mRNA structure for improving protein translation efficiency, and use thereof

Abstract

The present invention relates to an mRNA structure comprising: a gene encoding a target protein or peptide; and a 5' untranslated region (5'-UTR) comprising any one nucleotide sequence selected from the group consisting of SEQ ID NOS: 1 to 15 and linked upstream of the gene encoding the target protein or peptide, or a 3' untranslated region (3'-UTR) comprising any one nucleotide sequence selected from the group consisting of SEQ ID NOS: 16 to 35 and linked downstream of the gene encoding the target protein or peptide. The mRNA structure according to the present invention exhibits high mRNA stability and high expression efficiency of the target protein or peptide, and enables stable expression of the target protein within cells for an extended period of time, even when various target proteins, host cells, or delivery methods are used. Accordingly, the mRNA structure with enhanced protein translation efficiency according to the present invention can be advantageously utilized in various application fields related to target protein expression, such as therapeutic and vaccine development.

Description

mRNA structure for improving protein translation efficiency and use thereof

[0001] [Cross-reference with related applications]

[0002] This application claims the benefit of priority from Korean Patent Application No. 10-2023-0160554, filed November 20, 2023, the entire contents of which are incorporated herein by reference.

[0003] The present invention relates to an mRNA structure for improving protein translation efficiency and its use.

[0004]

[0005] Gene-based therapeutic and vaccine development technologies, when applied beyond the cellular level to target organisms, have often exhibited insufficient transcription and translation efficiency, resulting in insufficient therapeutic efficacy. This has been identified as a limitation of gene-based therapeutic development. Therefore, enhancing the intracellular and extracellular expression of antigenic proteins for the treatment of infectious diseases is a crucial requirement for the development of pharmaceuticals using artificial nucleic acid molecules. Furthermore, in the field of gene-based therapeutic and vaccine development, securing mRNA sequences with high stability and translation efficiency is also emerging as a prerequisite for mRNA-based therapeutics.

[0006] Meanwhile, mRNA is RNA that transfers the genetic information of DNA to the ribosome and expresses protein through the mRNA translation process. mRNA vaccines refer to medicines used for the prevention and treatment of cancer, infectious diseases, autoimmune diseases, etc., using the protein encoded by mRNA as an antigen. Since mRNA is produced in the cytoplasm, the probability of mutation due to infection or genomic DNA insertion is low, and since various modifications are possible within the cytoplasm, it can control the half-life or increase the amount of translated protein, thereby increasing intracellular stability. In addition, because it is easy to conduct in vitro experiments, rapid development and GMP production are possible, and mass production is easy, so mRNA therapeutics have the advantage of surpassing DNA therapeutics or virus therapeutics in terms of stability, efficiency, and productivity.

[0007] However, these mRNA vaccines require multifaceted research because the instability of RNA molecules requires technical elements for delivery into the cytoplasm, and after delivery of RNA molecules into the cytoplasm, damage to the RNA molecules or decreased translation efficiency due to excessive innate immune responses must be minimized.

[0008] Furthermore, the immune-activating properties of naked mRNA, which induce stimulation by adjuvants such as toll-like receptor agonists (TLR agonists), are known to conversely interfere with the transcriptional signaling pathway of mRNA, thereby inhibiting the expression of mRNA antigens in sufficient quantities to exhibit pharmacological activity. To address this issue, active efforts are underway to develop vaccines using modified mRNA with eliminated or weakened immunogenicity. However, in these cases, the low immunogenicity of mRNA also limits the induction of effective humoral and / or cellular immune responses.

[0009] Among the translation control mechanisms, the Kozak sequence in the 5'-untranslated region (5'-UTR) of mRNA plays an important role in the formation of the translation pre-initiation complex for translation regulation and the recognition of the start codon (AUG) by the ribosome. The 3'-untranslated region (3'-UTR) is located immediately after the stop codon in the coding region, and thus plays an important role in post-transcriptional regulation such as translation termination, mRNA stability, and regulatory RNA binding.

[0010] Against this backdrop, developing mRNA constructs that stably express target proteins while exhibiting low immunogenicity is crucial, but research remains limited. Therefore, the inventors of the present invention have developed a non-translated region that enhances the stability of the mRNA construct and enhances the expression efficiency of the target protein, thereby completing the mRNA construct of the present invention.

[0011]

[0012] The present invention aims to provide an mRNA construct comprising a gene encoding a target protein and a non-translated region that improves the translation efficiency of the target protein.

[0013] In addition, the present invention aims to provide an expression vector comprising the mRNA structure or a DNA complementary thereto.

[0014] In addition, the present invention aims to provide a transformant comprising the mRNA structure.

[0015] In addition, the present invention aims to provide a pharmaceutical composition comprising the mRNA structure.

[0016]

[0017] In order to achieve the above object, one aspect of the present invention provides an mRNA construct comprising: a gene encoding a target protein or peptide; a 5'untranslated region (5'-UTR) comprising any one base sequence selected from the group consisting of base sequences of SEQ ID NO: 1 to SEQ ID NO: 15 linked upstream of the gene encoding the target protein or peptide; or a 3'untranslated region (3'-UTR) comprising any one base sequence selected from the group consisting of base sequences of SEQ ID NO: 16 to SEQ ID NO: 35 linked downstream of the gene encoding the target protein or peptide.

