Nucleic acid aptamer, RNA molecule, pharmaceutical composition, and template DNA molecule

Abstract

Provided are a nucleic acid aptamer and an RNA molecule that allow for cap-independent translation.　Provided are an aptamer that is specifically recognized by a translation initiation factor and an RNA molecule that includes the aptamer. The aptamer includes a nucleic acid sequence represented by (i) GGCGNx1YACGNNx2(m1Ψ)NKCKCC(m1Ψ)CDGGGMGMGCC or (ii) GGGGCGMGNGAMKCG(m1Ψ)H(m1Ψ)CKMCYMNGGKMGACCC (in which S is C or G, R is A or G, W is A or m1Ψ (1-methylpseudouridine), M is C or A, K is G or m1Ψ, Y is m1Ψ or C, V is A, C, or G, B is m1Ψ, C, or G, H is A, C, or m1Ψ, D is A, G, or m1Ψ, N is A, m1Ψ, C, or G, and x1 and x2 are the same integer selected from 3 to 8, inclusive) or a nucleic acid sequence that results from replacing, deleting, inserting, or adding 1–5 nucleic acid bases to a nucleic acid sequence represented by (i) or (ii).

Description

Nucleic acid aptamers, RNA molecules, pharmaceutical compositions, and template DNA molecules

[0001] The present invention relates to a nucleic acid aptamer that functions as an RNA sequence that enables mRNA translation in a Cap-independent manner, an RNA molecule comprising the nucleic acid aptamer, a pharmaceutical composition containing the RNA molecule, and a template DNA molecule.

[0002] mRNA medicines can express proteins loaded onto mRNA molecules by administering artificial mRNA molecules into the body. mRNA medicines have attracted attention because they have a lower probability of insertion into the genome than DNA medicines, and are more efficient to introduce and easier to manufacture than protein medicines. In theory, mRNA medicines can express a variety of proteins simply by changing the gene sequence they carry, so they are expected to be applied not only to vaccines but also to enzyme replacement therapy and regenerative medicine.

[0003] One factor that has made mRNA medicines a reality is the discovery that nucleic acid modifications of mRNA molecules are useful (see, for example, Non-Patent Document 1). In particular, the introduction of N-1 methyl-pseudouridine (m1Ψ) into mRNA molecules is known to reduce immunogenicity and increase translation efficiency (see, for example, Non-Patent Document 2). This has made it clear that its practical application in mRNA medicines is effective. In currently available mRNA vaccines, the natural base uridine is also substituted with m1Ψ. However, the introduction of m1Ψ is limited to linear mRNA molecules that have a cap structure and undergo cap-dependent translation.

[0004] The virus-derived internal ribosome entry site (IRES) sequence enables cap-independent translation from mRNA molecules and has been utilized for bicistronic expression of two genes from a single mRNA molecule and translation from uncapped circular RNA molecules (see, for example, Non-Patent Documents 3 and 4).

[0005] Immunity. 2005 Aug;23(2):165-75. doi: 10.1016 / j.immuni.2005.06.008.J Control Release. 2015 Nov 10;217:337-44. doi: 10.1016 / j.jconrel.2015.08.051.Nat Biotechnol. 2023 Feb;41(2):262-272. doi: 10.1038 / s41587-022-01393-0.Mol Cell. 2019 May 2;74(3):508-520.e4. doi: 10.1016 / j.molcel.2019.02.015.

[0006] Although IRES-mediated cap-independent translation of mRNA molecules has attracted attention as a new tool for mRNA medicine, its application in medicine has yet to be achieved. A major factor behind this is the inability of IRESs to tolerate modification by m1Ψ (Non-Patent Document 4). Because the introduction of m1Ψ into an IRES leads to impaired translation initiation, the development of functional mRNA molecules containing m1Ψ has not been achieved to date. For the pharmaceutical application of bicistronic expression and circular RNA molecules, RNA molecules that retain translation activity even when containing m1Ψ are essential.

[0007] As a result of intensive research, the present inventors have found that a nucleic acid aptamer consisting of an artificial nucleic acid sequence having a specific sequence allows modification by m1Ψ and activates mRNA translation in a cap-independent manner, thereby completing the present invention. That is, the present invention includes the following aspects. [1] An aptamer that is specifically recognized by a translation initiation factor, the aptamer having the following formula (i) or (ii): GGCGN x1 YACGNN x2 (m1Ψ)NKCKCC(m1Ψ)CDGGGMGMGCC(GGCGN x1 YACGNN x2- SEQ ID NO: 1) (i) GGGGCGMGNGAMKCG(m1Ψ)H(m1Ψ)CKMCYMNGGKMGACCC (SEQ ID NO: 2) (ii) (in formula (i) or (ii), S is C or G, R is A or G, W is A or m1Ψ (1-methylpseudouridine), M is C or A, K is G or m1Ψ, Y is m1Ψ or C, V is A, C or G, B is m1Ψ, C or G, H is A, C or m1Ψ, D is A, G or m1Ψ, N is A, m1Ψ, C or G, and x1 and x2 are the same integer selected from 3 to 8), or a nucleic acid sequence in which 1 to 5 nucleic acid bases have been substituted, deleted, inserted or added in the nucleic acid sequence. [2] In formula (i), N x1 and N x2[1] The aptamer according to [1], wherein formula (i) is selected from SEQ ID NOs: 3 to 24, 43, and 44. [4] The aptamer according to [1], wherein formula (ii) is formula (ii) wherein GCGM and KCG(m1Ψ) form the first stem, (m1Ψ)CKMC and GKMGA form the second stem, and GG at the 5'-end and CC at the 3'-end form the third stem. [5] The aptamer according to [4], wherein formula (ii) is selected from SEQ ID NOs: 25 to 40. [6] The aptamer according to [1], wherein formula (i) is formula (ii) and the nucleic acid sequence having 1 to 5 nucleobases substituted, deleted, inserted, or added comprises SEQ ID NO: 45. [7] An RNA molecule comprising: (I) a translation activation region comprising the nucleic acid aptamer of [1]; and (II) a translation region located 3'-terminally closer to the nucleic acid aptamer, wherein the RNA molecule does not comprise a 5'-cap structure. [8] The RNA molecule of [7], wherein the translation region comprises a nucleic acid sequence encoding a detection protein, a therapeutic protein, or an RNA-binding protein. [9] The RNA molecule of [7], wherein the RNA molecule is a linear molecule comprising a Poly A sequence on the 3'-terminal side of the early translation region.

[10] The RNA molecule of [7], wherein the RNA molecule is a circular molecule comprising a Poly A sequence on the 3'-terminal side of the early translation region.

[11] A pharmaceutical composition comprising the RNA molecule of [7].

[12] A template DNA molecule comprising a sequence encoding the aptamer of [1].

[0008] The nucleic acid aptamer and mRNA molecule comprising the same according to the present invention allow modification by m1Ψ and enable cap-independent translation, thereby realizing the application of mRNA in pharmaceuticals.

