(3) Manufacturing [ka]

[0099] Example 1: Preparation of 2'-O-tertiary butyldimethylsilyl-3'mesyloxy-5'mesyloxyguanosine (1) 2'-O-tertiary-butyldimethylsilylguanosine was obtained commercially and used as the starting material without further treatment. 3.97 g (9.99 mmol) of 2'-O-tertiary-butyldimethylsilylguanosine was added to 91 mL of anhydrous pyridine and stirred for 10 minutes at 0°C while 2.32 mL (29.97 mmol) of methanesulfonyl chloride was gradually added. The reaction mixture was then stirred at 10-20°C for 3 hours. 270 mL of distilled water was gradually added at 0°C and stirred at 5-10°C for 1 hour. The resulting crystals were filtered, washed with distilled water, and vacuum dried to obtain 4.36 g (80% yield) of the target compound (1).

[0100] 1H NMR (400 MHz, DMSO-d6, 25℃): δ= 10.72 (s, 0.84H), 7.96 (s, 1H), 6.56 (1.65H), 5.80 (d, 1H), 5.15 (d, 1H), 4.96 (m, 1H), 4.56 ~ 4.62 (m, 3H, overlapped), 3.32 (m, 3H), 3.25 (m, 3H), 0.699 (m, 9H), -0.014 (m, 3H), -0.231 (m, 3H)

[0101] Example 2: Preparation of 7-methyl-3'-mesyloxyguanosine 5'-diphosphate triethylammonium salt (2) 4.2 g (7.59 mmol) of 2'-O-tertiary-butyldimethylsilyl-3'mesyloxy-5'mesyloxyguanosine (1) was dissolved in 33.6 mL of anhydrous acetonitrile, and 17.81 g (19.73 mmol) of tris(tetrabutylammonium) hydrogen pyrophosphate was added. The mixture was stirred at room temperature for 48 hours. Then, 3.71 mL (22.77 mmol) of triethylamine trihydrofluoride was gradually added at room temperature while stirring for 1 hour. The reaction solution was washed with 42 mL of dichloromethane and 42 mL of purified water, and the aqueous layer was separated. The separated aqueous layer was washed with 42 mL of dichloromethane and further separated. The pH of the separated aqueous layer was adjusted to 4.0 using glacial acetic acid. 28.79 mL (303.6 mmol) of dimethyl sulfate was gradually added to the reaction solution over 30 minutes, and the mixture was stirred at room temperature for 3 hours. The pH of the reaction mixture was maintained at 4.0±0.5 using 1 M sodium hydroxide. The reaction mixture was extracted three times with 126 mL of dichloromethane to remove unreacted dimethyl sulfate, and the aqueous layer was separated. The separated aqueous layer was titrated to pH 5.5 using 1 M sodium hydroxide, and the target compound was separated using a DEAE Sepharose column (140 x 210 mm) and lyophilized to obtain 1.68 g (30.0% yield) of the triethylammonium salt (2) of the target compound.

[0102] 1H NMR (400 MHz, D2O, 25℃): δ= 6.11 (d, 1H), 5.44 (dd, 1H), 5.02 (m, 1H), 4.74 (m, D2O overlapped, 1H), 4.23 (m, 2H), 4.1 (s, 3H), 3.32 (s, 3H), 3.19 ~ 3.13 (m, 12H), 1.28 ~ 1.18 (m, 18H) 31 P NMR (162 MHz, D2O, 25℃): δ= -7.13 (d, 1P), -10.77 (d, 1P)

[0103] Example 3: Preparation of 7-methyl-3'-mesyloxyguanosine 5'-diphosphate imidazolide (3) 1.0 g (1.36 mmol) of 7-methyl-3'-mesyloxyguanosine 5'-diphosphate triethylammonium salt (2) was dissolved in 40 mL of dimethylformamide, and 0.93 g (13.6 mmol) of imidazole and 1.50 g (6.8 mmol) of 2,2'-dipyridyl disulfide were added. 0.189 mL (1.36 mmol) of triethylamine and 1.78 g (6.8 mmol) of triphenylphosphine were added to the reaction mixture and stirred at room temperature for 2 hours. The reaction mixture was then added to a solution of 0.333 g (2.72 mmol) of sodium perchlorate in 320 mL of acetone. The mixture was cooled to 4°C, and the resulting crystals were filtered, washed with cold acetone, and dried under vacuum to obtain 0.83 g (100.0% yield) of the sodium salt of the target compound (3).

