Nucleic acid molecule comprising modified polyadenylation sequence

Abstract

Provided are: an mRNA molecule comprising a modified polyadenylation sequence; a composition for delivering, to an individual, the mRNA or a product expressed by the mRNA; a method for delivering, to a host cell, the mRNA or a product expressed by the mRNA; a DNA molecule that encodes the mRNA molecule; and use thereof.

Description

Nucleic acid molecule comprising a modified polyadenylation sequence

[0001] The present invention relates to a nucleic acid molecule comprising a modified polyadenylation sequence, a nucleic acid molecule encoding the same, and uses thereof.

[0002] RNA, for example, in vitro transcribed RNA (IVT-RNA), has been proposed as a therapeutic alternative to DNA. However, the injectability of RNA for clinical applications may be significantly limited by its short half-life.

[0003] Additionally, the IVT vector can be used as a template for in vitro transcription. The IVT vector can comprise a 5' RNA polymerase promoter, a 5'UTR, a 3'UTR, or a gene of interest flanked by a 5'UTR and a 3'UTR, and a 3' polyadenylation cassette comprising A nucleotides.

[0004] The 3' polyA of RNA is crucial for the nuclear transport of eukaryotic mRNA, RNA stability, and translation efficiency. It is known that the 3' polyA sequence shortens over time, and when sufficiently short, the RNA is subject to enzymatic degradation.

[0005] Therefore, even with the above-described prior art, there is a need for a nucleic acid molecule comprising a modified polyadenylation sequence, a nucleic acid molecule encoding the same, and a use thereof.

[0006] One aspect provides an mRNA molecule comprising a modified polyadenylation sequence.

[0007] Another aspect provides DNA encoding the mRNA.

[0008] Another aspect provides a method for propagating said DNA molecule.

[0009] Another aspect provides a method for obtaining RNA.

[0010] Another aspect provides a method for obtaining a polypeptide.

[0011] Another aspect provides a composition or kit for delivering to a subject an mRNA comprising the mRNA molecule described above or a product expressed thereby.

[0012] Another aspect provides a method of delivering mRNA or a product expressed thereby to a host cell, comprising the step of introducing the mRNA into the host cell.

[0013] A first aspect provides an mRNA molecule comprising a modified polyadenylation sequence, wherein the modified polyadenylation sequence comprises: (a) a first sequence comprising at least one microRNA binding sequence; (b) a second sequence of 20 or fewer consecutive A nucleotides, located 5' of the first sequence; and (c) a third sequence of at least 80 consecutive A nucleotides, located 3' of the first sequence.

[0014] As used herein, the term "polyadenyl sequence", "polyA", or "polyA tail" refers to a region of an mRNA downstream, for example directly downstream (i.e., 3') from a 3'UTR, that contains multiple consecutive adenosine monophosphates. The polyA tail may contain from 10 to 1000 adenosine monophosphates. For example, the polyA tail may comprise 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1,000 or more A nucleotides. The polyA tail may have, for example, an average molecular weight of 50 to 1000, 60 to 800, 80 to 600, 100 to 500, 50 to 500, 60 to 500, 70 to 500, 80 to 500, 90 to 500, 100 to 500, 50 to 400, 60 to 400, 70 to 400, 80 to 400, 90 to 400, 100 to 400, 50 to 300, 60 to 300, 70 to 300, 80 to 300, 90 to 300, 100 to 300, 50 to 200, 50 to 250, 60 to 200, 70 to It may comprise 200, 80 to 200, 90 to 200, 100 to 200, 50 to 100, 60 to 100, 70 to 100, 80 to 100, 90 to 100, or 150 to 250 consecutive A nucleotides.In living organisms, such as cells or organisms, the polyA tail may play a role in protecting mRNA from attack by enzymes, for example, enzymes in the cytoplasm, and assisting in transcription termination, nuclear export of mRNA, and translation.

[0015] Polyadenylation is the addition of a polyA sequence to primary transcript RNA. This polyA sequence consists of multiple adenosine monophosphates. In other words, a polyA sequence is a stretch of RNA containing only adenine bases. The polyA sequence is crucial for nuclear transport, translation, and mRNA stability. The polyA sequence shortens over time, and if sufficiently short, the mRNA can be degraded by enzymes.

[0016] In the mRNA molecule, the modified polyadenylation sequence may comprise one or more disruptions in the continuous A sequence, to which one or more microRNA binding sequences are linked. The modified polyadenylation sequence may perform the original function of the polyadenylation sequence equivalent to or superior to that of the unmodified polyadenylation sequence. The original function may be to assist in, for example, promote, the nuclear transport, translation, and stability of the mRNA.

[0017] In this specification, the term "microRNA" or "miRNA" is used in the sense known in the art. The microRNA may be a long non-coding RNA of 19-25 nucleotides that binds to an mRNA molecule and downregulates gene expression. The microRNA may bind to the 3'UTR of the mRNA. The microRNA may include a sequence complementary to the region of the target mRNA to which it binds. The complementary sequence may be a perfect or near perfect complementary sequence. The downregulation of gene expression may include downregulation by reducing the stability of the nucleic acid molecule or inhibiting translation.

[0018] The term "microRNA binding sequence" may be any nucleotide sequence to which a microRNA binds or associates. "Binding" may follow the traditional Watson-Crick hybridization rules or reflect any stable association of the microRNA with a target sequence at or adjacent to the microRNA binding sequence. The microRNA binding sequence may be a microRNA target sequence, a microRNA sequence, a microRNA seed sequence, or a combination thereof. The microRNA binding sequence may be a known sequence. The microRNA sequence comprises a "seed sequence." The seed sequence may be a sequence of the region of positions 2-8 of a mature microRNA. The seed sequence may have complete Watson-Crick complementarity to the microRNA target sequence. An example of the microRNA sequence may comprise positions 2-8 or 2-7 of a mature microRNA. The above microRNA sequence may comprise 7 nucleotides (e.g., nucleotides 2-8 of a mature microRNA), and the seed complementary site of the corresponding miRNA target may be flanked by an adenine (A) against position 1 of the microRNA. Alternatively, the above microRNA sequence may comprise 6 nucleotides (e.g., nucleotides 2-7 of a mature microRNA), and the seed complementary site of the corresponding miRNA target may be flanked by an adenine (A) against position 1 of the microRNA. The above microRNA binding sequence may be natural or artificially created.

[0019] In the first sequence among the above modified polyadenyl sequences, if two or more microRNA binding sequences are included, they may or may not be identical to each other. In addition, if two or more microRNA binding sequences are included, the one or more microRNA binding sequences may have microRNA target sequences, microRNA recognition sequences, or seed sequences thereof that are identical or not. For example, the two or more microRNA binding sequences may be arranged in the form of ABAB, AABB, or ABBA. The two or more microRNA binding sequences may be directly linked to each other without a spacer. In addition, the two or more microRNA binding sequences may be linked to each other via a spacer. The first sequence may be composed of one or more microRNA binding sequences. The spacer may be a modified or unmodified nucleotide sequence.

[0020] In the nucleic acid molecule, the microRNA may be tumor-specific. The microRNA may be upregulated or downregulated in tumor cells compared to normal cells. The microRNA binding sequence may bind to a tumor-specific miRNA, its seed sequence, or miRNA target sequence in vivo, thereby suppressing tumors.

[0021] MicroRNA miR-340, miR-133a, miR-34b, miR-19, miR-200, miR-15, miR-16, miR-1298, miR-663a, miR-449a, miR-138, miR-126, miR-10a, miR-10b, miR-140-3p, miR-140-5p, miR-204, miR-424, miR-34a, miR-193a-3p, miR-455-5p, miR-455-3p, miR-9, miR-10a, miR-148a, miR-488, miR-196a1, miR-182, miR-96, miR-193b, miR-27a, miR-196b, miR-10b, miR-29b2, miR-23a, miR-107, miR-542-3p, miR-93, miR-365a-4, miR-450a, miR-100, miR-105, miR-363, miR-105, miR-106b, miR-15b, miR-21, miR-376c, miR-93, miR-99b, miR-155, miR-33a, miR-876-3p, miR-362-3p, miR-25, let-7i, miR-423-3p, miR-16-2, miR-29a, miR-30d, miR-320, miR-181c, miR-128a, miR-21, let-7d, miR-450b-5p, miR-371-5p, miR-1245, miR-335, miR-492, miR-874, miR-30b, miR-193a-5p, miR-602, miR-346, miR-663, miR-25, miR-219-5p6, miR-184, miR-135a7, miR-584, miR-665, miR-638, miR-503, miR-628-3p, miR-381, miR-78, miR-92b, miR-149, miR-135b, miR-302d, miR-498, miR-766, miR-1389, miR-623, miR-519c-5p, miR-182, miR-494, miR-129-5p10, miR-513-5p, miR-200b, miR-634,It may be selected from the group consisting of miR-654-5p, miR-518b, miR-658, miR-373, miR-30c-2, miR-130a, miR-557, miR-551a, miR-637, miR-518c, miR-525-5p, miR-596, miR-552, miR-625, miR-183, miR-187, miR-544, miR-891a, miR-519e, miR-933, miR-939, miR-214, miR-671-5p, miR-137, miR-92b, miR-525-3p, miR-19a, and miR-409-5p. An example of the above microRNA may be downregulated in tumor cells. The downregulated miR may be miR-34a. However, the differential expression of miRs in cancer cells may vary depending on the cancer type.