[0009] Figure 1 is a diagram conceptually illustrating the secondary structure of the nucleic acid aptamer sequence represented by Sequence 1-WT in Table 1. Figure 2 is a diagram conceptually illustrating the secondary structure of the nucleic acid aptamer sequence represented by Sequence 2-WT in Table 2. Figure 3 shows the design of an mRNA molecule according to one embodiment of the present invention. mRNA (A) was prepared in which the nucleic acid aptamer sequence according to one embodiment of the present invention was introduced into the 5'-UTR. For comparison, an mRNA without a translation initiation sequence (B) and an IRES-containing mRNA (C) were prepared. An mRNA encoding the fluorescent reporter gene iRFP as a transfection marker and containing a translationally functional cap structure (ARCA) was prepared (D). Figure 4 shows the results of normalizing the EGFP fluorescence intensity with that of iRFP670 in HEK293FT cells transfected with mRNAs containing the Sequence 1-WT sequence shown in Table 1 and the Sequence 2-WT sequence shown in Table 2 in the 5'-UTR. Error bars in the figure indicate standard deviation (n = 3). Figure 5 shows the mRNA design used for mutation introduction analysis, structural stability analysis, and deletion / insertion analysis. Figure 6 shows the results of normalizing EGFP fluorescence intensity to iRFP670 fluorescence intensity in HEK293FT cells transfected with mRNA for point mutation introduction analysis into Sequence 1-WT. Error bars indicate standard deviation (n = 3). Figure 7 shows the results of normalizing EGFP fluorescence intensity to iRFP670 fluorescence intensity in HEK293FT cells transfected with mRNA for point mutation introduction analysis into Sequence 2-WT. Error bars indicate standard deviation (n = 3). Figure 8 shows the results of normalizing EGFP fluorescence intensity to iRFP670 fluorescence intensity in HEK293FT cells transfected with mRNA for structural stability analysis of Sequence 1-WT and Sequence 2-WT, and deletion / insertion analysis of Sequence 1-WT. In the figure, error bars indicate standard deviation (n = 3). Figure 9 shows the results of normalizing the luminescence intensity of Nluc by that of Fluc in HEK293FT cells transfected with circular mRNA carrying Sequence 2, with the No motif circular RNA used as a control. In the figure, error bars indicate standard deviation (n = 3).

[0010] Hereinafter, embodiments of the present invention will be described, but the present invention is not limited to the embodiments described below.

[0011] 1. Nucleic Acid Aptamer According to a first embodiment, the present invention relates to a nucleic acid aptamer that is specifically recognized by a translation initiation factor. The nucleic acid aptamer is represented by the following formula (i) or (ii): GGCGN x1 YACGNN x2 (m1Ψ)NKCKCC(m1Ψ)CDGGGMGMGCC (i) GGGGCGMGNGAMKCG(m1Ψ)H(m1Ψ)CKMCYMNGGKMGACCC (ii) (in formula (i) or (ii), S is C or G, R is A or G, W is A or m1Ψ (1-methylpseudouridine), M is C or A, K is G or m1Ψ, Y is m1Ψ or C, V is A, C or G, B is m1Ψ, C or G, H is A, C or m1Ψ, D is A, G or m1Ψ, N is A, m1Ψ, C or G, and x1 and x2 are the same integer selected from 3 to 8), or a nucleic acid sequence in which 1 to 5 nucleic acid bases have been substituted, deleted, inserted or added. N of the sequence represented by formula (i) x2 The sequence located on the 3'-terminal side of the formula ((m1Ψ)NKCKCC(m1Ψ)CDGGGMGMGCC) is represented by SEQ ID NO: 1. The sequence represented by formula (ii) is represented by SEQ ID NO: 2.

[0012] In formula (i), N x1 and N x2 forms the stem, KCKCC and GGMGM form the stem, and GG at the 5' end and CC at the 3' end form the stem. x1 and N x2"KCKCC" and "GGMGM" form a stem means that these sequences are completely complementary. "KCKCC" and "GGMGM" form a stem means that these sequences are completely complementary. In this specification, "completely complementary" means that the base pairs forming the stem are composed of an A-m1Ψ base pair, a GC base pair, or a G-m1Ψ base pair. When U is contained in place of m1Ψ, an AU base pair or a GU base pair may be contained. In this specification, N x1 and N x2 The stem formed by KCKCC and GGMGM is also referred to as the first stem of the nucleic acid aptamer of formula (i), the stem formed by GG at the 5'-end and CC at the 3'-end as the second stem of the nucleic acid aptamer of formula (i), and the stem formed by GG at the 5'-end and CC at the 3'-end as the third stem of the nucleic acid aptamer of formula (i). The second stem can also be referred to as the stem formed by the nucleobases 4 to 8 from the 3'-end and the nucleobases 13 to 17 from the 3'-end of formula (i). The stem formed by GG at the 5'-end and CC at the 3'-end is also referred to as the third stem of the nucleic acid aptamer of formula (i). The third stem can also be referred to as the stem formed by the nucleobases 1 to 2 from the 5'-end and the nucleobases 1 to 2 from the 3'-end of formula (i).

[0013] In formula (ii), GCGM and KCG(mΨ) form a stem, (mΨ)CKMC and GKMGA form a stem, and GG at the 5' end and CC at the 3' end form a stem. GCGM and KCG(mΨ) forming a stem means that they are completely complementary. (mΨ)CKMC and GKMGA forming a stem means that they are completely complementary. In this specification, the stem formed by GCGM and KCG(mΨ) is also referred to as the first stem of the nucleic acid aptamer of formula (ii), the stem formed by (mΨ)CKMC and GKMGA is also referred to as the second stem of the nucleic acid aptamer of formula (ii), and the stem formed by GG at the 5' end and CC at the 3' end is also referred to as the third stem of the nucleic acid aptamer of formula (ii). The first stem can also be referred to as a stem formed by the 4th to 7th bases from the 5' side and the 13th to 16th bases from the 3' side of formula (ii), and the second stem can also be referred to as a stem formed by the 18th to 22nd bases from the 5' side and the 27th to 31st bases from the 3' side of formula (ii). The stem formed by GG at the 5' end and CC at the 3' end is also referred to as the third stem of the nucleic acid aptamer of formula (ii). The third stem can also be referred to as a stem formed by the nucleobases at the 1st to 2nd bases from the 5' side and the 33rd to 34th bases from the 5' side of formula (ii).

[0014] The nucleic acid aptamer according to the first embodiment has a nucleic acid sequence that is specifically recognized by a translation initiation factor. The translation initiation factor is not particularly limited, but examples include, but are not limited to, eIF4, such as eIF4A, eIF4B, eIF4A, eIF4D, eIF4E, eIF4F, eIF4G, eIF4H, eIF4I, eIF4J, and eIF4K. The translation initiation factor may preferably be eIF4E. Specifically, the term "specifically recognized by a translation initiation factor" can be quantitatively defined by the binding constant between the translation initiation factor and the aptamer. More specifically, the equilibrium dissociation constant (K Ｄ) is 100 nM or less, preferably 80 nM or less. A nucleic acid aptamer having a nucleic acid sequence represented by formula (i) or (ii), and a nucleic acid sequence in which 1 to 5 nucleic acid bases have been substituted, deleted, inserted, or added, satisfy the above definition and are specifically recognized by a translation initiation factor. When the nucleic acid aptamer is introduced into the 5'-UTR of mRNA, it can activate translation independently of the 5' cap structure.

