UTR for improving RNA molecule translation efficiency and / or stability and application thereof

Abstract

Provided is a recombinant RNA molecule comprising a nucleotide sequence encoding a polypeptide of interest and 5 '-UTR and / or 3'-UTR, the RNA molecule having increased translation efficiency and / or stability, increasing the expression level of the polypeptide and / or protein. Also provided are vectors encoding the recombinant RNA molecules, pharmaceutical compositions comprising the recombinant RNA molecules, and methods of using the recombinant RNA molecules to treat or prevent disease.

Description

UTR for improving translation efficiency and / or stability of RNA molecules and its application Technical field:

[0001] The present invention belongs to the field of biotechnology and relates to a 5' and / or 3' untranslated region (UTR) for improving the translation efficiency and / or stability of RNA molecules, a nucleic acid molecule comprising the UTR, a vector comprising the UTR or the nucleic acid molecule, a pharmaceutical composition comprising the UTR, the nucleic acid molecule or the vector, and applications of the UTR, the nucleic acid molecule, the vector and the pharmaceutical composition. Background technology:

[0002] mRNA (messenger RNA) is a single-stranded RNA molecule that is manufactured using the genomic DNA in the cell nucleus as a template and then exported to the cytoplasm. It is translated in the ribosome to produce specific proteins to exert biological effects. mRNA has the potential to synthesize any protein. Due to its economical, safe, fast and flexible characteristics, mRNA drugs have huge application potential in the prevention of infectious diseases, cancer and rare diseases.

[0003] mRNA vaccines have already gained clinical application in the prevention of infectious diseases. Compared with recombinant protein subunit vaccines, inactivated vaccines, or DNA vaccines, mRNA vaccines offer several distinct advantages. First, because mRNA does not infect the body or integrate into genomic DNA, its safety is greatly improved. Second, the use of modified bases can reduce the inherent immunogenicity of mRNA molecules and minimize their degradation by the body, further improving the safety and stability of mRNA vaccines. Furthermore, the high yield of mRNA synthesis in vitro makes mRNA vaccines highly efficient, rapidly developable, cost-effective to manufacture, and easy to manage safely.

[0004] The untranslated region (UTR) of mRNA controls the translation, degradation and localization of genes, including the stem-loop structure, upstream start codon, upstream open reading frame, internal ribosome entry site and various cis-acting elements that bind to RNA-binding proteins.

[0005] UTRs play a crucial role in post-transcriptional regulation of gene expression, including regulating mRNA nuclear export and translation efficiency, subcellular localization, and stability. UTRs may also play other roles, such as the specific incorporation of the modified amino acid selenocysteine ​​at the UGA codon in mRNAs encoding selenoproteins, a process mediated by a conserved stem-loop structure in the 3'-UTR.

[0006] Those skilled in the art know that the translation efficiency of mRNA molecules directly affects the dosage and dosing interval of mRNA drugs (especially mRNA vaccines), ultimately affecting the bioavailability of mRNA drugs and determining the clinical application value of mRNA drugs. Although there are technical solutions for increasing the translation efficiency and stability of mRNA molecules, such as by adding untranslated regions (UTRs) to improve the translation efficiency of mRNA molecules, there is still a need for further improvement in improving the translation efficiency of mRNA molecules.

[0007] Therefore, there is a need to identify or design UTRs that can achieve higher mRNA translation efficiency and / or stability.

[0008] Summary of the Invention

[0009] To address the above problems, the inventors have identified a variety of 5'-UTRs and / or 3'-UTRs that can improve the translation efficiency and / or stability of mRNA molecules. The above-mentioned UTRs are universal core elements that give mRNA molecules containing the UTRs enhanced translation efficiency, significantly improving the expression of target genes and / or mRNA stability, and have broad application value in the industrialization of mRNA drugs.

[0010] In a first aspect, the present invention provides a recombinant RNA molecule comprising: (1) a first nucleotide sequence encoding a polypeptide and / or protein of interest; and (2) a second nucleotide sequence comprising a 5'-untranslated region (5'-UTR); the 5'-UTR comprising at least one selected from the following polynucleotides: (a): a 5'-UTR derived from at least one of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, HBB, MYSM1, LENG1, TMSB4X, CASP4, IFNA1, PGLYRP1, UCHL1, CPAMD8, TTR, APOA2, GH1, DTYMK, APOC2 and CDK7; (b): a fragment of the 5'-UTR in (a); (c): a variant of the 5'-UTR in (a); and (d): a variant of the fragment in (b); the first nucleotide sequence and the second nucleotide sequence do not naturally occur in the same RNA molecule.

[0011] In some embodiments, the gene is a human gene.

[0012] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: (e): a 5'-UTR derived from at least one of genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, and HBB; (f): a fragment of the 5'-UTR described in (e); (g): a variant of the 5'-UTR described in (e); and (h): a variant of the fragment described in (f).

[0013] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 1 to 21, a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 1 to 21, a variant of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 1 to 21, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 1 to 21; preferably, the variant of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 1 to 21, a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 1 to 21, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 1 to 21 have at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the RNA encoded by the polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 1 to 21.

[0014] In some embodiments, the second nucleotide sequence comprises at least one of: a 5'-UTR of at least two of the genes described in (a), a fragment of a 5'-UTR of at least two of the genes described in (a), a variant of a 5'-UTR of at least two of the genes described in (a), and a variant of a fragment of a 5'-UTR of at least two of the genes described in (a).

[0015] In some embodiments, the second nucleotide sequence comprises at least one of: at least two copies of the 5'-UTR in (a), at least two copies of a fragment of the 5'-UTR in (a), at least two copies of a variant of the 5'-UTR in (a), and at least two copies of a variant of the fragment of the 5'-UTR in (a).

[0016] In some embodiments, the recombinant RNA molecule further comprises at least one of a promoter, a 5'-cap structure, a 3'-UTR, and a poly(A) tail.

[0017] In some embodiments, the recombinant RNA molecule further comprises at least one of a 5'-cap structure, a 3'-UTR, and a poly(A) tail.

[0018] In some embodiments, the 5'-cap structure comprises m 7 GpppG、m2 7,3′-O GpppG、m 7 Gppp(5')N1 and m 7 Gppp(m 2′-O )N1. In some embodiments, the 3'-UTR comprises: i) a 3'-UTR derived from at least one of an albumin gene, an α-globin gene, a β-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen α gene; ii) a variant of the 3'-UTR in i); iii) at least one of a 3'-UTR derived from at least one of MPND, FBXW10, FBXW12, PGLYRP1, HPX, CDK7, APOC2, PFN1, RBP4, FTCD, NAAA, ALB, GSDMD, FBXL8, ORM1, CASP4, CHMP2A, LENG1, MYCBPAP, APOC1, GAPDH, HSPA8, APOA2, UCHL1, TSG101, NAE1, NFKB2, and GH1, a fragment, a variant, and a variant of a fragment thereof; preferably, the RNA encoded by the polynucleotide sequence as shown in at least one of SEQ ID NOs: 22 to 49, the sequence of SEQ ID NOs: 23 to 25, or the RNA encoded by the polynucleotide sequence as shown in at least one of SEQ ID NOs: 24 to 26. NO: 22-49, a fragment of an RNA encoded by the polynucleotides as shown in at least one of SEQ ID NOs: 22-49, a variant of an RNA encoded by the polynucleotides as shown in at least one of SEQ ID NOs: 22-49, and a variant of a fragment of an RNA encoded by the polynucleotides as shown in at least one of SEQ ID NOs: 22-49; preferably, the variant of the RNA encoded by the polynucleotides as shown in at least one of SEQ ID NOs: 22-49, the fragment of the RNA encoded by the polynucleotides as shown in at least one of SEQ ID NOs: 22-49, and the variant of the fragment of the RNA encoded by the polynucleotides as shown in at least one of SEQ ID NOs: 22-49 have at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 1 iv) at least two copies of a polynucleotide of i), ii) or iii); or v) at least two polynucleotides of the group consisting of polynucleotides of i) to iii).

[0019] In some embodiments, the nucleotides constituting the poly(A) tail comprise at least 20, at least 40, at least 80, at least 100, or at least 120 A nucleotides; preferably, the nucleotides constituting the poly(A) tail comprise at least 20, at least 40, at least 80, at least 100, or at least 120 consecutive A nucleotides. In some embodiments, the nucleotides constituting the poly(A) tail include one or more nucleotides other than A nucleotides; alternatively, the nucleotides constituting the poly(A) tail comprise two or more consecutive nucleotides other than A nucleotides.

[0020] In a second aspect, the present invention also provides another recombinant RNA molecule comprising: (1) a first nucleotide sequence encoding a polypeptide and / or protein of interest; and (2) a second nucleotide sequence comprising a 3'-untranslated region (3'-UTR); the 3'-UTR comprising at least one of the following polynucleotides: (a): derived from genes MPND, FBXW10, FBXW12, PGLYRP1, HPX, CDK7, APOC2, PFN1, RBP4, FTCD, NAAA, ALB, GSDMD, FB The invention relates to a method for preparing an RNA molecule comprising: (a) a fragment of the 3'-UTR of at least one of the genes XL8, ORM1, CASP4, CHMP2A, LENG1, MYCBPAP, APOC1, GAPDH, HSPA8, APOA2, UCHL1, TSG101, NAE1, and NFKB2; (b) a fragment of the 3'-UTR of (a); (c) a variant of the 3'-UTR of (a); and (d) a variant of the fragment of (b); the first nucleotide sequence and the second nucleotide sequence do not naturally occur in the same RNA molecule.

[0021] In some embodiments, the gene is a human gene.

[0022] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: (e): a 3'-UTR derived from at least one of the genes MPND, FBXW10, FBXW12, and PGLYRP1; (f): a fragment of the 3'-UTR in (e); (g): a variant of the 3'-UTR in (e); and (h): a variant of the fragment in (f). In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 22 to 48, a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 22 to 48, a variant of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 22 to 48, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 22 to 48; preferably, the variant of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 22 to 48, the fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 22 to 48, and the variant of a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 22 to 48, and the variant of a fragment of an RNA encoded by a polynucleotide having a sequence as shown in SEQ ID NOs: 22 to 48, The RNA encoded by at least one of the polynucleotides shown in NOs:22-48 has at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0023] In some embodiments, the second nucleotide sequence comprises at least one of: a 3'-UTR of at least two of the genes described in (a), a fragment of a 3'-UTR of at least two of the genes described in (a), a variant of a 3'-UTR of at least two of the genes described in (a), and a variant of a fragment of a 3'-UTR of at least two of the genes described in (a).

[0024] In some embodiments, the second nucleotide sequence comprises at least one of: at least two copies of the 3'-UTR in (a), at least two copies of a fragment of the 3'-UTR in (a), at least two copies of a variant of the 3'-UTR in (a), and at least two copies of a variant of the fragment of the 3'-UTR in (a).

[0025] In some embodiments, the recombinant RNA molecule further comprises at least one of a promoter, a 5'-cap structure, a 5'-UTR, and a poly(A) tail.

[0026] In some embodiments, the recombinant RNA molecule further comprises at least one of a 5'-cap structure, a 3'-UTR, and a poly(A) tail.

[0027] In some embodiments, the 5'-cap structure comprises m 7 GpppG、m2 7,3′-O GpppG、m 7 Gppp(5')N1 or m 7 Gppp(m 2′-O ) At least one of N1.

[0028] In some embodiments, the 5'-UTR comprises: i) at least one of a 5'-UTR derived from at least one of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, HBB, MYSM1, LENG1, TMSB4X, CASP4, IFNA1, PGLYRP1, UCHL1, CPAMD8, TTR, APOA2, GH1, DTYMK, APOC2, and CDK7, a fragment, a variant, and a variant of a fragment thereof; preferably, an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 1 to 21, a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 1 to 21, a variant of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 1 to 21, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 1 to 21; preferably, a variant of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 1 to 21, a variant of an RNA encoded by a polynucleotide having a sequence as shown in SEQ ID NOs: 1 to 21, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as shown in SEQ ID NOs: 1 to 21; NO: 1 to 21 as shown in at least one of the polynucleotides encoded fragments and the sequence as a variant of the RNA encoded by at least one of the polynucleotides encoded by SEQ ID NO: 1 to 21, and the sequence as a variant of the RNA encoded by at least one of the polynucleotides encoded by SEQ ID NO: 1 to 21, which has at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the RNA encoded by the polynucleotides encoded by at least one of SEQ ID NO: 1 to 21; ii) at least two copies of one of the polynucleotides in i); or iii) at least two polynucleotides in i).

[0029] In some embodiments, the nucleotides constituting the poly(A) tail comprise at least 20, at least 40, at least 80, at least 100, or at least 120 A nucleotides; preferably, the nucleotides constituting the poly(A) tail comprise at least 20, at least 40, at least 80, at least 100, or at least 120 consecutive A nucleotides. In some embodiments, the nucleotides constituting the poly(A) tail comprise one or more nucleotides other than A nucleotides.

[0030] In a third aspect, the present invention provides a DNA molecule encoding the recombinant RNA molecule of the present invention.

[0031] In a fourth aspect, the present invention provides a vector comprising the recombinant RNA molecule or DNA molecule of the present invention.

[0032] In a fifth aspect, the present invention provides a host cell comprising the recombinant RNA molecule, DNA molecule or vector of the present invention.

[0033] In a sixth aspect, the present invention provides a lipid nanoparticle comprising the recombinant RNA molecule of the present invention.

[0034] In a seventh aspect, the present invention provides a pharmaceutical composition comprising the recombinant RNA molecule of the present invention, the DNA molecule of the present invention, the vector of the present invention, the host cell of the present invention or the lipid nanoparticle of the present invention, and a pharmaceutically acceptable carrier.

[0035] In an eighth aspect, the present invention provides a vector comprising a first nucleotide sequence encoding a 5'-UTR and / or a second nucleotide sequence encoding a 3'-UTR, wherein:

[0036] The first nucleotide sequence comprises at least one of the following polynucleotides: (a): a polynucleotide encoding a 5'-UTR derived from at least one of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, HBB, MYSM1, LENG1, TMSB4X, CASP4, IFNA1, PGLYRP1, UCHL1, CPAMD8, TTR, APOA2, GH1, DTYMK, APOC2, and CDK7; (b): a polynucleotide encoding a fragment of the 5'-UTR in (a); (c): a polynucleotide encoding a variant of the 5'-UTR in (a); and (d): a polynucleotide encoding a variant of the fragment in (b);

[0037] The second nucleotide sequence comprises at least one of the following polynucleotides: (e): a polynucleotide encoding a 3'-UTR derived from at least one of the genes MPND, FBXW10, FBXW12, PGLYRP1, HPX, CDK7, APOC2, PFN1, RBP4, FTCD, NAAA, ALB, GSDMD, FBXL8, ORM1, CASP4, CHMP2A, LENG1, MYCBPAP, APOC1, GAPDH, HSPA8, APOA2, UCHL1, TSG101, NAE1 and NFKB2; (f): a polynucleotide encoding a fragment of the 3'-UTR described in (e); (g): a polynucleotide encoding a variant of the 3'-UTR described in (e); and (h): a polynucleotide encoding a variant of the fragment described in (f).

[0038] In some embodiments, the gene is a human gene.

[0039] In some embodiments, the first nucleotide sequence comprises a 5'-UTR derived from at least one of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, and HBB, or a variant thereof.

[0040] In some embodiments, the second nucleotide sequence comprises a 3'-UTR derived from at least one of the genes MPND, FBXW10, FBXW12, and PGLYRP1, or a variant thereof.

[0041] In some embodiments, the vector comprises the first nucleotide sequence and the second nucleotide sequence.

[0042] In some embodiments, the first nucleotide sequence comprises: i) a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1-21, a fragment of a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1-21, a variant of a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1-21, and a variant of a fragment of a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1-21; preferably, the variant of the polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1-21, the fragment of the polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1-21, and the variant of a fragment of the polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1-21 have at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1-21; ii) at least two copies of one polynucleotide of i); or iii) at least two polynucleotides of i).

[0043] In some embodiments, the second nucleotide sequence comprises: (1): a polynucleotide encoding a 3'-UTR derived from at least one of an albumin gene, an α-globin gene, a β-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen α gene; (2): a polynucleotide encoding a variant of the 3'-UTR in (1); (3): at least one of a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 22 to 48, a fragment of a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 22 to 48, a variant of a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 22 to 48, and a variant of a fragment of a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 22 to 48; preferably, the RNA encoded by the polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 22 to 48, the fragment encoded by the polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 22 to 48, and the variant of the fragment encoded by the polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 22 to 48, and the variant of the fragment encoded by the polynucleotide having a sequence as shown in SEQ ID NOs: 22 to 48. At least one of the polynucleotides shown in NO:22-48 has 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity; (4): at least two copies of a polynucleotide of (1), (2) or (3); or (5): at least two polynucleotides in the group consisting of the polynucleotides of (1) to (3).

[0044] In some embodiments, the vector further comprises a polynucleotide encoding a poly(A) tail. In some embodiments, the nucleotides comprising the poly(A) tail comprise at least 20, at least 40, at least 80, at least 100, or at least 120 A nucleotides; preferably, the nucleotides comprising the poly(A) tail comprise at least 20, at least 40, at least 80, at least 100, or at least 120 A nucleotides consecutively. In some embodiments, the nucleotides comprising the poly(A) tail comprise one or more nucleotides other than A nucleotides.

[0045] In a ninth aspect, the present invention provides a use of a recombinant RNA molecule, DNA molecule, vector, host cell, lipid nanoparticle or pharmaceutical composition of the present invention in the preparation of a drug; preferably, the drug is used for gene therapy, gene vaccination or protein replacement therapy.

[0046] In some embodiments, the drug is a nucleic acid drug, wherein the nucleic acid comprises at least one of RNA, messenger RNA (mRNA), DNA, a plasmid, ribosomal RNA (rRNA), single-stranded guide RNA (sgRNA), and Cas9 mRNA.