[0022] In the second sequence of the above modified polyadenyl sequences, the length of the A consecutive nucleotides may be any length that provides sufficient stability and / or translation to the mRNA obtained when transcribed, given the microRNA binding sequence and its number, and the length of the A consecutive nucleotides of the third sequence. For example, the length of the A consecutive nucleotides of the second sequence is 1 to 20, 2 to 20, 3 to 20, 4 to 20, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20, 10 to 20, 11 to 20, 12 to 20, 13 to 20, 14 to 20, 15 to 20, 16 to 20, 17 to 20, 18 to 20, 19 to 20, 20, 1 to 19, 2 to 19, 3 to 19, 4 to 19, 5 to 19, 6 to 19, 7 to 19, 8 to 19, 9 to 19, 10 to 19, 11 to 19, 12 to 19, 13 to 19, 14 to 19, 15 to 19, 16 to 19, 17 to 19, 18 to 19, 19, 1 to 18, 2 to 18, 3 to 18, 4 to 18, 5 to 18, 6 to 18, 7 to 18, 8 to 18, 9 to 18, 10 to 18, 11 to 18, 12 to 18, 13 to 18, 14 to 18, 15 to 18, 16 to 18, 17 to 18, 18, 1 to 17, 2 to 17, 3 to 17, 4 to 17, 5 to 17, 6 to 17, 7 to 17, 8 to 17, 9 to 17, 10 to 17, 11 to 17, 12 to 17, 13 to 17, 14 to 17, 15 to 17, 16 to 17, 17, 1 to 16, 2 to 16, 3 to 16, 4 to 16, 5 to 16, 6 to 16, 7 to 16, 8 to 16, 9 to 16, 10 to 16, 11 to 16, 12 to 16, 13 to 16, 14 to 16,15 to 16, 16, 1 to 15, 2 to 15, 3 to 15, 4 to 15, 5 to 15, 6 to 15, 7 to 15, 8 to 15, 9 to 15, 10 to 15, 11 to 15, 12 to 15, 13 to 15, 14 to 15, 15, 1 to 14, 2 to 14, 3 to 14, 4 to 14, 5 to 14, 6 to 14, 7 to 14, 8 to 14, 9 to 14, 10 to 14, 11 to 14, 12 to 14, 13 to 14, 14, 1 to 13, 2 to 13, 3 to 13, 4 to 13, 5 to 13, 6 to 13, 7 to 13, 8 to 13, 9 to 13, 10 to 13, 11 to 13, 12 to 13, 13, 1 to 12, 2 to 12, 3 to 12, 4 to 12, 5 to 12, 6 to 12, 7 to 12, 8 to 12, 9 to 12, 10 to 12, 11 to 12, 12, 1 to 11, 2 to 11, 3 to 11, 4 to 11, 5 to 11, 6 to 11, 7 to 11, 8 to 11, 9 to 11, 10 to 11, 11, 1 to 10, 2 to 10, 3 to 10, 4 to 10, 5 to 10, 6 to 10, 7 to 10, 8 to 10, 9 to 10, 10, 1 to 9, 2 to 9, 3 to 9, 4 to 9, 5 to 9, 6 to 9, 7 to 9, 8 to 9, 9, 1 to 8, 2 to 8, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 7 to 8, 8, 1 to 7, 2 to 7, 3 to 7, 4 to 7, 5 to 7, 6 to 7, 7, 1 to 6, 2 to 6, 3 to 6, 4 to 6, 5 to 6, 6, 1 to 5, 2 to 5, 3 to 5, 4 to 5, 5, 1 to 4, 2 to 4, 3 to 4, 4 to 4, 1 to 3, 2 to 3, 3, 1 to 2, 2,or may be one nucleotide.,

[0023] In the third sequence of the above modified polyadenylation sequence, the length of the A consecutive nucleotides may be any length that provides sufficient stability and / or translation to the mRNA obtained when transcribed, given the microRNA binding sequence and its number, and the length of the A consecutive nucleotides of the second sequence. For example, the length of the A consecutive nucleotides of the third sequence may be 80 to 150, 80 to 140, 80 to 130, 80 to 120, 80 to 110, 90 to 150, 90 to 140, 90 to 130, 90 to 120, or 90 to 110 nucleotides.

[0024] In the mRNA molecule, the length of the modified polyadenyl sequence may vary depending on the number of given microRNA binding sequences. For example, the length of the modified polyadenyl sequence may be increased by a length corresponding to the number of introduced microRNA binding sequences from the length of the polyadenyl sequence described for "polyadenyl sequence." For example, when the length of the polyadenyl sequence is 10 to 1000, 20 to 500, 30 to 500, 50 to 100, 80 to 160, 150 to 250, 100 to 300, 40 to 120, or 120 to 200 nucleotides, and when the microRNA binding sequence is 1, 2, 3, 4, and 5, the length of the modified polyadenyl sequence is 29 to 1025, 39 to 525, 49 to 525, 69 to 125, 99 to 185, 169 to 275, 119 to 325, 59 to 145, 139 to 225, 48 to 1050, 58 to 550, 68 to 550, 88 to 150, 118 to 210, 188 to 300, 138 to 350, 78 to 170, 158 to 250; 67 to 1075, 77 to 575, 87 to 575, 107 to 175, 137 to 235, 207 to 325, 157 to 375, 97 to 195, 177 to 275, 86 to 1100, 96 to 600, 106 to 600, 126 to 200, 156 to 260, 226 to 350, 176 to 400, 116 to 220, 196 to 300, 105 to 1125, 115 to 625, 125 to 625, 145 to 225, 175 to 285, 245 to 375, 195 to It may have a sequence of 425, 135 to 245, or 215 to 325 nucleotides.The length of the above modified polyadenyl sequence is described as an example and is not limited to the above length.

[0025] In the mRNA molecule, the one or more microRNA binding sequences may be located in a region of positions 21 to 39, 21 to 45, 21 to 70, 21 to 95, 21 to 120, 21 to 145, 11 to 39, 11 to 45, 11 to 70, 11 to 95, 11 to 120, or 11 to 145 of the modified polyadenylation sequence.

[0026] The mRNA molecule may comprise a sequence encoding a polypeptide or an expression control sequence.

[0027] The sequence encoding the polypeptide, the expression control sequence, and the modified polyadenylation sequence may be operably linked to each other.

[0028] As used herein, an "open reading frame (ORF)" or "a sequence encoding polypeptide" refers to a contiguous region of nucleic acid that begins with an initiation codon, for example, a methionine codon (ATG), and ends with a stop codon, for example, TAA, TAG, or TGA, and encodes a polypeptide.

[0029] As used herein, the term "operably linked" refers to the connection of nucleotide sequences on a single nucleic acid fragment such that one function is influenced by the other. For example, a promoter is operably linked to a coding sequence (e.g., an open reading frame (ORF)) if it can influence the expression of the coding sequence (i.e., if the coding sequence is under the transcriptional control of the promoter). The coding sequence may be operably linked to a regulatory sequence in either the sense or antisense direction. As used herein, unless otherwise stated, each sequence included in a nucleic acid molecule is referred to as being operably linked.

[0030] The expression control sequence may be a translation control sequence or a sequence involved in stabilizing RNA. The expression control sequence may be located upstream or downstream of the sequence encoding the polypeptide. The expression control sequence may be located downstream of the sequence encoding the polypeptide and upstream of the modified polyadenylation sequence.

[0031] The expression control sequence may be a Kozak sequence, a ribosome binding sequence, or a UTR. The UTR may be a 5'UTR, a 3'UTR, or a 5'UTR and a 3'UTR. The 5'UTR, the 3'UTR, or the 5'UTR and the 3'UTR may be homologous or heterologous to a sequence encoding a polypeptide. The 5'UTR may be a structured UTR. That is, the 5'UTR may have a three-dimensional structure such as a stem-loop structure.

[0032] In the above mRNA molecule, the 5'UTR may be a natural human p53 5'UTR or a natural human OX40L 5'UTR, the polypeptide may be a human p53 5'UTR or a human OX40L, and the 3'UTR may be a natural human cytochrome P450 2E1 3'UTR.

[0033] The 5'UTR or 3'UTR may be found in mRNA expressed in any tissue cell. The tissue may be a cancerous tissue. The cancer may be a hematological cancer or a solid cancer. The tissue may be liver, muscle, brain, breast, colon, lung, pancreas, ovary, skin, or blood. The tissue cell may be, for example, a liver cell, an endothelial cell, a muscle cell, a blood cell, or an adipose tissue cell. The above 5'UTR includes p53, OX40L, albumin, serum amyloid A, apolipoprotein A / B / E, transferrin, alpha fetoprotein, erythropoietin, factor VIII, MyoD, myosin, myoglobin, myogenin, Herculin, Tie-1, CD36, C / EBP, AML1, G-CSF, GM-CSF, CD11b, MSR, Fr-1, i-NOS, CD45, CD18, CD36, GLUT4, ACRP30, adiponectin, PA / B / C / D, ORM1, HPX, FGA, CYP2E1, C3, ASC, APOA2, ALB, AGXT, α-globin, β-globin, tyrosine It may be derived from an mRNA encoding a protein selected from the group consisting of tyrosine hydroxylase and collagen. The 5'UTR may be derived from an mRNA encoding, for example, human p53, human OX40L, or human C3 protein. In addition, the 3'UTR may be derived from an mRNA encoding human CYP450 2E1, human OX40L, or human C3 protein. The 3'UTR may be located 5' of the polyadenylation sequence.

[0034] The above expression control sequence may be a homologous or heterologous sequence with respect to the sequence encoding the polypeptide.

[0035] The polypeptide may be any polypeptide having anticancer activity. The polypeptide having anticancer activity may have anticancer activity by participating in the cell cycle regulation of cancer cells themselves, or may have anticancer activity through the activity of stimulating immune cells. The immune cells may include B cells, NK cells, T cells, or mast cells. The polypeptide having anticancer activity may not have antigenicity in the host cell or the organism containing the same. The polypeptide having anticancer activity may be an antibody, a hormone, a cytokine, an enzyme, a derivative thereof, or a combination thereof. Furthermore, the polypeptide having anticancer activity may inhibit the proliferation of cancer cells. The polypeptide may be a recombinant polypeptide, including a fusion protein. The sequence encoding the polypeptide may be codon-optimized. The codon optimization may vary depending on the host cell into which the nucleic acid molecule is introduced. Furthermore, the sequence encoding the polypeptide may include a degenerate sequence.

[0036] The polypeptide may exhibit a pharmacological effect by forming a trimer. The polypeptide may be fused with a factor that promotes trimer formation formed after translation. The factor may be a polypeptide that promotes trimer formation. The factor may be mannan-binding lectin (MBL) or a fragment thereof, or ferritin or a fragment thereof. The fragment may be an alpha helix region of MBL (position: "110-129" in the primary sequence of the protein). The fragment may be a heavy chain region of ferritin (position: "15-161" in the primary sequence of the protein). The polypeptide may be fused with one or more factors. The polypeptide may be fused with one or more factors via a linker. The linker may be a peptide linker. The peptide linker may be a linker comprising G4S. The above peptide linker may be a linker comprising G4S, (G4S)2, (G4S)3, (G4S)4, (G4S)5, or (G4S)6. The polypeptide may comprise a structure of polypeptide-linker-MBL-linker-ferritin or MBL-linker-polypeptide.

[0037] In the above mRNA molecule, the sequence encoding the polypeptide and the modified polyadenylation sequence may be transcribed under the control of a promoter to produce a common transcript. The modified polyadenylation sequence may be located at the 3' end of the common transcript.

[0038] The mRNA molecule may comprise one or more modified nucleotides. The modified nucleotides may be pseudouridine analogs. The pseudouridine may be 1-methylpseudouridine. The modified nucleotides may further comprise 5-methylcytidine. The mRNA molecule may have all U's replaced with 1-methylpseudouridine.

[0039] In the above mRNA molecule, the sequence encoding the polypeptide may be codon optimized.

[0040] Additionally, the mRNA may comprise one or more 5'-cap structures. As used herein, the term "cap" refers to a cap structure found at the 5' end of an mRNA molecule. The cap typically consists of a guanosine nucleotide linked to the mRNA via a 5' to 5' triphosphate linkage. This guanosine is methylated at position 7. The term "conventional 5'-cap" refers to a naturally occurring RNA 5'-cap, such as 7-methylguanosine (m7G). As used herein, the term "5'-cap" includes 5'-cap analogs that are modified to resemble an RNA cap structure and have the ability to stabilize RNA when attached, for example, in vivo and / or in cells. The one or more 5'-cap structures may be selected from the group consisting of 7-methylguanosine (m7G), ARCA, inosine, Nl-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine and 2-azido-guanosine. In the mRNA, the 5'-terminal GA may have a cap structure of m7(3'OMeG)(5')ppp(5')(2'OMeA). In the mRNA, all Us may be replaced with N1-methyl-pseudouridine. The mRNA may have all Us replaced with N1-methyl-pseudouridine and the 5'-terminal GA may have a cap structure of m7(3'OMeG)(5')ppp(5')(2'OMeA).