[0015] Specific examples of nucleic acid aptamers satisfying formula (i) according to this embodiment include SEQ ID NOs: 3 to 24 in Table 1 below. In Table 1, the sequence shown in SEQ ID NO: 3 is the basic sequence of the nucleic acid aptamer of formula (i) and is referred to as Sequence 1 or Sequence 1-WT. FIG. 1 is a conceptual diagram illustrating the secondary structure of Sequence 1-WT. Sequence 1-WT comprises a first stem and a first loop connecting it, a second stem and a second loop connecting it, and a third stem formed of two bases at the 5' end and two at the 3' end. The sequences shown in SEQ ID NOs: 4 to 24 are sequences in which a single-base point mutation (SEQ ID NOs: 4 to 17), a double-base point mutation (SEQ ID NOs: 18 to 23), or a triple-base point mutation (SEQ ID NO: 24) has been introduced based on Sequence 1 (SEQ ID NO: 3). The mutated bases are indicated in boxed letters.

[0016]

[0017] Specific examples of nucleic acid aptamers satisfying formula (ii) according to this embodiment include SEQ ID NOs: 25 to 40 in Table 2 below. In Table 2, the sequence shown in SEQ ID NO: 25 is the basic sequence of the nucleic acid aptamer of formula (ii) and is referred to as Sequence 2 or Sequence 2-WT. FIG. 2 is a conceptual diagram illustrating the secondary structure of Sequence 2-WT. Sequence 2 comprises a first stem and a first loop connecting it, a second stem and a second loop connecting it, and a third stem formed of two bases each at the 5' end and the 3' end. The sequences of SEQ ID NOs: 26 to 41 are sequences in which a single-base point mutation (SEQ ID NOs: 26 to 34), a two-base point mutation (SEQ ID NOs: 35 to 39), or a three-base point mutation (SEQ ID NO: 40) has been introduced based on Sequence 2 (SEQ ID NO: 25).

[0018]

[0019] Specific sequences that have been structurally stabilized or have had insertions or deletions introduced relative to Sequence 1 are shown in SEQ ID NOs: 41 to 45 below. Specific sequences that have been structurally stabilized relative to Sequence 2 are shown in SEQ ID NOs: 46 and 47 below.

[0020] In each of the nucleic acid sequences described above, sequences in which approximately 5% or less of the total number of m1Ψ are substituted with the natural base uridine (U) can also function as nucleic acid aptamers that are specifically recognized by translation initiation factors.

[0021] The nucleic acid aptamer according to the first embodiment of the present invention is a sequence that is specifically recognized by a translation initiation factor. Therefore, by introducing the nucleic acid aptamer into the 5'-UTR of an mRNA, the aptamer can be used as a translation initiation sequence in cap-independent mRNA translation, despite containing the modified base m1Ψ.

[0022] 2. RNA Molecule (Linear) According to a second embodiment, the present invention relates to an RNA molecule. The RNA molecule comprises the following regions (a) and (b), but does not comprise a 5' cap structure: (a) a translation activation region comprising the nucleic acid aptamer described in the first embodiment; (b) a translation region located on the 3' end side of the nucleic acid aptamer; Optionally, the RNA molecule may also comprise the following region (c): (c) a 5' end region located on the 5' end side of the nucleic acid aptamer; and (d) a first arbitrary nucleic acid sequence may be included between regions (a) and (c), and (e) a second arbitrary nucleic acid sequence may be included between regions (a) and (b).

[0023] The RNA molecule according to this embodiment is typically an mRNA in which a protein encoded by the translation region is translated in a cell or a cell-free translation system, and is an artificial mRNA synthesized in vitro. The structure and design of the RNA molecule according to this embodiment will be described below.

[0024] (a) Translation activation region The translation activation region is a region containing a nucleic acid sequence capable of activating a translation region independently of the 5' cap, and the nucleic acid sequence is a nucleic acid sequence consisting of the nucleic acid aptamer described in detail in Embodiment 1. The translation activation region is located on the 5' side of the translation region of (b).

[0025] (b) Translation Region The translation region is a region containing a nucleic acid sequence translated by a translation activation region, and typically contains a nucleic acid sequence encoding a protein. More specifically, the translation region contains, from the 5' end, an initiation codon, a nucleic acid sequence encoding the protein, and a termination codon. The term "protein" as used herein also includes protein fragments, fusion proteins, and peptides.

[0026] The protein may be any protein, and the type and number thereof are not limited. Desired proteins can be included depending on the use and purpose of the RNA molecule. For example, the protein may include a detection protein, a therapeutic protein, or an RNA-binding protein capable of binding to other RNA molecules. The detection protein, therapeutic protein, or RNA-binding protein is not limited to proteins that independently perform a detection, treatment, or binding function, but may also be a substance that performs the detection, treatment, or binding function together with a substance other than the protein encoded by the RNA molecule.

[0027] A detection protein refers to any protein that can be translated to display detectable information, and may be a protein that can be visualized and quantified by, or with the aid of, fluorescence, luminescence, or color.

[0028] Examples of fluorescent proteins include blue fluorescent proteins such as Sirius and EBFP; cyan fluorescent proteins such as mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan, and CFP; green fluorescent proteins such as TurboGFP, AcGFP, TagGFP, Azami-Green (e.g., hmAG1), ZsGreen, EmGFP, EGFP, GFP2, and HyPer; yellow fluorescent proteins such as TagYFP, EYFP, Venus, YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, and mBanana; and KusabiraOrange. (e.g., hmKO2) and mOrange; red fluorescent proteins such as TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, and mStrawberry; and near-infrared fluorescent proteins such as TurboFP602, mRFP1, JRed, KillerRed, mCherry, HcRed, KeimaRed (e.g., hdKeimaRed), mRasberry, and mPlum. An example of a photoprotein is, but is not limited to, aequorin.

[0029] Examples of proteins that assist fluorescence, luminescence, or color development include, but are not limited to, enzymes that decompose fluorescent, luminescent, or color precursors, such as luciferase, phosphatase, peroxidase, and β-lactamase. When using an RNA molecule containing a nucleic acid sequence encoding a protein that assists fluorescence, luminescence, or color development in its translation region, the molecule must be used in a manner that allows contact between the corresponding precursor and the protein produced by translation of the RNA molecule. For example, the precursor can be contacted with cells into which the RNA molecule has been introduced, or the corresponding precursor can be introduced into cells into which the RNA molecule has been introduced.

[0030] A therapeutic protein is a protein that can be used to treat, prevent, or diagnose diseases or conditions by affecting cellular function. Affecting cellular function includes increasing, decreasing, or maintaining a specific cellular function within a certain range. Examples of therapeutic proteins include, but are not limited to, cell proliferation proteins, cell death proteins, cell signaling factors, drug resistance genes, transcriptional regulators, translational regulators, differentiation regulators, reprogramming inducers, RNA-binding protein factors, chromatin regulators, membrane proteins, and fragments or complexes thereof. These proteins can also be said to be capable of displaying detectable information by affecting cellular function, and can therefore be considered both therapeutic and detection proteins. Other therapeutic proteins include, but are not limited to, enzymes, growth factors, antibodies, antigens, proteins constituting viruses or portions thereof, proteins that inhibit virus production, genome editing proteins, chimeric antigen receptors, and fragments or complexes thereof.