[0047] In some embodiments, the drug is used for the treatment and / or prevention of a disease; preferably, the disease is selected from the group consisting of: rare diseases, infectious diseases, cancer, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases, renal vascular diseases, and metabolic diseases; preferably, the cancer includes one or more of lung cancer, gastric cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, blood cancer or prostate cancer; the genetic disease includes one or more of hemophilia, thalassemia, and Gaucher's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1A shows a flowchart of the construction of plasmid D;

[0049] Figure 1B shows a schematic diagram of the construction and transformation of plasmid D;

[0050] Figure 2 shows a map of plasmid luciferase-pcDNA3;

[0051] Figure 3 shows a map of plasmid B;

[0052] Figure 4 shows a map of plasmid C;

[0053] Figure 5 shows a spectrum of plasmid D;

[0054] FIG6 shows the expression of luciferase in HEK293 cells after transfection of mRNA containing different 5'-UTRs in Example 5;

[0055] FIG7 shows the expression of luciferase in HEK293 cells after transfection of mRNA containing different 3'-UTRs in Example 6;

[0056] Figure 8 shows the expression of mRNAs containing the same 5'-UTR but different 3'-UTRs in mice;

[0057] FIG9 shows the expression of mRNAs containing the same 3'-UTR but different 5'-UTRs in mice.

[0058] Detailed Description of the Invention

[0059] 1. Definition

[0060] All patents, patent applications, scientific publications, manufacturer's instructions and guidelines, etc., cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such publication.

[0061] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, and microbiology used herein are terms widely used in the corresponding fields (see, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989). For a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0062] As used herein, the expressions "comprise," "include," "contain," and "have" are open ended and mean the inclusion of the listed elements, steps, or components but not the exclusion of other unlisted elements, steps, or components. The expression "consisting of excludes any element, step, or component not specified. The expression "consisting essentially of means that the scope is limited to the specified elements, steps, or components, plus optional elements, steps, or components that do not significantly affect the basic and novel properties of the claimed subject matter. It should be understood that the expressions "consisting essentially of" and "consisting of are encompassed within the meaning of the expression "comprising."

[0063] As used herein, unless the context indicates otherwise, the singular expressions "a," "an," "the," and similar references used in the context of describing the invention (especially in the context of the claims) should be interpreted to cover both the singular and the plural, The terms "one or more" or "at least one" encompass 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0064] The numerical ranges described herein should be understood to encompass any and all subranges contained therein. For example, the range "1 to 10" should be understood to include not only the explicitly stated values ​​of 1 and 10, but also any individual value (e.g., 2, 3, 4, 5, 6, 7, 8, and 9) and subranges (e.g., 1 to 2, 1.5 to 2.5, 1 to 3, 1.5 to 3.5, 2.5 to 4, 3 to 4.5, etc.) within the range of 1 to 10. This principle also applies to ranges that use only one value as a minimum or maximum value.

[0065] Unless otherwise stated, all methods described herein can be performed in any suitable order.

[0066] As used herein, the term "wild type" means that the sequence is naturally occurring and has not been artificially modified, including naturally occurring mutants. The term "fragment" or "fragment of a nucleic acid" refers to a portion of a nucleic acid. For example, a nucleic acid that is shortened at the 5' and / or 3' end. A fragment of a nucleic acid comprises at least 50%, 60%, 70% or 80% of the nucleic acid. Preferably, a fragment of a nucleic acid comprises at least 70% or 80% of the nucleotide residues from the nucleic acid. Preferably, at least 90%, 95%, 96%, 97%, 98% or 99% of the nucleotide residues. Typically, this can be a shorter portion of the full length of the nucleic acid.

[0067] The term "variant" in reference to nucleic acids refers to a nucleic acid variant, wherein at least one nucleotide of the nucleic acid variant is different from a reference nucleic acid (or "parent"). Compared to a reference nucleic acid, a variant nucleic acid includes single or multiple nucleotide deletions, additions, mutations and / or insertions, wherein: deletions include removing one or more nucleotides from a reference nucleic acid; additions include fusing one or more nucleotides (e.g., 1, 2, 3, 5, 10, 20, 30, 50 or more nucleotides) to the 5' and / or 3' end of the reference nucleic acid; mutations may include, but are not limited to, substitutions (e.g., at least one nucleotide is removed and another nucleotide is inserted in its place (e.g., transversions and conversions)); insertions include adding at least one nucleotide. The term "nucleic acid variant" as used herein includes naturally occurring variants and engineered variants. Therefore, "nucleic acid variants" as defined herein can be derived from, isolated from, related to, based on, or homologous to a reference nucleic acid sequence. "Nucleic acid variants" optionally have at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a corresponding naturally occurring (wild-type) nucleic acid or a homolog, fragment or derivative thereof; preferably at least 70%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, most preferably at least 95% or even 97% sequence identity. It will be understood that, with respect to nucleic acid molecules, the term "variant" includes degenerate nucleic acid sequences, wherein a degenerate nucleic acid sequence according to the present invention is a nucleic acid that differs from a reference nucleic acid in codon sequence due to the degeneracy of the genetic code.

[0068] In some embodiments, the nucleic acid variant is a variant of the 5'-UTR, a variant of the 3'-UTR, or a variant of ployA. In some embodiments, the mutation introduced into the nucleic acid variant can prevent the nucleic acid variant from being recognized by nucleases and cleaved, avoid binding to microRNA, or avoid forming complex secondary structures such as hairpin structures or G-quadruplexes. For example, the nucleic acid variant is a variant of the 3'-UTR of the ALB gene (GenBank accession number NM_000477.7), in which one "A" in the 3'-UTR of the ALB gene is mutated to a "C" to avoid being recognized by nucleases and cleaved.

[0069] As used herein, the term "% identity" or "% identity" refers to the percentage of identical nucleotides or amino acids in an optimal alignment between the sequences to be compared. The differences between the two sequences can be distributed over local regions (segments) or over the entire length of the sequences to be compared. The identity between the two sequences is usually determined after optimal alignment of a segment or "comparison window." Optimal alignment can be performed manually or with the aid of algorithms known in the art. Algorithms known in the art include, but are not limited to, the local homology algorithm described by Smith and Waterman, 1981, Ads App. Math. 2, 482 and Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, the similarity search method described by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or the similarity search method described by computer programs such as GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. For example, the percent identity of two sequences can be determined using the publicly available BLASTN or BLASTP algorithms available on the National Center for Biotechnology Information (NCBI) website.

[0070] "% identity" or "% homology" can be obtained by determining the number of identical positions corresponding to the sequences to be compared, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence), and multiplying this result by 100 to obtain % identity. In some embodiments, the degree of identity is given for a region of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%. In some embodiments, the degree of identity is given for the entire length of the reference sequence. Alignment for determining sequence identity can be performed using tools known in the art, preferably utilizing optimal sequence alignment, e.g., utilizing Align, utilizing standard settings, preferably EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0071] Preferably, a fragment or variant of a specific nucleic acid or a nucleic acid having a specific degree of identity with a specific nucleic acid preferably has at least one functional property of the specific nucleic acid and is preferably functionally equivalent to the specific nucleic acid, e.g. a nucleic acid that exhibits properties that are the same or similar to those of the specific nucleic acid.

[0072] As used herein, "nucleotide" includes deoxyribonucleotides, deoxyribonucleotides, deoxyribonucleotide derivatives, and ribonucleotide derivatives. As used herein, "ribonucleotide" is a constituent substance of ribonucleic acid (RNA), consisting of a base molecule, a pentose molecule, and a phosphate molecule. It refers to a nucleotide with a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. "Deoxyribonucleotide" is a constituent substance of deoxyribonucleic acid (DNA), also consisting of a base molecule, a pentose molecule, and a phosphate molecule. It refers to a nucleotide in which the hydroxyl group at the 2' position of the β-D-ribofuranosyl group is replaced by hydrogen. It is the main chemical component of chromosomes.

[0073] "Nucleotides" are generally referred to by single letters representing the bases therein: "A" or "A nucleotide" refers to adenine deoxyribonucleotide or adenine ribonucleotide containing adenine, "C" or "C nucleotide" refers to cytosine deoxyribonucleotide or cytosine ribonucleotide containing cytosine, "G" or "G nucleotide" refers to guanine deoxyribonucleotide or guanine ribonucleotide containing guanine, "U" or "U nucleotide" refers to uracil ribonucleotide containing uracil, and "T" or "T nucleotide" refers to thymine deoxyribonucleotide containing thymine.

[0074] As used herein, the term "nucleic acid" generally refers to a polymer comprising deoxyribonucleotides (deoxyribonucleic acid, referred to as DNA) or a polymer comprising ribonucleotides (ribonucleic acid, referred to as RNA) or any compound of a combination thereof. In addition, nucleic acids herein also include derivatives of nucleic acids. The term "derivatives of nucleic acids" includes chemical derivatization of nucleic acids on the bases, sugars or phosphates of the nucleotides, as well as nucleic acids containing non-natural nucleotides and nucleotide analogs. In addition, herein, nucleic acids can be in the form of single-stranded or double-stranded linear or covalently closed circular molecules.

[0075] "Polynucleotide sequence," "nucleic acid sequence," and "nucleotide sequence" are used interchangeably to refer to the order of nucleotides in a polynucleotide. Those skilled in the art will understand that a DNA coding strand (sense strand) and the RNA it encodes can be considered to have the same nucleotide sequence, with deoxythymidylate in the DNA coding strand sequence corresponding to uridine in the RNA sequence it encodes. DNA-encoded RNA refers to the RNA corresponding to the DNA, i.e., a polynucleotide in which all T nucleotides in the DNA are replaced with U nucleotides.

[0076] The polynucleotide may comprise one or more segments (nucleic acid fragments) (e.g., 1, 2, 3, 4, 5, 6, 7, 8 segments). For example, the polynucleotide may comprise a segment encoding a polypeptide of interest (e.g., a polypeptide and polypeptide antigen described herein). In a specific embodiment, the polynucleotide may comprise a segment encoding a polypeptide of interest and a regulatory segment (including but not limited to a segment for transcriptional regulation and translational regulation). In one embodiment, the regulatory segment comprises a polynucleotide corresponding to one or more of the following regulatory elements: a promoter, a 5' untranslated region (5'-UTR), a 3' untranslated region (3'-UTR), and a poly(A) tail.

[0077] The term "promoter" refers to a polynucleotide located upstream of the 5' end of the coding region of a gene. It contains conserved sequences required for specific binding of RNA polymerase and initiation of transcription, activating RNA polymerase, enabling accurate binding of RNA polymerase to the template DNA, and providing specificity for transcription initiation. Promoters can be derived from viruses, bacteria, fungi, plants, insects, and animals. Representative examples of promoters include the bacteriophage T7 promoter, the bacteriophage T3 promoter, the SP6 promoter, the lac operator promoter, the tac promoter, the SV40 late promoter, the SV40 early promoter, the RSV-LTR promoter, the CMV IE promoter, the SV40 early promoter, or the SV 40 late promoter and the CMV IE promoter. As used herein, the term "5' untranslated region" or "5'-UTR" refers to an RNA sequence located upstream of the coding sequence in an mRNA that is not translated into protein. The 5'-UTR in a gene typically begins at the transcription start site and ends at the nucleotide upstream of the translation start codon of the coding sequence. The 5'-UTR may contain elements that control gene expression, such as ribosome binding sites, 5'-terminal oligopyrimidine tracts, and translation initiation signals such as Kozak sequences. mRNA can be post-transcriptionally modified by adding a 5' cap. Therefore, the 5'-UTR in mature mRNA can also refer to the RNA sequence between the 5' cap and the start codon. As used herein, the term "3' untranslated region" or "3'-UTR" can be a portion of mRNA that is located downstream of the coding sequence and is not translated into protein. The 3'-UTR in mRNA is located between the stop codon and the poly(A) sequence of the coding sequence, for example, starting from the nucleotides downstream of the stop codon and ending at the nucleotides upstream of the poly(A) sequence.

[0078] As used herein, "5' or 3'-UTR derived from gene A" refers to the 5' or 3'-UTR of the mRNA of gene A. The 5' or 3'-UTR derived from gene A may be the entire 5' or 3'-UTR of the mRNA of gene A, or may be a partial 5' or 3'-UTR of the mRNA of gene A.

[0079] As used herein, the terms "poly(A) acid", "poly(A) sequence" and "poly(A) tail" are used interchangeably, and naturally occurring poly(A) sequences are typically composed of adenine ribonucleotides. According to the present invention, the term "modified poly(A) sequence" refers to a poly(A) sequence comprising nucleotides or nucleotide segments other than adenine ribonucleotides. The poly(A) sequence is typically located at the 3' end of the mRNA, such as the 3' end (downstream) of the 3'-UTR. As used herein, the term "5'-cap structure": the 5'-cap structure is typically located at the 5' end of the mature mRNA. In some embodiments, the 5'-cap structure is connected to the 5'-end of the mRNA via a 5'-5'-triphosphate bond. The 5'-cap structure is typically formed by modified (e.g., methylated) ribonucleotides (especially by guanine nucleotide derivatives). For example, m7GpppN (cap 0, or "cap0," is a cap structure formed by the 5'-phosphate group of the hnRNA reacting with the 5'-phosphate group of m7GTP under the action of guanylyltransferase to form a 5',5'-phosphodiester bond), where N is the terminal 5' nucleotide of the nucleic acid carrying the 5'-cap structure. In some embodiments, the 5'-cap structure includes, but is not limited to, cap 0, cap 1 (a cap structure formed by further methylating the 2'-OH group of the first nucleotide of the hnRNA based on cap 0, or "cap 1"), cap 2 (a cap structure formed by further methylating the 2'-OH group of the second nucleotide of the hnRNA based on cap 1, or "cap 2"), cap 4, cap 0 analogs, cap 1 analogs, cap 2 analogs, or cap 4 analogs.

[0080] As used herein, the term "expression" includes transcription and / or translation of a nucleotide sequence. Thus, expression can involve the production of transcripts and / or polypeptides. The term "transcription" refers to the process by which the genetic code in a DNA sequence is transcribed into RNA (transcript). The term "in vitro transcription" refers to the in vitro synthesis of RNA, particularly mRNA, in a cell-free system (e.g., in an appropriate cell extract) (see, e.g., Pardi N., Muramatsu H., Weissman D., Karikó K. (2013). In: Rabinovich P. (eds) Synthetic Messenger RNA and Cell Metabolism Modulation. Methods in Molecular Biology (Methods and Protocols), vol 969. Humana Press, Totowa, NJ.). A vector that can be used to produce a transcript is also referred to as a "transcription vector," which contains regulatory sequences required for transcription. The term "transcription" encompasses "in vitro transcription."

[0081] As used herein, the term "polypeptide" refers to a polymer comprising two or more amino acids covalently linked by peptide bonds. A "protein" may comprise one or more polypeptides, wherein the polypeptides interact with each other by covalent or non-covalent means.

[0082] As used herein, the term "host cell" refers to a cell for receiving, maintaining, replicating, expressing a polynucleotide or a vector. The term "host cell" includes prokaryotic cells (e.g., Escherichia coli) or eukaryotic cells (e.g., yeast cells and insect cells). For example, cells from humans, mice, hamsters, pigs, goats, primates. The cell can be derived from a variety of tissue types and includes primary cells and cell lines. Some specific examples include keratinocytes, peripheral blood leukocytes, bone marrow stem cells, and embryonic stem cells. In other embodiments, the host cell is an antigen presenting cell, particularly a dendritic cell, a monocyte, or a macrophage. The nucleic acid can be present in a host cell in a single copy or in several copies. In some embodiments, the host cell can be a cell expressing a polypeptide of the present invention therein.

[0083] As used herein, the term "recombinant" or "recombinant" means "produced by genetic engineering." Preferably, in the context of the present invention, a "recombinant material," such as a recombinant RNA molecule, is non-naturally occurring. As used herein, the term "naturally occurring" or "naturally occurring" refers to the fact that a material can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including a virus) and can be isolated from a source in nature and has not been intentionally modified by man in an experiment is naturally occurring.

[0084] In the context of the present invention, the term "plasmid" generally refers to a circular DNA molecule, but the term can also encompass linearized DNA molecules. Specifically, the term "plasmid" also encompasses molecules obtained by, for example, digesting a circular plasmid with a restriction enzyme, thereby converting the circular plasmid molecule into a linear molecule and linearizing the circular plasmid. Plasmids can replicate, i.e., amplify the genetic information stored as chromosomal DNA in the cell, and can be used for cloning, i.e., for amplifying genetic information in bacterial cells. Preferably, the DNA plasmid is a medium copy or high copy plasmid, more preferably a high copy plasmid. Examples of such high copy plasmids include, for example, pUC and pTZ plasmids or any other plasmid (e.g., pMB1, pCoIE1) comprising a replication origin that supports high copies of the plasmid.

[0085] The terms "treat" and "treatment" and the like are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. Thus, the treatment of the present invention may relate to the treatment of a disease state, but may also relate to prophylactic treatment in terms of completely or partially preventing a disease or its symptoms. Preferably, the term "treat" is understood to be therapeutic in terms of partially or completely curing a disease and / or attributing to the adverse effects and / or symptoms of the disease. Treatment may also be prophylactic or preventive treatment, i.e., measures taken to prevent a disease, e.g., to prevent the onset of infection and / or disease.

[0086] Herein, some elements of the present invention will be described. These elements are listed together with specific embodiments, however, it should be understood that they can be combined in any manner and in any number to produce additional embodiments. The different described examples and preferred embodiments should not be interpreted as limiting the present invention to only the embodiments explicitly described. This specification should be understood to support and include embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. In addition, unless the context indicates otherwise, any permutation and combination of all described elements in the present invention should be deemed to be disclosed by the specification of the present invention. For example, if in one embodiment, the 5'-UTR of the recombinant nucleic acid molecule includes a polynucleotide with a sequence as shown in SEQ ID NO: 1, and if in another embodiment, the 3'-UTR of the recombinant nucleic acid molecule includes a polynucleotide with a sequence as shown in SEQ ID NO: 22, then the following scheme is also an embodiment claimed in the present invention: the 5'-UTR of the recombinant nucleic acid molecule includes a polynucleotide with a sequence as shown in SEQ ID NO: 1, and the 3'-UTR of the recombinant nucleic acid molecule includes a polynucleotide with a sequence as shown in SEQ ID NO: 22.

[0087] 2. Improving the translation of the 5'-UTR

[0088] The inventors unexpectedly discovered that incorporating the 5'-UTR of gene PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, HBB, MYSM1, LENG1, TMSB4X, CASP4, IFNA1, PGLYRP1, UCHL1, CPAMD8, TTR, APOA2, GH1, DTYMK, APOC2, or CDK7 into mRNA can increase the translation efficiency of the coding sequence.