[0104] 1 H NMR (400 MHz, D2O, 25℃): δ= 7.999 (s, 1H), 7.32 (s, 1H), 7.07 (s, 1H), 6.06 (d, 1H), 5.29 (m, 1H), 4.90 (m, 1H), 4.67 (m, 1H), 4.21 ~ 4.08 (m, 2H), 4.05 (s, 3H), 3.29 (s, 3H) 31 P NMR (162 MHz, D2O, 25℃): δ= -11.41 (d, 1P), -19.61 (d, 1P)

[0105] Examples 4 to 7: Preparation of pA(2'OMe)pG(7) [ka]

[0106] Example 4: Preparation of N2-isobutyryl-2',3'-diacetoxy-guanosine (4) 2.4 g (3.66 mmol) of N2-isobutyryl-5'-O-DMT guanosine was dissolved in 12 ml of dichloromethane, followed by the addition of 1.47 ml (18.3 mmol) of pyridine and 1.78 ml (18.3 mmol) of acetic anhydride. The mixture was stirred at room temperature for 5 hours. 24 ml of ethyl acetate was added to the reaction mixture for extraction, followed by washing with 14.4 ml of saturated aqueous sodium bicarbonate, 14.4 ml of 20% aqueous citric acid, and 14.4 ml of distilled water. The organic layer was dried over sodium sulfate, and the residue obtained after vacuum distillation was dried under nitrogen for 1 day. 36 ml of 3% aqueous trichloroacetic acid was added to the dried mixture, and the mixture was reacted at room temperature for 3 hours. 24 ml of methanol was added, and the mixture was stirred for an additional 2 hours and 30 minutes. The reaction mixture was vacuum distilled, and 36 ml of dichloromethane and 18 ml of distilled water were added. The mixture was then neutralized by the addition of saturated aqueous sodium bicarbonate. The organic layer was separated, dried over sodium sulfate, and then vacuum distilled. The residue after vacuum distillation was dissolved in 12 ml of ethyl acetate and then slowly added to 108 ml of hexane at room temperature to crystallize. The resulting crystals were filtered, washed three times with 12 ml of hexane, and then dried in vacuo to obtain 1.43 g of the target compound (4) (yield 89.6%).

[0107] LC-MS (ESI, m / z) = 438.16 [M + H + ]

[0108] Example 5: Preparation of (N2-isobutyryl-2',3'-diacetoxy-guanosinyl)-N6-benzoyl-2'-methoxy-adenosinylcyanoethyl phosphate (5) 5'-O-DMT-N6-benzoyl-2'-methoxy-adenosine amidite was obtained commercially and used as the starting material without further treatment. 1.43 g (3.28 mmol) of N2-isobutyryl-2',3'-diacetoxy-guanosine (4) and 3.78 g (4.26 mmol) of 5'-O-DMT-N6-benzoyl-2'-methoxy-adenosine amidite were dissolved in 28.4 mL of 1H-tetrazole (0.45 M acetonitrile solution (12.79 mmol)) and stirred at room temperature for 1 hour. 544 mg (2.15 mmol) of iodine was dissolved in 56.7 mL of a tetrahydrofuran:distilled water:pyridine mixture (v:v:v, 7:2:1), and the solution was added to the reaction mixture and stirred for 45 minutes. To the reaction mixture, 7.2 ml of 10% aqueous sodium thiosulfate solution was added, followed by 43 ml of dichloromethane and 14.3 ml of distilled water, followed by separation of the organic layer. The separated organic layer was dried over sodium sulfate, distilled under reduced pressure, and then dried under nitrogen for one day. 21.5 ml of 3% aqueous trichloroacetic acid solution was added to the dried mixture and reacted at room temperature for 3 hours. 14.3 ml of methanol was added and stirred for an additional 2 hours and 30 minutes. The reaction mixture was distilled under reduced pressure, and 21.5 ml of dichloromethane and 10.7 ml of distilled water were added, followed by neutralization with saturated aqueous sodium bicarbonate solution. The separated organic layer was washed with distilled water, dried over sodium sulfate, and distilled under reduced pressure. The residue obtained after vacuum distillation was dissolved in 28.6 ml of dichloromethane and slowly added to 86 ml of methyl tertiary butyl ether at room temperature to crystallize. The resulting crystals were filtered and dried under vacuum for one day, yielding 2.7 g of the target compound (5) (yield 87.8%).