[0031] For example, cell proliferation proteins function as markers by allowing only the cells that express them to proliferate and identifying the proliferated cells. Cell death proteins cause cell death in the cells that express them, killing cells that either contain or do not contain a specific molecule (target substance), thereby functioning as markers indicating cell viability. Cell signaling factors function as markers by allowing cells that express them to emit specific biological signals and identifying these signals. Examples of cell death proteins include, but are not limited to, RNA enzymes such as barnase derived from Bacillus amyloliquefaciens, toxins such as HokB, Fst, GhoT (membrane disruption), HipA (inhibition of nucleic acid elongation by phosphorylation), RelE, YafO, VapC, MazF, MqsR, PemKHicA (endonuclease), FicT (adenylation), oc (phosphorylation), CcdB, ParE (gyrase inhibitor), Tact (inhibitor of translation), and cbtA (inhibitor of cytoskeletal protein), and apoptosis-inducing proteins such as Bax and Bim. Translational regulatory factors, for example, function as markers by recognizing and binding to the tertiary structure of specific RNAs and controlling the translation of other mRNAs into proteins. The translational regulatory factors include 5R1, 5R2 (Nat Struct Biol. 1998 Jul; 5(7):543-6), B2 (Nat Struct Mol Biol. 2005 Nov;12(11):952-7), Fox-1 (EMBO J. 2006 Jan 11;25(1):163-73), GLD-1 (J Mol Biol. 2005 Feb 11;346(1):91-104), Hfq (EMBO J. 2004 Jan 28;23(2):396-405), HuD (Nat Struct Biol. 2001 Feb;8(2):141-5), SRP19 (RNA. 2005 Jul;11(7):1043-50), and L1 (Nat Struct Biol. 2003 Feb;10(2):104-8.), L11 (Nat Struct Biol.2000 Oct;7(10):834-7.)、L18(Biochem J. 2002 Mar 15;362(Pt 3):553-60)、L20(J Biol Chem. 2003 Sep 19;278(38):36522-30.)、L23(J Biomol NMR. 2003 Jun;26(2):131-7)、L25(EMBO J. 1999 Nov 15;18(22):6508-21.)、L30(Nat Struct Biol. 1999 Dec;6(12):1081-3.)、LicT(EMBO J. 2002 Apr 15;21(8):1987-97.)、MS2 coat(FEBS J. 2006 Apr;273(7):1463-75.)、Nova-2(Cell. 2000 Feb 4;100(3):323-32)、Nucleocapsid(J Mol Biol. 2000 Aug 11;301(2):491-511.)、Nucleolin(EMBO J. 2000 Dec 15;19(24):6870-81.)、p19(Cell. 2003 Dec 26;115(7):799-811)、L7Ae(RNA. 2005 Aug;11(8):1192-200.)、PAZ(PiWi Argonaut and Zwille)(Nat Struct Biol. 2003 Dec;10(12):1026-32.)、RnaseIII(Cell. 2006 Jan 27;124(2):355-66)、RR1-38(Nat Struct Biol. 1998 Jul;5(7):543-6.)、S15(EMBO J. 2003 Apr 15;22(8):1898-908.)、S4(J Biol Chem. 1979 Mar 25;254(6):1775-7.)、S8(J Mol Biol. 2001 Aug 10;311(2):311-24.)、SacY(EMBO J. 1997 Aug 15;16(16):5019-29.)、SmpB(J Biochem (Tokyo). 2005 Dec;138(6):729-39)、snRNP U1A(Nat Struct Biol. 2000 Oct;7(10):834-7.)、SRP54(RNA.2005 Jul;11(7):1043-50), Tat (Nucleic Acids Res. 1996 Oct 15;24(20):3974-81), ThrRS (Nat Struct Biol. 2002 May;9(5):343-7), TIS11d (Nat Struct Mol Biol. 2004 Mar;11(3):257-64), Virp1 (Nucleic Acids Res. 2003 Oct 1;31(19):5534-43), Vts1P (Nat Struct Mol Biol. 2006 Feb;13(2):177-8), and λN (Cell. 1998 Apr 17;93(2):289-99).

[0032] The translation region may include a nucleic acid sequence encoding a fusion protein consisting of a combination of two or more of the above-mentioned proteins, or may include a self-cleaving sequence between the two or more proteins, so that the translated proteins function as separate protein molecules.

[0033] (c) 5'-terminal region The 5'-terminal region is an optional region, and may have a structure that prevents the degradation of the RNA molecule by exoribonucleases, such as a structure in which the OH group at the 5'-terminal is blocked, such as G(5')ppp(5')A (A-Cap), G(5')ppp(5')G (G-Cap), Cl 6 G(5')ppp(5')G,m 1 G(5')ppp(5')G and the like can be used, but are not limited to these specific sequences.

[0034] The 5'-terminal region of the RNA molecule does not contain a 5'-cap structure. A 5'-cap structure refers to a structure present at the 5'-terminal of an RNA molecule and known as a modified structure recognized by translation initiation factors. Known 5'-cap structures include, but are not limited to, 7-methylguanosine 5'-phosphate (Cap structure), Ambion's Anti-Reverse Cap Analog (ARCA), New England Biolabs' m7G(5')ppp(5')G RNA Cap Structure Analog, and TriLink's CleanCap. Note that the nucleic acid aptamer according to the first embodiment is a "modified structure recognized by translation initiation factors," but does not fall under the category of a 5'-cap structure.

[0035] (d) First Arbitrary Nucleic Acid Sequence A first arbitrary nucleic acid sequence may be contained within the region corresponding to the 5'-UTR of the RNA molecule, between the translation activation region (a) and the optional 5'-terminal region (c). The first arbitrary nucleic acid sequence may be a nucleic acid sequence of one or more bases, for example, a nucleic acid sequence of about 1 to 200 bases, preferably about 50 to 120 bases. The first arbitrary nucleic acid sequence may be a sequence that simply functions as a spacer, or a sequence having a specific function, and is not particularly limited.

[0036] (e) Second Arbitrary Nucleic Acid Sequence A second arbitrary nucleic acid sequence may be contained within the region corresponding to the 5'-UTR of the RNA molecule, between the translation activation region (a) and the start codon of the translation region (b). The second arbitrary nucleic acid sequence may be a nucleic acid sequence of one or more bases, for example, about 1 to 150 bases, or in some cases, about 10 to 30 bases. The second arbitrary nucleic acid sequence may be a sequence that simply functions as a spacer, or may be a sequence having a specific function, and is not particularly limited.

[0037] (f) Poly A sequence The region corresponding to the 3'-UTR of the RNA molecule, i.e., the region 3' to the translated region (b), contains a poly A sequence. The total length of A in the poly A sequence may be 50 mer or more, and nucleic acid bases other than A may be included in the middle.

[0038] In the RNA molecule according to this embodiment, the sugar residue (ribose) of each nucleotide may be modified for purposes such as reducing cytotoxicity, as long as the translation effect of the RNA molecule according to this embodiment is not impaired. As described in the first embodiment, region (a) contains mΨ instead of the natural base uridine. Regions (b) and (c), as well as the nucleic acid sequence that may be optionally contained, also contain mΨ instead of the natural base uridine. However, in regions (b), (c), and the optional nucleic acid sequence, it is sufficient that mΨ accounts for 95% or more of the total number of uridine bases or modified bases thereof, and in some cases, less than 5% of the natural base uridine or pseudouridine (Ψ) may be contained.