[0041] The 5'-cap can be cap0, cap1, or cap2. In cap0, the riboses of the first and second cap-proximal nucleotides of the mRNA both contain a 2'-hydroxyl. In cap1, the riboses of the first and second cap-proximal nucleotides of the mRNA contain a 2'-methoxy and a 2'-hydroxyl, respectively. In cap2, the riboses of the first and second cap-proximal nucleotides of the mRNA both contain a 2'-methoxy.

[0042] Providing RNA having a 5'-cap or 5'-cap analogue can be accomplished by in vitro transcription of a DNA template in the presence of a 5'-cap or 5'-cap analogue. In this case, the 5'-cap or 5'-cap analogue can be introduced into the RNA strand produced co-transcriptionally. Alternatively, for example, the RNA can be produced by in vitro transcription and then post-transcriptionally produced using a capping enzyme, such as the capping enzyme of vaccinia virus.

[0043] The above microRNA binding sequence may be tumor-specific. The microRNA may be upregulated or downregulated in tumor cells compared to normal cells. The microRNA binding sequence may bind to a tumor-specific miRNA, its seed sequence, or miRNA target sequence in vivo, thereby suppressing the tumor.

[0044] MicroRNA miR-340, miR-133a, miR-34b, miR-19, miR-200, miR-15, miR-16, miR-1298, miR-663a, miR-449a, miR-138, miR-126, miR-10a, miR-10b, miR-140-3p, miR-140-5p, miR-204, miR-424, miR-34a, miR-193a-3p, miR-455-5p, miR-455-3p, miR-9, miR-10a, miR-148a, miR-488, miR-196a1, miR-182, miR-96, miR-193b, miR-27a, miR-196b, miR-10b, miR-29b2, miR-23a, miR-107, miR-542-3p, miR-93, miR-365a-4, miR-450a, miR-100, miR-105, miR-363, miR-105, miR-106b, miR-15b, miR-21, miR-376c, miR-93, miR-99b, miR-155, miR-33a, miR-876-3p, miR-362-3p, miR-25, let-7i, miR-423-3p, miR-34b, miR-16-2, miR-29a, miR-30d, miR-320, miR-181c, miR-128a, miR-21, let-7d, miR-450b-5p, miR-371-5p, miR-1245, miR-335, miR-492, miR-874, miR-30b, miR-193a-5p, miR-602, miR-346, miR-663, miR-25, miR-219-5p6, miR-184, miR-135a7, miR-584, miR-665, miR-638, miR-503, miR-628-3p, miR-381, miR-78, miR-92b, miR-149, miR-135b, miR-302d, miR-498, miR-766, miR-1389, miR-623, miR-519c-5p, miR-182, miR-494, miR-129-5p10, miR-513-5p, miR-200b,It may be selected from the group consisting of miR-634, miR-654-5p, miR-518b, miR-658, miR-373, miR-30c-2, miR-130a, miR-557, miR-551a, miR-637, miR-518c, miR-525-5p, miR-596, miR-552, miR-625, miR-183, miR-187, miR-544, miR-891a, miR-519e, miR-933, miR-939, miR-214, miR-671-5p, miR-137, miR-92b, miR-525-3p, miR-19a, and miR-409-5p. An example of the above microRNA may be downregulated in tumor cells. The downregulated miR may be miR-34a. However, the differential expression of miRs in cancer cells may vary depending on the cancer type.

[0045] The mRNA may further comprise a chain-terminating nucleoside. The chain-terminating nucleoside may be selected from the group consisting of 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxytidine, 2',3'-dideoxynucleoside, 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'-dideoxyguanosine, 2',3'-dideoxytidine, 2'-deoxynucleoside, and -O-methylnucleoside. The 3' tail region may comprise a three-dimensional structure, for example, a stem-loop structure.

[0046]

[0047] The second aspect provides a composition for delivering to a subject an mRNA molecule or a product expressed by the mRNA molecule, comprising the above-described mRNA molecule as an active ingredient.

[0048] A third aspect provides a kit for delivering to a subject an mRNA molecule comprising the above-described mRNA molecule as an active ingredient or a product expressed thereby.

[0049] In the second and third aspects, the composition or kit may be for treating cancer. The cancer may be a hematological cancer or a solid cancer.

[0050] The mRNA molecule may encode a polypeptide. The polypeptide may be any polypeptide having anticancer activity. The polypeptide having anticancer activity is the same as described in the first aspect.

[0051] The composition can be administered to a subject and the RNA (e.g., mRNA) can be translated in vivo to produce a polypeptide.

[0052] The composition may be contacted with a cell, tissue or organism in an “effective amount”.

[0053] The effective amount can be determined based on the target tissue, target cells, administration vehicle, physical characteristics of the polynucleotide (e.g., size and content of modified nucleosides), and other components of the composition.

[0054] The composition may be administered in combination with other prophylactic or therapeutic compounds. The prophylactic or therapeutic compound may be, for example, an anticancer agent. As used herein, the anticancer agent may be any known agent.

[0055] The composition may be administered intramuscularly, subcutaneously, intradermally, nasally, or pulmonary. The composition may be administered locally to tissues containing cancer. The composition may be administered locally to tissues containing cells with elevated levels of cancer-specific miRNAs, either intracellularly or extracellularly. The composition may be administered locally to tissues containing cells with upregulated or downregulated cancer-specific miRNAs, either intracellularly or extracellularly, compared to normal cells.

[0056] The composition may include a pharmaceutically acceptable carrier, diluent, or excipient. The polynucleotide in the composition may be formulated or complexed with the carrier, diluent, or excipient. The carrier, diluent, or excipient may be one known in the art.

[0057] As used herein, the term "active ingredient" refers to the composition or a polynucleotide contained therein, for example, RNA, including mRNA. The RNA may, for example, encode a polypeptide having anticancer activity.

[0058] The relative amounts of the active ingredient, pharmaceutically acceptable carrier, excipients, and / or other additional ingredients included in the composition may vary depending on the identity, size, and / or condition of the subject to be treated and the route of administration. For example, the composition may comprise from 0.1% to 100%, for example, from 0.5% to 50%, from 1.0% to 30%, from 5.0% to 80%, or greater than 80% (w / w) of the active ingredient.

[0059] The composition may be formulated as a nanoparticle. The nanoparticle may be known to be used for delivering a polynucleotide, such as mRNA, to a cell. For example, the nanoparticle may be a lipid nanoparticle (LNP). The composition may be formulated as a lipid nanoparticle (LNP) or may be bound to an LNP. The binding may include binding within or to the surface of an LNP. For example, the composition may be formulated within a lipid polycation complex or may be bound to a lipid polycation complex. The lipid polycation complex is also referred to as a cationic lipid nanoparticle. The polycation may include a cationic polypeptide such as MC3, Lipid 319, C12-200, 5A2-SC8, 306Oi10, Moderna Lipid 5, Acuitas A9, SM-102, ALC-0315, Arcturus Lipid 2,2(8,8) 4C CH3, Genevant CL1, polylysine, polyornithine, and / or polyarginine. Additionally, the composition may be formulated or bound to lipid nanoparticles comprising a sterol such as distearoylphosphatidylcholine (DSPC), 1,2 distearoyl-sn-glycerol-3-phosphocholine (DSPC), dipalmitoyl phosphatidylcholine (DPPC), cholesterol, or a non-cationic lipid such as dioleoyl phosphatidylethanolamine (DOPE).Additionally, the composition may be formulated or bound to lipid nanoparticles comprising PEG-lipids such as 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000-DMG), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), and polyethylene glycol dimethacrylate (PEG-DMA).

[0060] The lipid nanoparticle formulation can include a cationic lipid, a phospholipid, a sterol such as cholesterol, a PEG-lipid, or a combination thereof. The lipid nanoparticle formulation can include, for example, a cationic lipid, a phospholipid, a sterol such as cholesterol, and a PEG-lipid. Additionally, the lipid nanoparticle can include a PEG-modified lipid, a non-cationic lipid, a sterol, and an ionizable lipid, or a combination thereof. The lipid nanoparticle can include 0.5 to 15 mol % PEG-modified lipid, 5 to 25 mol % non-cationic lipid, 25 to 55 mol % sterol, and 20 to 60 mol % ionizable lipid. The PEG-modified lipid may be 1,2 dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG2000-DMG), the non-cationic lipid may be 1,2 distearoyl-sn-glycerol-3-phosphocholine (DSPC), the sterol may be cholesterol, and the ionizable cationic lipid may be compound 1 having the following structure.

[0061] (Compound 1)

[0062]

[0063] The PEG-modified lipid may be compound 2 (ALC-0159) having the following structure, the non-cationic lipid may be 1,2 distearoyl-sn-glycerol-3-phosphocholine (DSPC), the sterol may be cholesterol, and the ionizable cationic lipid may be compound 3 (ALC-0315) having the following structure.

[0064] (Compound 2)

[0065]

[0066] (Compound 3)

[0067]

[0068] A fourth aspect provides a method of delivering the mRNA or a product expressed thereby to a subject, comprising the step of introducing the mRNA molecule into a host cell.

[0069] In the above method, the host cell may be any cell derived from an organism. The organism may be a mammal, including a human. The cell may be, for example, a stem cell, a germ cell, or a somatic cell. In the above method, the host cell may be a cancer cell, an antigen-presenting cell including a dendritic cell, a monocyte, or a macrophage. The introducing step may be locally administered to a tissue where cancer exists. The introducing step may be locally administered a nucleic acid molecule or an mRNA molecule to a tissue where cells with high levels of cancer-specific miRNA exist, either intracellularly or extracellularly. The administration may be locally administered a nucleic acid molecule or an mRNA molecule to a tissue where cells with upregulated or downregulated cancer-specific miRNA exist, either intracellularly or extracellularly, compared to normal cells.

[0070] The method may comprise administering the mRNA to a subject. The administration may be parenteral or oral. The administration may be intramuscular, subcutaneous, intradermal, nasal, or intrapulmonary. The subject may be a mammal, including a human. The administering step may be local administration to a tissue where cancer exists. The administering step may be local administration of a polynucleotide, for example, an mRNA molecule, intracellularly or extracellularly to a tissue where cells with high levels of cancer-specific miRNA exist. The administration may be local administration of a polynucleotide, for example, an mRNA molecule, intracellularly or extracellularly to a tissue where cells with upregulated or downregulated cancer-specific miRNA exist compared to normal cells.

[0071] The method may involve producing the polypeptide in a cell to inhibit cancer cell proliferation. Furthermore, the method may be for treating a disease in a subject. The polypeptide may be a cancer cell antigen protein.

[0072] The sixth aspect provides a DNA molecule encoding said mRNA.

[0073] The DNA molecule may comprise a nucleotide sequence for introducing a transcribable nucleotide sequence and an expression control sequence. The expression control sequence may be a transcription control sequence, a translation control sequence, or a sequence involved in stabilizing RNA. The expression control sequence may be located upstream or downstream of the nucleotide sequence for introducing the transcribable nucleotide sequence. The expression control sequence may be located downstream of the nucleotide sequence for introducing the transcribable nucleotide sequence and upstream of the modified polyadenylation sequence. The nucleotide sequence for introducing the transcribable nucleotide sequence may be a cloning site. The cloning site may be a multiple cloning site. The cloning site may include a recognition site, a cleavage site, or a combination thereof for a restriction enzyme.