[0089] The full names of the above genes are shown below.

[0090] PPIA: peptidylprolyl isomerase A;

[0091] HPX: hemopexin;

[0092] FTCD:formimidoyltransferase cyclodeaminase;

[0093] CDK5RAP3:CDK5 regulatory subunit associated protein 3;

[0094] HSPA8:heat shock protein family A(Hsp70)member 8;

[0095] HBA1:hemoglobin subunit alpha 1;

[0096] HBB:hemoglobin subunit beta;

[0097] MYSM1:Myb like,SWIRM and MPN domains 1;

[0098] LENG1:leukocyte receptor cluster member 1;

[0099] TMSB4X:thymosin beta 4 X-linked;

[0100] CASP4:caspase 4;

[0101] IFNA1:interferon alpha 1;

[0102] PGLYRP1:peptidoglycan recognition protein 1;

[0103] UCHL1:ubiquitin C-terminal hydrolase L1;

[0104] CPAMD8:C3 and PZP like,alpha-2-macroglobulin domain containing 8;

[0105] TTR:transthyretin;

[0106] APOA2:apolipoprotein A2;

[0107] GH1:growth hormone 1);

[0108] DTYMK: deoxythymidylate kinase;

[0109] APOC2: apolipoprotein C2;

[0110] CDK7: cyclin-dependent kinase 7.

[0111] Therefore, the present invention provides a 5'-UTR comprising at least one selected from the following polynucleotides: (a): a 5'-UTR derived from at least one gene of PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, HBB, MYSM1, LENG1, TMSB4X, CASP4, IFNA1, PGLYRP1, UCHL1, CPAMD8, TTR, APOA2, GH1, DTYMK, APOC2 and CDK7; (b): a fragment of the 5'-UTR described in (a); (c): a variant of the 5'-UTR described in (a); and (d): a variant of the fragment described in (b).

[0112] In some embodiments, the gene is a eukaryotic gene.

[0113] In some embodiments, the gene is a chordate gene.

[0114] In some embodiments, the gene is a vertebrate gene.

[0115] In some embodiments, the gene is a mammalian gene.

[0116] In some embodiments, the gene is a primate gene.

[0117] In some embodiments, each of the genes is independently a gene of Homo sapiens, a gene of bonobo (Pan paniscus), a gene of chimpanzee (Pan troglodytes), or a gene of western lowland gorilla (Gorilla gorilla gorilla).

[0118] In some embodiments, the gene is a human gene.

[0119] In some embodiments, the gene is human PPIA gene, human HPX gene, bonobo HPX gene, human FTCD gene, human CDK5RAP3 gene, western lowland gorilla CDK5RAP3 gene, human HSPA8 gene, human HBA1 gene, human HBB gene, human MYSM1 gene, bonobo MYSM1 gene, human LENG1 gene, human TMSB4X gene, human CASP4 gene, human IFNA1 gene, human PGLYRP1 gene, western lowland gorilla PGLYRP1 gene, human UCHL1 gene, human CPAMD8 gene, chimpanzee CPAMD8 gene, human TTR gene, western lowland gorilla TTR gene, human APOA2 gene, human GH1 gene, human DTYMK gene, bonobo DTYMK gene, human APOC2 gene, chimpanzee APOC2 gene, and human CDK7 gene.

[0120] In some embodiments, the GenBank accession number of the PPIA gene is The GenBank accession number of the HPX gene is BC137057.1; the GenBank accession number of the FTCD gene is NM_006657.3; the GenBank accession number of the CDK5RAP3 gene is AK223387.1; the GenBank accession number of the HSPA8 gene is NM_006597.6; the GenBank accession number of the HBA1 gene is NM_000558.5; the GenBank accession number of the HBB gene is NM_000518.5; the GenBank accession number of the MYSM1 gene is NM_001085487.2; the GenBank accession number of the LENG1 gene is NM_024316.3; the GenBank accession number of the TMSB4X gene is NM_021109.4; the GenBank accession number of the CASP4 gene is NM_ 001225.3; the GenBank accession number of the IFNA1 gene is NM_024013.3; the GenBank accession number of the PGLYRP1 gene is NM_005091.2; the GenBank accession number of the UCHL1 gene is NG_012931.1; the GenBank accession number of the CPAMD8 gene is NG_054892.1; the GenBank accession number of the TTR gene is NM_000371.3; the GenBank accession number of the APOA2 gene is NM_001643.2; the GenBank accession number of the GH1 gene is NM_000515.5; the GenBank accession number of the DTYMK gene is NM_001165031.1; the GenBank accession number of the APOC2 gene is NM_000483.4; and the GenBank accession number of the CDK7 gene is AY130859.1.

[0121] In some embodiments, the polynucleotides encoding the 5'-UTRs of the human genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, HBB, MYSM1, LENG1, TMSB4X, CASP4, IFNA1, PGLYRP1, UCHL1, CPAMD8, TTR, APOA2, GH1, DTYMK, APOC2, and CDK7 are as shown in Table 1.

[0122] Table 1. Identified 5'-UTRs that improve translation:

[0123] Thus, in some embodiments, the 5'-UTR comprises at least one of the following polynucleotides: an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21, a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21, a variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21. In some embodiments, the fragment, variant, or variant of the fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21 is at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the RNA encoded by the polynucleotide having a sequence as set forth in one of SEQ ID NOs: 1 to 21. In some embodiments, a fragment, variant, or variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides inserted, added, deleted, or substituted compared to an RNA encoded by one of SEQ ID NOs: 1 to 21. It should be noted that, herein, "a fragment, variant, or variant of a fragment of A" is an abbreviation for a fragment of A, a variant of A, or a variant of a fragment of A. For example, "a fragment, variant, or variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21" refers to a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21, a variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21, or a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21.

[0124] In preferred embodiments, the gene is selected from at least one of PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, and HBB. In some embodiments, the 5'-UTR comprises at least one of the following polynucleotides: 1) an RNA encoded by a polynucleotide as set forth in SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6; 2) a fragment of the RNA described in 1); 3) a variant of the RNA described in 1); and a variant of the fragment described in 2). In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide of SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the RNA encoded by the polynucleotide of SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide of SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotide insertions, additions, deletions, or substitutions compared to the RNA encoded by the polynucleotide of SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6.

[0125] The 5'-UTR of the present invention may comprise two or more tandem 5'-UTRs from the above genes, fragments of the 5'-UTRs of the above genes, variants of the 5'-UTRs of the above genes, or variants of the fragments of the 5'-UTRs of the above genes.

[0126] In some embodiments, the 5'-UTR comprises at least one of the following polynucleotides: (a): 5'-UTRs derived from at least two of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, HBB, MYSM1, LENG1, TMSB4X, CASP4, IFNA1, PGLYRP1, UCHL1, CPAMD8, TTR, APOA2, GH1, DTYMK, APOC2, and CDK7; (b): fragments of the 5'-UTRs of at least two of the genes in (a); (c): variants of the 5'-UTRs of at least two of the genes in (a); and (d): variants of the fragments in (b). In some embodiments, the 5'-UTR comprises at least one of the following polynucleotides: (e): a 5'-UTR derived from at least two genes of PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, and HBB; (f): a fragment of the 5'-UTR described in (e); (g): a variant of the 5'-UTR described in (e); and (h): a variant of the fragment described in (f).

[0127] In some embodiments, the 5'-UTR comprises at least one of the following polynucleotides: an RNA encoded by a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 1 to 21, a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 1 to 21, a variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 1 to 21, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 1 to 21. In some embodiments, the fragment, variant, or variant of the fragment of an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 1 to 21 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the RNA encoded by the polynucleotide having a sequence as set forth in one of SEQ ID NOs: 1 to 21. In some embodiments, the fragment, variant, or variant of the fragment of the RNA encoded by the polynucleotide whose sequence is as shown in one of SEQ ID NOs: 1 to 21 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides inserted, added, deleted or substituted compared to the RNA encoded by the polynucleotide whose sequence is as shown in one of SEQ ID NOs: 1 to 21.

[0128] In some embodiments, the 5'-UTR includes at least one of the following polynucleotides: an RNA encoded by a polynucleotide having a sequence as shown in at least two of SEQ ID NOs: 9, 7, 18, 12, 8, 1 and 6, a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least two of SEQ ID NOs: 9, 7, 18, 12, 8, 1 and 6, a variant of an RNA encoded by a polynucleotide having a sequence as shown in at least two of SEQ ID NOs: 9, 7, 18, 12, 8, 1 and 6, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least two of SEQ ID NOs: 9, 7, 18, 12, 8, 1 and 6. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide of SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the RNA encoded by the polynucleotide of SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide of SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotide insertions, additions, deletions, or substitutions compared to the RNA encoded by the polynucleotide of SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6.

[0129] In some embodiments, the 5'-UTR comprises at least one of the following polynucleotides: a) at least two copies of a 5'-UTR derived from at least one of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, HBB, MYSM1, LENG1, TMSB4X, CASP4, IFNA1, PGLYRP1, UCHL1, CPAMD8, TTR, APOA2, GH1, DTYMK, APOC2, and CDK7; b) at least two copies of a fragment of the 5'-UTR of at least one of the genes in a); c) at least two copies of a variant of the 5'-UTR of at least one of the genes in a); and d) at least two copies of a variant of a fragment of the 5'-UTR of at least one of the genes in a). In some embodiments, the 5'-UTR comprises at least two copies of a 5'-UTR derived from at least one of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, and HBB. Preferably, the at least two copies are two copies, three copies, four copies, five copies, six copies, seven copies, eight copies or nine copies.

[0130] In some embodiments, the 5'-UTR comprises at least one of the following polynucleotides: at least two copies of an RNA encoded by a polynucleotide having a sequence as set forth in any one of SEQ ID NOs: 1 to 21, at least two copies of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in any one of SEQ ID NOs: 1 to 21, at least two copies of a variant of an RNA encoded by a polynucleotide having a sequence as set forth in any one of SEQ ID NOs: 1 to 21, and at least two copies of a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in any one of SEQ ID NOs: 1 to 21. Preferably, the at least two copies are two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, or nine copies. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide having a sequence as set forth in one of SEQ ID NOs: 1 to 21 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the RNA encoded by the polynucleotide having a sequence as set forth in one of SEQ ID NOs: 1 to 21. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide having a sequence as set forth in one of SEQ ID NOs: 1 to 21 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides inserted, added, deleted, or substituted compared to the RNA encoded by the polynucleotide having a sequence as set forth in one of SEQ ID NOs: 1 to 21.

[0131] In some embodiments, the 5'-UTR comprises at least one of the following polynucleotides: at least two copies of an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 9, 7, 18, 12, 8, 1, and 6; at least two copies of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 9, 7, 18, 12, 8, 1, and 6; at least two copies of a variant of an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 9, 7, 18, 12, 8, 1, and 6; and at least two copies of a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 9, 7, 18, 12, 8, 1, and 6. Preferably, the at least two copies are two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, or nine copies. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide having a sequence as set forth in one of SEQ ID NOs: 9, 7, 18, 12, 8, 1, and 6 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NOs: 9, 7, 18, 12, 8, 1, or 6. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NOs: 9, 7, 18, 12, 8, 1, and 6 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotide insertions, additions, deletions, or substitutions compared to the RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NOs: 9, 7, 18, 12, 8, 1, or 6.

[0132] 3. Improving the translation of the 3'-UTR

[0133] The inventors unexpectedly discovered that incorporation of a 3'-UTR from the gene MPND, FBXW10, FBXW12, PGLYRP1, HPX, CDK7, APOC2, PFN1, RBP4, FTCD, NAAA, ALB, GSDMD, FBXL8, ORM1, CASP4, CHMP2A, LENG1, MYCBPAP, APOC1, GAPDH, HSPA8, APOA2, UCHL1, TSG101, NAE1, or NFKB2 into mRNA can increase the translation efficiency of the coding sequence.

[0134] The full names of the above genes are shown below.

[0135] MPND: MPN domain containing;

[0136] FBXW10:F-box and WD repeat domain containing 10;

[0137] FBXW12:F-box and WD repeat domain containing 12;

[0138] PGLYRP1:peptidoglycan recognition protein 1;

[0139] HPX:hemopexin;

[0140] CDK7:cyclin dependent kinase 7;

[0141] APOC2:apolipoprotein C2;

[0142] PFN1:profilin 1;

[0143] RBP4:retinol binding protein 4;

[0144] FTCD:formimidoyltransferase cyclodeaminase;

[0145] NAAA:N-acylethanolamine acid amidase;

[0146] ALB:albumin;

[0147] GSDMD:gasdermin D;

[0148] FBXL8:F-box and leucine rich repeat protein 8;

[0149] ORM1:orosomucoid 1;

[0150] CASP4:caspase 4;

[0151] CHMP2A:charged multivesicular body protein 2A;

[0152] LENG1:leukocyte receptor cluster member 1;

[0153] MYCBPAP: MYCBP associated protein;

[0154] APOC1: apolipoprotein C1;

[0155] GAPDH: glyceraldehyde-3-phosphate dehydrogenase;

[0156] HSPA8: heat shock protein family A (Hsp70) member 8;

[0157] APOA2: apolipoprotein A2;

[0158] UCHL1: ubiquitin C-terminal hydrolase L1;

[0159] TSG101: tumor susceptibility 101;

[0160] NAE1: NEDD8 activating enzyme E1 subunit 1;

[0161] NFKB2: nuclear factor kappa B subunit 2.

[0162] Therefore, the present invention provides a 3'-UTR comprising at least one of the following polynucleotides: (a): a 3'-UTR derived from at least one gene of MPND, FBXW10, FBXW12, PGLYRP1, HPX, CDK7, APOC2, PFN1, RBP4, FTCD, NAAA, ALB, GSDMD, FBXL8, ORM1, CASP4, CHMP2A, LENG1, MYCBPAP, APOC1, GAPDH, HSPA8, APOA2, UCHL1, TSG101, NAE1 and NFKB2; (b): a fragment of the 3'-UTR described in (a); (c): a variant of the 3'-UTR described in (a); and (d): a variant of the fragment described in (b).

[0163] In some embodiments, the gene is a eukaryotic gene.

[0164] In some embodiments, the gene is a chordate gene.

[0165] In some embodiments, the gene is a vertebrate gene.

[0166] In some embodiments, the gene is a mammalian gene.

[0167] In some embodiments, the gene is a primate gene.

[0168] In some embodiments, each of the genes is independently a gene of Homo sapiens, a gene of bonobo (Pan paniscus), a gene of chimpanzee (Pan troglodytes), or a gene of western lowland gorilla (Gorilla gorilla gorilla).

[0169] In some embodiments, the gene is a human gene.

[0170] In some embodiments, the gene is a human MPND gene, a bonobo MPND gene, a human FBXW10 gene, a human FBXW12 gene, a bonobo FBXW12 gene, a human PGLYRP1 gene, a human HPX gene, a human CDK7 gene, a human APOC2 gene, a human PFN1 gene, a human RBP4 gene, a human FTCD gene, a human NAAA gene, a human ALB gene, a human GSDMD gene, a human FBXL8 gene, a bonobo FBXL8 gene, a western lowland gorilla FB At least one of the XL8 gene, human ORM1 gene, human CASP4 gene, human CHMP2A gene, bonobo CHMP2A gene, human LENG1 gene, bonobo LENG1 gene, human MYCBPAP gene, human APOC1 gene, human GAPDH gene, human HSPA8 gene, human APOA2 gene, human UCHL1 gene, human TSG101 gene, human NAE1 gene, bonobo NAE1 gene, human NFKB2 gene and bonobo NFKB2 gene.

[0171] In some embodiments, the GenBank accession number of the MPND gene is NM_001300862.1; the GenBank accession number of the FBXW10 gene is NM_001267586.2; the GenBank accession number of the FBXW12 gene is NM_001159929.1; the GenBank accession number of the PGLYRP1 gene is NM_005091.3; the GenBank accession number of the HPX gene is AH002827.2; the GenBank accession number of the CDK7 gene is AY130859.1; and the GenBank accession number of the APOC2 gene is NM_0004 83.5; the GenBank accession number of PFN1 gene is NM_005022.4; the GenBank accession number of RBP4 gene is NM_006744.4; the GenBank accession number of FTCD gene is NM_006657.3; the GenBank accession number of NAAA gene is NM_001363719.2; the GenBank accession number of ALB gene is NM_000477.7; the GenBank accession number of GSDMD gene is NM_024736.7; the GenBank accession number of FBXL8 gene is NM _018378.2; the GenBank accession number of ORM1 gene is NM_000607.4; the GenBank accession number of CASP4 gene is NM_001225.3; the GenBank accession number of CHMP2A gene is NM_198426.3; the GenBank accession number of LENG1 gene is NM_024316.3; the GenBank accession number of MYCBPAP gene is NM_032133.6; the GenBank accession number of APOC1 gene is NG_012859.1; the GenBank accession number of GAPDH gene is The GenBank accession number of the HSPA8 gene is NM_006597.6; the GenBank accession number of the APOA2 gene is NM_001643.2; the GenBank accession number of the UCHL1 gene is NG_012931.1; the GenBank accession number of the TSG101 gene is NM_006292.4; the GenBank accession number of the NAE1 gene is NM_003905.4; and the GenBank accession number of the NFKB2 gene is NM_001261403.3.

[0172] In some embodiments, the polynucleotides encoding the 3'-UTRs of human genes MPND, FBXW10, FBXW12, PGLYRP1, HPX, CDK7, APOC2, PFN1, RBP4, FTCD, NAAA, ALB, GSDMD, FBXL8, ORM1, CASP4, CHMP2A, LENG1, MYCBPAP, APOC1, GAPDH, HSPA8, APOA2, UCHL1, TSG101, NAE1, and NFKB2 are as shown in Table 2.

[0173] Table 2. Identified 3'-UTRs that Improve Translation

[0174] Thus, in some embodiments, the 3'-UTR comprises at least one of the following polynucleotides: an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 22 to 48, a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 22 to 48, a variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 22 to 48, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 22 to 48. In some embodiments, the fragment, variant, or variant of the fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 22 to 48 is at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 22 to 48. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 22 to 48 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides inserted, added, deleted, or substituted compared to the RNA encoded by the polynucleotide having a sequence as shown in one of SEQ ID NOs: 22 to 48.