[0018] In addition, in order to achieve the above purpose, another aspect of the present invention provides an expression vector comprising a base sequence of the mRNA structure or a DNA base sequence complementary thereto.

[0019] In addition, to achieve the above purpose, another aspect of the present invention provides a transformant comprising the mRNA structure.

[0020] In addition, to achieve the above purpose, another aspect of the present invention provides a pharmaceutical composition comprising the mRNA structure.

[0021] The above pharmaceutical composition may be a vaccine or a gene therapy agent.

[0022]

[0023] The mRNA construct according to the present invention exhibits high mRNA stability and protein expression efficiency, and can stably express a target protein within cells for a long period of time, even when using a variety of target proteins, introduction cells, and introduction methods. Furthermore, the mRNA construct comprising the RNA construct with enhanced protein translation efficiency according to the present invention exhibits excellent immunogenicity, making it useful in various applications related to target protein expression, such as therapeutic agent and vaccine development.

[0024] However, the effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

[0025]

[0026] Figure 1 is a schematic diagram showing the process of screening the non-translated region of the present invention.

[0027] Figure 2 shows the results of measuring the GC / AT content ratio and length for the non-translated region library.

[0028] Figure 3a is a schematic diagram showing the process of selecting and improving the untranslated region of the present invention from a 3'untranslated region library.

[0029] Figure 3b is a schematic diagram showing the process of selecting and improving the untranslated region of the present invention from a 5'untranslated region library.

[0030] Figure 4 shows the 3'-UTR selection process of the present invention and the results of confirming the iv in vitro mRNA expression ability using an mRNA construct including the nine selected 3'-UTRs. #1 in Figure 4 represents the 3'-UTR of SEQ ID NO: 18, #2 represents the 3'-UTR of SEQ ID NO: 16, #3 represents the 3'-UTR of SEQ ID NO: 21, #4 represents the 3'-UTR of SEQ ID NO: 22, #5 represents the 3'-UTR of SEQ ID NO: 23, #6 represents the 3'-UTR of SEQ ID NO: 25, #7 represents the 3'-UTR of SEQ ID NO: 27, #8 represents the 3'-UTR of SEQ ID NO: 17, and #9 represents the 3'-UTR of SEQ ID NO: 32.

[0031] Figure 5a shows the results of confirming the iv in vitro mRNA expression ability using an mRNA construct including the selected 3'-UTR and the 5'-UTR of the present invention. 5A of Figure 5a represents the 5'-UTR of SEQ ID NO: 1, 5B represents the 5'-UTR of SEQ ID NO: 2, 1A represents the 5'-UTR of SEQ ID NO: 3, 1B represents the 5'-UTR of SEQ ID NO: 4, 1 represents the 5'-UTR of SEQ ID NO: 5, and 5 represents the 5'-UTR of SEQ ID NO: 9. In Fig. 5a, 1-2 represents structure 1, 5-2 represents structure 2, 1A-2 represents structure 3, 1B-2 represents structure 4, 5A-2 represents structure 5, 5B-2 represents structure 6, #2 represents structure 7, 1-8 represents structure 8, 5-8 represents structure 9, 1A-8 represents structure 10, 1B-8 represents structure 11, 5A-8 represents structure 12, 5B-8 represents structure 13, and #2 represents structure 14.

[0032] Figure 5b shows the results of confirming the iv vivo mRNA expression ability using the above mRNA structure.

[0033] Figures 6a and 6b show the results of immunogenicity evaluation of a vaccine using the mRNA structure of the present invention.

[0034]

[0035] Hereinafter, the present invention will be described in detail.

[0036]

[0037] 1. mRNA structure of the present invention

[0038] One aspect of the present invention provides an mRNA construct comprising: a gene encoding a target protein or peptide; a 5'untranslated region (5'-UTR) comprising any one base sequence selected from the group consisting of base sequences of SEQ ID NO: 1 to SEQ ID NO: 15 linked upstream of the gene encoding the target protein or peptide; or a 3'untranslated region (3'-UTR) comprising any one base sequence selected from the group consisting of base sequences of SEQ ID NO: 16 to SEQ ID NO: 35 linked downstream of the gene encoding the target protein or peptide;

[0039] In addition, in the present invention, the mRNA structure may include a gene encoding the target protein or peptide; the mRNA structure may include both a 5'-UTR linked upstream of the gene encoding the target protein or peptide, and a 3'-UTR linked downstream of the gene encoding the target protein or peptide.

[0040] In the present invention, the target protein or peptide is a protein or peptide that a person skilled in the art wishes to express or produce in large quantities, and may include all proteins and peptides that are intended to be expressed using the mRNA construct of the present invention. For example, in the present invention, the target protein or peptide may be at least one selected from the group consisting of antigens, viral proteins, enzymes, chimeric antigen receptors (CARs), cell receptors, and serum proteins, but is not limited thereto.

[0041] The above untranslated region (UTR) is a part of mRNA excluding the coding region. When it exists in the 5' part of the coding region, it is called 5'-UTR, and when it exists in the 3' part of the coding region, it is called 3'-UTR. It affects the transcriptional attenuation or translation of mRNA, and also plays a role in the translational control of the coding region. The 5'-UTR refers to the mRNA from the transcription start site to just before the translation start codon, and includes a ribosome-binding site for the ribosome to bind to and start translation of the coding region. It is a sequence that is complementary to the base sequence of 16s RNA in the ribosome that constitutes the ribosome and is located 3 to 10 bases above the start codon of the coding region. The above 3'-UTR is located immediately after the stop codon of the coding region, so it affects post-transcriptional regulation such as translation termination, mRNA stability, and regulatory RNA binding. Specifically, it acts as a binding site for regulatory RNA and affects polyadenylation, translation efficiency, transport to the nucleus or cytoplasm, and mRNA stability.