[0074] As used herein, the term "transcription" refers to the process by which the genetic code of a DNA sequence is transcribed into RNA. This RNA can then be translated into protein. The term "transcription" includes in vitro transcription. The term "in vitro transcription" relates to the process by which RNA, particularly mRNA, is synthesized in vitro in a cell-free system. Cloning vectors are used to produce transcripts. These cloning vectors are also commonly referred to as transcription vectors. The term "vector" includes transcription vectors. The RNA can be, for example, in vitro-transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of a DNA template. The promoter controlling transcription can be any promoter for any RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly cDNA, and introduced into a suitable vector for in vitro transcription.

[0075] The above expression control sequence may be a promoter, a ribosome binding sequence, a 5'-untranscribed sequence, or a UTR.

[0076] As used herein, the term "promoter" or "promoter region" refers to a DNA sequence upstream (5') of the coding sequence of a gene, which controls the expression of the coding sequence by providing a recognition and binding site for RNA polymerase. The promoter region may further include recognition or binding sites for additional factors involved in regulating the transcription of the gene. A promoter can control the transcription of a prokaryotic or eukaryotic gene. A promoter can be "inducible" and initiate transcription in response to an inducer, or "constitutive" if transcription is not controlled by an inducer. In the absence of an inducer, little or no transcription of the gene may occur under the control of the inducible promoter. In the presence of the inducer, transcription of the gene may increase under the control of the inducible promoter.

[0077] The promoter may be homologous or heterologous to the sequence encoding the polypeptide. The promoter may be derived from a mammal, including a human. The promoter may be derived from a mammal other than a human. The promoter may be derived from a plant, a microorganism, or a virus. The microorganism may be yeast or bacteria. The bacteria may be gram-positive or gram-negative bacteria. The bacteria may belong to the genus Escherichia. The promoter may be a T7, T3, or SP6 promoter. The promoter may be operable in a microorganism of the genus Escherichia. The microorganism may be Escherichia coli.

[0078] In the above DNA molecule, the promoter may be a T7 promoter and the microRNA may be microRNA-34a.

[0079] In the above DNA molecule, the sequence encoding the polypeptide and the modified polyadenylation sequence can be transcribed under the control of a promoter to produce a common transcript. The modified polyadenylation sequence may be located at the 3' end of the common transcript.

[0080] As used herein, the term "nucleic acid capable of being transcribed to produce a common transcript" refers to nucleic acid sequences that are operably linked to each other such that, when transcribed, an RNA molecule is formed comprising transcripts in which the nucleic acid sequences are covalently linked to each other. The transcription may be, where appropriate, transcription that occurs under the control of a promoter, for example, after linearization of the nucleic acid molecule, for example, a closed circular nucleic acid molecule, or after restriction enzyme digestion of the nucleic acid molecule. The common transcript may, where appropriate, be separated by a sequence located between the nucleic acid sequences.

[0081] The above DNA molecule may be a closed circular molecule or a linear molecule.

[0082] The DNA molecule may further comprise one or more selected from the group consisting of (i) a reporter gene, (ii) a selectable marker, and (iii) a proliferation origin. The nucleic acid molecule may be a vector. The vector may be a cloning vector or an expression vector.

[0083] The above DNA molecule, after linearization, may be suitable for in vitro transcription of mRNA.

[0084] In the above DNA molecule, the nucleic acid sequence for introducing the transcribable nucleic acid sequence may be a cloning site. The nucleic acid molecule may comprise a nucleic acid molecule for introducing the transcribable nucleic acid sequence, which includes a multiple cloning site.

[0085] As used herein, the term "cloning site" refers to a restriction enzyme recognition or cleavage site sequence. The cloning site may include, for example, Hind IIII, Xho I, Nhe I, and Eco RI restriction sites. The nucleic acid molecule may further include a cloning site at a different location from the nucleic acid sequence for introducing the above-mentioned transcribable nucleic acid sequence. The nucleic acid molecule may include a cloning site in the expression control sequence. The nucleic acid molecule may include a cloning site downstream of the modified polyadenylation sequence. The downstream of the modified polyadenylation sequence may be adjacent to the modified polyadenylation sequence or located within 20 nucleotides.

[0086] In this specification, the term "expression" is used in a general sense and includes the production of RNA or RNA and protein. Such expression also includes partial expression of nucleic acids. Furthermore, such expression may be transient or stable. With respect to RNA, the terms "expression" or "translation" refer to the process in a cell's ribosomes whereby a strand of messenger RNA directs the assembly of an amino acid sequence to produce a peptide or protein.

[0087] A seventh aspect provides a method of propagating a nucleic acid molecule, comprising the steps of: (i) providing the DNA molecule described above; and (ii) propagating the nucleic acid molecule in a cell.

[0088] In the above method, the cell may be a host cell. The term "host cell" refers to any cell into which a foreign nucleic acid can be transformed or transduced. The host cell includes a prokaryotic cell (e.g., Escherichia coli) or a eukaryotic cell (e.g., yeast cells and insect cells). The cell may be an animal, plant, or microbial cell. The animal may be a mammal. The mammals include humans, mice, rats, hamsters, pigs, and primates. The cell may be derived from multiple tissue cells, including primary cells and cell lines. The microorganism may be a yeast or a bacterium. The bacterium may be a gram-positive or gram-negative bacterium. The bacterium may belong to the genus Escherichia. The cell may be E. coli.

[0089] In the above method, the proliferating step may include a step of transforming a cell with the nucleic acid molecule and a step of culturing the transformed cell.

[0090] The method may comprise a step of isolating the nucleic acid molecule from the cell after proliferation.

[0091] The eighth aspect provides a method for obtaining RNA, comprising the steps of (i) amplifying a nucleic acid molecule according to the method of the seventh aspect; and (ii) in vitro transcribing the nucleic acid molecule into RNA as a template.

[0092] The term "in vitro transcription" or "RNA in vitro transcription" refers to the process by which RNA, including mRNA, is synthesized in a cell-free system, i.e., in vitro. Cloning vector DNA, including plasmid DNA vectors, can be used as templates for the production of RNA transcripts. These cloning vectors are also commonly referred to as transcription vectors. RNA can be obtained by DNA-dependent in vitro transcription of a suitable DNA template. The DNA template may be a linearized plasmid DNA template. The promoter controlling RNA in vitro transcription may be any promoter for a DNA-dependent RNA polymerase. The DNA-dependent RNA polymerase may be T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, or a combination thereof. The DNA template for RNA in vitro transcription can be obtained by cloning a nucleic acid, particularly cDNA, and introducing it into a vector for RNA in vitro transcription. The vector for RNA in vitro transcription may be circular plasmid DNA. The above cDNA can be obtained by reverse transcription of mRNA or chemical synthesis.

[0093] A ninth aspect provides a method for obtaining a polypeptide, comprising the steps of: (i) obtaining an mRNA encoding a polypeptide according to the method of the eighth aspect; and (ii) translating the mRNA into a polypeptide.

[0094] The above method may comprise, prior to step (i), a step of cleaving the nucleic acid molecule.

[0095] The tenth aspect provides an RNA molecule obtained by transcribing the nucleic acid molecule of the sixth aspect. The RNA molecule may be an mRNA molecule. The RNA molecule may be an RNA molecule obtained according to the method of the third aspect.

[0096] mRNA according to the daily pattern is relatively stable and has high translation efficiency, so it can be used to efficiently translate mRNA.

[0097] DNA molecules with different modalities can be used to produce RNA with increased stability.

[0098] According to the method of multiplying DNA molecules according to the following aspects, DNA molecules can be efficiently multiplied.

[0099] According to a method of obtaining RNA according to different aspects, RNA can be obtained efficiently.

[0100] According to a method for obtaining a polypeptide according to another aspect, a polypeptide can be obtained efficiently.

[0101] Compositions or kits for delivering mRNA or a product expressed thereby to a subject according to different aspects may be used to treat cancer.

[0102] According to a method of delivering mRNA or a product expressed thereby to an individual according to another aspect, mRNA or a product expressed thereby can be efficiently delivered to an individual.

[0103] Figure 1 is a drawing showing a vector obtained through the same process as Examples 1 and 2.

[0104] Figure 2 is a drawing showing a vector including a T7 promoter and 5'UTR and a target protein ORF between HindIII and Xhol restriction enzyme sites in the template vector of Figure 1.

[0105] FIG. 3 is a diagram showing a vector for p53 gene expression, p53-polyA-miR34a, which includes a modified polyadenylation sequence including 5'UTR, p53 gene, 3'UTR, and miR34a.

[0106] Figure 4 is a diagram showing the results of confirming the effect of miRNA-34a on the expression of mRNA introduced into cells through Western blot.

[0107] Figure 5a is a diagram showing the degree of growth inhibition of normal BEAS-2B cells by p53-polyA-miR34a mRNA.

[0108] Figure 5b is a diagram showing the degree of growth inhibition of lung cancer cells H1373 by p53-polyA-miR34a mRNA.

[0109] Figure 5c is a diagram showing the degree of growth inhibition of lung cancer cells H1299 by p53-polyA-miR34a mRNA.

[0110] Figure 6 is a drawing showing a template vector for transcription of the produced OX40L or fusion protein gene thereof.

[0111] The present invention will be described in more detail below through examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0112] Example 1: Cloning of 3'UTR sequences

[0113] In this example, 3'UTR was cloned to increase the expression level and sequence stability of a gene encoding mRNA. The 3'UTR used was the 3'UTR sequence of human cytochrome P450 2E1. The 3'UTR includes XhoI and NheI restriction enzyme sequences at the 5'-end and 3'-end, respectively, so that it can be used to recombine nucleic acids such as the transcribable nucleic acid sequence or polyadenylation sequence. The transcribable nucleic acid sequence may be an open reading frame (ORF). The 3'UTR sequence can be linked to the 3'-end of the open reading frame through the XhoI restriction enzyme site at the 5'-end.

[0114] A recombinant 3'UTR sequence containing an XhoI restriction enzyme sequence, a 3'UTR sequence of human cytochrome P450 2E1, and a NheI restriction enzyme sequence was synthesized by an in vitro nucleic acid synthesis method. The obtained recombinant 3'UTR sequence is DNA. The recombinant 3'UTR sequence and the template vector were digested with XhoI and NheI restriction enzymes, respectively, and then the two sequences were linked using DNA ligase. The template vector was pUC57-Kan. R Vector (self-made) was used. pUC57-Kan R The vector is a pUC57 vector containing Kanamycin as an antibiotic resistance gene. The antibiotic resistance gene is a selectable marker. As a result, a template vector containing a recombinant 3'UTR sequence was constructed. Table 1 shows the sequence encoding the 3'UTR sequence of human cytochrome P450 2E1.