[0109] LC-MS (ESI, m / z) = 938.28 [M + H + ]

[0110] Example 6: p(OCE)2A bz (2'OMe)p(OCE)G ib Preparation of (2',3'OAc) (6) 2 g (2.13 mmol) of (N2-isobutyryl-2',3'-diacetoxy-guanosinyl)-N6-benzoyl-2'-methoxy-adenosinylcyanoethyl phosphate (5) and 1.11 mL (4.26 mmol) of bis(2-cyanoethyl)-N,N-diisopropylphosphoramidite were dissolved in 9.47 mL of 1H-tetrazole (0.45 M acetonitrile solution (4.26 mmol)) and stirred at room temperature for 30 minutes. 811 mg (3.2 mmol) of iodine was dissolved in 30 mL of a 7:2:1 mixture of tetrahydrofuran (THH), distilled water, and pyridine (v:v:v). The solution was then added to the reaction mixture and stirred for 30 minutes. 20 mL of 10% aqueous sodium thiosulfate was added to the reaction mixture, followed by the addition of 100 mL of dichloromethane and 40 mL of distilled water. The organic layer was then separated. The separated organic layer was dried over sodium sulfate and distilled under reduced pressure. 27 ml of dichloromethane was added to the concentrated solution, which was then dissolved and slowly added to 133 ml of methyl tertiary butyl ether at room temperature to crystallize. The resulting crystals were filtered and dried under vacuum for one day to obtain 2.2 g of the target compound (6) (yield 92.2%).

[0111] LC-MS (ESI, m / z) = 1124.30 [M + H + ]

[0112] Example 7: Preparation of pA(2'OMe)pG(7) p(OCE)2A bz (2'OMe)p(OCE)G ib 2 g (1.78 mmol) of (2',3'OAc) (6) was added to a mixture of 44 mL of methanol and 44 mL of concentrated ammonia and stirred at 50-55°C for 24 hours. Upon completion of the reaction, the solvent was distilled under reduced pressure, and 20 mL of methanol was added to the concentrate, which was then distilled three times. The concentrate was dissolved in distilled water again and purified using a DEAE Sepharose column and reverse-phase chromatography to obtain 1.13 g (70% yield) of the triethylammonium salt of the target compound (7).

[0113] 1H NMR (400 MHz, D2O, 25℃): δ= 8.44 (s, 1H), 8.12 (s, 1H), 7.90 (s, 1H), 6.07 (m, 1H), 5.80 (m, 1H), 4.46 ~ 4.01 (m, 8H), 3.45 (m, 3H) LC-MS (ESI, m / z) = 707.13 [M + H+]

[0114] Example 8: 7m G (3’OMs) pppA (2’OMe) Preparation of pG(8) [ka]