[0039] Furthermore, to enhance the resistance of the RNA molecule to nucleases and hydrolysis, the phosphate groups (e.g., terminal phosphate residues) contained in the RNA molecule may be modified as long as the function of the RNA molecule according to this embodiment is not impaired. For example, the phosphate group P(O)O may be substituted with P(O)S (thioate), P(S)S (dithioate), P(O)NR2 (amidate), P(O)R, R(O)OR', CO or CH2 (formacetal), or 3'-amine (-NH-CH2-CH2-) (wherein each R or R' is independently H or substituted or unsubstituted alkyl (e.g., methyl, ethyl)). Examples of linking groups include -O-, -N-, and -S-, and adjacent nucleotides can be linked via these linking groups.

[0040] Once the molecular structure and nucleic acid sequence of the RNA molecule according to this embodiment have been determined as described above, those skilled in the art can synthesize it using any known genetic engineering method. According to one embodiment, the RNA molecule according to this embodiment can be obtained as a synthetic RNA molecule by an in vitro synthesis method using a template DNA containing a promoter sequence. RNA molecules that do not contain a 5' cap structure can be produced by omitting the capping step in a conventional method for synthesizing mRNA molecules. The ability to obtain a synthetic RNA molecule as designed using a simple method is one advantage of the present invention.

[0041] According to the linear RNA molecule of the second embodiment of the present invention, the artificial RNA molecule containing N1-methylpseudouridine (N1mΨ) enables cap-independent translation, thereby enabling the production of RNA molecules with low immunogenicity and high protein translation efficiency, which are useful as mRNA medicines.

[0042] 3. RNA Molecule (Circular) According to a third embodiment, the present invention relates to a circular RNA molecule. The circular RNA molecule includes the following regions (A) and (B): (A) a translation activation region including the nucleic acid aptamer described in the first embodiment; and (B) a translation region located on the 3'-end side of the nucleic acid aptamer.

[0043] The circular RNA molecule comprises, in the 5' to 3' direction, (A) a translation activation region and (B) a translation region operably linked in this order, and may optionally further comprise (C) a poly A sequence, (D) a junction stabilizing sequence, and (E) a spacer sequence.

[0044] The (A) translation activation region, (B) translation region, and (C) poly A sequence may be the same as the (a) translation activation region, (b) translation region, and (f) poly A sequence of the linear RNA molecule according to embodiment 3. The poly A sequence may or may not be present, but if present, the total length of As may be 30 mer or more, preferably 80 mer or more, and in either case, nucleic acid bases other than As may be included in the middle.

[0045] (D) Junction Stabilizing Sequence: A junction stabilizing sequence is a sequence derived from a sequence introduced to increase the reaction efficiency during the circularization process, and may be included in circular RNA molecules that are circularized using ligase, in particular. The junction stabilizing sequence may be, for example, a sequence that forms a stem-loop structure of 50 to 80 bases in length and includes a junction point for circularization. The junction stabilizing sequence is divided into two parts in the linear RNA molecule that serves as the precursor of the circular RNA molecule, and is provided at (A) the 5'-end of the translation activation region and (B) the translating region or (C) the 3'-end of the polyA sequence. Details will be described later. However, circularization of linear RNA molecules can be performed without a junction stabilizing sequence, and a junction stabilizing sequence is not essential.

[0046] (E) Spacer Sequence The circular RNA molecule may contain a spacer sequence that does not fall into any of the above categories between the (A) translation activation region, (B) translation region, and optionally, the (C) poly A sequence and (D) connection stabilizing sequence. The spacer sequence is preferably a sequence that does not inhibit the function of the aptamer sequence of the translation activation region, a sequence used for circularization of group I introns, or the poly A sequence, and is, for example, preferably 1 to 100 bases long, more preferably 20 to 80 bases long.

[0047] In the circular RNA molecule, the above (A) translation activation region and (B) translation region are operably linked, and when the RNA molecule is introduced into a cell and the translation activation region is recognized by a translation initiation factor, translation of the protein encoded by the translation region is initiated.

[0048] In one embodiment, the circular RNA molecule preferably comprises the following in the 5' to 3' direction, taking the (A) translation activation region as the starting point: [(A) translation activation region] - [(B) translation region] - [(C) polyA sequence] - [(D) connection stabilizing sequence], where the 3' end of [(D) connection stabilizing sequence] is connected to the 5' end of [(A) translation activation region]. The [(C) polyA sequence], the [(D) connection stabilizing sequence], or both may be omitted. In such cases, the 3' end of [(B) translation region] may be connected to the 5' end of [(A) translation activation region], or the 3' end of [(C) polyA sequence] may be connected to the 5' end of [(A) translation activation region]. Furthermore, the (E) spacer sequence may be included between the 3' end of [(D) connection stabilizing sequence] and the 5' end of [(A) translation activation region].

[0049] In the circular RNA molecule, as in the linear RNA molecule, region (A) contains m1Ψ instead of the natural base uridine. Regions (B) to (E) also contain m1Ψ instead of the natural base uridine. The proportion of natural bases contained may be the same as in the second embodiment. Furthermore, as in the second embodiment, any other modified base may be contained, and a modified phosphate group may also be contained.

[0050] The circular RNA molecule of this embodiment can be produced using any circularization technique once the molecular structure and nucleic acid sequence are determined as described above. A specific method for producing the circular RNA molecule of this embodiment in vitro includes the following steps: (1) obtaining a linear RNA molecule containing a linear nucleic acid sequence containing at least (A) a translation activation region and (B) a translation region, as determined as described above; and (2) circularizing the linear RNA molecule or a precursor RNA molecule obtained therefrom.

[0051] In the first step of synthesizing a linear RNA molecule, a linear RNA molecule is designed comprising a nucleic acid sequence containing at least (A) and (B), which are components of a circular RNA molecule. Preferably, (A) is located at the 5' end of the linear RNA molecule, (B) is located at the 3' end, and optionally, a (C) polyA sequence is located at the 3' end of (B). When a (D) connection stabilizing sequence is included, a sequence having a stem-loop structure that serves as the connection stabilizing sequence is bisected, for example, at the base portion forming the loop or at a base portion near the loop, and the 5'-end sequence of the connection stabilizing sequence is located at the 3' end of (B) or (C). Furthermore, the 3'-end sequence of the bisected connection stabilizing sequence is located at the 5' end of (A). Next, a linear RNA molecule comprising a nucleic acid sequence containing (A) and (B), and optionally (C) and / or (D) and / or (E), can be obtained by in vitro synthesis using a template DNA containing a promoter sequence as a template.

[0052] The second step can be performed using any nucleic acid circularization technique. One example is a circularization method using ligase. In this case, a linear RNA molecule is designed and prepared, comprising a divided 5'-end fragment of [(D) junction stabilizing sequence] at the 3' end and a divided 3'-end fragment at the 5' end. The triphosphate at the 5' end of the prepared linear RNA molecule is removed with anthratic phosphatase to form a hydroxyl group, and the 5' end is then converted to a monophosphate state with T4 polynucleotide kinase. The hydroxyl group at the 3' end and the phosphoryl group at the 5' end are then ligated using a ligase such as T4 RNA ligase to prepare a circular RNA. If unreacted linear RNA molecules remain, they can be treated with terminator exonuclease or RNase R, an enzyme that degrades mRNA from the termini, or purified by gel or HPLC to obtain the desired circular RNA molecule.