[0115] Sequence number 3'UTR sequence 1GTGTGTGGAGGACACCCTGAACCCCCCGCTTTCAAACAAGTTTTCAAATTGTTTGAGGTCAGGATTTCTCAAACTGATTCCTTTCTTTGCATATGAGTATTTGAAAATAAATATTTTCCCAGAATATAAATAAATCATCACATGATTATTTTAACTAT

[0116] In Table 1, the sequence of sequence number 1 is the 3'UTR natural sequence of the human cytochrome P450 2E1 gene.

[0117] Example 2: Cloning of a continuous adenosine sequence containing a miRNA binding sequence.

[0118] In this example, a recombinant template vector was constructed in which a modified polyadenylation sequence was linked to the 3'-end of the 3'UTR sequence from the recombinant 3'UTR-containing template vector constructed in Example 1. The modified polyadenylation sequence is intended to increase the stability and expression level of the gene encoding mRNA.

[0119] The modified polyadenyl sequence includes a nucleotide sequence complementary to miRNA-34a in the middle region of an unmodified 100 nt polyadenyl sequence. Specifically, the modified polyadenyl sequence has nucleotide sequences 1 to 22 complementary to miRNA-34a introduced between positions 20 and 21 of the unmodified polyadenyl sequence.

[0120] In addition, the modified polyadenylation sequence was made available for recombination by linking NheI and EcoRI restriction enzyme sequences to the 5'-end and 3'-end, respectively. The recombinant modified polyadenylation sequence containing the NheI restriction enzyme sequence, the modified polyadenylation sequence, and the EcoRI restriction enzyme sequence was synthesized by an in vitro nucleic acid synthesis method. The obtained recombinant sequence is DNA.

[0121] The resulting recombinant modified polyadenyl sequence and the recombinant 3'UTR-containing template vector constructed in Example 1 were digested with NheI and EcoRI restriction enzymes, respectively, and the two sequences were ligated using DNA ligase to construct a 3'UTR-modified polyadenyl sequence-containing template vector. Table 2 shows the nucleotide sequence of the modified polyadenyl sequence.

[0122] Sequence number poly A tail sequence 2AAAAAAAAAAAAAAAAAAAAAAACAACCAGCTAAGACACTGCCAAA ...

[0123] The nucleotides underlined in the sequences described in Table 2 are complementary sequences to miRNA-34a, i.e., miRNA-34a binding sequences. Fig. 1 is a drawing showing a vector obtained by the same process as Examples 1 and 2. As shown in Fig. 1, the vector includes a 3'UTR linked to the 5' of a modified polyadenylation sequence including a miRNA-34a binding sequence in the region of nucleotides 21-42 of the vector. The vector includes HindIII and XhoI restriction enzyme recognition sites upstream of the 3'UTR, so that it can be used to introduce a target protein ORF linked to the 3' end of the 5'UTR. That is, the vector can be a platform vector used to clone ORF. The 5'UTR-ORF is linked to the T7 promoter upstream thereof. The vector also includes an antibiotic resistance marker (Kan R ) and the E. coli replication origin (ColE1 ori). The nucleotide sequence of the template vector as shown in Fig. 1 is shown in Table 3.

[0124] Figure 2 is a drawing showing a vector including a T7 promoter and 5'UTR and a target protein ORF between HindIII and Xhol restriction enzyme sites in the template vector of Figure 1.

[0125]

[0126] The nucleotides indicated in capital letters and underlined in the sequence of the template vector of SEQ ID NO: 3 in Table 3 are HindIII restriction enzyme recognition sequences. The nucleotides indicated in bold letters and capital letters in the sequence of the template vector of SEQ ID NO: 3 in Table 3 are XhoI restriction enzyme recognition sequences. The nucleotides indicated in lowercase letters in the sequence of the template vector of SEQ ID NO: 3 in Table 3 are the human cytochrome P450 2E1 3'UTR native sequence (SEQ ID NO: 1).

[0127] The nucleotides in bold in lowercase letters in the sequence of the template vector of sequence number 3 described in Table 3 are NheI restriction enzyme recognition sequences.

[0128] The nucleotides underlined in lowercase letters among the sequences of the template vector of sequence number 3 described in Table 3 are miRNA-34a binding sequences complementary to miRNA-34a.

[0129] The nucleotides indicated in capital letters in the sequence of the template vector of sequence number 3 described in Table 3 are the EcoRI restriction enzyme recognition sequence.

[0130] Sequence number 4 listed in Table 3 is the sequence of sequence number 3 excluding the restriction enzyme recognition site.

[0131] Example 3: Construction of a p53 mRNA transcription vector

[0132] In this example, a template vector to be used for p53 mRNA transcription was produced using the vector platform as shown in Figure 1 produced in Example 2.

[0133] Example 3-1. Cloning of 5'UTR sequence and native p53 gene sequence

[0134] A recombinant gene containing a 5' restriction enzyme recognition site, a T7 promoter sequence, a 5'UTR, an open reading frame, and a 3' restriction enzyme recognition site was synthesized by an in vitro nucleic acid synthesis method. The 5' restriction enzyme recognition site and the 3' restriction enzyme recognition site are HindIII and XhoI restriction enzyme sequences, respectively. The 5'UTR is the sequence indicated in bold in Table 4 below. The open reading frame (ORF) is a nucleotide sequence encoding human native p53. As a result, a recombinant sequence in which a HindIII recognition site, a T7 promoter sequence, a 5'UTR, a p53 ORF, and an XhoI recognition site are linked was obtained.

[0135] The above-obtained recombinant sequence and the template vector of Figure 1 constructed in Example 2 were each digested with restriction enzymes HindIII and XhoI, and the two sequences were ligated using DNA ligase. As a result, a transcription vector containing a 5'UTR, a native p53 ORF, a cytochrome P450 2E1 3'UTR, and a modified polyadenylation sequence was constructed (hereinafter referred to as "p53-polyA-miR34a"). As a control, p53-polyA-miR34a was used in which the modified polyadenylation sequence was replaced with an unmodified polyA containing 100 As (hereinafter referred to as "p53-polyA").

[0136] Table 4 shows the nucleotides of the recombinant sequence linked to the HindIII recognition site, T7 promoter sequence, 5'UTR, p53 ORF, and XhoI recognition site. The HindIII and XhoI recognition sites were attached to both ends of the linked recombinant sequence obtained.

[0137] Sequence number 5'UTR and native p53 gene sequence 5TAATACGACTCACTATA 6AGTGCTCCCCATCCAACTAAACTGTCCCTCTGTCCGAACTAAACTGCACTGTCCCAGCACC 7TCCGCGAATTGAACGAGGCCCTGGAGTTGAAAGACGCTCAAGCCGGAAAGGAACCAGGTGGGTCTAGGGCCCACTCAAGCCATCTGAAGAGTAAGAAAGGCCAGTCCACTTCCAGGCACAAAAAGTTAATGTTCAAGACGGAGGGTCCTGACTCTGAC

[0138] In the sequences described in Table 4, the underlined nucleotides in SEQ ID NO: 5 represent the T7 promoter portion involved in the initiation of mRNA transcription. In addition, the bold nucleotides in SEQ ID NO: 6 are the 5'UTR sequence. In addition, SEQ ID NO: 7 is the nucleotide sequence downstream of the 5'UTR that encodes the native p53 protein. Figure 3 is a drawing showing a vector for p53 gene expression, p53-polyA-miR34a, which includes a 5'UTR, a p53 gene, a 3'UTR, and a modified polyadenylation sequence including miR34a. Table 5 shows the nucleotide sequence of each element among the products obtained by in vitro transcription of the obtained transcription vector. Table 6 shows the amino acid sequence encoded by the p53 gene included in the obtained transcription vector.

[0139] Sequence number region mRNA sequence 85'UTRAGUGCUCCCCAUCCAACUAAACUGUCCCUCUGUCCGAACUAAACUGCACUGUCCCAGCACC9 natural type p53 ProteinUCCGCGAAUUGAACGAGGCCCUGGAGUUGAAAGACGCUCAAGCCGGAAAGGAACCAGGUGGGUCUAGGGCCCACUCAAGCCAUCUGAAGAGUAAGAAAGGCCAGUCCACUUCCAGGCACAAAAAGUUAAUGUUCAAGACGGAGGGUCCUGACUCUGACCUCGAG103' UTRGUGUGUGGAGGACACCCUGAACCCCCCGCUUUCAAACAAGUUUUCAAAUUGUUUGAGGUCAGGAUUUCUCAAACUGAUUCCUUUCUUUGCAUAUGAGUAUUUGAAAAUAAAUAUUUUCCCAGAAUAUAAAUAAAUCAUCACAUGAUUAUUUUAACUAUGCUAGC11PolyA TailAAAAAAAAAAAAAAAAAAAAAAACAACCAGCUAAGACACUGCCAAA ... ProteinUCCGCGAAUUGAACGAGGCCCUGGAGUUGAAAGACGCUCAAGCCGGAAAGGAACCAGGUGGGUCUAGGGCCCACUCAAGCCAUCUGAAGAGUAAGAAAGGCCAGUCCACUUCCAGGCACAAAAAGUUAAUGUUCAAGACGGAGGGUCCUGACUCUGAC133' UTRGUGUGUGGAGGACACCCUGAACCCCCCGCUUUCAAACAAGUUUUCAAAUUGUUUGAGGUCAGGAUUUCUCAAACUGAUUCCUUUCUUUGCAUAUGAGUAUUUGAAAAUAAAUAUUUUCCCAGAAUAUAAAUAAAUCAUCACAUGAUUAUUUUAACUAU14PolyATailAAAAAAAAAAAAAAAAAAAAAAACAACCAGCUAAGACACUGCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0140] The underlined nucleotides in the native p53 protein region of SEQ ID NO: 9 listed in Table 5 are XhoI restriction enzyme recognition sequences. The underlined nucleotides in the 3'UTR region of SEQ ID NO: 10 listed in Table 5 are NheI restriction enzyme recognition sequences. The underlined nucleotides in the polyA tail region of SEQ ID NO: 11 listed in Table 5 are SapI restriction enzyme recognition sequences. The bold nucleotides in the polyA tail region of SEQ ID NO: 11 listed in Table 5 are part of the EcoRI restriction enzyme recognition sequence.

[0141] Sequence numbers 12 to 14 listed in Table 5 are sequences excluding the restriction enzyme recognition portion from sequence numbers 9 to 11.

[0142] SEQ ID NO: Natural p53 protein amino acid SEQ ID NO:15MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSSVPSQKTYQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHL IRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD

[0143] In Table 6, sequence number 15 is the amino acid sequence obtained by translation of sequence number 12.

[0144] Example 3-2. Confirmation of p53 mRNA expression in H1299 cells

[0145] An in vitro transcription reaction was performed by incubating a mixture containing each of the p53-polyA-miR34a vector and the p53-polyA vector produced in Example 3-1, thereby obtaining p53-polyA-miR34a mRNA and p53-polyA mRNA. The p53-polyA-miR34a mRNA includes the 5'UTR of SEQ ID NOs: 8, 9, 10, and 11, the p53 ORF, the 3'UTR, and polyA-miR34a.

[0146] The obtained p53-polyA-miR34a mRNA or p53-polyA mRNA and miRNA-34a were delivered to H1299 cells, and expression was confirmed. Synthetic miRNA-34a was used. H1299 cells are a human non-small cell lung carcinoma cell line derived from lymph nodes. H1299 cells do not express the p53 protein due to homozygous partial deletion of the p53 gene (hereinafter referred to as "p53-null lung cancer cells").