[0175] In a preferred embodiment, the gene is selected from at least one of MPND, FBXW10, FBXW12, and PGLYRP1. In some embodiments, the 3'-UTR comprises at least one of the following polynucleotides: an RNA encoded by a polynucleotide with a sequence as set forth in SEQ ID NO: 24, 22, 23, or 25, a fragment of an RNA encoded by a polynucleotide with a sequence as set forth in SEQ ID NO: 24, 22, 23, or 25, a variant of an RNA encoded by a polynucleotide with a sequence as set forth in SEQ ID NO: 24, 22, 23, or 25, and a variant of a fragment of an RNA encoded by a polynucleotide with a sequence as set forth in SEQ ID NO: 24, 22, 23, or 25. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide of SEQ ID NO: 24, 22, 23, or 25 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the RNA encoded by the polynucleotide of SEQ ID NO: 24, 22, 23, or 25. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide of SEQ ID NO: 24, 22, 23, or 25 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotide insertions, additions, deletions, or substitutions compared to the RNA encoded by the polynucleotide of SEQ ID NO: 24, 22, 23, or 25.

[0176] The 3'-UTR sequence of the present invention may comprise two or more tandem 3'-UTRs from the above genes, fragments of the 3'-UTRs of the above genes, variants of the 3'-UTRs of the above genes, or variants of the 3'-UTR fragments of the above genes.

[0177] In some embodiments, the 3'-UTR comprises at least one of the following polynucleotides: (a): 3'-UTRs derived from at least two of the genes MPND, FBXW10, FBXW12, PGLYRP1, HPX, CDK7, APOC2, PFN1, RBP4, FTCD, NAAA, ALB, GSDMD, FBXL8, ORM1, CASP4, CHMP2A, LENG1, MYCBPAP, APOC1, GAPDH, HSPA8, APOA2, UCHL1, TSG101, NAE1, and NFKB2; (b): fragments of the 3'-UTRs of at least two of the genes in (a); (c): variants of the 3'-UTRs of at least two of the genes in (a); and (d): variants of the fragments in (b). In some embodiments, the 3'-UTR comprises at least one of the following polynucleotides: (e): a 3'-UTR derived from at least two genes of the genes MPND, FBXW10, FBXW12, and PGLYRP1; (f): a fragment of the 3'-UTR described in (e); (g): a variant of the 3'-UTR described in (e); and (h): a variant of the fragment described in (f).

[0178] In some embodiments, the 3'-UTR comprises at least one of the following polynucleotides: an RNA encoded by at least two of the polynucleotides set forth in SEQ ID NOs: 22 to 48, a fragment of an RNA encoded by at least two of the polynucleotides set forth in SEQ ID NOs: 22 to 48, a variant of an RNA encoded by at least two of the polynucleotides set forth in SEQ ID NOs: 22 to 48, and a variant of a fragment of an RNA encoded by at least two of the polynucleotides set forth in SEQ ID NOs: 22 to 48. In some embodiments, the fragment, variant, or variant of the fragment of an RNA encoded by one of the polynucleotides set forth in SEQ ID NOs: 22 to 48 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an RNA encoded by a polynucleotide set forth in SEQ ID NOs: 22 to 48. In some embodiments, a fragment, variant, or variant of an RNA encoded by a polynucleotide set forth in one of SEQ ID NOs: 22 to 48 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide insertions, additions, deletions, or substitutions compared to the RNA encoded by the polynucleotide set forth in one of SEQ ID NOs: 22 to 48.

[0179] In some embodiments, the 3'-UTR comprises at least one of the following polynucleotides: an RNA encoded by at least two of the polynucleotides shown in sequences of SEQ ID NOs: 24, 22, 23 and 25, a fragment of an RNA encoded by at least two of the polynucleotides shown in sequences of SEQ ID NOs: 24, 22, 23 and 25, a variant of an RNA encoded by at least two of the polynucleotides shown in sequences of SEQ ID NOs: 24, 22, 23 and 25, and a variant of a fragment of an RNA encoded by at least two of the polynucleotides shown in sequences of SEQ ID NOs: 24, 22, 23 and 25. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide of sequence SEQ ID NO: 24, 22, 23, or 25 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the RNA encoded by the polynucleotide of sequence SEQ ID NO: 24, 22, 23, or 25. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide of sequence SEQ ID NO: 24, 22, 23, or 25 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotide insertions, additions, deletions, or substitutions compared to the RNA encoded by the polynucleotide of sequence SEQ ID NO: 24, 22, 23, or 25.

[0180] In some embodiments, the 3'-UTR comprises at least one of the following polynucleotides: a): at least two copies of a 3'-UTR derived from one of the genes MPND, FBXW10, FBXW12, PGLYRP1, HPX, CDK7, APOC2, PFN1, RBP4, FTCD, NAAA, ALB, GSDMD, FBXL8, ORM1, CASP4, CHMP2A, LENG1, MYCBPAP, APOC1, GAPDH, HSPA8, APOA2, UCHL1, TSG101, NAE1, and NFKB2; b): at least two copies of a fragment of the 3'-UTR of at least one of the genes in a); c): at least two copies of a variant of the 3'-UTR of at least one of the genes in a); and d): at least two copies of a variant of a fragment of the 3'-UTR of at least one of the genes in a). In some embodiments, the 3'-UTR comprises at least one of the following polynucleotides: e): at least two copies of a 3'-UTR derived from at least one of the genes MPND, FBXW10, FBXW12, and PGLYRP1; f): at least two copies of a fragment of a 3'-UTR derived from at least one of the genes in e); g): at least two copies of a variant of a 3'-UTR derived from at least one of the genes in e); and h): at least two copies of a variant of the fragment in f). Preferably, the at least two copies are two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, or nine copies.

[0181] In some embodiments, the 3'-UTR sequence comprises at least one of the following polynucleotides: at least two copies of an RNA encoded by a polynucleotide having a sequence as set forth in any one of SEQ ID NOs: 22 to 48, two copies of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in any one of SEQ ID NOs: 22 to 48, two copies of a variant of an RNA encoded by a polynucleotide having a sequence as set forth in any one of SEQ ID NOs: 22 to 48, and two copies of a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in any one of SEQ ID NOs: 22 to 48. Preferably, the at least two copies are two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, or nine copies. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide having a sequence as set forth in any of SEQ ID NOs: 22 to 48 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the RNA encoded by the polynucleotide having a sequence as set forth in any of SEQ ID NOs: 22 to 48. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide having a sequence as set forth in any of SEQ ID NOs: 22 to 48 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides inserted, added, deleted, or substituted compared to the RNA encoded by the polynucleotide having a sequence as set forth in any of SEQ ID NOs: 22 to 48.

[0182] In some embodiments, the 3'-UTR comprises at least one of the following polynucleotides: at least two copies of an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 24, 22, 23, and 25, at least two copies of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 24, 22, 23, and 25, at least two copies of a variant of an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 24, 22, 23, and 25, and at least two copies of a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 24, 22, 23, and 25. Preferably, the at least two copies are two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, or nine copies. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide having a sequence as set forth in one of SEQ ID NOs: 24, 22, 23, and 25 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NOs: 24, 22, 23, or 25. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NOs: 24, 22, 23, and 25 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotide insertions, additions, deletions, or substitutions compared to the RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NOs: 24, 22, 23, or 25.

[0183] IV. Recombinant RNA molecules comprising the 5' and / or 3'-UTR of the present invention

[0184] The present invention provides a recombinant RNA molecule comprising the 5' and / or 3'-UTR identified in the present invention that improves translation of a coding sequence.

[0185] In some embodiments, the recombinant RNA molecule comprises a first nucleotide sequence encoding a polypeptide and / or protein of interest and a second nucleotide sequence comprising a 5'-UTR, wherein the second nucleotide sequence comprises at least one selected from the following polynucleotides: (a): a 5'-UTR derived from at least one of genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, HBB, MYSM1, LENG1, TMSB4X, CASP4, IFNA1, PGLYRP1, UCHL1, CPAMD8, TTR, APOA2, GH1, DTYMK, APOC2 and CDK7; (b): a fragment of the 5'-UTR described in (a); (c): a variant of the 5'-UTR described in (a); and (d): a variant of the fragment described in (b), wherein the first nucleotide sequence and the second nucleotide sequence do not naturally occur in the same RNA molecule.

[0186] In some embodiments, the gene is a human gene. In some embodiments, the first nucleotide sequence encodes at least one polypeptide of interest. For example, one, two, three, four, five, six, seven, eight, nine or ten polypeptides of interest. In some embodiments, the first nucleotide sequence encodes at least one protein of interest. For example, one, two, three, four, five, six, seven, eight, nine or ten proteins of interest. In some embodiments, the first nucleotide sequence encodes at least one polypeptide of interest and at least one protein of interest. For example, one, two, three, four, five, six, seven, eight, nine or ten polypeptides of interest and one, two, three, four, five, six, seven, eight, nine or ten proteins of interest.

[0187] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21, a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21, a variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21. In some embodiments, the fragment, variant, or variant of the fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21 is at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the RNA encoded by the polynucleotide having a sequence as set forth in one of SEQ ID NOs: 1 to 21. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 1 to 21 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides inserted, added, deleted or substituted compared to the RNA encoded by the polynucleotide having a sequence as shown in one of SEQ ID NOs: 1 to 21.

[0188] In preferred embodiments, the gene is selected from at least one of PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, and HBB. In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: 1) an RNA encoded by a polynucleotide as set forth in SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6; 2) a fragment of the RNA described in 1); 3) a variant of the RNA described in 1); and 4) a variant of the fragment described in 2). In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide of SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the polynucleotide of SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide of SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotide insertions, additions, deletions, or substitutions compared to the RNA encoded by the polynucleotide of SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6.

[0189] The second nucleotide sequence may comprise two or more tandem 5'-UTRs from the above genes, fragments of the 5'-UTRs of the above genes, variants of the 5'-UTRs of the above genes, or variants of the fragments of the 5'-UTRs of the above genes.

[0190] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: (a): 5'-UTRs derived from at least two of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, HBB, MYSM1, LENG1, TMSB4X, CASP4, IFNA1, PGLYRP1, UCHL1, CPAMD8, TTR, APOA2, GH1, DTYMK, APOC2, and CDK7; (b): fragments of the 5'-UTRs of at least two of the genes in (a); (c): variants of the 5'-UTRs of at least two of the genes in (a); and (d): variants of the fragments in (b). In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: (e): a 5'-UTR derived from at least two genes of genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, and HBB; (f): a fragment of the 5'-UTR described in (e); (g): a variant of the 5'-UTR described in (e); and (h): a variant of the fragment described in (f).

[0191] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: RNA encoded by a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 1 to 21, a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 1 to 21, a variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 1 to 21, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 1 to 21. In some embodiments, the second nucleotide sequence comprises RNA encoded by a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 1 to 21, a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 1 to 21, a variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 1 to 21, or a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 1 to 21. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide of one of SEQ ID NOs: 1 to 21 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the RNA encoded by the polynucleotide of one of SEQ ID NOs: 1 to 21. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide of one of SEQ ID NOs: 1 to 21 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides inserted, added, deleted, or substituted compared to the RNA encoded by the polynucleotide of one of SEQ ID NOs: 1 to 21.

[0192] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: an RNA encoded by a polynucleotide having a sequence as shown in at least two of SEQ ID NOs: 9, 7, 18, 12, 8, 1 and 6, a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least two of SEQ ID NOs: 9, 7, 18, 12, 8, 1 and 6, a variant of an RNA encoded by a polynucleotide having a sequence as shown in at least two of SEQ ID NOs: 9, 7, 18, 12, 8, 1 and 6, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least two of SEQ ID NOs: 9, 7, 18, 12, 8, 1 and 6. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide of SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the RNA encoded by the polynucleotide of SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide of SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotide insertions, additions, deletions, or substitutions compared to the RNA encoded by the polynucleotide of SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6.

[0193] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: a): at least two copies of a 5'-UTR derived from at least one of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, HBB, MYSM1, LENG1, TMSB4X, CASP4, IFNA1, PGLYRP1, UCHL1, CPAMD8, TTR, APOA2, GH1, DTYMK, APOC2, and CDK7; b): at least two copies of a fragment of the 5'-UTR of at least one of the genes in a); c): at least two copies of a variant of the 5'-UTR of at least one of the genes in a); and d): at least two copies of a variant of a fragment of the 5'-UTR of at least one of the genes in a). In some embodiments, the second nucleotide sequence comprises at least two copies of a 5'-UTR derived from one of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, HBB, MYSM1, LENG1, TMSB4X, CASP4, IFNA1, PGLYRP1, UCHL1, CPAMD8, TTR, APOA2, GH1, DTYMK, APOC2, and CDK7. In some embodiments, the 5'-UTR sequence comprises at least one of the following polynucleotides: a): at least two copies of a 5'-UTR derived from at least one of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, and HBB; b): at least two copies of a fragment of the 5'-UTR of at least one of the genes in a); c): at least two copies of a variant of the 5'-UTR of at least one of the genes in a); and d): at least two copies of a variant of a fragment of the 5'-UTR of at least one of the genes in a). In some embodiments, the 5'-UTR sequence comprises at least two copies of a 5'-UTR derived from one of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, and HBB. Preferably, the at least two copies are two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, or nine copies.

[0194] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: at least two copies of an RNA encoded by a polynucleotide having a sequence as set forth in any one of SEQ ID NOs: 1 to 21, at least two copies of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in any one of SEQ ID NOs: 1 to 21, at least two copies of a variant of an RNA encoded by a polynucleotide having a sequence as set forth in any one of SEQ ID NOs: 1 to 21, and at least two copies of a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in any one of SEQ ID NOs: 1 to 21. Preferably, the at least two copies are two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, or nine copies. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide having a sequence as set forth in one of SEQ ID NOs: 1 to 21 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the RNA encoded by the polynucleotide having a sequence as set forth in one of SEQ ID NOs: 1 to 21. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide having a sequence as set forth in one of SEQ ID NOs: 1 to 21 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides inserted, added, deleted, or substituted compared to the RNA encoded by the polynucleotide having a sequence as set forth in one of SEQ ID NOs: 1 to 21.

[0195] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: at least two copies of an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 9, 7, 18, 12, 8, 1, and 6, two copies of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 9, 7, 18, 12, 8, 1, and 6, two copies of a variant of an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 9, 7, 18, 12, 8, 1, and 6, and two copies of a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 9, 7, 18, 12, 8, 1, and 6. Preferably, the at least two copies are two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, or nine copies. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide having a sequence as set forth in one of SEQ ID NOs: 9, 7, 18, 12, 8, 1, and 6 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NOs: 9, 7, 18, 12, 8, 1, or 6. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NOs: 9, 7, 18, 12, 8, 1, and 6 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotide insertions, additions, deletions, or substitutions compared to the RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NOs: 9, 7, 18, 12, 8, 1, or 6.

[0196] In some embodiments, the recombinant RNA molecule is an mRNA molecule. In some embodiments, the recombinant RNA molecule further comprises at least one of a promoter, a 5'-cap structure, a 3'-UTR, and a poly(A) tail. In some embodiments, the recombinant RNA molecule further comprises at least one of a 5'-cap structure, a 3'-UTR, and a poly(A) tail.

[0197] In some embodiments, the 5'-cap structure includes but is not limited to m 7 GpppG、m2 7,3′-O GpppG、m 7 Gppp(5')N1 and m 7 Gppp(m 2′-O)N1. "m7G" represents 7-methylguanosine cap nucleoside, "ppp" represents the triphosphate bond between the 5' carbon of the cap nucleoside and the first nucleotide of the primary RNA transcript, N1 is the 5' most nucleotide, "G" represents guanosine nucleoside, and "m 7 ” represents the methyl group at the 7-position of guanine, “m 2′-O ” represents the methyl group at the 2′-O position of the nucleotide.

[0198] In some embodiments, the 3'-UTR comprises: i) a nucleotide sequence derived from the 3'-UTR of at least one of the albumin gene, the α-globin gene, the β-globin gene, the tyrosine hydroxylase gene, the lipoxygenase gene, and the collagen α gene, or a variant thereof; ii) a variant of the 3'-UTR in i); iii) at least one of the 3'-UTRs, fragments, variants, and variants of fragments thereof, derived from at least one of the genes MPND, FBXW10, FBXW12, PGLYRP1, HPX, CDK7, APOC2, PFN1, RBP4, FTCD, NAAA, ALB, GSDMD, FBXL8, ORM1, CASP4, CHMP2A, LENG1, MYCBPAP, APOC1, GAPDH, HSPA8, APOA2, UCHL1, TSG101, NAE1, NFKB2, and GH1; preferably, the RNA encoded by the polynucleotides as shown in SEQ ID NOs: 22 to 49, the RNA encoded by the polynucleotides as shown in SEQ ID NOs: 23 to 25, the RNA encoded by the polynucleotides as shown in SEQ ID NOs: 24 to 26, the RNA encoded by the polynucleotides as shown in SEQ ID NOs: 25 to 27, the RNA encoded by the polynucleotides as shown in SEQ ID NOs: 26 to 27 At least one of a fragment of an RNA encoded by a polynucleotide as set forth in SEQ ID NOs: 22 to 49, a variant of an RNA encoded by a polynucleotide as set forth in SEQ ID NOs: 22 to 49, and a variant of a fragment of an RNA encoded by a polynucleotide as set forth in SEQ ID NOs: 22 to 49. In some embodiments, the fragment, variant, or variant of an RNA encoded by a polynucleotide as set forth in SEQ ID NOs: 22 to 49 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an RNA encoded by a polynucleotide as set forth in SEQ ID NOs: 22 to 49; iv) at least two copies of one polynucleotide sequence of i), ii), or iii); or v) at least two polynucleotides of the group consisting of the polynucleotides of i) to iii). Preferably, the at least two copies are two copies, three copies, four copies, five copies, six copies, seven copies, eight copies or nine copies.