[0042] Accordingly, the 5'-UTR and / or 3'-UTR of the present invention may have an activity that increases the translation efficiency of a gene encoding a target protein or peptide, the stability of a gene encoding a target protein or peptide, or a combination thereof.

[0043] In addition, the base sequence of SEQ ID NOs: 1 to 35 includes a polynucleotide having a base sequence substantially identical to the base sequence of SEQ ID NOs: 1 to 35 and a variant or active fragment thereof. The polynucleotide having a substantially identical base sequence refers to a polynucleotide including a base sequence having a sequence homology of about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% or more, about 95% or more, about 97% or more, about 98% or more, or about 99% or more to the base sequence of SEQ ID NOs: 1 to 35, respectively.

[0044] The mRNA construct of the present invention is intended for use in transformation, preferably temporary transformation, to express a target protein or peptide only for a certain period of time without inserting a gene into the chromosome of a host cell. While it is important for the mRNA to be structurally stable and to express the protein with high efficiency for a long period of time, it is ultimately an essential condition for achieving the purpose of transformation to be able to express a protein that fully exhibits the original function of the target protein with high efficiency.

[0045] Accordingly, the present inventors have produced an mRNA construct including a nucleic acid sequence including a 5'-UTR including any one base sequence selected from the group consisting of base sequences of SEQ ID NO: 1 to SEQ ID NO: 15 and / or a 3'-UTR including any one base sequence selected from the group consisting of base sequences of SEQ ID NO: 16 to SEQ ID NO: 35, together with a target protein, and confirmed that when the protein is expressed, mRNA stability and protein translation efficiency are increased, enabling more efficient long-term expression of the target protein or peptide.

[0046] Specifically, in the mRNA structure of the present invention, the 5'-UTR may include any one base sequence selected from the group consisting of base sequences of SEQ ID NO: 1 to SEQ ID NO: 5 and SEQ ID NO: 9, and the 3'-UTR may include a base sequence of SEQ ID NO: 16 or SEQ ID NO: 17.

[0047] In particular, in the mRNA structure of the present invention, the 5'-UTR may include a base sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and the 3'-UTR may include a base sequence of SEQ ID NO: 16 or SEQ ID NO: 17.

[0048] In one embodiment of the present invention, in the mRNA structure, the 5'-UTR may include a base sequence of SEQ ID NO: 1, and the 3'-UTR may include a base sequence of SEQ ID NO: 16.

[0049] In one embodiment of the present invention, the mRNA structure may have the 5'-UTR including the base sequence of SEQ ID NO: 2, and the 3'-UTR including the base sequence of SEQ ID NO: 16.

[0050] In one embodiment of the present invention, the mRNA structure may have the 5'-UTR including the base sequence of SEQ ID NO: 2 and the 3'-UTR including the base sequence of SEQ ID NO: 17.

[0051] In a specific embodiment of the present invention, an mRNA construct of the present invention was produced, which includes any one of the base sequences of SEQ ID NO: 1 to SEQ ID NO: 5 and SEQ ID NO: 9 in the 5'-UTR of mRNA, which is an upstream region of a target protein or peptide, and includes the base sequence of SEQ ID NO: 16 or SEQ ID NO: 17 in the 3'-UTR of mRNA, which is a downstream region of the target protein or peptide, and the COVID-19 mRNA vaccine BNT162b2 sold by BioNTech and the COVID-19 mRNA vaccine mRNA-1273 sold by Moderna were used as controls for the mRNA construct for expressing the target protein. As a result, when the mRNA construct of the present invention was used to express the target protein luciferase in vitro, the fluorescence value was measured to be higher than that of the two controls, confirming that the expression efficiency of the target protein was excellent. Therefore, the mRNA structure of the present invention can stably express a target protein or peptide in a cell for a long period of time, and can be effectively used in various application fields related to target protein expression, such as anticancer immunotherapy and vaccine development.

[0052] In the present invention, the region encoding the target protein or peptide and the mRNA structure are composed of a nucleotide sequence, and the "nucleotide sequence" refers to a polymeric substance containing a plurality of nucleotide units, specifically, a polymer in which a plurality of nucleotide units are linked to each other by phosphodiester bonds of a sugar / phosphate backbone, and can be used interchangeably with the terms "polynucleotide," "nucleic acid," and "nucleic acid molecule." The polynucleotide is a biopolymer essential to living organisms, and may be RNA or DNA that encodes genetic information through a unique base sequence. The polynucleotide is isolated, artificially synthesized, or non-naturally occurring or engineered, and the "non-naturally occurring or engineered" means a state created by artificial modification rather than a state in which it exists as it is in nature. Here, the artificial modification may be to mimic the structure of natural mRNA, specifically, mature mRNA, to enhance the expression of the target antigen in a cell.