[0115] 0.55 g of magnesium chloride was added to 27.5 mL of dimethylformamide and dissolved. 7m 0.6 g (0.99 mmol) of G(3'OMs)(3) and 0.5 g (0.55 mmol) of pA(2'OMe)pG(7) were added and stirred at room temperature for 24 hours. After completion of the reaction, 275 mL of 25 mM ethylenediaminetetraacetic acid solution was added dropwise to terminate the reaction. The mixture was cooled to room temperature and neutralized with 1 M aqueous sodium bicarbonate solution. The reaction solution was purified using a DEAE Sepharose column (140 x 210 mm), distilled, and dried under vacuum. The dried solid was dissolved in 2.5 mL of distilled water and added to a solution of 299 mg (2.44 mmol) of sodium perchlorate in 15 mL of acetone. The mixture was cooled to 4 °C, and the resulting crystals were filtered, washed with cold acetone, and dried under vacuum to obtain 0.43 g of the sodium salt of the target compound (8) (60.0% yield).

[0116] 1H NMR (400 MHz, D2O, 25℃): δ= 8.28 (s, 1H), 8.05 (s, 1H), 7.89 (s, 1H), 5.92 (d, 1H), 5.82 (d, 1H), 5.77 (d, 1H), 5.33 (m, 1H), 4.59 ~ 4.12 (m, overlapped, 13H), 4.00 (s, 3H), 3.39 (s, 3H), 3.29 (s, 3H) 31 P NMR (162 MHz, D2O, 25℃): δ= -0.27 (s, 1P), -10.88 (dd, 2P), -22.29 (t, 1P). LC-MS (ESI, m / z) = 1224.13 [M + H + ]

[0117] Experimental Example 1: In vitro transcription (IVT) of capped luciferase mRNA via co-transcriptional capping For the mRNA synthesis reaction using the compound of Example 8 as a cap analog, 100 ng / ml firefly luciferase, DNA transcription template (containing poly A 101), 5 mM ATP, CTP, GTP, me 1 A transcription reaction mixture containing φTP (1-N-Methyl-Pseudouridine 5'-Triphosphate), 4 mM of the compound of Example 8, 10 U / ml T7 RNA polymerase (New England Biolab #dy1670), 1 U / ml RNase inhibitor protein, 0.01 U / ml inorganic pyrophosphatase, 40 mM Tris HCl (pH 8.0), 20 mM magnesium acetate, 1 mM spermidine, and 10 mM DTT was prepared. As a comparative example, a trimer cap analogue m7G (Trilink) was used instead of the compound of Example 8. (3’OMe) pppA (2’OMe)pG was added under the same conditions to prepare transcription reaction mixtures. Each prepared transcription reaction mixture was incubated at 37°C for 1 to 4 hours. To terminate the reaction, 10 mM Tris·HCl (pH 7.5), 2.5 mM magnesium chloride, 0.1 mM calcium chloride, and 100 U / mL DNase I (Thermo Fisher catalog #2270A) were added to the reaction mixture and incubated at 25°C for 1 hour. The terminated mRNA was purified using the New England Biolabs Monarch® RNA Cleanup Kit or reverse-phase high-performance liquid chromatography according to the manufacturer's instructions. The concentration of the purified mRNA was measured using a Thermo Scientific Nanodrop, and the total amount of mRNA was calculated by multiplying the concentration by the reaction volume. To calculate the mRNA IVT yield, the total amount of mRNA synthesized using each cap analog was divided by the amount of L-DNA added to quantify the IVT yield. Specifically, an IVT yield of 100 means that 100 μg of mRNA was synthesized using 1 μg of L-DNA. This can be calculated using Equation 1 below.

[0118] Then, Trilink's trimer cap analog, 7m G (3’OMe) pppA (2’OMe) The in vitro transcription (IVT) yield value (Equation 1) for pG was designated as 100%, and the in vitro transcription (IVT) yield value (Equation 1) for each example compound was calculated as follows: 7m G (3’OMe) pppA (2’OMe) Divide by the pG yield value (Equation 1) and then multiply by 100. 7m G (3’OMe) pppA (2’OMe) The in vitro transcription (IVT) yield of Example 8 relative to pG was expressed as a percentage (%), which can be calculated by the following formula 2.