[0053] Techniques for circularizing linear RNA molecules are not limited to methods using ligases, and various known techniques can be used. For example, they can be performed using a trans-splicing-based method (Du, Y., Zuber, PK, Xiao, H. et al. Efficient circular RNA synthesis for potent rolling circle translation. Nat. Biomed. Eng (2024). https: / / doi.org / 10.1038 / s41551-024-01306-3). They can also be performed using a self-splicing intron (WO2024 / 010028, Wesselhoeft RA., et al. Nat. Commun. 9(1), 2629 (2018) and JP2021-526792). Furthermore, linear RNA molecules can be circularized via functional groups at their 3' and / or 5' ends by chemical ligation. Additionally, circular permuted group II introns can be used to circularize linear RNA. Circular mRNA molecules can be produced by any method that can circularize RNA molecules, in addition to the methods specifically described herein.

[0054] According to the third embodiment of the present invention, the circular RNA molecule contains N1-methylpseudouridine (N1mΨ), enabling cap-independent translation. As a result, it is possible to obtain an RNA molecule with low immunogenicity, high protein translation efficiency, and improved stability, which is useful as an mRNA medicine.

[0055] 4. Pharmaceutical Composition According to a fourth embodiment, the present invention relates to a pharmaceutical composition, in particular a pharmaceutical composition comprising an RNA molecule according to the second or third embodiment.

[0056] The pharmaceutical composition according to this embodiment may be a pharmaceutical composition used to treat, prevent, or diagnose a specific disease in a multicellular organism by activating protein translation in the organism. The multicellular organism is preferably a mammal (e.g., human, mouse, monkey, pig, rat, etc.), more preferably human.

[0057] The pharmaceutical composition according to this embodiment can be widely applied to diseases for which a therapeutic effect is expected by altering a specific function in cells.

[0058] The RNA molecule contained in the pharmaceutical composition for cancer treatment can be, for example, an RNA molecule having a translation region encoding a protein that kills cancer cells. Such a pharmaceutical composition is expected to have a therapeutic effect when administered to a subject suffering from cancer, and to have a recurrence prevention effect when administered to a subject after cancer treatment. Therefore, the pharmaceutical composition according to this embodiment can be suitably used as a cancer treatment agent or recurrence prevention agent.

[0059] As the RNA molecule contained in the pharmaceutical composition for virus-infected cells, for example, an RNA molecule having a translation region encoding a protein useful for killing and removing viruses can be used.

[0060] The pharmaceutical composition according to the present embodiment may contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a substance commonly used in the pharmaceutical technology field that is added to facilitate the formulation of a pharmaceutical composition and its application to a living body without inhibiting or suppressing its function. Examples of carriers include excipients, binders, disintegrants, fillers, emulsifiers, flow regulators, lubricants, and stabilizers.

[0061] The pharmaceutical composition according to this embodiment can be introduced into cells in vivo. Any commonly used method for introducing RNA into cells in vivo can be used. For example, in mammals, RNA molecules can be directly introduced into cells using intramuscular injection, subcutaneous injection, intravenous injection, intra-articular injection, or other methods. The RNA can also be introduced by being supported on a drug delivery carrier known in the art. Examples of such carriers include polymer nanoparticles, polymer micelles, dendrimers, liposomes, viral nanoparticles, and carbon nanotubes (see, for example, Cho K. et al., Clin Cancer Res. 2008 Mar 1;14(5):1310-6).

[0062] The present invention will be described in more detail below with reference to examples, which are not intended to limit the scope of the present invention.

[0063] [Example 1] [mRNA Design] mRNA encoding the fluorescent reporter gene EGFP was used. The translation initiation sequences used were Sequence 1-WT (Table 1) and Sequence 2-WT (Table 2). Both sequences were confirmed to specifically recognize the translation initiation factor eIf4e. mRNAs were designed with these translation initiation sequences in the 5'-UTR (Figure 3(A)). For comparison, mRNAs containing a natural IRES (Figure 3(B)), mRNA lacking a translation initiation sequence (Figure 3(C)), and mRNA with a translationally functional cap structure (Figure 3(D)) were prepared. As a transfection marker, mRNA encoding the fluorescent reporter gene iRFP and with a translationally functional cap structure (ARCA) was used (Figure 3(D)).

[0064] [Construction of IVT template DNA] Template DNA for in vitro transcription was amplified by PCR (TOYOBO) using appropriate oligonucleotides (Eurofin). The forward primer for IVT template synthesis was a T7 promoter (Primer-F1, SEQ ID NO: 48), and the reverse primer was a poly(T)-tailed primer (Primer-R1, SEQ ID NO: 49). The T7 promoter and poly(A) tail were added during PCR amplification. After amplification, the template plasmid was digested with DpnI (TOYOBO) and purified using the Monarch PCR & DNA Cleanup kit (New England Biolab: NEB). The sequences of the template plasmid DNAs are shown in SEQ ID NO: 50 (template Sequence 1-WT) for Sequence 1 and SEQ ID NO: 51 (template Sequence 2-WT) for Sequence 2.

[0065] [MRNA Preparation] mRNA was prepared from the IVT template DNA described above using the MEGAScript T7 kit (Thermo Fisher Scientific). Regarding the template sequence, N1-methyl-pseudouridine-triphosphate (m1ΨTP: TriLink) was used instead of uridine-triphosphate (UTP). Capping was performed with G(5')ppp(5')A RNA Cap Structure Analog (Acap; New England Biolabs). For transcription of IRES-containing mRNA, UTP and Acap were used. For transcription of m7G-capped mRNA, m1ΨTP and Anti Reverse Cap Analog, 3'-O-Me-m7G(5')ppp(5')G (ARCA; TriLink) were used. The reaction mixture was incubated at 37°C for 6 hours, and then TURBO deoxyribonuclease (DNase; Thermo Fisher Scientific) was added and incubated for an additional 30 minutes at 37°C. The resulting RNA was purified using Monarch RNA Cleanup Columns (NEB). The fragments were then treated with Antarctic Phosphatase (NEB) and purified again using Monarch RNA Cleanup Columns.

[0066] [Cell culture] HEK293FT cells were cultured in DMEM medium supplemented with 10% FBS (JBS), 1x MEM Non-Essential amino acid solution (Thermo Fisher Scientific), 1 mM sodium pyruvate (Sigma-Aldrich), and 1 mM L-glutamine (Thermo Fisher Scientific).

[0067] [mRNA transfection] The day before transfection, HEK293FT cells were transfected into a multi-well plate (24 wells) with 0.75 × 10 cells per well. 5 -1.0×10 5Cells were seeded and transfected with transcribed RNA using Lipofectamine MessengerMAX (Thermo Fisher Scientific) according to the manufacturer's instructions. For all experimental conditions, 300 pmol of RNA was transfected per cell. ARCA-capped mRNA encoding iRFP670 was cotransfected as a transfection control. Cells were then cultured for 24 hours.

[0068] [Translation Activity Assessment] The medium from HEK293FT cells was washed once with phosphate-buffered saline (PBS) and incubated at 37°C for approximately 5 minutes with Trypsin-EDTA (Thermo Fisher Scientific). After confirming cell detachment from the well bottom, the cells were resuspended in DMEM supplemented with the indicated additives. The fluorescence intensity of EGFP and iRFP670 was measured using a flow cytometer (Cytoflex: Beckman Coulter), and the ratio of EGFP to iRFP fluorescence intensity was calculated. These results demonstrated that Sequence 1 and Sequence 2 had sufficient activity to initiate translation from N1-methyl-pseudouridine-modified mRNA (Figure 4). While some of the sequences shown have lower translation activity than ARCA, their translation activity is sufficiently high compared to natural IRESs, making them suitable for use as "translation initiation sequences."