[0199] In some embodiments, the nucleotides comprising the poly(A) tail comprise at least 20, at least 40, at least 80, at least 100, or at least 120 A nucleotides; the nucleotides comprising the poly(A) tail comprise at least 20, at least 40, at least 80, at least 100, or at least 120 consecutive A nucleotides. In some embodiments, the nucleotides comprising the poly(A) tail comprise one or more nucleotides other than A nucleotides. In some embodiments, the poly(A) tail comprises two or more consecutive nucleotides other than A nucleotides, wherein the first and last nucleotides in the sequence of two or more consecutive nucleotides are nucleotides other than A nucleotides. Preferably, the poly(A) tail is truncated, i.e., m consecutive A nucleotides and n consecutive A nucleotides are joined by a linker sequence consisting of p non-A nucleotides, where m, n, and p are positive integers. Preferably, m is 30, n is 70, and p is 10.

[0200] In some embodiments, the recombinant RNA molecule comprises a first nucleotide sequence encoding a polypeptide and / or protein of interest and a second nucleotide sequence comprising a 3'-UTR, wherein the second nucleotide sequence comprises at least one of the following polynucleotides: (a): a 3'-UTR derived from at least one of the genes MPND, FBXW10, FBXW12, PGLYRP1, HPX, CDK7, APOC2, PFN1, RBP4, FTCD, NAAA, ALB, GSDMD, FBXL8, ORM1, CASP4, CHMP2A, LENG1, MYCBPAP, APOC1, GAPDH, HSPA8, APOA2, UCHL1, TSG101, NAE1, and NFKB2; (b): a fragment of the 3'-UTR in (a); (c): a variant of the 3'-UTR in (a); and (d): a variant of the fragment in (b); wherein the first and second nucleotide sequences do not naturally occur in the same RNA molecule.

[0201] In some embodiments, the gene is a human gene.

[0202] In some embodiments, the first nucleotide sequence encodes at least one polypeptide of interest. For example, one, two, three, four, five, six, seven, eight, nine, or ten polypeptides of interest. In some embodiments, the first nucleotide sequence encodes at least one protein of interest. For example, one, two, three, four, five, six, seven, eight, nine, or ten proteins of interest. In some embodiments, the first nucleotide sequence encodes at least one polypeptide of interest and at least one protein of interest. For example, one, two, three, four, five, six, seven, eight, nine, or ten polypeptides of interest and one, two, three, four, five, six, seven, eight, nine, or ten proteins of interest.

[0203] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 22 to 48, a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 22 to 48, a variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 22 to 48, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 22 to 48. In some embodiments, the fragment, variant, or variant of the fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 22 to 48 is at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the RNA encoded by the polynucleotide having a sequence as set forth in one of SEQ ID NOs: 22 to 48. In some embodiments, a fragment, variant, or variant of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 22 to 48 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides inserted, added, deleted, or substituted with the RNA encoded by the polynucleotide having a sequence as shown in one of SEQ ID NOs: 22 to 48.

[0204] In a preferred embodiment, the gene is selected from at least one of MPND, FBXW10, FBXW12, and PGLYRP1. In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: an RNA encoded by a polynucleotide with a sequence as set forth in SEQ ID NO: 24, 22, 23, or 25, a fragment of an RNA encoded by a polynucleotide with a sequence as set forth in SEQ ID NO: 24, 22, 23, or 25, a variant of an RNA encoded by a polynucleotide with a sequence as set forth in SEQ ID NO: 24, 22, 23, or 25, and a variant of a fragment of an RNA encoded by a polynucleotide with a sequence as set forth in SEQ ID NO: 24, 22, 23, or 25. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide of SEQ ID NO: 24, 22, 23, or 25 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the RNA encoded by the polynucleotide of SEQ ID NO: 24, 22, 23, or 25. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide of SEQ ID NO: 24, 22, 23, or 25 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotide insertions, additions, deletions, or substitutions compared to the RNA encoded by the polynucleotide of SEQ ID NO: 24, 22, 23, or 25.

[0205] The 3'-UTR in the second nucleotide sequence in the recombinant RNA molecule of the present invention may comprise two or more tandem 3'-UTRs from the above genes, fragments of the 3'-UTRs of the above genes, variants of the 3'-UTRs of the above genes, or variants of the 3'-UTR fragments of the above genes.

[0206] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: (a): 3'-UTRs derived from at least two of the genes MPND, FBXW10, FBXW12, PGLYRP1, HPX, CDK7, APOC2, PFN1, RBP4, FTCD, NAAA, ALB, GSDMD, FBXL8, ORM1, CASP4, CHMP2A, LENG1, MYCBPAP, APOC1, GAPDH, HSPA8, APOA2, UCHL1, TSG101, NAE1, and NFKB2; (b): fragments of the 3'-UTRs of at least two of the genes in (a); (c): variants of the 3'-UTRs of at least two of the genes in (a); and (d): variants of the fragments in (b). In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: (e): a 3'-UTR derived from at least two genes of the genes MPND, FBXW10, FBXW12, and PGLYRP1; (f): a fragment of the 3'-UTR described in (e); (g): a variant of the 3'-UTR described in (e); and (h): a variant of the fragment described in (f).

[0207] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: an RNA encoded by a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 22 to 48, a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 22 to 48, a variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 22 to 48, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 22 to 48. In some embodiments, the fragment, variant, or variant of the fragment of an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 22 to 48 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the RNA encoded by the polynucleotide having a sequence as set forth in one of SEQ ID NOs: 22 to 48. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide having a sequence as shown in one of SEQ ID NOs: 22 to 48 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides inserted, added, deleted, or substituted compared to the RNA encoded by the polynucleotide having a sequence as shown in one of SEQ ID NOs: 22 to 48.

[0208] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: RNA encoded by at least two of the polynucleotides shown in sequences SEQ ID NO: 24, 22, 23 and 25, fragments of RNA encoded by at least two of the polynucleotides shown in sequences SEQ ID NO: 24, 22, 23 and 25, variants of RNA encoded by at least two of the polynucleotides shown in sequences SEQ ID NO: 24, 22, 23 and 25, and variants of fragments of RNA encoded by at least two of the polynucleotides shown in sequences SEQ ID NO: 24, 22, 23 and 25. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide of SEQ ID NO: 24, 22, 23, or 25 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the RNA encoded by the polynucleotide of SEQ ID NO: 24, 22, 23, or 25. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide of SEQ ID NO: 24, 22, 23, or 25 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides inserted, added, deleted, or substituted compared to the RNA encoded by the polynucleotide of SEQ ID NO: 24, 22, 23, or 25.

[0209] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: a): at least two copies of a 3'-UTR derived from at least one of the genes MPND, FBXW10, FBXW12, PGLYRP1, HPX, CDK7, APOC2, PFN1, RBP4, FTCD, NAAA, ALB, GSDMD, FBXL8, ORM1, CASP4, CHMP2A, LENG1, MYCBPAP, APOC1, GAPDH, HSPA8, APOA2, UCHL1, TSG101, NAE1, and NFKB2; b): at least two copies of a fragment of the 3'-UTR of at least one of the genes in a); c): at least two copies of a variant of the 3'-UTR of at least one of the genes in a); and d): at least two copies of a variant of a fragment of the 3'-UTR of at least one of the genes in a). In some embodiments, the second nucleotide sequence comprises at least two copies of a nucleotide sequence derived from the 3'-UTR of at least one of the genes MPND, FBXW10, FBXW12, and PGLYRP1. Preferably, the at least two copies are two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, or nine copies. In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: at least two copies of an RNA encoded by a polynucleotide having a sequence as set forth in any of SEQ ID NOs: 22-48, at least two copies of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in any of SEQ ID NOs: 22-48, at least two copies of a variant of an RNA encoded by a polynucleotide having a sequence as set forth in any of SEQ ID NOs: 22-48, and at least two copies of a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in any of SEQ ID NOs: 22-48. Preferably, the at least two copies are two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, or nine copies. In some embodiments, the fragment, variant, or variant of the fragment of the RNA encoded by the polynucleotide whose sequence is shown as one of SEQ ID NOs: 22 to 48 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the RNA encoded by the polynucleotide whose sequence is shown as one of SEQ ID NOs: 22 to 48.In some embodiments, a fragment, variant, or variant of an RNA encoded by a polynucleotide having a sequence as shown in one of SEQ ID NOs: 22 to 48 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide insertions, additions, deletions, or substitutions compared to the RNA encoded by the polynucleotide having a sequence as shown in one of SEQ ID NOs: 22 to 48.

[0210] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: at least two copies of an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 24, 22, 23, and 25, at least two copies of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 24, 22, 23, and 25, at least two copies of a variant of an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 24, 22, 23, and 25, and at least two copies of a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 24, 22, 23, and 25. Preferably, the at least two copies are two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, or nine copies. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide having a sequence as set forth in one of SEQ ID NOs: 24, 22, 23, and 25 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NOs: 24, 22, 23, or 25. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NOs: 24, 22, 23, and 25 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotide insertions, additions, deletions, or substitutions compared to the RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NOs: 24, 22, 23, or 25.

[0211] In some embodiments, the recombinant RNA molecule is an mRNA molecule. In some embodiments, the recombinant RNA molecule further comprises at least one of a promoter, a 5'-cap structure, a 5'-UTR, and a poly(A) tail.

[0212] In some embodiments, the recombinant RNA molecule further comprises at least one of a 5'-cap structure, a 5'-UTR, and a poly(A) tail.

[0213] In some embodiments, the 5'-cap structure includes but is not limited to m 7 GpppG、m2 7,3′-O GpppG、m 7 Gppp(5')N1 or m 7 Gppp(m 2′-O )N1. "m7G" represents 7-methylguanosine cap nucleoside, "ppp" represents the triphosphate bond between the 5' carbon of the cap nucleoside and the first nucleotide of the primary RNA transcript, N1 is the 5' most nucleotide, "G" represents guanosine nucleoside, and "m 7 ” represents the methyl group at the 7-position of guanine, “m 2′-O ” represents the methyl group at the 2′-O position of the nucleotide.

[0214] In some embodiments, the 5'-UTR comprises: i) a 5'-UTR derived from at least one of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, HBB, MYSM1, LENG1, TMSB4X, CASP4, IFNA1, PGLYRP1, UCHL1, CPAMD8, TTR, APOA2, GH1, DTYMK, APOC2, and CDK7, a fragment, a variant, or a variant of a fragment thereof; preferably, at least one of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 1 to 21, a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 1 to 21, a variant of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 1 to 21, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 1 to 21; a variant of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 1 to 21, a variant of an RNA encoded by a polynucleotide having a sequence as shown in SEQ ID NOs: 1 to 21, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as shown in SEQ ID NOs: 1 to 21. NO: 1 to 21, and variants of the fragment of the RNA encoded by at least one of the polynucleotides as shown in SEQ ID NO: 1 to 21, which have at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the RNA encoded by at least one of the polynucleotides as shown in SEQ ID NO: 1 to 21; ii) at least two copies of at least one of the polynucleotides as shown in i); or iii) at least two of the polynucleotides as shown in i). Preferably, the at least two copies are two copies, three copies, four copies, five copies, six copies, seven copies, eight copies or nine copies.

[0215] In some embodiments, the nucleotides comprising the poly(A) tail comprise at least 20, at least 40, at least 80, at least 100, or at least 120 A nucleotides; preferably, the nucleotides comprising the poly(A) tail comprise at least 20, at least 40, at least 80, at least 100, or at least 120 consecutive A nucleotides. Preferably, in some embodiments, the poly(A) tail comprises one or more nucleotides other than A nucleotides. In some embodiments, the poly(A) tail comprises two or more consecutive nucleotides other than A nucleotides, wherein the first and last nucleotides in the sequence of two or more consecutive nucleotides are nucleotides other than A nucleotides. Preferably, the poly(A) tail is a truncated polyA, i.e., m consecutive A nucleotides and n consecutive A nucleotides are joined by a linker sequence consisting of p non-A nucleotides, where m, n, and p are positive integers. Preferably, m is 30, n is 70, and p is 10.

[0216] In some embodiments, the DNA sequence corresponding to the poly(A) tail is shown in SEQ ID NO:53.

[0217] In some embodiments, the recombinant RNA molecule is no longer than 50,000 nt. Preferably, the recombinant RNA molecule is no longer than 40,000 nt, 30,000 nt, 20,000 nt, 10,000 nt, 9,000 nt, 8,000 nt, or 6,000 nt. In some embodiments, the recombinant RNA molecule is 500 nt to 50,000 nt. In some embodiments, the recombinant RNA molecule is 1,000 nt to 40,000 nt, 1,000 nt to 30,000 nt, 1,500 nt to 10,000 nt, or 1,500 nt to 8,000 nt.

[0218] In some embodiments, the recombinant RNA molecule comprises a first nucleotide sequence encoding a polypeptide and / or protein of interest, a second nucleotide sequence comprising a 5'-UTR, and a third nucleotide sequence comprising a 3'-UTR, wherein the second nucleotide sequence comprises at least one of a 5'-UTR, a fragment, a variant, and a variant of a fragment thereof, derived from at least one of genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, HBB, MYSM1, LENG1, TMSB4X, CASP4, IFNA1, PGLYRP1, UCHL1, CPAMD8, TTR, APOA2, GH1, DTYMK, APOC2, and CDK7, wherein the third nucleotide sequence comprises a 5'-UTR, a fragment, a variant, and a variant of a fragment thereof, derived from at least one of genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, HBB, MYSM1, LENG1, TMSB4X, CASP4, IFNA1, PGLYRP1, UCHL1, CPAMD8, TTR, APOA2, GH1, DTYMK, APOC2, and CDK7. The nucleotide sequence comprises at least one of a 3'-UTR derived from at least one of the genes MPND, FBXW10, FBXW12, PGLYRP1, HPX, CDK7, APOC2, PFN1, RBP4, FTCD, NAAA, ALB, GSDMD, FBXL8, ORM1, CASP4, CHMP2A, LENG1, MYCBPAP, APOC1, GAPDH, HSPA8, APOA2, UCHL1, TSG101, NAE1, NFKB2, and GH1, a fragment, a variant, or a variant of a fragment thereof, and wherein the first nucleotide sequence and at least one of the second nucleotide sequence and the third nucleotide sequence do not naturally occur in the same RNA molecule.

[0219] Compared to the 5'-UTR or 3'-UTR derived from at least one of the above genes, fragments, variants, or variants of fragments thereof alone, the combination of the 5'-UTR derived from at least one of the above genes, fragments, variants, or variants of fragments thereof and the 3'-UTR derived from at least one of the above genes, fragments, variants, or variants of fragments thereof can further improve the translation efficiency and / or stability of the mRNA molecule and increase the expression level of the polypeptide and / or protein of interest, for example, by 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold, etc.

[0220] It should be noted that, herein, "A, a fragment, a variant, or a variant of a fragment thereof" is an abbreviation for A, a fragment of A, a variant of A, or a variant of a fragment of A. For example, "a 5'-UTR derived from at least one of the genes PPIA, HPX, and FTCD, a fragment, a variant, or a variant of a fragment thereof" refers to a 5'-UTR derived from at least one of the genes PPIA, HPX, and FTCD, a fragment derived from the 5'-UTR of at least one of the genes PPIA, HPX, and FTCD, a variant of the 5'-UTR of at least one of the genes PPIA, HPX, and FTCD, or a variant of a fragment derived from the 5'-UTR of at least one of the genes PPIA, HPX, and FTCD. Herein, "at least one of A, a fragment, a variant, and a variant of a fragment thereof" is an abbreviation for at least one of the group consisting of A, a fragment of A, a variant of A, and a variant of a fragment of A. For example, "at least one of a 5'-UTR derived from at least one of genes PPIA, HPX, and FTCD, a fragment, a variant, and a variant of a fragment thereof" means at least one of a 5'-UTR derived from at least one of genes PPIA, HPX, and FTCD, a fragment derived from a 5'-UTR of at least one of genes PPIA, HPX, and FTCD, a variant of a 5'-UTR derived from at least one of genes PPIA, HPX, and FTCD, and a variant of a fragment derived from a 5'-UTR of at least one of genes PPIA, HPX, and FTCD.

[0221] In some embodiments, the gene is a human gene.

[0222] In some embodiments, the first nucleotide sequence encodes at least one polypeptide of interest. For example, one, two, three, four, five, six, seven, eight, nine, or ten polypeptides of interest. In some embodiments, the first nucleotide sequence encodes at least one protein of interest. For example, one, two, three, four, five, six, seven, eight, nine, or ten proteins of interest. In some embodiments, the first nucleotide sequence encodes at least one polypeptide of interest and at least one protein of interest. For example, one, two, three, four, five, six, seven, eight, nine, or ten polypeptides of interest and one, two, three, four, five, six, seven, eight, nine, or ten proteins of interest.

[0223] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21, a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21, a variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21. In some embodiments, the second nucleotide sequence comprises one of the following polynucleotides: an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21, a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21, a variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the RNA encoded by the polynucleotide having a sequence as set forth in one of SEQ ID NOs: 1 to 21. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides inserted, added, deleted, or substituted compared to the RNA encoded by the polynucleotide having a sequence as set forth in one of SEQ ID NOs: 1 to 21.

[0224] In preferred embodiments, the gene is selected from at least one of PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, and HBB. In some embodiments, the second nucleotide sequence comprises at least one of the following nucleotides: 1) an RNA encoded by the polynucleotide set forth in SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6; 2) a fragment of the RNA set forth in 1); 3) a variant of the RNA set forth in 1); and 4) a variant of the fragment set forth in 2). In some embodiments, the second nucleotide sequence comprises one of the following nucleotides: 1) an RNA encoded by the polynucleotide set forth in SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6; 2) a fragment of the RNA set forth in 1); 3) a variant of the RNA set forth in 1); and 4) a variant of the fragment set forth in 2). In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide of SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the RNA encoded by the polynucleotide of SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide of SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotide insertions, additions, deletions, or substitutions compared to the RNA encoded by the polynucleotide of SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6.

[0225] The 5'-UTR in the second nucleotide sequence in the recombinant RNA molecule of the present invention may comprise two or more tandem 5'-UTRs from the above genes, fragments of the 5'-UTRs of the above genes, variants of the 5'-UTRs of the above genes, or variants of the 5'-UTR fragments of the above genes.