[0119]

number

[0120]

number

[0121] Example 8 or 7m G (3’OMe) pppA (2’OMe) The in vitro transcription (IVT) yield values for pG were run in triplicate and the significance was verified via unpaired t-tests. The results are shown in Figure 1.

[0122] According to the results in Figure 1, the in vitro transcription (IVT) yield of Example 8 compound was 100% of that of the conventional third-generation cap analog. 7m G (3’OMe) pppA (2’OMe) It was confirmed that the in vitro transcription (IVT) yield of pG was almost identical. Therefore, the compound according to one embodiment is a conventional third-generation cap analog. 7m G (3’OMe) pppA (2’OMe) It was confirmed that it can be used to synthesize 5'-capped mRNA with a yield equivalent to that of pG.

[0123] Experimental Example 2: Translation of capped luciferase mRNA in HEK293T cells In Experimental Example 1, the compound of Example 8 or 7m G (3’OMe) pppA (2’OMe) The translation activity of capped luciferase mRNA produced by in vitro transcription (IVT) of each pG was evaluated in human embryonic kidney 293T cells (HEK293T). HEK293T cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin at 37°C under a 5% CO2 atmosphere. 5HEK293T cells were plated at 1 / well in a 6-well plate. The next day, cells were transfected with 100 ng of mRNA per well using a transfection reagent (messengerMAX lipofectamine (Invitrogen catalog #LMRNA003)). (Tube A: Dilute 0.3 μL of transfection reagent in 5 μL of complex medium (Opti-MEM (Life Technologies)) and incubate for 10 minutes at room temperature, as recommended by the transfection reagent supplier. Tube B: Dilute 0.3 μL of the transfection reagent in 10 μL of Opti-MEM and incubate for 10 minutes at room temperature.) A 200ng solution was prepared by diluting. The solutions in tubes A and B were mixed and incubated at room temperature for 5 minutes. A vehicle control was incubated in the same manner, excluding the capped luciferase mRNA. The incubated mixture was then used to transfect cells. After transfection, cells were harvested at 1, 4, 6, 12, 24, and 48 hours, and luciferase activity was measured using the Dual-Glo® Luciferase Assay Kit (Promega catalog #E2910) according to the manufacturer's recommendations (75μl of phosphate buffer solution (PBS) was added after removing the medium from the transfected cells). Then, 75μl of Dual-Glo® Reagent was added, mixed, and incubated for 10 minutes. Firefly luciferase activity was then measured using a luminometer (Perkin Elmer). The luminescence of firefly luciferase was measured. Then, 75 μl of Dual-Glo® Stop&Glo® Reagent was added and incubated for 10 minutes, after which the luminescence of Renilla luciferase was measured again. The ratio of the firefly luciferase luminescence to the Renilla luciferase luminescence (control) was calculated, and the luciferase activity (relative light units (RLU)) was normalized for each time point and is shown in Figure 2.All luciferase activity measurements were performed independently three times, and significance was verified using one-way ANOVA and Tukey's post hoc tests. To estimate the total luciferase protein expression from 1 hour to 48 hours after transfection, the AUC (area under the curve) was calculated using GraphPad Prism. To obtain the total peak area, the luciferase activity values at each time point were assigned to the Y-axis and the corresponding time values to the X-axis, resulting in the curve shown in Figure 2. Then, using Prism's trapezoid rule, the area under the curve was calculated based on the area under the curve between the mRNA capped with Example 8 compound and the mRNA capped with Example 8 compound. 7m G (3’OMe) pppA (2’OMe) The total luciferase area value for the pG-capped mRNA was calculated and is shown in Figure 3. The formula for calculating the area value in Prism is as follows:

[0124]

number

[0125] The mRNA capped with the compound according to Example 8 was synthesized using conventional 7m G (3’OMe) pppA (2’OMe) The mRNA capped with pG showed significantly higher luciferase activity from 6 to 12 hours after transfection (see Figure 2). Furthermore, due to the increased protein expression at the early stage of transfection, the mRNA capped with Example 8 also showed significantly higher overall protein expression levels. 7m G (3’OMe) pppA (2’OMe) It was confirmed that the mRNA was higher than that capped with pG (see Figure 3).