[0147] Specifically, H1299 cells were cultured in 3x10 5Cells were seeded into each well of a 6-well plate at a volume of 2 mL medium per well and incubated at 37°C for 24 h. RPMI 1640 (Gibco, Cat No. 22400-089) was used as the medium. Next, the p53-polyA-miR34a mRNA or p53-polyA mRNA and Lipofectamine™ MessengerMAX™ Transfection Reagent (Thermo Scientific™, Cat No. LMRNA001) were mixed at a ratio of 1 μl per 1 μg of mRNA and incubated for 20 minutes. 1 μg of the resulting reaction product and miRNA-34a were added to each well of a 6-well plate. miRNA-34a was added at concentrations of 0 nM, 10 nM, and 25 nM, respectively. The reaction mixture was incubated at 37°C for 24 h. After completion of the incubation, lysis buffer, M-PER TM (Mammalian Protein Extraction Reagent)(Thermo Scientific TM , Cat No. 78501) was added to each well at a volume of 100 μl / well to rupture the cells and extract proteins, and then Pierce TM BCA Protein Assay Kit (Thermo Scientific TM , Cat No. 23225) was used to quantify BCA protein. The obtained sample was diluted with lysis buffer and 5x reducing dye (10% SDS, 0.5% bromophenol blue, 50% glycerol, 0.5% 2-mercaptoethanol, and 250 mM tris, pH 6.8) to equalize the protein concentration. Western blot experiments were performed on the obtained diluted sample using anti-p53 monoclonal antibody (CST, Cat No. #2527) and anti-β-actin (abcam, Cat No. ab8227).

[0148] Figure 4 is a diagram showing the results of Western blot analysis to determine the effect of miRNA-34a on the expression of mRNA introduced into cells. As shown in Figure 4, p53 expression was significantly reduced in p53-polyA-miR34a mRNA compared to p53-polyA mRNA. This indicates that p53 expression is reduced by the modified polyA containing the miR34a binding site.

[0149] That is, since p53-polyA mRNA does not have a miRNA-34a binding site at the polyA position, there was no difference in p53 expression even when the concentration of miRNA-34a composite was increased. On the other hand, p53-polyA-miR34a mRNA has a site at the polyA position where miRNA-34a can bind, so p53 expression decreased in proportion to the concentration of the added miRNA-34a composite. This indicates that miRNA-34a normally binds to the miRNA-34a binding site inserted at the polyA position to regulate p53 expression. However, the claimed invention is not limited by a specific mechanism.

[0150] Example 3-3. Confirmation of cell growth inhibition in BEAS-2B, H1373, and H1299 cells.

[0151] An in vitro transcription reaction was performed by incubating a mixture containing the p53-polyA-miR34a vector produced in Example 3-1, thereby obtaining p53-polyA-miR34a mRNA. The p53-polyA-miR34a mRNA includes the 5'UTR of SEQ ID NOs: 8, 9, 10, and 11, the p53 ORF, the 3'UTR, and polyA-miR34a.

[0152] The obtained p53-polyA-miR34a mRNA was delivered to normal and cancer cells, and its cell growth inhibitory activity was confirmed. BEAS-2B human bronchial epithelial cells were used as normal cells, and H1373 and H1299 cells were used as cancer cells. H1373 cells are a lung adenocarcinoma cell line in which the p53 gene is mutated and expression is not detected by Western blot.

[0153] Specifically, BEAS-2B, H1373, and H1299 cells were treated with 3x10 3 Cells / 200 μL medium / well, 3x10 3 cells / 200 μL medium / well, and 5x10 3 Cells were seeded into each well of a 98-well plate at a volume of 200 μL medium / well and incubated at 37°C for 24 hours. For BEAS-2B cells, BEBM (Lonza, Cat No. cc-3171) medium was used, and for H1373 and H1299 cells, RPMI 1640 (Gibco, Cat No. 22400-089) medium was used.

[0154] Next, the p53-polyA-miR34a mRNA and Lipofectamine™ MessengerMAX™ Transfection Reagent (Thermo Scientific™, Cat No. LMRNA001) were mixed at a ratio of 1 μL per 1 μg of p53-polyA-miR34a mRNA and incubated for 20 minutes. 0.2 μL of the resulting reaction product was added to each well of a 98-well plate. At this time, p53-polyA-miR34a mRNA was added at 4 μg / mL and 8 concentrations, each of which was 1 / 4 lower than 4 μg / mL. The cells in the medium contained in each well of the resulting plate were cultured at 37°C for 3 days, and then cell viability was measured using the WST-8 cell viability assay kit (BIOMAX, Cat No. QM5000) reagent.

[0155] After the culture was completed, the WST-8 reagent was mixed with the cell medium at a ratio of 1:10 and reacted for 2 hours. The absorbance at a wavelength of 450 nm was measured using Graphpad Prism. TM GI using software 50 The value was derived. GI 50 represents the half maximal growth inhibition concentration.

[0156] Figure 5a is a diagram showing the degree of growth inhibition of normal BEAS-2B cells by p53-polyA-miR34a mRNA.

[0157] Figure 5b is a diagram showing the degree of growth inhibition of lung cancer cells H1373 by p53-polyA-miR34a mRNA.

[0158] Figure 5c is a diagram showing the degree of growth inhibition of lung cancer cells H1299 by p53-polyA-miR34a mRNA.

[0159] As shown in Figures 5a, 5b, and 5c, the mRNA into which the miRNA binding site was introduced did not significantly inhibit cell growth in normal cells, but inhibited cell growth specifically in cancer cells.

[0160] Example 4. Construction of OX40L mRNA transcription vector

[0161] In this example, a template vector to be used for OX40L mRNA transcription was produced using the vector platform (Fig. 1) produced in Example 2.

[0162] A recombinant gene containing a 5' restriction enzyme recognition site, a T7 promoter sequence, a 5'UTR, an open reading frame (ORF), and a 3' restriction enzyme recognition site was synthesized by in vitro nucleic acid synthesis. The 5' restriction enzyme recognition site and the 3' restriction enzyme recognition site are HindIII and XhoI restriction enzyme sequences, respectively. The open reading frame is a sequence encoding a fusion protein of native OX40L, the alpha helix region of OX40L-MBL-ferritin, or a fusion protein of MBL-OX40L, respectively. The heavy chain region of native ferritin was used as ferritin. MBL stands for mannan binding lectin, and a sequence with the carbohydrate recognition domain removed was used.

[0163] As a result, a recombinant sequence was obtained in which a HindIII recognition site, a T7 promoter sequence, a 5'UTR, each of the above-mentioned ORFs, and an XhoI recognition site were linked. The fusion protein includes a linker sequence between each fusion partner. The linker sequence is a G4S, i.e., GGGGS sequence.

[0164] Next, the obtained recombinant sequence and the template vector of Figure 1 produced in Example 2 were each cut by treatment with restriction enzymes HindIII and XhoI, and the two sequences were linked using DNA ligase.

[0165] As a result, a transcription vector containing the 5'UTR, each of the above-mentioned ORFs, the cytochrome P450 2E1 3'UTR, and a modified polyadenylation sequence was constructed. Table 7 shows the nucleotides of the recombinant sequences linked to the T7 promoter sequence, the 5'UTR, the OX40L ORF, the fusion protein ORF of the alpha helix region of OX40L-MBL-ferritin, or the fusion protein ORF of MBL-OX40L. The HindIII recognition site and XhoI recognition site sequences are not included in the 5'UTR and ORF, respectively, and were used only for cloning.

[0166] 서열번호영역뉴클레오티드 서열5T7 프로모터TAATACGACTCACTATA65'UTRAGTGCTCCCCATCCAACTAAACTGTCCCTCTGTCCGAACTAAACTGCACTGTCCCAGCACC16신호 서열ATGACAAGGTTGACGGTCCTGGCATTGCTTGCCGGGCTTTTGGCGAGTTCTCGGGCG17OX40L의 세포외 도메인AGCAGTTCACCTGCAAAGGACCCCCCAATCCAGCGACTGAGAGGCGCAGTCACCCGGTGTGAAGATGGACAGCTTTTTATATCATCTTACAAGAATGAGTATCAGACTATGGAGGTTCAAAATAACTCCGTGGTTATAAAGTGCGACGGGCTTTACATCATCTATCTGAAGGGGTCCTTTTTCCAGGAGGTCAAGATAGATTTGCACTTCAGGGAAGACCATAACCCCATTTCTATTCCTATGCTTAATGACGGACGACGGATCGTATTTACTGTTGTGGCTTCTCTCGCTTTTAAGGACAAGGTCTACCTTACCGTGAATGCCCCAGACACACTTTGCGAGCACCTCCAGATCAACGACGGAGAGCTTATCGTTGTTCAACTTACACCAGGTTATTGCGCTCCAGAGGGGAGCTATCACTCCACCGTCAACCAGGTACCCCTT18G4S 링커*2GGTGGAGGCGGTTCAGGCGGAGGTGGCTCT19MBL의 알파 헬릭스AGCGAGAGAAAGGCCCTTCAGACAGAAATGGCACGCATAAAAAAATGGCTTACATTCAGT31G4S링커GGTGGAGGCGGTTCA20페리틴CAGGACTCCGAAGCAGCAATAAACCGACAAATAAATCTGGAGTTGTACGCATCCTACGTTTACCTGTCCATGTCTTACTACTTCGATAGAGACGATGTCGCCCTCAAAAACTTCGCCAAGTATTTCCTGCATCAGTCCCATGAAGAGAGAGAGCACGCAGAGAAACTGATGAAACTTCAGAACCAACGGGGGGGCAGGATCTTTCTGCAAGACATTAAGAAGCCTGATTGCGATGACTGGGAATCTGGACTTAATGCCATGGAGTGTGCCCTGCACCTCGAAAAAAATGTGAACCAGAGCCTGTTGGAATTGCATAAGCTGGCAACCGATAAGAACGATCCACATCTTTGCGATTTCATCGAAACCCACTATCTCAATGAACAAGTGAAGGCAATTAAGGAGCTGGGCGATCATGTAACAAATTTGCGAAAAATGGGTGCC5T7 프로모터TAATACGACTCACTATA65'UTRAGTGCTCCCCATCCAACTAAACTGTCCCTCTGTCCGAACTAAACTGCACTGTCCCAGCACC16신호 서열ATGACAAGGTTGACGGTCCTGGCATTGCTTGCCGGGCTTTTGGCGAGTTCTCGGGCG21MBLTCTGGCTCTCAGACTTGTGAAGATACCCTTAAGACTTGCAGTGTCATAGCCTGTGGGCGAGACGGGCGCGATGGACCCAAGGGCGAAAAAGGAGAACCAGGGCAAGGGCTCAGAGGACTGCAAGGTCCACCTGGTAAATTGGGGCCACCTGGAAGCGTGGGGTCCCCAGGAAGTCCAGGCCCAAAAGGTCAGAAAGGGGACCACGGCGATAATCGAGCCATTGAGGAAAAACTCGCAAATATGGAGGCCGAAATTAGGATTCTGAAGAGTAAGTTGCAGCTGACCAACAAGCTCCATGCTTTTAGCATGGGC18G4S 링커*2GGTGGAGGCGGTTCAGGCGGAGGTGGCTCT17OX40L의 세포외DomainAGCAGTTCACCTGCAAAGGACCCCCCAATCCAGCGACTGAGAGGCGCAGTCACCCGGTGTGAAGATGGACAGCTTTTTATATCATCTTACAAGAATGAGTATCAGACTATGGAGGTTCAAAATAACTCCGTGGTTATAAAGTGCGACGGGCTTTACATCATCTATCTGAAGGGGTCCTTTTTCCAGGAGGTCAAGATAGATTTGCACTTCAGGGAAGACC ATAACCCCATTTCTATTCCTATGCTTAATGACGGACGGACGGATCGTATTTACTGTTGTGGCTTCTCTCGCTTTTAAGGACAAGGTCTACCTTACCGTGAATGCCCCAGACACACTTTGCGAGCACCTCCAGATCAACGACGGAGAGCTTATCGTTGTTCAACTTACACCAGGTTATTGCGCTCCAGAGGGGAGCTATCACTCCACCGTCAACCAGGTACCCCTT