[0226] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: (a): 5'-UTRs derived from at least two of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, HBB, MYSM1, LENG1, TMSB4X, CASP4, IFNA1, PGLYRP1, UCHL1, CPAMD8, TTR, APOA2, GH1, DTYMK, APOC2, and CDK7; (b): fragments of the 5'-UTRs of at least two of the genes in (a); (c): variants of the 5'-UTRs of at least two of the genes in (a); and (d): variants of the fragments in (b).

[0227] In some embodiments, the second nucleotide sequence comprises one of the following polynucleotides: (a): 5'-UTRs derived from at least two of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, HBB, MYSM1, LENG1, TMSB4X, CASP4, IFNA1, PGLYRP1, UCHL1, CPAMD8, TTR, APOA2, GH1, DTYMK, APOC2, and CDK7; (b): fragments of the 5'-UTRs of at least two of the genes in (a); (c): variants of the 5'-UTRs of at least two of the genes in (a); and (d): variants of the fragments in (b). In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: (e): a 5'-UTR derived from at least two of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, and HBB; (f): a fragment of the 5'-UTR described in (e); (g): a variant of the 5'-UTR described in (e); and (h): a variant of the fragment described in (f). In some embodiments, the second nucleotide sequence comprises one of the following polynucleotides: (e): a 5'-UTR derived from at least two of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, and HBB; (f): a fragment of the 5'-UTR described in (e); (g): a variant of the 5'-UTR described in (e); and (h): a variant of the fragment described in (f).

[0228] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: RNA encoded by a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 1 to 21, a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 1 to 21, a variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 1 to 21, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 1 to 21. In some embodiments, the second nucleotide sequence comprises RNA encoded by a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 1 to 21, a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 1 to 21, a variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 1 to 21, or a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 1 to 21. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide of one of SEQ ID NOs: 1 to 21 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the RNA encoded by the polynucleotide of one of SEQ ID NOs: 1 to 21. In some embodiments, the fragment, variant, or variant of the RNA encoded by at least two of SEQ ID NOs: 1 to 21 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides inserted, added, deleted, or substituted compared to the RNA encoded by the polynucleotide of one of SEQ ID NOs: 1 to 21.

[0229] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: an RNA encoded by a polynucleotide having a sequence as shown in at least two of SEQ ID NOs: 9, 7, 18, 12, 8, 1 and 6, a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least two of SEQ ID NOs: 9, 7, 18, 12, 8, 1 and 6, a variant of an RNA encoded by a polynucleotide having a sequence as shown in at least two of SEQ ID NOs: 9, 7, 18, 12, 8, 1 and 6, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least two of SEQ ID NOs: 9, 7, 18, 12, 8, 1 and 6. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide of SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the RNA encoded by the polynucleotide of SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide of SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotide insertions, additions, deletions, or substitutions compared to the RNA encoded by the polynucleotide of SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6.

[0230] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: a): at least two copies of a 5'-UTR derived from at least one of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, HBB, MYSM1, LENG1, TMSB4X, CASP4, IFNA1, PGLYRP1, UCHL1, CPAMD8, TTR, APOA2, GH1, DTYMK, APOC2, and CDK7; b): at least two copies of a fragment of the 5'-UTR of at least one of the genes in a); c): at least two copies of a variant of the 5'-UTR of at least one of the genes in a); and d): at least two copies of a variant of a fragment of the 5'-UTR of at least one of the genes in a).

[0231] In some embodiments, the second nucleotide sequence comprises at least two copies of a 5'-UTR derived from one of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, HBB, MYSM1, LENG1, TMSB4X, CASP4, IFNA1, PGLYRP1, UCHL1, CPAMD8, TTR, APOA2, GH1, DTYMK, APOC2, and CDK7. In some embodiments, the 5'-UTR sequence comprises at least two copies of a 5'-UTR derived from at least one of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, and HBB, at least two copies of a fragment of a 5'-UTR derived from at least one of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, and HBB, at least two copies of a variant of a 5'-UTR derived from at least one of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, and HBB, or at least two copies of a variant of a fragment of a 5'-UTR derived from at least one of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, and HBB. In some embodiments, the 5'-UTR sequence comprises at least two copies of a 5'-UTR derived from one of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, and HBB, at least two copies of a fragment of a 5'-UTR derived from one of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, and HBB, at least two copies of a variant of a 5'-UTR derived from one of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, and HBB, or at least two copies of a variant of a fragment of a 5'-UTR derived from one of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, and HBB. Preferably, the at least two copies are two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, or nine copies.

[0232] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: at least two copies of an RNA encoded by a polynucleotide having a sequence as set forth in any one of SEQ ID NOs: 1 to 21, at least two copies of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in any one of SEQ ID NOs: 1 to 21, at least two copies of a variant of an RNA encoded by a polynucleotide having a sequence as set forth in any one of SEQ ID NOs: 1 to 21, and at least two copies of a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in any one of SEQ ID NOs: 1 to 21. Preferably, the at least two copies are two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, or nine copies. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide having a sequence as set forth in one of SEQ ID NOs: 1 to 21 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the RNA encoded by the polynucleotide having a sequence as set forth in one of SEQ ID NOs: 1 to 21. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide having a sequence as set forth in one of SEQ ID NOs: 1 to 21 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides inserted, added, deleted, or substituted compared to the RNA encoded by the polynucleotide having a sequence as set forth in one of SEQ ID NOs: 1 to 21.

[0233] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: at least two copies of an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 9, 7, 18, 12, 8, 1, and 6, two copies of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 9, 7, 18, 12, 8, 1, and 6, two copies of a variant of an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 9, 7, 18, 12, 8, 1, and 6, and two copies of a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 9, 7, 18, 12, 8, 1, and 6. Preferably, the at least two copies are two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, or nine copies. In some embodiments, in some embodiments, the fragment, variant, or variant of the fragment of the RNA encoded by the polynucleotide whose sequence is as shown in one of SEQ ID NOs: 9, 7, 18, 12, 8, 1, and 6 has a sequence of at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the RNA encoded by the polynucleotide whose sequence is shown in SEQ ID NOs: 9, 7, 18, 12, 8, 1, or 6. In some embodiments, a fragment, variant, or variant of an RNA encoded by a polynucleotide having a sequence as shown in one of SEQ ID NOs: 9, 7, 18, 12, 8, 1, and 6 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotide insertions, additions, deletions, or substitutions compared to an RNA encoded by a polynucleotide having a sequence as shown in SEQ ID NOs: 9, 7, 18, 12, 8, 1, or 6.

[0234] In some embodiments, the third nucleotide sequence comprises at least one of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 22 to 49, a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 22 to 49, a variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 22 to 49, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 22 to 49. In some embodiments, the third nucleotide sequence comprises at least one of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 22 to 48, a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 22 to 48, a variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 22 to 48, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 22 to 48. In some embodiments, a fragment, variant, or variant of an RNA encoded by a polynucleotide having a sequence as set forth in any of SEQ ID NOs: 22 to 49 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an RNA encoded by a polynucleotide having a sequence as set forth in any of SEQ ID NOs: 22 to 49. In some embodiments, a fragment, variant, or variant of an RNA encoded by a polynucleotide having a sequence as set forth in any of SEQ ID NOs: 22 to 49 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotide insertions, additions, deletions, or substitutions compared to an RNA encoded by a polynucleotide having a sequence as set forth in any of SEQ ID NOs: 22 to 49.

[0235] In preferred embodiments, the gene is selected from at least one of MPND, FBXW10, FBXW12, and PGLYRP1. In some embodiments, the gene is selected from one of MPND, FBXW10, FBXW12, and PGLYRP1. In some embodiments, the third nucleotide sequence comprises at least one of the following polynucleotides: an RNA encoded by a polynucleotide with a sequence as set forth in SEQ ID NO: 24, 22, 23, or 25; a fragment of an RNA encoded by a polynucleotide with a sequence as set forth in SEQ ID NO: 24, 22, 23, or 25; a variant of an RNA encoded by a polynucleotide with a sequence as set forth in SEQ ID NO: 24, 22, 23, or 25; and a variant of a fragment of an RNA encoded by a polynucleotide with a sequence as set forth in SEQ ID NO: 24, 22, 23, or 25. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide of SEQ ID NO: 24, 22, 23, or 25 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the RNA encoded by the polynucleotide of SEQ ID NO: 24, 22, 23, or 25. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide of SEQ ID NO: 24, 22, 23, or 25 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotide insertions, additions, deletions, or substitutions compared to the RNA encoded by the polynucleotide of SEQ ID NO: 24, 22, 23, or 25.

[0236] The 3'-UTR in the third nucleotide sequence of the recombinant molecule of the present invention may comprise two or more tandem 3'-UTRs from the above genes, fragments of the 3'-UTRs of the above genes, variants of the 3'-UTRs of the above genes, or variants of the 3'-UTR fragments of the above genes.

[0237] In some embodiments, the third nucleotide sequence comprises at least one of the following polynucleotides: (a): 3'-UTRs derived from at least two of the genes MPND, FBXW10, FBXW12, PGLYRP1, HPX, CDK7, APOC2, PFN1, RBP4, FTCD, NAAA, ALB, GSDMD, FBXL8, ORM1, CASP4, CHMP2A, LENG1, MYCBPAP, APOC1, GAPDH, HSPA8, APOA2, UCHL1, TSG101, NAE1, NFKB2, and GH1; (b): fragments of the 3'-UTRs of at least two of the genes in (a); (c): variants of the 3'-UTRs of at least two of the genes in (a); and (d): variants of the fragments of the 3'-UTRs of at least two of the genes in (a). In some embodiments, the third nucleotide sequence comprises at least one of the following polynucleotides: (e): a 3'-UTR derived from at least two genes of the genes MPND, FBXW10, FBXW12, and PGLYRP1; (f): a fragment of the 3'-UTR in (e); (g): a variant of the 3'-UTR in (e); and (h): a variant of the fragment in (f).

[0238] In some embodiments, the third nucleotide sequence comprises at least one of the following polynucleotides: RNA encoded by polynucleotides having sequences as set forth in at least two of SEQ ID NOs: 22 to 49, fragments of RNA encoded by polynucleotides having sequences as set forth in at least two of SEQ ID NOs: 22 to 49, variants of RNA encoded by polynucleotides having sequences as set forth in at least two of SEQ ID NOs: 22 to 49, and variants of fragments of RNA encoded by polynucleotides having sequences as set forth in at least two of SEQ ID NOs: 22 to 49. In some embodiments, the third nucleotide sequence comprises at least one of the following polynucleotides: RNA encoded by polynucleotides having sequences as set forth in at least two of SEQ ID NOs: 22 to 48, fragments of RNA encoded by polynucleotides having sequences as set forth in at least two of SEQ ID NOs: 22 to 48, variants of RNA encoded by polynucleotides having sequences as set forth in at least two of SEQ ID NOs: 22 to 48, and variants of fragments of RNA encoded by polynucleotides having sequences as set forth in at least two of SEQ ID NOs: 22 to 48. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide having a sequence as set forth in any of SEQ ID NOs: 22 to 49 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the RNA encoded by the polynucleotide having a sequence as set forth in any of SEQ ID NOs: 22 to 49. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide having a sequence as set forth in any of SEQ ID NOs: 22 to 49 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides inserted, added, deleted, or substituted compared to the RNA encoded by the polynucleotide having a sequence as set forth in any of SEQ ID NOs: 22 to 49.

[0239] In some embodiments, the third nucleotide sequence comprises at least one of the following polynucleotides: RNA encoded by at least two of the polynucleotides shown in sequences SEQ ID NO: 24, 22, 23 and 25, fragments of RNA encoded by at least two of the polynucleotides shown in sequences SEQ ID NO: 24, 22, 23 and 25, variants of RNA encoded by at least two of the polynucleotides shown in sequences SEQ ID NO: 24, 22, 23 and 25, and variants of fragments of RNA encoded by at least two of the polynucleotides shown in sequences SEQ ID NO: 24, 22, 23 and 25. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide of SEQ ID NO: 24, 22, 23, or 25 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the RNA encoded by the polynucleotide of SEQ ID NO: 24, 22, 23, or 25. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide of SEQ ID NO: 24, 22, 23, or 25 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides inserted, added, deleted, or substituted compared to the RNA encoded by the polynucleotide of SEQ ID NO: 24, 22, 23, or 25.

[0240] In some embodiments, the third nucleotide sequence comprises at least one of the following polynucleotides: a): at least two copies of a 3'-UTR derived from at least one of the genes MPND, FBXW10, FBXW12, PGLYRP1, HPX, CDK7, APOC2, PFN1, RBP4, FTCD, NAAA, ALB, GSDMD, FBXL8, ORM1, CASP4, CHMP2A, LENG1, MYCBPAP, APOC1, GAPDH, HSPA8, APOA2, UCHL1, TSG101, NAE1, NFKB2, and GH1; b): at least two copies of a fragment of the 3'-UTR of at least one of the genes in a); c): at least two copies of a variant of the 3'-UTR of at least one of the genes in a); and d): at least two copies of a variant of a fragment of the 3'-UTR of at least one of the genes in a). In some embodiments, the second nucleotide sequence comprises at least two copies of a 3'-UTR derived from at least one of the genes MPND, FBXW10, FBXW12, and PGLYRP1. Preferably, the at least two copies are two, three, four, five, six, seven, eight, or nine copies. In some embodiments, the third nucleotide sequence comprises at least one of the following polynucleotides: at least two copies of an RNA encoded by a polynucleotide having a sequence as set forth in any of SEQ ID NOs: 22-49, at least two copies of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in any of SEQ ID NOs: 22-49, at least two copies of a variant of an RNA encoded by a polynucleotide having a sequence as set forth in any of SEQ ID NOs: 22-49, and at least two copies of a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in any of SEQ ID NOs: 22-49. In some embodiments, the third nucleotide sequence comprises at least one of the following polynucleotides: at least two copies of an RNA encoded by a polynucleotide having a sequence as set forth in any one of SEQ ID NOs: 22 to 49, at least two copies of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in any one of SEQ ID NOs: 22 to 49, at least two copies of a variant of an RNA encoded by a polynucleotide having a sequence as set forth in any one of SEQ ID NOs: 22 to 49, and at least two copies of a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in any one of SEQ ID NOs: 22 to 49. Preferably, the at least two copies are two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, or nine copies.In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide having a sequence as set forth in any of SEQ ID NOs: 22 to 49 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the RNA encoded by the polynucleotide having a sequence as set forth in any of SEQ ID NOs: 22 to 49. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide having a sequence as set forth in any of SEQ ID NOs: 22 to 49 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides inserted, added, deleted, or substituted compared to the RNA encoded by the polynucleotide having a sequence as set forth in any of SEQ ID NOs: 22 to 49.

[0241] In some embodiments, the third nucleotide sequence comprises at least one of the following polynucleotides: at least two copies of an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 24, 22, 23, and 25, at least two copies of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 24, 22, 23, and 25, at least two copies of a variant of an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 24, 22, 23, and 25, and at least two copies of a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 24, 22, 23, and 25. Preferably, the at least two copies are two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, or nine copies. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide having a sequence as set forth in one of SEQ ID NOs: 24, 22, 23, and 25 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NOs: 24, 22, 23, or 25. In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NOs: 24, 22, 23, and 25 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotide insertions, additions, deletions, or substitutions compared to the RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NOs: 24, 22, 23, or 25.

[0242] In some embodiments, the nucleotide sequence of the 5'-UTR is shown in SEQ ID NO:55.

[0243] In some embodiments, the recombinant RNA molecule comprises a first nucleotide sequence encoding a polypeptide and / or protein of interest, a 3'-UTR according to any of the above embodiments, and a 5'-UTR having a nucleotide sequence as shown in SEQ ID NO:55.

[0244] In some embodiments, the recombinant RNA molecule comprises a first nucleotide sequence encoding a polypeptide and / or protein of interest, a 3'-UTR sequence as shown in one of SEQ ID NOs: 22-48, and a 5'-UTR sequence as shown in SEQ ID NO:55.

[0245] In some embodiments, the recombinant RNA molecule comprises a first nucleotide sequence encoding a polypeptide and / or protein of interest, a 3'-UTR derived from at least one of genes FBXW10, FBXW12, MPND, and PGLYRP1, and a 5'-UTR having a nucleotide sequence as shown in SEQ ID NO:55.

[0246] In some embodiments, the recombinant RNA molecule comprises a first nucleotide sequence encoding a polypeptide and / or protein of interest, a 3'-UTR sequence as shown in one of SEQ ID NOs: 22-25, and a 5'-UTR sequence as shown in SEQ ID NO:55.

[0247] In some embodiments, the 3'-UTR is a 3'-UTR derived from the gene COP1 (CARD only protein).

[0248] In some embodiments, the nucleotide sequence of the 3'-UTR is shown in SEQ ID NO:56.

[0249] In some embodiments, the recombinant RNA molecule comprises a first nucleotide sequence encoding a polypeptide and / or protein of interest, a 5'-UTR according to any of the above embodiments, and a 3'-UTR having a nucleotide sequence as shown in SEQ ID NO:56.

[0250] In some embodiments, the recombinant RNA molecule comprises a first nucleotide sequence encoding a polypeptide and / or protein of interest, a 5'-UTR sequence as shown in one of SEQ ID NOs: 1-21, and a 3'-UTR of the gene COP1 (CARD only protein).

[0251] In some embodiments, the recombinant RNA molecule comprises a first nucleotide sequence encoding a polypeptide and / or protein of interest, a 5'-UTR sequence as shown in one of SEQ ID NOs: 1-21, and a 3'-UTR sequence as shown in SEQ ID NO:56.

[0252] In some embodiments, the recombinant RNA molecule comprises a first nucleotide sequence encoding a polypeptide and / or protein of interest, a 5'-UTR derived from at least one of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, and HBB, and a 3'-UTR derived from the gene COPl.

[0253] In some embodiments, the recombinant RNA molecule comprises a first nucleotide sequence encoding a polypeptide and / or protein of interest, a 5'-UTR sequence as shown in one of SEQ ID NOs: 1, 6-9, 12 and 18, and a 3'-UTR sequence as shown in SEQ ID NO: 56.

[0254] In some embodiments, the recombinant RNA molecule is an mRNA molecule. In some embodiments, the recombinant RNA molecule further comprises at least one of a 5'-cap structure and a poly(A) tail.