[0053] In the present invention, "mRNA (Messenger RNA)" refers to RNA that is transcribed from a DNA template and transmits the genetic information of the DNA to ribosomes in the cytoplasm. In eukaryotic organisms, the transcription process takes place in the nucleus of a cell and involves the processing of premature RNA. Specifically, this process is called post-transcriptional modification and includes processes such as splicing, 5'-capping, polyadenylation, and export from the nucleus or mitochondria. As a result of this process, mature mRNA is generated, which includes a nucleotide sequence that can be translated into the amino acid sequence of a specific peptide or protein. Generally, the mature mRNA may optionally include a 5'-cap, a 5'-UTR, an open reading frame, a 3'-UTR, and a poly A tail. The mRNA may be synthesized by any of several known methods, for example, the mRNA may be synthesized via in vitro transcription (IVT).

[0054] In the present invention, the “5’cap” is a structure located at the 5’ terminal region that affects the stability and expression efficiency of a polynucleotide, and can generally be formed by a modified nucleotide, particularly a derivative of a guanine nucleotide.

[0055] In addition, in the present invention, the "poly A tail" is a structure located at the 3'-terminal region that delays the degradation process of RNA exo-nuclease and extends the stability and in vivo half-life of the polynucleotide, thereby affecting expression efficiency, and can generally be formed by a plurality of adenine nucleotide sequences. In one embodiment, the poly A tail can be comprised of 20 to 200 adenines, for example, 20 to 190, 20 to 170, 20 to 150, 20 to 130, 20 to 110, 20 to 90, 20 to 70, 20 to 50, 20 to 30, 30 to 190, 30 to 170, 30 to 150, 30 to 130, 30 to 110, 30 to 90, 30 to 70, or 30 to 50 repetitive adenine nucleotide sequences. Additionally, the poly A tail can be comprised of a plurality of units connected by a linker.

[0056] The mRNA construct of the present invention may further include, without limitation, a region capable of enhancing the protein expression rate from the mRNA. For example, the construct may further include a region that directs ribosomes, a region associated with translation initiation or promotion of mRNA, a region associated with transport of mRNA out of the nucleus, a region associated with binding to the endoplasmic reticulum membrane, a region comprising an endoplasmic reticulum retention signal (ER retention signal) sequence, or a region comprising an endoplasmic reticulum signal sequence.

[0057]

[0058] 2. Utilization of the above mRNA structure

[0059] As described above, the mRNA construct of the present invention exhibits high expression efficiency in cells and can therefore be utilized for the expression of a target protein or peptide.

[0060] Another aspect of the present invention provides an expression vector comprising the base sequence of the mRNA structure or a DNA base sequence complementary thereto.

[0061] Since the overlapping description is the same as that described above in ‘1. mRNA structure’, the description thereof is omitted.

[0062] The expression vector of the present invention is an expression vector, and the expression vector refers to a genetic construct that includes essential regulatory elements operably linked to an insert so that the insert is expressed in a cell.

[0063] In the present invention, the term “operably linked” means a state in which a region encoding a target protein or peptide is linked to a regulatory element in a manner that allows expression of the nucleotide sequence.

[0064] In the present invention, the expression vector can be produced and purified using standard recombinant DNA technology. The type of the expression vector is not particularly limited as long as it has the function of expressing a desired gene and producing a desired protein in various host cells of prokaryotic and eukaryotic cells, but may be a vector that has a promoter that exhibits strong activity and a strong expression ability while being capable of producing a target protein or peptide in large quantities, but is not limited thereto. For example, the expression vector may be a vector composed of plasmids, cosmids, artificial chromosomes, liposomes, retrovirus, adenovirus, adenovirus-associated virus (AAV), vaccinia virus, herpes virus, lentivirus, or spumavirus, but is not limited thereto.

[0065] In addition, the expression vector may include, but is not limited to, a promoter, an initiation codon, a gene encoding a target protein or peptide, and a stop codon terminator. In addition, it may appropriately include, but is not limited to, DNA encoding a signal peptide, an enhancer sequence, untranslated regions on the 5th and 3rd sides of the desired gene, a selectable marker region, or a replicable unit.

[0066] The expression vector of the present invention can stably express a target protein or peptide in a transformant for a long period of time by including an mRNA structure including the 5'-UTR and / or 3'-UTR, and thus can be effectively used in various application fields related to the expression and production of target proteins, such as anticancer immunotherapy and vaccine development.

[0067] In addition, another aspect of the present invention provides a transformant comprising the mRNA construct.

[0068] The above transformant refers to a transformant into which the mRNA structure has been introduced into a host cell. The above transformant refers to a host cell into which a foreign genetic material has been introduced through transfection or transduction, and in the present invention, the above transformant may refer to a host cell into which the mRNA structure of the present invention or an expression vector containing the base sequence of the mRNA structure of the present invention and a DNA base sequence complementary thereto has been introduced.

[0069] The transformant of the present invention can effectively express a target protein or peptide by being transformed with the mRNA construct or expression vector of the present invention.

[0070] In the present invention, the above “introduction” means a process of transforming a host cell by transfecting or transducing a foreign genetic material into the host cell, and the host cell may be, for example, an immune cell, an epithelial cell, a muscle cell, a kidney cell, a tumor cell, etc.

[0071] The exogenous genetic material may be introduced into cells by physical, chemical, or biological means. Physical means may include gene guns, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc., and chemical means may include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, LNPs, and liposomes. Furthermore, biological means may include the use of DNA or RNA vectors.