[0126] Based on the results shown in Figures 2 and 3, it was found that a capping analogue according to one embodiment, in which a sulfonyl-containing group is introduced instead of a methyl group at the 3' hydroxyl group of methylguanosine, has an improved protein expression enhancing effect compared to conventional commercialized cap analogues.

Claims

1. A compound of Formula 1 below, or a pharmaceutically acceptable salt thereof: Formula 1 【Chemical 1】 where n=0, 1 or 2; Y １ , Y ２ and Y ３ are each independently O or S; R １ is C １～６ Alkyl or CH ２ Ph, R ２ is H or a sulfonyl-containing group; R 3 is a sulfonyl-containing group, wherein said sulfonyl-containing groups are independently selected from the group consisting of mesyl, esyl, triflyl, tresyl, tosyl, brosyl, nosyl, and dansyl; R ４ and R ５ are each independently OH or methoxy; R ６ is OH or a mononucleotide or oligonucleotide having 1 to 7 bases, Z and Z' are each independently a natural, modified or unnatural nucleoside base.

2. 10. The compound of claim 1, having the structure of formula 1a: Formula 1a 【Chemistry 2】 where n=0, 1 or 2; X １ , X ２ , X ３ and X ４ are each independently absent, a monovalent metal ion, or a combination thereof, and in this case, when any of X 1 , X 2 , X 3 and X 4 is absent, OX exists as O − , and X １ , X ２ , X ３ and X ４ is selected so that the compound of Formula 1 is electroneutral; Y １ , Y ２ and Y ３ are each independently O or S; R １ is C １～６ Alkyl or CH ２ Ph, R ２ is H or a sulfonyl-containing group; R 3 is a sulfonyl-containing group, wherein said sulfonyl-containing groups are independently selected from the group consisting of mesyl, esyl, triflyl, tresyl, tosyl, brosyl, nosyl, and dansyl; R ４ and R ５ are each independently OH or methoxy; R ６ is OH or a mononucleotide or oligonucleotide having 1 to 7 bases, Z and Z' are each independently a natural, modified or unnatural nucleoside base.

3. The monovalent metal ion is Na ＋ , Li ＋ and K. ＋ 3. The compound of claim 2, selected from the group consisting of: or a pharmaceutically acceptable salt thereof.

4. n=0, 1 or 2; X １ , X ２ , X ３ and X ４ are each independently absent or Na; Y １ , Y ２ and Y ３ are each independently O or S; R １ is C １～６ is alkyl, R ２ is H or a sulfonyl-containing group; R 3 is a sulfonyl-containing group, each of said sulfonyl-containing groups being independently selected from the group consisting of mesyl, esyl, and tosyl; R ４ and R ５ are each independently OH or methoxy; R ６ is OH or a mononucleotide or oligonucleotide having 1 to 3 bases, 3. The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein Z and Z' are each independently a natural nucleoside base.

5. 10. The compound of claim 1, selected from the group consisting of compounds of the following formula and pharmaceutically acceptable salts thereof: ７ｍＧ (3'OEsyl) pppA (2'OMe) pG 【Chemistry 3】 、 ７ｍＧ (3'OMesyl) pppA (2'OMe) pG 【Chemistry 4】 and ７ｍＧ (3'OTosyl) pppA (2'OMe) pG 【Chemistry 5】 。

6. 6. An mRNA 5'-capped with a cap analogue, which is a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof.

7. A composition for producing 5'-capped mRNA, comprising a cap analogue which is a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof.

8. A pharmaceutical composition comprising an mRNA 5'-capped with a cap analogue that is a compound described in any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

9. A cell containing the 5'-capped mRNA of claim 6.

10. A kit for producing 5'-capped mRNA, comprising a cap analogue which is a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof.

Images