[0069] [Example 2] [mRNA design] Sequence 1-WT (Figure 1), Sequence 2-WT (Figure 2), and sequences with mutations introduced into these were prepared (Tables 1 and 2). These sequences were introduced into the 5'-UTR of mRNA. Figure 5 is a diagram conceptually illustrating the structure of mRNA containing these sequences, and the aptamer sequences in Tables 1 and 2 were introduced into the "Translation initiation sequence" portion of the diagram. EGFP was introduced into the ORF as in Example 1. For comparison, an mRNA with a translation-functional cap structure (ARCA) was prepared.

[0070] [Construction of IVT template DNA] Template DNA for in vitro transcription containing SEQ ID NOS: 4 to 22 shown in Table 1 and SEQ ID NOS: 26 to 37 shown in Table 2 was constructed in the same manner as in Example 1. The primers used for synthesis of the template DNA are shown in SEQ ID NOS: 48 (Primer-F1) and 49 (Primer-R1). The sequences of the plasmid DNAs used as templates for PCR are SEQ ID NO: 52 (template Sequence1-mutant1), SEQ ID NO: 53 (template Sequence1-mutant2), SEQ ID NO: 54 (template Sequence1-mutant3), SEQ ID NO: 55 (template Sequence1-mutant4), SEQ ID NO: 56 (template Sequence1-mutant5), SEQ ID NO: 57 (template Sequence1-mutant6), SEQ ID NO: 58 (template Sequence1-mutant7), SEQ ID NO: 59 (template Sequence1-mutant8), SEQ ID NO: 60 (template Sequence1-mutant9), SEQ ID NO: 61 (template Sequence1-mutant10), SEQ ID NO: 62 (template Sequence1-mutant11), SEQ ID NO: 63 (template Sequence1-mutant12), SEQ ID NO: 64 (template Sequence1-mutant13), SEQ ID NO: 65 (template Sequence1-mutant14), SEQ ID NO: 66 (template Sequence1-mutant15), SEQ ID NO: 67 (template Sequence1-mutant16), SEQ ID NO: 68 (template Sequence1-mutant17), SEQ ID NO: 69 (template Sequence1-mutant18), SEQ ID NO: 70 (template Sequence 1-mutant 19), SEQ ID NO: 71 (template Sequence 2-mutant 1), SEQ ID NO: 72 (template Sequence 2-mutant 2), SEQ ID NO: 73 (template Sequence 2-mutant 3), SEQ ID NO: 74 (templateThe templates are shown in SEQ ID NO: 75 (template Sequence2-mutant5), SEQ ID NO: 76 (template Sequence2-mutant6), SEQ ID NO: 77 (template Sequence2-mutant7), SEQ ID NO: 78 (template Sequence2-mutant8), SEQ ID NO: 79 (template Sequence2-mutant9), SEQ ID NO: 80 (template Sequence2-mutant10), SEQ ID NO: 81 (template Sequence2-mutant11), and SEQ ID NO: 82 (template Sequence2-mutant12). Template DNAs for in vitro transcription of SEQ ID NO: 23 and SEQ ID NO: 24 shown in Table 1, and SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40 shown in Table 2 were constructed using SEQ ID NO: 50 (template Sequence1-WT) as a PCR template. The primers used for synthesis of template DNA are shown in SEQ ID NO: 83 (Primer Sequence 1-mutant 20), SEQ ID NO: 84 (Primer Sequence 1-mutant 21), SEQ ID NO: 85 (Primer Sequence 2-mutant 13), SEQ ID NO: 86 (Primer Sequence 2-mutant 14), SEQ ID NO: 87 (Primer Sequence 2-mutant 15), and SEQ ID NO: 49 (Primer-R1).

[0071] [Preparation of mRNA, cell culture, transfection] mRNA was prepared in the same manner as in Example 1.

[0072] [Translation Activity Evaluation] HEK293FT cells cultured in the same manner as in Example 1 were transfected with mRNA and cultured for 24 hours. Then, the fluorescence intensities of EGFP and iRFP670 were measured using a flow cytometer (Cytoflex: Beckman Coulter) in the same manner as in Example 1, and the ratio of EGFP fluorescence intensity to iRFP fluorescence intensity was calculated. The results are shown in Figures 6 and 7. These results revealed that the mutated sequences have the activity of initiating translation from N1-methyl-pseudouridine-modified mRNA. These sequences were also shown to have sufficient performance as "translation initiation sequences."

[0073] [Example 3] [mRNA design] Sequence 1 was prepared with structural stabilization, or with insertion or deletion (Table 3), and Sequence 2 was prepared with structural stabilization. These sequences were introduced into the 5'-UTR of mRNA. As in Example 1, EGFP was introduced into the ORF. As in Example 1, an mRNA with a translationally functional cap structure (ARCA) was prepared as a comparative mRNA.

[0074] [Construction of IVT template DNA] Template DNA for in vitro transcription of the structurally stabilized sequence was constructed in the same manner as in Example 1 using the sequences shown in SEQ ID NO: 88 (template Sequence 1-stb1), SEQ ID NO: 89 (template Sequence 1-stb2), SEQ ID NO: 90 (template Sequence 2-stb1), and SEQ ID NO: 91 (template Sequence 2-stb2) as templates. Template DNA for in vitro transcription with an insertion or deletion introduced into Sequence 1 was synthesized using SEQ ID NO: 50 (template Sequence 1-WT) as a template. Primers used were SEQ ID NO: 92 (Primer Sequence 1-variant A), SEQ ID NO: 93 (Primer Sequence 1-variant B), SEQ ID NO: 94 (Primer Sequence 1-variant C), and SEQ ID NO: 49 (Primer-R1).

[0075] [Preparation of mRNA] mRNA was prepared from the IVT template DNA described above using the MEGAScript T7 kit (Thermo Fisher Scientific). Regarding the template sequence, N1-methyl-pseudouridine-triphosphate (m1ΨTP: TriLink) was used instead of uridine-triphosphate. G(5')ppp(5')A RNA Cap Structure Analog (Acap; NEB) was used for capping. Uridine-triphosphate (UTP) and Acap were used for transcription of IRES-containing mRNA. m1ΨTP and Anti-Reverse Cap Analog, 3'-O-Me-m7G(5')ppp(5')G (ARCA; TriLink) were used for transcription of m7G-capped mRNA. The reaction mixture was incubated at 37°C for 6 hours, and then TURBO deoxyribonuclease (DNase; Thermo Fisher Scientific) was added and incubated for an additional 30 minutes at 37°C. The resulting RNA was purified using Monarch RNA Cleanup Columns (NEB). The fragments were then treated with Antarctic Phosphatase (NEB) and purified again using Monarch RNA Cleanup Columns.

[0076] [Translation Activity Evaluation] HEK293FT cells cultured as in Example 1 were transfected with mRNA and cultured for 24 hours. Then, as in Example 1, the fluorescence intensities of EGFP and iRFP670 were measured using a flow cytometer (Cytoflex: Beckman Coulter), and the ratio of EGFP fluorescence intensity to iRFP fluorescence intensity was calculated. The results are shown in Figure 8. These results revealed that the aptamers shown in Table 3, which contain structure-stabilizing or insertion / deletion mutations added to Sequence 1 or Sequence 2, have the activity of initiating translation from N1-methyl-pseudouridine-modified mRNA. These sequences were also shown to have sufficient performance as "translation initiation sequences."

