EP-4737569-A1 - C(5)-HALOGENATED PYRIMIDINE RIBONUCLEOTIDE-BOUND MRNA AND METHOD FOR INCREASING PROTEIN EXPRESSION USING SAME

EP4737569A1EP 4737569 A1EP4737569 A1EP 4737569A1EP-4737569-A1

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.

Inventors

SEO, YOUNG JUN
CHABUNGBAM, Dhurbachandra Singh
ALOM, Kazi Morshed
THOKCHOM, Simander Singh

Assignees

INDUSTRIAL COOPERATION FOUNDATION JEONBUK NATIONAL UNIVERSITY

Dates

Publication Date: 20260506
Application Date: 20240621

Claims (12)

An mRNA construct comprising a modified nucleotide in which the fifth carbon of a pyrimidine base of cytidine or uridine is substituted with a halogen.
The mRNA construct according to claim 1, wherein the modified nucleotide is substituted with one selected from the group consisting of fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).
The mRNA construct according to claim 1, wherein the modified nucleotide is represented by one selected from the group consisting of the following Chemical Formulas 1 to 4:
A composition for RNA synthesis comprising a modified nucleotide in which the fifth carbon of a pyrimidine base of cytidine or uridine is substituted with a halogen.
The composition for RNA synthesis according to claim 4, wherein the modified nucleotide is represented by one selected from the group consisting of the following Chemical Formulas 1 to 4:
The composition for RNA synthesis according to claim 4, wherein the composition further comprises pseudouridine or methylpseudouridine.
A method for increasing protein expression efficiency using the mRNA construct according to claim 1.
The method according to claim 7, wherein the mRNA construct comprises a compound represented by one selected from the group consisting of the following Chemical Formulas 1 to 4:
The method according to claim 7, wherein the protein expression efficiency is increased through cap-dependent translation.
The method according to claim 7, wherein the mRNA construct further comprises pseudouridine or methylpseudouridine.
The method according to claim 10, wherein the mRNA construct comprises a compound represented by Chemical Formula 1 and pseudouridine:
The method according to claim 7, wherein the protein is an antigen protein.

Description

TECHNICAL FIELD The present invention relates to C(5)-halogenated pyrimidine ribonucleotide-bound mRNA and to a technique for increasing protein expression using the same. BACKGROUND ART To treat the COVID-19 pandemic that has been spreading worldwide, mRNA vaccines have played a pivotal role. The main components of mRNA vaccines are modified nucleosides (pseudouridine, ψ) and methyl nucleosides (1-methylpseudouridine, m1ψ). The introduced mRNA can evade innate immune responses and express antigens with high efficiency, possesses many advantages such as rapid protein production, and avoids harmful chromosomal integration-a serious adverse effect of DNA-based vaccines. Accordingly, many research institutions have conducted preclinical and clinical studies for the therapeutic application of mRNA, and have demonstrated therapeutic efficacy using modified mRNA platforms. Exogenous mRNA introduced into cells is recognized by various pattern recognition receptors (PRRs) present in endosomes or the cytosol, which induces activation of the type I interferon (IFN-I) pathway and causes an excessive immune response that inhibits expression of antigen proteins. However, it is known that mRNA into which chemically modified nucleosides-pseudouridine and 1-methylpseudouridine-are introduced can evade activation of TLR7, TLR8, and other innate immune sensors, thereby reducing type I interferon activation efficiency and increasing the efficiency of protein expression. Modified nucleosides are analogs other than the conventional A, U, G, and C; and mRNA into which such modified nucleic acids are introduced may exhibit slightly different secondary-structure modifications even for the same sequence, may not be recognized by RNA pattern recognition receptors so as to evade excessive immunity, and is also known to provide different efficiencies in terms of RNA structural stability. Through research, it has been demonstrated that, by singly or doubly modifying bases such as N6-methyladenosine (m6A), pseudouridine (ψ), 5-methylcytidine (m5C), 2-thiouridine (s2U), and 5-methyluridine (m5U), the TLR-mediated immunogenicity of mRNA is suppressed, thereby lowering the immunogenicity of in vitro-transcribed mRNA; and by substituting uridine with pseudouridine, the stability of mRNA and high translational capacity have been demonstrated. These two research results were important endeavors that changed the perception and reality regarding mRNA and have recently contributed to the development of modified mRNA as therapeutics. In addition, for therapeutic applications of mRNA, many researchers have reported the effects of modified mRNA in various therapeutic fields using m5C/ψ or m5C/s2U-modified mRNA; and to utilize it in various therapeutic fields, it is important to develop new and diverse modified mRNA. However, the present inventors screened various commercially available purine- and pyrimidine-modified mRNAs for protein expression, but most did not exhibit efficient transcription and translation efficiency. Accordingly, in an effort to identify modified nucleotides capable of increasing protein expression efficiency, the present inventors focused on C(5) modification of pyrimidines, among pyrimidine modifications, which can produce more stable mRNA due to higher in vivo stability and bioavailability. Thus, it was confirmed that, when modified nucleotides in which halogens are substituted at the fifth carbon of the cytidine and uridine pyrimidine bases are introduced into an mRNA construct, excessive immune responses are evaded and the expression efficiency of antigen proteins can be increased. In addition, unlike the usual decrease in transcription efficiency when using modified nucleotides, it was confirmed that, when modified nucleotides in which halogens are substituted at the fifth carbon of the cytidine and uridine pyrimidine bases were used, transcription efficiency did not decrease, and the present invention was completed. DETAILED DESCRIPTION OF THE INVENTION TECHNICAL PROBLEM The problem to be solved by the present invention is to provide a method for increasing protein expression efficiency using an mRNA construct in which C(5)-halogenated pyrimidine ribonucleotides are incorporated. TECHNICAL SOLUTION In order to solve the above-described technical problem, in one embodiment of the present invention, there is provided an mRNA construct into which a modified nucleotide, in which the fifth carbon of a pyrimidine base of cytidine or uridine is substituted with a halogen, is introduced. In another embodiment, there is provided a composition for RNA synthesis comprising the modified nucleotide. In an embodiment of the present invention, there is also provided a method for increasing protein expression efficiency using mRNA into which the modified nucleotide is introduced. In the present invention, the composition for RNA synthesis may further comprise pseudouridine or methylpseudouridine. In the present invention, the method ma