[0167] Figure 6 is a drawing showing a template vector for transcription of the OX40L or fusion protein gene produced. As shown in Figure 6, the template vector includes a modified polyadenylation sequence containing a promoter-5'UTR-OX40L ORF-CYP450 2E1 3'UTR-miRNA-34a binding sequence. In addition, the template vector includes ColE1 ori and Kan R Contains genes. Table 8 shows the nucleotide sequence of each element among the products obtained by in vitro transcription of the obtained transcription vector. Table 9 shows the amino acid sequence encoded by each ORF included in the obtained transcription vector.

[0168] 물질서열번호영역mRNA 서열185'UTRAGUGCUCCCCAUCCAACUAAACUGUCCCUCUGUCCGAACUAAACUGCACUGUCCCAGCACC22신호 서열AUGACAAGGUUGACGGUCCUGGCAUUGCUUGCCGGGCUUUUGGCGAGUUCUCGGGCG23OX40L의 세포외 도메인AGCAGUUCACCUGCAAAGGACCCCCCAAUCCAGCGACUGAGAGGCGCAGUCACCCGGUGUGAAGAUGGACAGCUUUUUAUAUCAUCUUACAAGAAUGAGUAUCAGACUAUGGAGGUUCAAAAUAACUCCGUGGUUAUAAAGUGCGACGGGCUUUACAUCAUCUAUCUGAAGGGGUCCUUUUUCCAGGAGGUCAAGAUAGAUUUGCACUUCAGGGAAGACCAUAACCCCAUUUCUAUUCCUAUGCUUAAUGACGGACGACGGAUCGUAUUUACUGUUGUGGCUUCUCUCGCUUUUAAGGACAAGGUCUACCUUACCGUGAAUGCCCCAGACACACUUUGCGAGCACCUCCAGAUCAACGACGGAGAGCUUAUCGUUGUUCAACUUACACCAGGUUAUUGCGCUCCAGAGGGGAGCUAUCACUCCACCGUCAACCAGGUACCCCUU24G4S 링커*2GGUGGAGGCGGUUCAGGCGGAGGUGGCUCU25MBL의 알파 헬릭스AGCGAGAGAAAGGCCCUUCAGACAGAAAUGGCACGCAUAAAAAAAUGGCUUACAUUCAGU32G4S링커GGUGGAGGCGGUUCA26페리틴CAGGACUCCGAAGCAGCAAUAAACCGACAAAUAAAUCUGGAGUUGUACGCAUCCUACGUUUACCUGUCCAUGUCUUACUACUUCGAUAGAGACGAUGUCGCCCUCAAAAACUUCGCCAAGUAUUUCCUGCAUCAGUCCCAUGAAGAGAGAGAGCACGCAGAGAAACUGAUGAAACUUCAGAACCAACGGGGGGGCAGGAUCUUUCUGCAAGACAUUAAGAAGCCUGAUUGCGAUGACUGGGAAUCUGGACUUAAUGCCAUGGAGUGUGCCCUGCACCUCGAAAAAAAUGUGAACCAGAGCCUGUUGGAAUUGCAUAAGCUGGCAACCGAUAAGAACGAUCCACAUCUUUGCGAUUUCAUCGAAACCCACUAUCUCAAUGAACAAGUGAAGGCAAUUAAGGAGCUGGGCGAUCAUGUAACAAAUUUGCGAAAAAUGGGUGCC273'UTRCUCGAGGUGUGUGGAGGACACCCUGAACCCCCCGCUUUCAAACAAGUUUUCAAAUUGUUUGAGGUCAGGAUUUCUCAAACUGAUUCCUUUCUUUGCAUAUGAGUAUUUGAAAAUAAAUAUUUUCCCAGAAUAUAAAUAAAUCAUCACAUGAUUAUUUUAACUAUGCUAGC11폴리A 테일AAAAAAAAAAAAAAAAAAAAACAACCAGCUAAGACACUGCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAGAGCGAAUU285'UTRAGUGCUCCCCAUCCAACUAAACUGUCCCUCUGUCCGAACUAAACUGCACUGUCCCAGCACC22신호서열AUGACAAGGUUGACGGUCCUGGCAUUGCUUGCCGGGCUUUUGGCGAGUUCUCGGGCG28MBLUCUGGCUCUCAGACUUGUGAAGAUACCCUUAAGACUUGCAGUGUCAUAGCCUGUGGGCGAGACGGGCGCGAUGGACCCAAGGGCGAAAAAGGAGAACCAGGGCAAGGGCUCAGAGGACUGCAAGGUCCACCUGGUAAAUUGGGGCCACCUGGAAGCGUGGGGUCCCCAGGAAGUCCAGGCCCAAAAGGUCAGAAAGGGGACCACGGCGAUAAUCGAGCCAUUGAGGAAAAACUCGCAAAUAUGGAGGCCGAAAUUAGGAUUCUGAAGAGUAAGUUGCAGCUGACCAACAAGCUCCAUGCUUUUAGCAUGGGC24G4S 링커*2GGUGGAGGCGGUUCAGGCGGAGGUGGCUCU23OX40L의 세포외도메인AGCAGUUCACCUGCAAAGGACCCCCCAAUCCAGCGACUGAGAGGCGCAGUCACCCGGUGUGAAGAUGGACAGCUUUUUAUAUCAUCUUACAAGAAUGAGUAUCAGACUAUGGAGGUUCAAAAUAACUCCGUGGUUAUAAAGUGCGACGGGCUUUACAUCAUCUAUCUGAAGGGGUCCUUUUUCCAGGAGGUCAAGAUAGAUUUGCACUUCAGGGAAGACCAUAACCCCAUUUCUAUUCCUAUGCUUAAUGACGGACGACGGAUCGUAUUUACUGUUGUGGCUUCUCUCGCUUUUAAGGACAAGGUCUACCUUACCGUGAAUGCCCCAGACACACUUUGCGAGCACCUCCAGAUCAACGACGGAGAGCUUAUCGUUGUUCAACUUACACCAGGUUAUUGCGCUCCAGAGGGGAGCUAUCACUCCACCGUCAACCAGGUACCCCUU273'UTRCUCGAGGUGUGUGGAGGACACCCUGAACCCCCCGCUUUCAAACAAGUUUUCAAAUUGUUUGAGGUCAGGAUUUCUCAAACUGAUUCCUUUCUUUGCAUAUGAGUAUUUGAAAAUAAAUAUUUUCCCAGAAUAUAAAUAAAUCAUCACAUGAUUAUUUUAACUAUGCUAGC11폴리A 테일AAAAAAAAAAAAAAAAAAAAACAACCAGCUAAGACACUGCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAGAGCGAAUU385'UTRAGUGCUCCCCAUCCAACUAAACUGUCCCUCUGUCCGAACUAAACUGCACUGUCCCAGCACC22신호 서열AUGACAAGGUUGACGGUCCUGGCAUUGCUUGCCGGGCUUUUGGCGAGUUCUCGGGCG23OX40L의 세포외도메인AGCAGUUCACCUGCAAAGGACCCCCCAAUCCAGCGACUGAGAGGCGCAGUCACCCGGUGUGAAGAUGGACAGCUUUUUAUAUCAUCUUACAAGAAUGAGUAUCAGACUAUGGAGGUUCAAAAUAACUCCGUGGUUAUAAAGUGCGACGGGCUUUACAUCAUCUAUCUGAAGGGGUCCUUUUUCCAGGAGGUCAAGAUAGAUUUGCACUUCAGGGAAGACCAUAACCCCAUUUCUAUUCCUAUGCUUAAUGACGGACGACGGAUCGUAUUUACUGUUGUGGCUUCUCUCGCUUUUAAGGACAAGGUCUACCUUACCGUGAAUGCCCCAGACACACUUUGCGAGCACCUCCAGAUCAACGACGGAGAGCUUAUCGUUGUUCAACUUACACCAGGUUAUUGCGCUCCAGAGGGGAGCUAUCACUCCACCGUCAACCAGGUACCCCUU24G4S 링커*2GGUGGAGGCGGUUCAGGCGGAGGUGGCUCU25MBL의 알파 헬릭스AGCGAGAGAAAGGCCCUUCAGACAGAAAUGGCACGCAUAAAAAAAUGGCUUACAUUCAGU24G4s링커GGUGGAGGCGGUUCA26페리틴CAGGACUCCGAAGCAGCAAUAAACCGACAAAUAAAUCUGGAGUUGUACGCAUCCUACGUUUACCUGUCCAUGUCUUACUACUUCGAUAGAGACGAUGUCGCCCUCAAAAACUUCGCCAAGUAUUUCCUGCAUCAGUCCCAUGAAGAGAGAGAGCACGCAGAGAAACUGAUGAAACUUCAGAACCAACGGGGGGGCAGGAUCUUUCUGCAAGACAUUAAGAAGCCUGAUUGCGAUGACUGGGAAUCUGGACUUAAUGCCAUGGAGUGUGCCCUGCACCUCGAAAAAAAUGUGAACCAGAGCCUGUUGGAAUUGCAUAAGCUGGCAACCGAUAAGAACGAUCCACAUCUUUGCGAUUUCAUCGAAACCCACUAUCUCAAUGAACAAGUGAAGGCAAUUAAGGAGCUGGGCGAUCAUGUAACAAAUUUGCGAAAAAUGGGUGCC133'UTRGUGUGUGGAGGACACCCUGAACCCCCCGCUUUCAAACAAGUUUUCAAAUUGUUUGAGGUCAGGAUUUCUCAAACUGAUUCCUUUCUUUGCAUAUGAGUAUUUGAAAAUAAAUAUUUUCCCAGAAUAUAAAUAAAUCAUCACAUGAUUAUUUUAACUAU14폴리A 테일AAAAAAAAAAAAAAAAAAAAACAACCAGCUAAGACACUGCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA485'UTRAGUGCUCCCCAUCCAACUAAACUGUCCCUCUGUCCGAACUAAACUGCACUGUCCCAGCACC22신호서열AUGACAAGGUUGACGGUCCUGGCAUUGCUUGCCGGGCUUUUGGCGAGUUCUCGGGCG28MBLUCUGGCUCUCAGACUUGUGAAGAUACCCUUAAGACUUGCAGUGUCAUAGCCUGUGGGCGAGACGGGCGCGAUGGACCCAAGGGCGAAAAAGGAGAACCAGGGCAAGGGCUCAGAGGACUGCAAGGUCCACCUGGUAAAUUGGGGCCACCUGGAAGCGUGGGGUCCCCAGGAAGUCCAGGCCCAAAAGGUCAGAAAGGGGACCACGGCGAUAAUCGAGCCAUUGAGGAAAAACUCGCAAAUAUGGAGGCCGAAAUUAGGAUUCUGAAGAGUAAGUUGCAGCUGACCAACAAGCUCCAUGCUUUUAGCAUGGGC24G4S 링커*2GGUGGAGGCGGUUCAGGCGGAGGUGGCUCU23OX40L의 세포외도메인AGCAGUUCACCUGCAAAGGACCCCCCAAUCCAGCGACUGAGAGGCGCAGUCACCCGGUGUGAAGAUGGACAGCUUUUUAUAUCAUCUUACAAGAAUGAGUAUCAGACUAUGGAGGUUCAAAAUAACUCCGUGGUUAUAAAGUGCGACGGGCUUUACAUCAUCUAUCUGAAGGGGUCCUUUUUCCAGGAGGUCAAGAUAGAUUUGCACUUCAGGGAAGACCAUAACCCCAUUUCUAUUCCUAUGCUUAAUGACGGACGACGGAUCGUAUUUACUGUUGUGGCUUCUCUCGCUUUUAAGGACAAGGUCUACCUUACCGUGAAUGCCCCAGACACACUUUGCGAGCACCUCCAGAUCAACGACGGAGAGCUUAUCGUUGUUCAACUUACACCAGGUUAUUGCGCUCCAGAGGGGAGCUAUCACUCCACCGUCAACCAGGUACCCCUU133'UTRGUGUGUGGAGGACACCCUGAACCCCCCGCUUUCAAACAAGUUUUCAAAUUGUUUGAGGUCAGGAUUUCUCAAACUGAUUCCUUUCUUUGCAUAUGAGUAUUUGAAAAUAAAUAUUUUCCCAGAAUAUAAAUAAAUCAUCACAUGAUUAUUUUAACUAU14폴리A 테일AAAAAAAAAAAAAAAAAAAAACAACCAGCUAAGACACUGCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0169] In Table 8, the extracellular domain of OX40L is, for example, the OX40 receptor binding domain. In Table 8, G4S linker*2 represents G4SG4S. The underlined nucleotides in the 3'UTR region of SEQ ID NO: 27 in Table 8 are XhoI restriction enzyme recognition sequences. The bold nucleotides in the 3'UTR region of SEQ ID NO: 27 in Table 8 are NheI restriction enzyme recognition sequences. The underlined nucleotides in the polyA tail region of SEQ ID NO: 11 in Table 8 are SapI restriction enzyme recognition sequences.