[0077] Example 4 Design of Circular mRNA We designed circular mRNAs containing Sequence 2 as the translation activation region, as well as linear mRNAs for their preparation. Three types of circular mRNA structures were prepared: a molecule containing Sequence 2 and a translation region but no Poly A (ΔpA); a molecule containing Sequence 2, a translation region, and a 30-base Poly A, with the 3' end of Poly A attached to the 5' end of Sequence 2 (pA30); and a molecule in which the Poly A of pA30 was replaced with an 80-base Poly A (pA80). Nanoluc® was introduced into the ORF. All designs included a connection stabilizing sequence. For comparison, circular mRNAs without aptamers (No-motif) were designed for each of the three structures.

[0078] [Construction of IVT template DNA] The sequences of the plasmid DNA used as templates for in vitro transcription of linear RNA, which serves as a precursor to circular RNA, were constructed in the same manner as in Example 1, using the sequences shown in SEQ ID NO: 95 (pTY-953(pUC19_No-motif_Nluc)) and SEQ ID NO: 96 (pFI-0213(pUC19_Sequence2_Nluc)) as templates. The primers used were SEQ ID NO: 97 (FIO-0425), SEQ ID NO: 98 (FIO-0270), SEQ ID NO: 99 (FIO-0457), and SEQ ID NO: 100 (FIO-0248). The sequences used in PCR for IVT template synthesis are shown below.

[0079]

[0080] The obtained IVT template sequences are shown in SEQ ID NO: 101 (No-motif_ΔpA), SEQ ID NO: 102 (No-motif_pA30), SEQ ID NO: 103 (No-motif_pA80), SEQ ID NO: 104 (Sequence2_ΔpA), SEQ ID NO: 105 (Sequence2_pA30), and SEQ ID NO: 106 (Sequence2_pA80).

[0081] [mRNA Preparation] mRNA was prepared by transcription from the IVT template DNA described above using the MEGAScript T7 kit (Thermo Fisher Scientific). N1-methyl-pseudouridine-triphosphate (m1ΨTP: TriLink) was used instead of uridine-triphosphate in the template sequence. The reaction mixture was incubated at 37°C for 6 hours, and then TURBO deoxyribonuclease (DNase; Thermo Fisher Scientific) was added and incubated at 37°C for an additional 30 minutes. The resulting RNA was purified using Monarch RNA Cleanup Columns (NEB). It was then treated with Antarctic Phosphatase (NEB) and purified again with Monarch RNA Cleanup Columns to obtain dephosphorylated linear RNA. It was then treated with T4 Polynucleotide Kinase (TaKaRa) and purified again with Monarch RNA Cleanup Columns to obtain the precursor monophosphorylated linear RNA. The ends of linear mRNA were ligated using T4 RNA Ligase 2 (NEB), and the resulting RNA was purified using Monarch RNA Cleanup Columns. Circular RNA was then synthesized and purified by degrading the remaining linear RNA with Terminator exonuclease (epicentre) and RNase R (Applied Biological Materials), enzymes that degrade mRNA from the terminus.

[0082] [Translation Activity Evaluation] HEK293FT cells were cultured as in Example 1 and transfected with mRNA encoding circular NLuc and mRNA encoding Fluc (transfection control). 24 hours later, luminescence from Fluc and Nanoluc was measured using the Nano-Glo® Dual-Luciferase® Reporter Assay System (Promega). The Nluc intensity was normalized by Nanoluc / Fluc, and relative values ​​were calculated by setting the Nomotif-ΔpA (no pA) value to 1. The results are shown in Figure 9. These results demonstrate that Sequence 2, even when incorporated into circular RNA, performs satisfactorily as a "translation initiation sequence." This provides a new means for efficiently inducing cap-independent translation in circular RNAs containing m1Ψ modifications and other mRNA molecules, which was difficult to achieve with conventional IRES sequences. The nucleic acid aptamer of the present invention has the remarkable property of maintaining translational activity while tolerating m1Ψ modification. This opens up an innovative path to the development of next-generation mRNA drugs, such as circular RNAs incorporating m1Ψ modifications, for which the introduction of m1Ψ modifications has previously been a substantial barrier. By applying this technology, sustained and highly efficient protein expression in cells can be expected, while enjoying the significant benefits of m1Ψ modifications, such as reduced immunogenicity and improved stability of RNA molecules. Therefore, the present invention overcomes the limitations of conventional technology and is expected to significantly contribute to the widespread application of more effective and safer mRNA drugs.

Claims

1. An aptamer that is specifically recognized by a translation initiation factor, the aptamer having the following formula (i) or (ii): GGCGN x1 YACGNN x2 (m1Ψ)NKCKCC(m1Ψ)CDGGGMGMGCC(GGCGN x1 YACGNN x2 - SEQ ID NO: 1) (i) GGGGCGMGNGAMKCG(m1Ψ)H(m1Ψ)CKMCYMNGGKMGACCC (SEQ ID NO: 2) (ii) (in formula (i) or (ii), S is C or G, R is A or G, W is A or m1Ψ (1-methylpseudouridine), M is C or A, K is G or m1Ψ, Y is m1Ψ or C, V is A, C or G, B is m1Ψ, C or G, H is A, C or m1Ψ, D is A, G or m1Ψ, N is A, m1Ψ, C or G, and x1 and x2 are the same integer selected from 3 to 8), or a nucleic acid sequence in which 1 to 5 nucleic acid bases have been substituted, deleted, inserted or added in the nucleic acid sequence.

2. In formula (i), N x1 and N x2 The aptamer of claim 1, wherein: forms the first stem; KCKCC and GGMGM form the second stem; and GG at the 5' end and CC at the 3' end form the third stem.

3. The aptamer of claim 2, wherein formula (i) is selected from SEQ ID NOs: 3 to 24, 43, and 44.

4. The aptamer described in claim 1, wherein in formula (ii), GCGM and KCG(m1Ψ) form the first stem, (m1Ψ)CKMC and GKMGA form the second stem, and GG at the 5' end and CC at the 3' end form the third stem.

5. The aptamer described in claim 4, wherein formula (ii) is selected from SEQ ID NOs: 25 to 40.

6. The aptamer described in claim 1, wherein the nucleic acid sequence in which 1 to 5 nucleic acid bases have been substituted, deleted, inserted or added in formula (i) comprises SEQ ID NO:

45.

7. An RNA molecule comprising: (I) a translation activation region containing the nucleic acid aptamer described in claim 1; and (II) a translation region located 3'-terminally closer to the nucleic acid aptamer, and not containing a 5' cap structure.

8. The RNA molecule of claim 7, wherein the translated region comprises a nucleic acid sequence encoding a detection protein, a therapeutic protein, or an RNA-binding protein.

9. The RNA molecule according to claim 7, wherein the RNA molecule is a linear molecule comprising a poly A sequence on the 3' end of the early translated region.

10. The RNA molecule according to claim 7, wherein the RNA molecule is a circular molecule comprising a Poly A sequence on the 3'-end side of the early translation region.

11. A pharmaceutical composition comprising the RNA molecule of claim 7.

12. A template DNA molecule comprising a sequence encoding the aptamer of claim 1.

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