[0072] In this specification, transfection and transduction may be used interchangeably, but both can be broadly interpreted as the transformation of a host cell by transferring foreign genetic material to the host cell.

[0073] Meanwhile, the above transfection can be classified as stable transfection if the injected foreign genetic material is inserted into the chromosomal DNA of the host cell or is in a form that can replicate independently, and as transient transfection if not.

[0074] In addition, the mRNA construct of the present invention, which can be transfected into cells as described above, can be used as an active ingredient of a pharmaceutical composition depending on the target protein.

[0075] For example, if the target protein is a virus- or bacteria-derived protein, the pharmaceutical composition comprising the mRNA construct of the present invention may be a vaccine. In this case, the target virus- or bacteria-derived protein is expressed with high efficiency by the mRNA construct within the subject injected with the pharmaceutical composition of the present invention, and the virus- or bacteria-derived protein thus expressed is recognized as an antigen within the subject, inducing an immune response, thereby functioning as a vaccine.

[0076] The above vaccine means a substance containing a component that is effective in preventing infection or reinfection by a pathogen or antigen, reducing the severity of symptoms or eliminating symptoms, or substantially or completely eliminating a disease caused by the pathogen or antigen by inducing an immune response to a specific antigen in an individual.

[0077] The above antigen refers to a molecule capable of inducing an immune response in a host organism, and in the present invention, the antigen may be at least one selected from the group consisting of cells, viruses, proteins, peptides, nucleic acids, oligonucleotides, carbohydrates, and lipids, and may include all of those derived from microorganisms including viruses, bacteria, or fungi, and those derived from cancer cells.

[0078] In the present invention, when the antigen is derived from a virus, the virus may be, for example, influenzavirus, coronavirus, flavivirus, calicivirus, respiratory syncytial virus (RSV), rotavirus, parvovirus, picornavirus, pestivirus, rhabdovirus, birnavirus, retrovirus, herpesvirus, etc.

[0079] In a specific embodiment of the present invention, a construct comprising the SARS-CoV-2 spike protein of the Pfizer vaccine as a target protein antigen was encapsulated in LNPs and injected into mice, followed by immunogenicity evaluation. As a result, the construct of the present invention was confirmed to exhibit superior immunogenic efficacy, with increased immunoglobulin production and T cell responses compared to BioNTech's existing mRNA vaccine.

[0080] The vaccine composition of the present invention can be prophylactically administered to an individual prior to infection with the pathogen or prior to the onset of a disease, and can also be therapeutically administered after infection with the pathogen or after the onset of a disease.

[0081] Meanwhile, the vaccine composition of the present invention can be administered by an appropriate method such as intramuscular injection, subcutaneous injection, intradermal injection, intraperitoneal injection, nasal administration, oral administration, transdermal administration, or oral administration, depending on the purpose.

[0082] In addition, if the target protein is a causal protein deficient in a specific disease such as cancer or a genetic disease, the pharmaceutical composition comprising the mRNA construct of the present invention may be a gene therapy agent. In this case, the target protein, which is a causal protein deficient in the specific disease, is expressed with high efficiency by the mRNA construct in a subject injected with the pharmaceutical composition of the present invention, and the deficiency of the causal protein is complemented by the high-efficiency expression of the causal protein, thereby treating the specific disease. Therefore, any disease that can be treated by complementing the deficient gene can be treated by the pharmaceutical composition of the present invention without any particular limitation.

[0083] In particular, the diseases most suitable for application as gene therapy as described above are monogenic genetic diseases caused by a single gene defect, such as spinal muscular atrophy (SMA), a rare neuromuscular disease, and cystic fibrosis (CF), Duchenne muscular dystrophy (DMD), Friedreich's ataxia (FA), hemophilia, Rett syndrome, Fabry disease, and sickle cell disease.

[0084] When the mRNA structure of the present invention is used as an active ingredient of a pharmaceutical composition as described above, the pharmaceutical composition may further include a delivery means for delivering the mRNA structure of the present invention.

[0085] Representative examples of the above delivery means include nanoparticles such as liposomes or LNPs (lipid nanoparticles), and the liposomes or LNPs include cationic lipids, non-cationic lipids, or neutral lipids, and may additionally include other lipids such as PEG (polyethylene glycol) or cholesterol. In addition, exosomes may also be used as nanoparticles that serve as the above delivery means, and cationic biocompatible polymers or cationic peptides (e.g., protamine, cell penetrating peptides (CPPs)) that can provide improved stability by electrostatically interacting with the mRNA structure of the present invention may be used as other delivery means, but the present invention is not limited thereto, and any and all means known in the technical field to which the present invention pertains that can deliver the mRNA structure may be used.

[0086] In addition, the pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers. The pharmaceutically acceptable carrier must be compatible with the active ingredient of the present invention, and may be used as a mixture of saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into an injectable formulation such as an aqueous solution, suspension, or emulsion. In particular, it is preferable to provide the composition in a lyophilized form. A method commonly known in the art to which the present invention pertains can be used to manufacture a lyophilized formulation, and a stabilizer for lyophilization may be added. Furthermore, it can be preferably formulated according to each disease or ingredient using an appropriate method in the field or a method disclosed in Remington's pharmaceutical Science (Mack Publishing company, Easton PA).