[0170] The nucleotides in bold in the polyA tail region of sequence number 11 listed in Table 8 are part of the EcoRI restriction enzyme recognition sequence.

[0171] The sequences of substances 3 and 4 described in Table 8 represent the sequences excluding the restriction enzyme recognition sites shown in substances 1 and 2.

[0172] OX40 ligand (OX40L) and OX40 are classical members of the tumor necrosis factor ligand family and the tumor necrosis factor receptor family, respectively. OX40L and OX40 are involved in costimulation and differentiation of T cells. Like other tumor necrosis factor ligands, OX40L is a type II transmembrane protein. Soluble human OX40L assembles to form trimers and binds to OX40, but is inactive when unassembled. Substances 1 and 3 are fusion proteins of the following structure when transcribed and translated: the extracellular domain of OX40L-the alpha helix of G4S linker*2-MBL-the G4S linker-ferritin. That is, the extracellular domain of soluble OX40L is connected to the alpha helix of MBL and ferritin via a linker, respectively. The above fusion protein comprises a trimeric unit in which three fusion proteins are assembled, and these units can combine with each other to form an oligomer, for example, an oligomer composed of eight units. These oligomers have the activity of binding to OX40 and activating OX40. This can facilitate the interaction between the extracellular domain of OX40L and OX40 compared to when soluble OX40L is expressed alone. In the above fusion protein, the alpha helix of MBL may promote the formation of the trimeric unit of the fusion protein. Furthermore, in the above fusion protein, ferritin may promote the formation of oligomers by causing the trimeric units of the formed fusion protein to combine with each other. The oligomer may have a cage-like form. In the above fusion protein, the extracellular domain of OX40L may be any polypeptide comprising the extracellular domain of OX40L, for example, full-length OX40L. However, the claimed invention should not be construed as being limited to these specific mechanisms.

[0173] Substances 2 and 4 are fusion proteins having the extracellular domain structure of MBL-G4S linker*2-OX40L when transcribed and translated. That is, the extracellular domain of soluble OX40L is a fusion protein linked to MBL via a linker. The fusion protein forms a trimeric unit in which three fusion proteins are assembled, and these units can bind to each other to form an oligomer, for example, an oligomer composed of 1, 2, 4, or more of the trimer units. These oligomers can bind to OX40 and activate OX40. This can facilitate the interaction between the extracellular domain of OX40L and OX40 more than when soluble OX40L is expressed alone. In the fusion protein, MBL may promote the formation of a trimeric unit of the fusion protein. Furthermore, in the fusion protein, MBL may promote the formation of an oligomer by causing the trimeric units of the formed fusion protein to bind to each other. The above oligomer may have a bouquet-like structure. In the above fusion protein, the extracellular domain of OX40L may be any polypeptide comprising the extracellular domain of OX40L, for example, full-length OX40L. However, the claimed invention should not be construed as being limited to this specific mechanism.

[0174] Sequence number amino acid sequence

[0175] In Table 9, sequence numbers 29 and 30 are amino acid sequences obtained by translation of sequences of substances 3 and 4, respectively.

Claims

1. An mRNA molecule comprising a modified polyadenyl sequence, wherein the modified polyadenyl sequence is (a) a first sequence comprising one or more microRNA binding sequences; (b) a second sequence of 20 or fewer consecutive A nucleotides, located 5' of the first sequence; and (c) an mRNA molecule comprising a third sequence of at least 80 consecutive A nucleotides, located 3' of the first sequence.

2. An mRNA molecule according to claim 1, wherein the microRNA binding sequence of the first sequence is tumor-specific. 3.청구항 2에 있어서, 상기 microRNA는 miRNA-34a, miR-340, miR-133a, miR-34b, miR-19, miR-200, miR-15, miR-16, miR-1298, miR-663a, miR-449a, miR-138, miR-126, miR-10a, miR-10b, miR-140-3p, miR-140-5p, miR-204, miR-424, miR-193a-3p, miR-455-5p, miR-455-3p, miR-9, miR-10a, miR-148a, miR-488, miR-196a1, miR-182, miR-96, miR-193b, miR-27a, miR-196b, miR-10b, miR-29b2, miR-23a, miR-107, miR-542-3p, miR-93, miR-365a-4, miR-450a, miR-100, miR-105, miR-363, miR-105, miR-106b, miR-15b, miR-21, miR-376c, miR-93, miR-99b, miR-155, miR-33a, miR-876-3p, miR-362-3p, miR-25, let-7i, miR-423-3p, miR-16-2, miR-29a, miR-30d, miR-320, miR-181c, miR-128a, miR-21, let-7d, miR-450b-5p, miR-371-5p, miR-1245, miR-335, miR-492, miR-874, miR-30b, miR-193a-5p, miR-602, miR-346, miR-663, miR-25, miR-219-5p6, miR-184, miR-135a7, miR-584, miR-665, miR-638, miR-503, miR-628-3p, miR-381, miR-78, miR-92b, miR-149, miR-135b, miR-302d, miR-498, miR-766, miR-1389, miR-623, miR-519c-5p, miR-182, miR-494, miR-129-5p10, miR-513-5p,miR-200b,miR-634,miR-654-5p,miR-518b,miR-658,miR-373,miR-30c-2,miR-130a,miR-557,miR-551a,miR-637,miR-518c,miR-525-5p,miR-596,miR-552,miR-625,miR-183 an mRNA selected from the group consisting of miR-187, miR-544, miR-891a, miR-519e, miR-933, miR-939, miR-214, miR-671-5p, miR-137, miR-92b, miR-525-3p, miR-19a, and miR-409-5p Molecule., 4. An mRNA molecule comprising one or more modified nucleotides according to claim 1.

5. An mRNA molecule according to claim 1, further comprising at least one sequence selected from the group consisting of a sequence encoding a polypeptide and an expression control sequence.

6. An mRNA molecule according to claim 5, wherein the expression control sequence comprises at least one selected from the group consisting of a Kozak sequence, a 5'UTR, and a 3'UTR.

7. In claim 6, the 5'UTR is p53, OX40L, albumin, serum amyloid A, apolipoprotein A / B / E, transferrin, alpha fetoprotein, erythropoietin, factor VIII, MyoD, myosin, myoglobin, myogenin, Herculin, Tie-1, CD36, C / EBP, AML1, G-CSF, GM-CSF, CD11b, MSR, Fr-1, i-NOS, CD45, CD18, CD36, GLUT4, ACRP30, adiponectin, PA / B / C / D, ORM1, HPX, FGA, CYP2E1, C3, ASC, APOA2, ALB, AGXT, α-globin, β-globin, An mRNA molecule derived from an mRNA encoding a protein selected from the group consisting of tyrosine hydroxylase and collagen.

8. In claim 6, the 3'UTR is p53, OX40L, albumin, serum amyloid A, apolipoprotein A / B / E, transferrin, alpha fetoprotein, erythropoietin, factor VIII, MyoD, myosin, myoglobin, myogenin, Herculin, Tie-1, CD36, C / EBP, AML1, G-CSF, GM-CSF, CD11b, MSR, Fr-1, i-NOS, CD45, CD18, CD36, GLUT4, ACRP30, adiponectin, PA / B / C / D, ORM1, HPX, FGA, CYP2E1, C3, ASC, APOA2, ALB, AGXT, α-globin, β-globin, An mRNA molecule derived from an mRNA encoding a protein selected from the group consisting of tyrosine hydroxylase and collagen.

9. An mRNA molecule according to claim 1, wherein the modified polyadenylation sequence comprises 90 to 1125 nucleotides.

10. A composition for delivering to a subject an mRNA or a product expressed thereby, comprising the mRNA of any one of claims 1 to 9 as an active ingredient.

11. A method for delivering mRNA or a product expressed thereby to a host cell, comprising the step of introducing the mRNA of any one of claims 1 to 9 into the host cell.

12. A DNA molecule encoding the mRNA molecule of any one of claims 1 to 9.

13. A DNA molecule according to claim 12, further comprising an expression control sequence operably linked to the DNA molecule, wherein the expression control sequence comprises a T7, T3, or SP6 promoter.

14. A DNA molecule according to claim 12, which is a nucleic acid structure or vector.

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