[0255] In some embodiments, the 5'-cap structure includes but is not limited to m 7 GpppG、m2 7,3′-O GpppG、m 7 Gppp(5')N1 or m 7 Gppp(m 2′-O )N1. "m7G" represents 7-methylguanosine cap nucleoside, "ppp" represents the triphosphate bond between the 5' carbon of the cap nucleoside and the first nucleotide of the primary RNA transcript, N1 is the 5' most nucleotide, "G" represents guanosine nucleoside, and "m 7 ” represents the methyl group at the 7-position of guanine, “m 2′-O ” represents the methyl group at the 2′-O position of the nucleotide.

[0256] In some embodiments, the nucleotides constituting the poly(A) tail comprise at least 20, at least 40, at least 80, at least 100, or at least 120 A nucleotides. Preferably, the nucleotides constituting the poly(A) tail comprise at least 20, at least 40, at least 80, at least 100, or at least 120 A nucleotides consecutively. In some embodiments, the poly(A) tail comprises one or more nucleotides other than A nucleotides. In some embodiments, the poly(A) tail comprises two or more consecutive nucleotides other than A nucleotides, wherein the first and last nucleotides in the sequence of two or more consecutive nucleotides are nucleotides other than A nucleotides. Preferably, the poly(A) tail is truncated, i.e., m consecutive A nucleotides and n consecutive A nucleotides are reconnected by a linker sequence consisting of p non-A nucleotides, wherein m, n, and p are positive integers. Preferably, m is 30, n is 70, and p is 10.

[0257] In some embodiments, the DNA sequence corresponding to the poly(A) tail is shown in SEQ ID NO:53.

[0258] In some embodiments, the recombinant RNA molecule is no longer than 50,000 nt. Preferably, the recombinant RNA molecule is no longer than 40,000 nt, 30,000 nt, 20,000 nt, 10,000 nt, 9,000 nt, 8,000 nt, or 6,000 nt. In some embodiments, the recombinant RNA molecule is 500 nt to 50,000 nt. In some embodiments, the recombinant RNA molecule is 1,000 nt to 40,000 nt, 1,000 nt to 30,000 nt, 1,500 nt to 10,000 nt, or 1,500 nt to 8,000 nt.

[0259] In some embodiments, the recombinant RNA molecule of any of the above embodiments comprises modified nucleosides. In some embodiments, the recombinant DNA molecule comprises at least one of modified uridine, modified cytidine, modified adenosine, and modified guanosine.

[0260] In some embodiments, the modified nucleoside is a modified uridine. In some embodiments, 0.1% to 100% of the uridines in the recombinant RNA molecule are modified. Preferably, 80% to 100% of the uridines are modified. Preferably, 100% of the uridines are modified. Exemplary modified uridines include pseudouridine (ψ), N1-methyl pseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), uridine-5-oxyethyl acid (cmo5U), uridine-5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio -uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine Uridine (τm5s2U), 1-taurine methyl-4-thio-pseudouridine, 5-methyl-uridine (m5U, i.e., with the nucleobase deoxythymine), 1-methyl-pseudouridine (m1ψ), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3 -carboxypropyl) pseudouridine (acp3ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl- Uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-methoxyformylvinyl)uridine (5-(2-carbomethoxyvinyl)uridine) and 5-[3-(1-E-propenylamino)uridine.

[0261] In some embodiments, the modified nucleoside is a modified cytidine. In some embodiments, 0.1% to 100% of the cytidines in the recombinant RNA molecule are modified. Preferably, 80% to 100% of the cytidines are modified. Preferably, 100% of the cytidines are modified. Exemplary modified cytidines include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl-cytidine (ac4C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-cytidine, 5-methyl-cytidine, 6-aza-cytidine, 5-methyl-cytidine, 6-aza-cytidine, 6-methyl-cytidine, 4-acetyl-cytidine, 5-acetyl-cytidine, 6 ... iso-zebulin, 5-methyl-zebulin, 5-aza-2-thio-zebulin, 2-thio-zebulin, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), α-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl Cm), 2'-F-arabino-cytidine, 2'-F-cytidine and 2'-OH-arabino-cytidine.

[0262] In some embodiments, the modified nucleoside is a modified adenosine. In some embodiments, 0.1% to 100% of the adenosines in the recombinant RNA molecule are modified. Preferably, 80% to 100% of the adenosines are modified. Preferably, 100% of the adenosines are modified. Exemplary modified adenosines include 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl -adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-glycylcarbamoyl-adenosine (g6A), N6-threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxynorvalylcarbamoyl-adenosine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio -adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m62Am), 1,2'-O-dimethyl-adenosine (m1Am), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-arabino-adenosine, 2'-F-adenosine, 2'-OH-arabino-adenosine and N6-(19-amino-pentaoxahonadecyl)-adenosine.

[0263] In some embodiments, the modified nucleoside is a modified guanosine. In some embodiments, 0.1% to 100% of the guanosine in the recombinant RNA molecule is modified. Preferably, 80% to 100% of the guanosine is modified. Preferably, 100% of the guanosine is modified. Exemplary modified guanosines include inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methyl wyosine (mimG), 4-demethyl-wyosine (imG-14), iso-wyosine (imG2), yW, peroxyyW (o2yW), hydroxyyW (OHyW), undermodified hydroxyyW (OHyW*), 7-deaza-guanosine, queuosine (Q), epoxy queuosine (o Q), galactosyl-braided guanosine (galQ), mannosyl-braided guanosine (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), archaeosine (G+), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m1G), N2-methyl-guanosine (m2G), N2,N2-dimethyl-guanosine (m22G), N2,7-dimethyl-guanosine (m2,7G), N2,N2,7-dimethyl-guanosine (m2,2,7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl guanosine (m2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m22Gm), 1-methyl-2'-O-methyl-guanosine (m1Gm), N2,7-dimethyl-2'-O-methyl-guanosine (M2,7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m1Im), 2'-O-ribosylguanosine (phosphate) (Gr(p)), 1-thioguanosine, O6-methyl-guanosine, 2'-F-arabinoguanosine and 2'-F-guanosine.

[0264] In some embodiments, in any of the above embodiments, the polypeptide and / or protein of interest refers to a therapeutically or pharmaceutically active polypeptide or protein having a therapeutic or preventive effect, whose function in or near a cell is necessary or beneficial, for example, a protein whose deficiency or defective form causes a disease, and providing such a protein can modulate or prevent the disease, or a protein whose presence in or near a cell is beneficial to the body. The polypeptide or protein may comprise a complete protein or a functional variant thereof.

[0265] In any of the above embodiments, the nucleotide sequence encoding the polypeptide and / or protein of interest or the expressed peptide and / or protein comprises or is one or more of the following: (a) an antigen; (b) a therapeutic protein or polypeptide, a fragment, fragment or variant thereof; and (c) other polypeptides or proteins.

[0266] In some embodiments, the peptide and / or protein expressed by the nucleotide sequence encoding the polypeptide and / or protein of interest comprises or is an antigen.

[0267] In some embodiments, the antigen expressed by the nucleotide sequence encoding the polypeptide and / or protein of interest is derived from one or more of the following: (1) a pathogenic antigen, a fragment, a variant, or a variant of a fragment thereof, (2) a tumor antigen, a fragment, a variant, or a variant of a fragment thereof, (3) an allergic antigen, a fragment, a variant, or a variant of a fragment thereof, (4) an autoimmune self-antigen, a fragment, a variant, or a variant of a fragment thereof.

[0268] In some embodiments, pathogenic antigens are derived from pathogenic organisms that are capable of eliciting an immune response in a subject (e.g., a mammalian subject, further e.g., a human). In some embodiments, the pathogenic organisms include or are one or more of the following: bacteria, viruses, fungi, and protozoa (e.g., single-cell organisms, multicellular organisms).

[0269] In some embodiments, the pathogenic antigen comprises or is a surface antigen, fragment, variant, or variant of a fragment thereof, such as a protein, fragment (e.g., an external portion of a surface antigen), variant, or variant of a fragment thereof located on the surface of a virus, bacteria, or protozoa.

[0270] In some embodiments, the pathogenic antigen comprises or is derived from a polypeptide or protein of a pathogen associated with an infectious disease.

[0271] In some embodiments, the pathogenic antigen is selected from but not limited to the group consisting of antigens derived from pathogens described on pages 21 to 35 of WO2018 / 078053A1, antigens derived from pathogens described on page 57, paragraph 3 to page 63, paragraph 2 of WO2019 / 077001A1, antigens derived from pathogens described on page 32, line 26 to page 34, line 27 of WO2013 / 120628A1, and antigens described on page 34, line 29 to page 59, line 5 of WO2013 / 120628A1.

[0272] In some embodiments, the pathogen of the pathogenic antigen is selected from but not limited to one or more of the following: scabies, Babesia, Leishmania, Gnatostoma, Ancylostoma braziliensis, Ancylostoma duodenale, Strongyloides stercoralis, Trichuris trichuris, Toxocara canis, Toxocara felis, Toxoplasma gondii, Trypanosoma brucei, Trypanosoma cruzi, Brugia malayi, Onchocerca volvulus, Bancroftian filariasis, Tapeworm, Taenia solium, Echinococcus, Ascaris lumbricoides, Dikaryotic amoeba fragilis, Naegleria fowleri, fowleri), Necator americanus, Paragonimus (e.g., Paragonimus westermani), Clonorchis sinensis, Plasmodium (e.g., P. falciparum, P. vivax, P. malariae, or P. ovale), Pneumocystis jiroveci, Metagonid fluke, Trichinella spiralis, Trichomonas vaginalis, Giardia intestinalis, Schistosoma japonicum, Geotrichum candidum, human yeast, Bartonella henselae, Wernicke's black yeast (Hortaea Wernicke), Aspergillus, Malassezia, Vibrio cholerae, Acinetobacter baumannii, Paracoccidioides brasiliensis, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Nocardia asteroides, Nocardia schenckii, Sporothrix schenckii, Staphylococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Trichophyton, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Ureaplasma urealyticum, Salmonella, Francisella tularensis, Fusobacterium, Mycobacterium leprae, Mycobacterium leprae disseminated, Mycobacterium tuberculosis, Mycobacterium ulcerans, Shigella, and Cryptococcus hemolyticus. Bacillus anthracis, Bacillus cereus, Histoplasma capsulatum, Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Burkholderia, Brucella, Burkholderia (e.g., Burkholderia cepacia, Burkholderia mallei, Burkholderia pseudomallei), Campylobacter, Candida (e.g., Candida albicans), Chlamydomonas pneumoniae, Corynebacterium diphtheriae, Coxiella burnetii, Clostridium (e.g., Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium perfringens), Clostridium tetani, Coccidioides, Ehrlichia chaffeensis, Ehrlichia eiewingii), Ehrlichia, extraintestinal pathogenic Escherichia coli, Kingella, Klebsiella granulomatosa, Anaplasmas (e.g., Anaplasma phagocytophilum), Leptospira, Borrelia burgdorferi, Treponema pallidum, Rickettsiae (e.g., Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia typhi), Chlamydia psittaci, Chlamydia trachomatis, kuru prion, Lassa virus (LASV), Legionella pneumophila, Listeria monocytogenes, Enterococci, dermatophytes, Escherichia coli O157:H7 and O104:H4, Fasciola hepatica and Fasciola gigantica, enteroviruses (e.g., Coxsackie virus Viruses include: A virus, enterovirus 71 (EV71), FFI prions, CJD prions, Epstein-Barr virus (EBV), feline immunodeficiency virus (FIV), flavivirus, GSS prions, Guanarito virus, Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori, Bunyaviridae, Caliciviridae, Astroviridae, coronavirus, Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium spp., cytomegalovirus (CMV), BK virus, dengue virus, Ebola virus (EBOV), herpes simplex virus (HSV), human immunodeficiency virus (HIV), Human papillomavirus (HPV), influenza virus, rabies virus, norovirus, Nipah virus, Henlepa virus (Henkley virus-Nipah virus), hepatitis A virus, hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus, hepatitis E virus, human bocavirus (HBoV), human metapneumovirus (hMPV), human parainfluenza virus (HPIV), Japanese encephalitis virus, JC virus, Junin virus, yellow fever virus, MERS coronavirus, lymphocytic choriomeningitis virus (LCMV), Machupo virus, Marburg virus virus, measles virus, human molluscum contagiosum virus (MCV), mumps virus, parvovirus B19, Mycoplasma pneumoniae orthomyxovirus, poliovirus, rhinovirus, Rift Valley fever virus, rotavirus, rubella virus, Sabia virus, SARS coronavirus (e.g., SARS-CoV-2), nCoV-2019 coronavirus, Sin Nombre virus, hantavirus, vaccinia virus, respiratory syncytial virus (RSV), tick-borne encephalitis virus (TBEV), varicella-zoster virus (VZV), Venezuelan equine encephalitis virus, West Nile virus, western equine encephalitis virus, and Zika virus.

[0273] In some embodiments, the pathogenic antigen includes or is one or more of the following:

[0274] (1) one or more of the following proteins of SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus or SARS coronavirus (SARS-CoV) including spike protein (S), envelope protein (E), membrane protein (M) or nucleocapsid protein (N); (2) one or more of the following proteins of MERS coronavirus including spike protein (S), spike S1 segment (S1), envelope protein (E), membrane protein (M) or nucleocapsid protein (N); (3) one or more of the following proteins of human papillomavirus (e.g., HPV16): replication protein E1, regulatory protein E2, protein E3, protein E4, protein E5, protein E6, protein E7, protein E8, major capsid protein L1 and minor capsid protein L2; (4) one or more of the following proteins of human parainfluenza virus (HPIV / PIV) (e.g., hPIV-1, hPIV-2, hPIV-3 or hPIV-4 serotype): fusion protein (F), hemagglutinin neuraminidase , glycoprotein (G), matrix protein (M), phosphoprotein (P), nucleocapsid protein, fusion glycoprotein F0, F1 or F2, recombinant PIV3 / PIV1 fusion glycoprotein, C protein, D protein, viral replicase (L) and non-structural V protein; (5) one or more of the following proteins of human metapneumovirus (hMPV): fusion (F) glycoprotein, glycoprotein (G), phosphoprotein (P), and nucleocapsid protein; (6) one or more of the following proteins of influenza virus: hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), M1 protein, M2 protein, NS1 protein, NS2 protein (NEP protein: nuclear export protein), PA protein, PB1 protein (polymerase basic 1 protein), PB1-F2 protein and PB2 protein; (7) one or more of the following proteins of rabies virus: nucleoprotein (N), large structural protein (L), phosphoprotein (P), matrix protein (M) and glycoprotein (G); (8) one or more of the following proteins of human immunodeficiency virus: HIV p24 antigen, HIV envelope proteins (Gp120, Gp41, Gp160), polyprotein GAG, negative factor protein Nef, transcriptional transactivator Tat and Brec1; (9) one or more of the following proteins of Chlamydia trachomatis: major outer membrane protein MOMP, possible outer membrane protein PMPC, outer membrane complex protein B OmcB, heat shock protein Hsp60 HSP10, protein IncA, type III secretion system protein, ribonucleotide reductase small chain protein NrdB, plasmid protein Pgp3, Chlamydia outer protein N CopN, antigen CT521, antigen CT425, antigen CT043, antigen TC0052, antigen TC0189, antigen TC0582, antigen TC0660, antigen TC0726, antigen TC0816, antigen TC0828;(10) One or more of the following proteins of cytomegalovirus (CMV / HCMV): pp65 antigen, membrane protein pp15, capsid proximal tegument protein pp150, protein M45, DNA polymerase UL54, helicase UL105, glycoprotein gM, glycoprotein gN, glycoprotein H, glycoprotein B gB, protein UL83, protein UL94, protein UL99, HCMV glycoprotein (selected from gH-gL, gB, gO, gN and gM), HCMV protein (selected from UL83, UL123, UL128, UL130 and UL131A), epidermal protein pp150 (pp150), tegument protein pp65 / submatrix phosphoprotein (pp65), envelope glycoprotein M (UL100), regulatory protein IE1 (UL123), envelope protein (UL128), envelope glycoprotein (130), envelope protein (UL131A), envelope glycoprotein B (UL55), structural glycoprotein N gpUL73 (UL73), structural glycoprotein O gpUL74 (UL74); (11) one or more of the following proteins of dengue virus: capsid protein C, pre-membrane protein prM, membrane protein M, envelope protein E (domain I, domain II, domain II), protein NS1, protein NS2A, protein NS2B, protein NS3, protein NS4A, protein 2K, protein NS4B, protein NS5; (12) one or more of the following proteins of EBOV virus: EBOV glycoprotein (GP), surface EBOV GP, wild-type EBOV pro GP, mature EBOV GP, secreted wild-type EBOV pro GP, secreted mature EBOV GP, EBOV nucleoprotein (NP), RNA polymerase L and EBOV matrix protein (selected from VP35, VP40, VP24 and VP30); (13) one or more of the following proteins of hepatitis B virus (HBV): hepatitis B surface antigen HBsAg, hepatitis B core antigen HbcAg, polymerase, protein Hbx, pre-S2 middle surface protein, surface protein L, large S protein, viral protein VP1, viral protein VP2, viral protein VP3, and viral protein VP4;(14) One or more of the following proteins of respiratory syncytial virus (RSV): fusion protein F, F protein, nucleoprotein N, matrix protein M, matrix protein M2-1, matrix protein M2-2, phosphoprotein P, small hydrophobic protein SH, major surface glycoprotein G, polymerase L, nonstructural protein 1 NS1, nonstructural protein 2 NS2, RSV attachment protein (G) (glycoprotein G), fusion (F) glycoprotein (glycoprotein F), nucleoprotein (N), phosphoprotein (P), large polymerase protein (L), matrix proteins (M, M2), small hydrophobic protein (SH), nonstructural protein 1 (NS1), nonstructural protein 2 (NS2), membrane-bound RSV F protein, membrane-bound DS Cavl (stable prefusion RSV F protein); (15) one or more of the following proteins of Mycobacterium tuberculosis: secretory antigen SssA (Staphylococcus spp., staphylococcal food poisoning); secretory antigen SssA (Staphylococcus spp., such as Staphylococcus aureus, staphylococcal infection); molecular chaperone DnaK, cell surface lipoprotein Mpt83, lipoprotein P23, phosphate transport system permeabilization protein pstA, 14 kDa antigen, fibronectin binding protein C FbpC1, alanine dehydrogenase TB43, glutamine synthetase 1, ESX-1 protein, protein CFP10, TB10.4 protein, protein MPT83, protein MTB12, protein MTB8, Rpf-like protein, protein MTB32, protein MTB39, crystal protein, heat shock protein HSP65, and protein PST-S; (16) one or more of the following proteins of yellow fever virus: genomic polyprotein, protein E, protein M, capsid protein C, protease NS3, protein NS1, protein NS2A, protein AS2B, protein NS4A, protein NS4B, protein NS5; (17) cyclosporin; and (18) one or more of the following proteins of Zika virus: Zika virus capsid protein (c), Zika virus premembrane protein (prM), Zika virus pr protein (pr), Zika virus membrane protein (M), Zika virus envelope protein (E), Zika virus nonstructural protein, Zika virus prME antigen, Zika virus capsid protein, premembrane / membrane protein, ZIKV envelope protein, ZIKV nonstructural protein 1, ZIKV nonstructural protein 2A, ZIKV nonstructural protein 2B, ZIKV nonstructural protein 3, ZIKV nonstructural protein 4A, ZIKV nonstructural protein 4B, ZIKV nonstructural protein 5, and Zika virus envelope protein (e). ;

[0275] In some embodiments, the tumor antigen is selected from but not limited to the group consisting of the tumor antigens described in WO2018 / 078053A1, pages 47-51.