[0087] The content and administration method of the active ingredients, etc. included in the above pharmaceutical composition can be determined by a person skilled in the art based on the symptoms and severity of the disease of a typical patient. Furthermore, the composition can be formulated in various forms, such as powders, tablets, capsules, liquids, injections, ointments, and syrups, and can also be provided in unit-dose or multi-dose containers, such as sealed ampoules and bottles.

[0088] In addition, the pharmaceutical composition can be administered orally or parenterally. The route of administration of the composition according to the present invention is not limited to these, but for example, bronchial, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, enteral, sublingual, or topical administration is possible. The dosage of the composition according to the present invention varies depending on the patient's body weight, age, sex, health condition, diet, administration time, method, excretion rate, or disease severity, and can be easily determined by a person skilled in the art. In addition, the pharmaceutical composition can be formulated into a suitable dosage form using a known technique for clinical administration.

[0089]

[0090] Hereinafter, the present invention will be described in detail by examples.

[0091] However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

[0092]

[0093] [Example 1]

[0094] Design of mRNA constructs containing various untranslated regions

[0095] In order to design an mRNA construct that can most effectively express various target proteins or peptides, green fluorescent protein (GFP) containing the nucleotide sequence of SEQ ID NO: 36 was selected as the target protein, and then about 2.5 million 5'-UTR libraries and 3'-UTR libraries were created using random nucleotides, and various mRNA constructs were created by connecting the 5'-UTR to the upstream of the GFP and connecting the 3'-UTR to the downstream of the GFP (Fig. 1). Next, using luciferase containing the nucleotide sequence of SEQ ID NO: 38 as the target protein, the fluorescence values ​​of the mRNA constructs containing the luciferase, the 5'-UTR library, and the 3'-UTR library were measured to determine the protein expression ability. Meanwhile, for the 5'-UTR library and 3'-UTR library, the length of UTR and the GC / AT base ratio were measured, and the results were additionally considered in the selection of the non-translated region of the present invention (Fig. 2).

[0096] First, in order to select the 5'-UTR of the present invention, the 5'-UTR region with the highest ribosome binding and translation efficiency was discovered through ribosome profiling. As a result, as shown in Fig. 3b, the top 11 species that were shown to bind the most to ribosomes were first selected from a 5'-UTR library of approximately 2.5 million species. Next, an mRNA expression experiment was performed on the 11 species selected in the first round, and the top 2 species showing the highest mRNA expression were secondarily selected, and these were improved to derive 4 species of 5'-UTR. Specifically, in the case of 5'-UTR, it is known that the shorter the sequence length, the less the mRNA structure is generated, which helps ribosome scanning. Therefore, the sequence length of the two 5'-UTRs previously selected in the second round was reduced to 50 to 65 nt and improved. In addition, the sequence was replaced and improved so that the degree of mRNA twisting could be minimized through the RNA secondary structure prediction program (RNA fold). In addition, the GC content was adjusted to a level similar to that of the mRNA vaccines of Moderna and Pfizer, which have been recognized for their stability, and the sequence was replaced and improved so that the ratio of uracil (U) was 20% or less. At this time, the start codon AUG sequence was replaced to remove the upstream open reading frame (uORF) that suppresses mRNA expression. Through the above improvement process, four types of 5'-UTRs were produced. Therefore, a total of 15 sequences (SEQ ID NO: 1 to SEQ ID NO: 15) of the 11 5'-UTRs selected above and the 4 improved 5'-UTR sequences above were selected as the 5'-UTR of the present invention.

[0097] Next, in order to select the 3'-UTR of the present invention, a 3'-UTR region showing high mRNA stability and long half-life was discovered using an mRNA half-life assay. As a result, as shown in Fig. 3a, the top 20 sequences (SEQ ID NOs: 16 to 35) showing long half-lives were first selected as the 3'-UTR of the present invention. Furthermore, the mtRNR1-AES 3'-UTR region of the BNT162b2 vector was replaced with the sequence of the 3'-UTR library, and a vector containing enhanced green fluorescent protein (eGFP) in the CDS (coding sequence) region was constructed. The mRNA vector constructed as described above was transfected into primary human nasal epithelial cells (HNEPC), and the cells were harvested at 0, 1, 2, 4, and 8 hours after transfection, and RNA was isolated for next-generation sequencing (NGS). In the NGS analysis, more than 500,000 unique sequences were identified, and among them, 9 3'-UTR sequences (SEQ ID NOs: 16, 18, 21, 22, 23, 25, 27, 32, and 33) showing the longest mRNA half-lives were selected for the second round after comparing mRNA levels at various time points in the NGS data.

[0098] Thereafter, the 5'-UTR region of any one of SEQ ID NOs. 1 to 15 was connected upstream of the gene encoding the target protein or peptide, and the 3'-UTR region of any one of SEQ ID NOs. 16 to 35 was connected downstream of the luciferase gene, thereby producing the mRNA construct of the present invention.

[0099]

[0100] [Example 2]

[0101] [2-1] Confirmation of stability and mRNA expression ability of mRNA structure including 3'-UTR of the present invention

[0102] Thereafter, in order to evaluate the stability and protein expression of the nine 3'-UTR regions (SEQ ID NOs: 16, 17, 18, 21, 22, 23, 25, 27, and 32) improved or selected through the above Example 1, a luciferase reporter gene assay was performed using a NanoLuc-hPEST reporter gene expression vector. The NanoLuc-hPEST contains a C-terminal PEST domain with a half-life of 2 hours, and can measure mRNA stability and expression due to the 3'-UTR. The expression vector was introduced into two cell lines (DC2.4 (mouse dendritic cells) and C2C12 (mouse muscle cells)) and transformed. By measuring the fluorescence value expressed in the transformants, the mRNA expression ability due to the UTR of the present invention was confirmed. Additionally, the total protein expression level over time after the transformation was measured using the luciferase reporter gene analysis. As controls, the COVID-19 mRNA vaccine BNT162b2, sold by BioNTech, and the COVID-19 mRNA vaccine mRNA-1273, sold by Moderna, were used as mRNA constructs for expressing the target protein.

[0103] As a result, as shown in Fig. 4, when an mRNA construct including the 3'-UTR sequences of SEQ ID NOs: 16, 17, 18, 21, 22, 25, 27, and 32 (#1 to #4 and #6 to #9 in Fig. 4) was used, luciferase activity was observed, and in particular, when an mRNA construct including the 3'-UTR sequence of SEQ ID NO: 16 (#2 in Fig. 4a) and the 3'-UTR sequence of SEQ ID NO: 17 (#8 in Fig. 4a) was used, it was confirmed that significantly improved protein expression and stability were exhibited compared to the control group.

[0104] [2-2] Confirmation of stability and mRNA expression ability of mRNA structure including 3'-UTR and 5'-UTR of the present invention (in vitro)

[0105] In the above Example 2-1, the 3'-UTR sequence of SEQ ID NO: 16 or the 3'-UTR sequence of SEQ ID NO: 17 (#8 in FIG. 4a) confirmed to have excellent protein expression and stability were combined with five 5'-UTRs (SEQ ID NOs: 1 to 5 and SEQ ID NO: 9) among the 5'-UTR regions of SEQ ID NOs: 1 to 15 improved or selected through the above Example 1, thereby producing 14 mRNA constructs of Table 1 below, which have luciferase as the target protein. The five 5'-UTRs were selected and used by analyzing polysome-rich, GC content, G-quadruplex complex structure, upstream open reading frames (uORF), and uridine ratio through polysome profiling and NGS analysis from the 5'-UTR library of Example 1. While polysome-rich, constructs 7 and 14 were constructed as mRNA constructs comprising the 3'-UTR region of the present invention linked downstream to the Pfizer / BioNTech vaccine mRNA (Pfizer / BioNTech) comprising the sequence of SEQ ID NO: 37.

[0106] Structure 5'-UTR 3'-UTR 1 SEQ ID NO: 5 (Fig. 5, No. 1) SEQ ID NO: 16 (Fig. 5, No. 2) 2 SEQ ID NO: 9 (Fig. 5, No. 5) SEQ ID NO: 16 (Fig. 5, No. 2) 3 SEQ ID NO: 3 (Fig. 5, No. 1A) SEQ ID NO: 16 (Fig. 5, No. 2) 4 SEQ ID NO: 4 (Fig. 5, No. 1B) SEQ ID NO: 16 (Fig. 5, No. 2) 5 SEQ ID NO: 1 (Fig. 5, No. 5A) SEQ ID NO: 16 (Fig. 5, No. 2) 6 SEQ ID NO: 2 (Fig. 5, No. 5B) SEQ ID NO: 16 (Fig. 5, No. 2) 7 SEQ ID NO: 16 (Fig. 5, No. 2) 8 SEQ ID NO: 5 (Fig. 5, No. 1) SEQ ID NO: 17 (Fig. 5, No. 8) 9 SEQ ID NO: 9 (Fig. 5, No. 5) SEQ ID NO: 17 (8 in FIG. 5) 10 SEQ ID NO: 3 (1A in FIG. 5) SEQ ID NO: 17 (8 in FIG. 5) 11 SEQ ID NO: 4 (1B in FIG. 5) SEQ ID NO: 17 (8 in FIG. 5) 12 SEQ ID NO: 1 (5A in FIG. 5) SEQ ID NO: 17 (8 in FIG. 5) 13 SEQ ID NO: 2 (5B in FIG. 5) SEQ ID NO: 17 (8 in FIG. 5) 14 - SEQ ID NO: 17 (8 in FIG. 5)

[0107] An expression vector containing the constructs produced as in Example 2-1 was prepared, and the vector was introduced into one cell line (DC2.4 (mouse dendritic cells) and C2C12 (mouse muscle cells)) and transformed. By measuring the fluorescence value expressed in the transformant, the mRNA expression ability due to the UTR of the present invention was confirmed. In addition, the total protein expression amount over time after the transformation was measured. At this time, as a control group, the COVID-19 mRNA vaccine BNT162b2 sold by BioNTech and the COVID-19 mRNA vaccine mRNA-1273 sold by Moderna were used as the mRNA constructs for expressing the target protein. As a result, as shown in Fig. 5a, luciferase activity was observed in all cases where constructs 1 to 14 including the UTR of the present invention or a combination thereof were used, and in particular, the fluorescence values ​​were measured to be significantly higher in constructs 5, 6, and 13 than in the control group, indicating excellent mRNA expression ability.

[0108] [2-3] Confirmation of mRNA expression ability (in vivo)

[0109] In Example 2-2, mRNA constructs 5, 6, and 13, which were confirmed to have excellent mRNA expression ability, were encapsulated inside lipid nanoparticles (LNPs) and then injected into C57BL / 6J mice via intramuscular injection (IM). The LNP used in the Pfizer COVID-19 vaccine was used as the LNP, and specifically, an LNP composed of ionizable lipid (ALC0315), cholesterol, polyethylene glycol (PEG), and helper lipid was used. After 6 hours and 24 hours of injection of the mRNA constructs, the radiance changes and total fluorescence emission (Total Flux) appearing in the mice were measured using an in vivo bioluminescence imaging (BLI) device to confirm the mRNA expression ability. At this time, the COVID-19 mRNA vaccine BNT162b2 sold by BioNTech was used as a control group.

[0110] As a result, as shown in Fig. 5b, when the mRNA construct of the present invention was injected, the Total Flux value was measured to be higher than that of the control group, and in particular, when construct 13 was injected, the level of luciferase expression was the highest, confirming that the mRNA construct of the present invention has excellent mRNA expression ability.

[0111]

[0112] [Example 3]

[0113] Evaluation of in vivo immunogenicity of the mRNA construct of the present invention

[0114] In the construct 13 produced in the above Example 2-2, the immunogenicity of the construct was evaluated in order to increase the stability of mRNA and protein expression efficiency and to produce an mRNA construct more optimized for protein expression. The spike protein of SARS-CoV-2 was selected as the target protein, and a construct was produced that included a region encoding the spike protein, a 5'-UTR region of SEQ ID NO: 2 upstream of the region, and a 3'-UTR region of SEQ ID NO: 17 downstream. At this time, the COVID-19 mRNA vaccine BNT162b2 sold by BioNTech was used as a control. In order to evaluate the immunogenicity of the mRNA construct of the present invention, the construct was respectively entrapped inside two types of nanoparticles (ALC03415 and H9T6 LNP in FIG. 6) and then injected intramuscularly (IM) into mice for the first time (Day 0) and the second time (Day 21). After the first injection, mouse blood was obtained on Day 20 and Day 34, and the spleen of the mouse was isolated on Day 35 to compare the humoral and cellular immune responses in the mouse. Specifically, the absorbance (OD) of the antibody proteins immunoglobulin G (IgG) and immunoglobulin A (IgA) was measured using an enzyme-linked immunosorbent assay. 450 ) was measured, and the number of interferon-gamma secreting cells (IFN-gamma secreting cells) was measured.

[0115] As a result, as shown in Figures 6a and 6b, it was confirmed that when the construct of the present invention was injected, a strong humoral response and T cell response were exhibited compared to the control group. Therefore, it was confirmed that the construct of the present invention, when used as a vaccine, has an excellent ability to produce antibodies and induce an immune response.

[0116]

[0117] Although representative embodiments of the present application have been described above as examples, the scope of the present application is not limited to the specific embodiments described above, and a person with ordinary knowledge in the relevant field will be able to make appropriate changes within the scope described in the claims of the present application.

Claims

1. A gene encoding a target protein or peptide; and An mRNA structure comprising a 5'untranslated region (5'-UTR) comprising any one base sequence selected from the group consisting of base sequences of SEQ ID NO: 1 to SEQ ID NO: 15 linked upstream of a gene encoding the target protein or peptide, or a 3'untranslated region (3'-UTR) comprising any one base sequence selected from the group consisting of base sequences of SEQ ID NO: 16 to SEQ ID NO: 35 linked downstream of a gene encoding the target protein or peptide.

2. In claim 1, The above mRNA structure is An mRNA construct comprising both a 5'-UTR linked upstream of a gene encoding the target protein or peptide and a 3'-UTR linked downstream of a gene encoding the target protein or peptide.

3. In claim 1, The above 5'-UTR comprises any one base sequence selected from the group consisting of base sequences of SEQ ID NO: 1 to SEQ ID NO: 5 and SEQ ID NO: 9, An mRNA structure, wherein the 3'-UTR comprises a base sequence of SEQ ID NO: 16 or SEQ ID NO:

17.

4. In claim 3, The above 5'-UTR contains the base sequence of sequence number 1, An mRNA structure, wherein the above 3'-UTR comprises a base sequence of sequence number 16.

5. In claim 3, The above 5'-UTR contains the base sequence of sequence number 2, An mRNA structure, wherein the above 3'-UTR comprises a base sequence of sequence number 16.

6. In claim 3, The above 5'-UTR contains the base sequence of sequence number 2, An mRNA structure, wherein the above 3'-UTR comprises a base sequence of sequence number 17.

7. In claim 1, An mRNA structure wherein the 5'-UTR or the 3'-UTR has an activity of increasing the translation efficiency of a gene encoding a target protein, the stability of a gene encoding a target protein, or a combination thereof.

8. An expression vector comprising the mRNA construct of any one of claims 1 to 7 or a DNA sequence complementary thereto.

9. A transformant comprising the mRNA construct of any one of claims 1 to 7.

10. A pharmaceutical composition for preventing or treating a microbial infectious disease, comprising the mRNA construct of any one of claims 1 to 7.

11. In claim 10, A pharmaceutical composition wherein the above pharmaceutical composition is a vaccine.

12. A pharmaceutical composition for preventing or treating cancer or a genetic disease, comprising the mRNA construct of any one of claims 1 to 7.

13. In claim 12, The pharmaceutical composition above is a pharmaceutical composition which is a gene therapy agent.

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