[0276] In some embodiments, the antigens expressed by the nucleotide sequence encoding the polypeptide and / or protein of interest include or are allergic antigens and autoimmune self-antigens. In some embodiments, the allergic antigens and autoimmune self-antigens are derived from or selected from the group of antigens described on pages 59 to 73 of WO2018 / 078053A1, but are not limited thereto.

[0277] In some embodiments, the antigens expressed by the nucleotide sequences encoding the polypeptides and / or proteins of interest are listed on pages 48 to 51 of WO 2018 / 078053 A1.

[0278] In some embodiments, the polypeptide and / or protein expressed by the nucleotide sequence encoding the polypeptide and / or protein of interest comprises or is a therapeutic protein or polypeptide.

[0279] In some embodiments, the therapeutic protein or polypeptide includes or is one or more of the following:

[0280] (1) Enzyme replacement therapy for the treatment of metabolic, endocrine or amino acid disorders or therapeutic proteins or polypeptides for replacing missing, defective or mutated proteins; (2) Therapeutic proteins or polypeptides for the treatment of blood diseases, circulatory system diseases, respiratory system diseases, infectious diseases or immune deficiencies; (3) Therapeutic proteins or polypeptides for the treatment of cancer or tumor diseases; (4) Therapeutic proteins or polypeptides for hormone replacement therapy; (5) Therapeutic proteins or polypeptides for reprogramming somatic cells into pluripotent stem cells or totipotent stem cells; (6) Therapeutic proteins or polypeptides used as adjuvants or immunostimulants; (7) Therapeutic proteins or polypeptides as therapeutic antibodies; (8) Therapeutic proteins or polypeptides as gene editing agents; (9) Therapeutic proteins or polypeptides for the treatment or prevention of liver diseases selected from the group consisting of liver fibrosis, cirrhosis and liver cancer; and (10) Therapeutic proteins or polypeptides for the treatment or prevention of rare diseases.

[0281] In some embodiments, the enzyme replacement therapy for treating metabolic, endocrine or amino acid disorders or the therapeutic protein or polypeptide for replacing a missing, deleted or mutant protein comprises or is one or more of the following: acid sphingomyelinase, fatty acid, aglycosidase beta, leucosidase, α-galactosidase A, α-glucosidase, α-L-iduronidase, α-N-acetylglucosaminidase, amphiregulin, angiopoietin (Ang1, Ang2, Ang3, Ang4, ANGPTL2, ANG PTL3, ANGPTL4, ANGPTL5, ANGPTL6, ANGPTL7), ATPase, Cu(2+)-transporting β polypeptide (ATP7B), argininosuccinate synthetase (ASS1), beta-cells, β-glucuronidase, bone morphogenetic proteins (BMPs) (BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP15), CLN6 protein, epidermal growth factor (EG F), epigenetic proteins, epigenetic regulators, fibroblast growth factors (FGF, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-16, FGF-17, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-2 3), fumarylacetoacetate hydrolase (FAH), sulfatase, growth hormone-releasing peptide, glucocerebrosidase, GM-CSF, heparin-binding EGF-like growth factor (HB-EGF), hepatocyte growth factor HGF, hepatopoietin, human albumin, increased albumin loss, iduronate sulfatase (iduronate-2-sulfatase), integrins αVβ3, αVβ5, and α5β1, uduronate sulfatase, laronidase, N-acetylgalactosamine-4-sulfatase (rhASB;Sulfatase, arylsulfatase A (ARSA), arylsulfatase B (ARSB)), N-acetylglucosamine-6-sulfatase, nerve growth factor (NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin 4 / 5 (NT-4 / 5), neuregulins (NRG1, NRG2, NRG3, NRG4), neuropilin (NRP-1, NRP-2), obestatin, phenylalanine hydroxylase (PAH), phenylalanine aminohydrolase (PAL), platelet-derived growth factor (PDGF (PDFF-A, PDGF-B, PDGF-C, PDGF-D)), TGFβ receptors (endothelin, TGFβ1 receptor, TGFβ2 receptor, TGFβ3 receptor), thrombopoietin (THPO) (megakaryocyte growth and development factor (MGDF)), transforming growth factors (TGF (TGF-a, TGF-β (TGFβ1, TGFβ2 and TGFβ3))), VEGF (VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and PIGF) , nesiritide, trypsin, adrenocorticotropic hormone (ACTH), atrial natriuretic peptide (ANP), cholecystokinin, gastrin, leptin, oxytocin, somatostatin, vasopressin (antidiuretic hormone), calcitonin, exenatide, growth hormone (GH), growth hormone, insulin, insulin-like growth factor 1 IGF-1, mecarsiphene ester, IGF-1 analog, pegvisomant, pramlintide, teriparatide (human parathyroid hormone residues 1-34), becaplermin, dibotermin-α (bone morphogenetic protein 2), histrelin acetate (gonadotropin-releasing hormone GnRH), octreotide, hepatocyte nuclear factor 4 alpha (HNF4A), CCAAT / enhancer binding protein alpha (CEBPA), fibroblast growth factor 21 (FGF21), extracellular matrix protease or human collagenase MMP1, hepatocyte growth factor (HGF), TNF-related apoptosis-inducing ligand (TRAIL), opioid growth factor receptor-like 1 (OGFRL1), clostridial type II collagenase, relaxin 1 (RLN1), relaxin 2 (RLN2), relaxin 3 (RLN3), and palifermin (keratinocyte growth factor; KGF).

[0282] In some embodiments, the therapeutic protein or polypeptide for treating metabolic or endocrine diseases is selected from the proteins or polypeptides described in Table A (in combination with Table C) of WO2017 / 191274.

[0283] In some embodiments, the therapeutic protein or polypeptide for treating a blood disorder, a circulatory system disorder, a respiratory system disorder, a cancer or tumor disorder, an infectious disease, or an immunodeficiency comprises or is one or more of the following: alteplase (tissue plasminogen activator; tPA), anistreplase, antithrombin III (AT-III), bivalirudin, darbepoetin-α, drotrecogin-α (activated protein C), erythropoietin, epoetin alfa-α, erythropoietin, erthropoyetin, factor IX, factor VIIa, factor VIII, recombinant hirudin, protein C concentrate, reteplase (tP A deletion mutant protein), streptokinase, tenecteplase, urokinase, angiostatin, anti-CD22 immunotoxin, denileukin, immunocyanine, MPS (zinc finger protein), aflibercept, endostatin, collagenase, human deoxyribonuclease I, deoxyribonuclease, hyaluronidase, papain, L-asparaginase, PEG-asparaginase, rasburicase, human chorionic gonadotropin (HCG), human follicle-stimulating hormone (FSH), luteinizing hormone-α, prolactin, α-1-proteinase inhibitor, lactase, pancreatic enzymes (lipase, amylase, protease), adenosine deaminase (bovine PEG-ADA), abatacept, alefacept, anakinra, etanercept, interleukin-1 (IL-1 receptor antagonist), anakinra, thymosin nonapeptide, TNF-α antagonist, enfuvirtide, and thymosin α1.

[0284] In some embodiments, the therapeutic protein or polypeptide for treating cancer or tumor disease includes or is one or more of the following: cytokines, chemokines, suicide gene products, immunogenic proteins or peptides, apoptosis inducers, angiogenesis inhibitors, heat shock proteins, tumor antigens, β-catenin inhibitors, STING pathway activators, checkpoint regulators, innate immune activators, antibodies, dominant negative receptors and decoy receptors, myeloid-derived suppressor cell (MDSCs) inhibitors, IDO pathway inhibitors, and proteins or peptides that bind to apoptosis inhibitors;

[0285] In some embodiments, the hormones in the therapeutic protein or polypeptide for hormone replacement therapy include one or more of the following: estrogen, progesterone, progesterone, and testosterone.

[0286] In some embodiments, therapeutic proteins for reprogramming somatic cells into pluripotent or totipotent stem cells include one or more of the following: Oct-3 / 4, Sox gene family (e.g., Sox1, Sox2, Sox3, and Sox15), Klf family (e.g., Klf1, Klf2, Klf4, and Klf5), Myc family (e.g., c-Myc, L-Myc, and N-Myc), Nanog, and LIN28.

[0287] In some embodiments, the therapeutic protein or polypeptide used as an adjuvant or immunostimulatory protein includes or is one or more of the following: human adjuvant proteins, in particular pattern recognition receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11; NOD1, NOD2, NOD3, NOD4, NOD5, NALP1, NALP2, NALP3, NALP4, NALP5, NALP6, NALP6, NALP7, NALP7, NALP8, NALP9, NALP10, NALP11, NALP12, NALP13, NALP14; IPAF, NAIP, CIITA, RIG-I, MDA5 and LGP2, signal transducers of TLR signals (including adaptor proteins (such as Trif and Cardif), components of small GTPases signals (such as RhoA, Ras, Rac1, Cdc42, Rab, etc.), components of PIP signals (such as PI3K, Src kinase, etc.), components of MyD88-dependent signals (such as MyD88, IRAK1, IRAK2, IRAK4, TIRAP, TRAF6, etc.), components of MyD88-independent signals (such as TICAM1, TICAM2, TRAF6, TBK1, IRF3, TAK1, IRAK1, etc.), etc.); activated kinases (such as Akt, MEKK1, MKK1, MKK3, MKK4, MKK6, MKK7, ERK1, ERK2, GSK3, PKC kinase, PKD kinase, GSK3 kinase, JNK, p 38MAPK, TAK1, IKK, TAK1, etc.); activated transcription factors (such as NF-kB, c-Fos, c-Jun, c-Myc, CREB, AP-1, Elk-1, ATF2, IRF-3, IRF-7, heat shock proteins (such as HSP10, HSP60, HSP65, HSP70, HSP75 and HSP90), gp96, fibrinogen, fibronectin type III repeat extra structure domain, etc.); components of the complement system (e.g., C1q, MBL, C1r, C1s, C2b, Bb, D, MASP-1, MASP-2, C4b, C3b, C5a, C3a, C4a, C5b, C6, C7, C8, C9, CR1, CR2, CR3, CR4, C1qR, C1INH, C4bp, MCP, DAF, H, I, P, CD59, etc.); cell surface proteins that induce target genes (e.g., β-defensins).In some embodiments, the human auxiliary proteins include one or more of the following: trif, flt-3 ligand, Gp96 or fibronectin, cytokines that induce or enhance innate immune responses (e.g., IL-1α, IL-1R1, IL1β, IL-2, IL-6, IL-7, IL-8, IL-9, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21, IL-23, TNFα, IFNα, IFNβ, IFNγ, GM-CSF, G-CSF, M-CSF), chemokines (e.g., IL-8, IP-10, MCP-1, MIP-1α, RANTES, Eotaxin, CCL21), cytokines released by macrophages (e.g., IL-1, IL-6, IL-8, IL-12, TNF-α, etc.).

[0288] In some embodiments, therapeutic proteins or polypeptides used as adjuvants or immunostimulators include one or more of the following: bacterial (adjuvant) proteins, protozoan (adjuvant) proteins, viral (adjuvant) proteins, fungal (adjuvant) proteins, and animal-derived proteins.

[0289] In some embodiments, the bacterial (adjuvant) protein includes one or more of the following: bacterial heat shock proteins or chaperones (including Hsp60, Hsp70, Hsp90, Hsp100); Gram-negative bacterial OmpA (outer membrane protein); OspA; bacterial porins (e.g., OmpF); bacterial toxins (e.g., pertussis toxin (PT) of Bordetella pertussis, pertussis adenylate cyclase toxins CyaA and CyaC of Bordetella pertussis, pertussis toxin PT-9K / 129G mutant, pertussis adenylate cyclase toxins CyaA and CyaC of Bordetella pertussis, tetanus toxin, cholera toxin (CT), cholera toxin The B subunit of toxin, cholera toxin CTK63 mutant, CTE112K mutant of CT, Escherichia coli heat-labile enterotoxin (LT), heat-labile enterotoxin with reduced toxicity (LTB), B subunit of Escherichia coli heat-labile enterotoxin mutant (e.g., LTK63, LTR72); phenol-soluble regulatory protein; Helicobacter pylori neutrophil-activating protein (HP-NAP); surfactant protein D; Borrelia burgdorferi outer surface protein A lipoprotein, Ag38 (38 kDa antigen) of Mycobacterium tuberculosis; bacterial fimbriae proteins (e.g., fimbriae proteins of Gram-negative bacteria) and surfactant protein A, and bacterial flagellin.

[0290] In some embodiments, the protozoan (adjuvant) proteins include one or more of the following: Tc52 from Trypanosoma cruzi, PFTG from Trypanosoma gondii, protozoan heat shock proteins, LeIF from Leishmania, and spectra-like proteins from Toxoplasma gondii.

[0291] In some embodiments, the viral (adjuvant) proteins include one or more of the following: respiratory syncytial virus fusion glycoprotein (F protein), MMT virus envelope protein, mouse leukemia virus protein, and wild-type measles virus hemagglutinin protein.

[0292] In some embodiments, the fungal (adjuvant) protein comprises a fungal immunomodulatory protein (FIP, eg, LZ-8).

[0293] In some embodiments, the animal-derived protein comprises keyhole limpet hemocyanin (KLH).

[0294] In some embodiments, the polypeptide and / or protein expressed by the nucleotide sequence encoding the polypeptide and / or protein of interest comprises or is a therapeutic protein or polypeptide that is a therapeutic antibody, for example, one or more of the cytokines, chemokines, suicide enzymes and gene products, apoptosis inducers, endogenous angiogenesis inhibitors, heat shock proteins, tumor antigens, innate immune activators, and antibodies against proteins associated with tumor or cancer development listed in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, Table 10, Table 11, and Table 12 of WO2016 / 170176A1.

[0295] 5. DNA molecules or vectors encoding the recombinant RNA molecules of the 5' and / or 3'-UTR of the present invention

[0296] The present invention provides a DNA molecule encoding the 5'-UTR, 3'-UTR and / or recombinant RNA molecule of the present invention. The present invention also provides a vector comprising the recombinant RNA molecule or DNA molecule of the present invention and a host cell comprising the recombinant RNA molecule, DNA molecule and / or vector of the present invention.

[0297] The present invention also provides a vector comprising a first nucleotide sequence encoding a 5'-UTR and / or a second nucleotide sequence encoding a 3'-UTR, wherein: the first nucleotide sequence comprises at least one of the following polynucleotides: (a): a polynucleotide encoding a 5'-UTR derived from at least one gene of PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, HBB, MYSM1, LENG1, TMSB4X, CASP4, IFNA1, PGLYRP1, UCHL1, CPAMD8, TTR, APOA2, GH1, DTYMK, APOC2 and CDK7; (b): a polynucleotide encoding a fragment of the 5'-UTR in (a); (c): a polynucleotide encoding a variant of the 5'-UTR in (a); and (d): a polynucleotide encoding a fragment of the fragment in (b). a polynucleotide encoding a variant of the 3'-UTR of at least one gene selected from the group consisting of MPND, FBXW10, FBXW12, PGLYRP1, HPX, CDK7, APOC2, PFN1, RBP4, FTCD, NAAA, ALB, GSDMD, FBXL8, ORM1, CASP4, CHMP2A, LENG1, MYCBPAP, APOC1, GAPDH, HSPA8, APOA2, UCHL1, TSG101, NAE1, and NFKB2; (f) a polynucleotide encoding a fragment of the 3'-UTR of (e); (g) a polynucleotide encoding a variant of the 3'-UTR of (e); and (h) a polynucleotide encoding a variant of the fragment of (f).

[0298] In some embodiments, the gene is a human gene.

[0299] In some embodiments, the first nucleotide sequence comprises at least one of the following polynucleotides: a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1-21, a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1-21, a variant of a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1-21, and a variant of a fragment of a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1-21. In some embodiments, the fragment, variant, or variant of the fragment of the polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1-21 is at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide having a sequence as set forth in one of SEQ ID NOs: 1-21. In some embodiments, a fragment, variant, or variant of a fragment of a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide insertions, additions, deletions, or substitutions compared to a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 1 to 21.

[0300] In preferred embodiments, the gene is selected from at least one of PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, and HBB. In some embodiments, the first nucleotide sequence comprises at least one of the following polynucleotides: 1) a polynucleotide as set forth in SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6; 2) a fragment of the polynucleotide as set forth in 1); 3) a variant of the polynucleotide as set forth in 1); and 4) a variant of the fragment as set forth in 2). In some embodiments, the fragment, variant, or variant of the polynucleotide as set forth in SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the polynucleotide as set forth in SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6. In some embodiments, a fragment, variant, or variant of a polynucleotide having a sequence as shown in SEQ ID NO: 9, 7, 18, 12, 8, 1 or 6 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide insertions, additions, deletions or substitutions compared to a polynucleotide having a sequence as shown in SEQ ID NO: 9, 7, 18, 12, 8, 1 or 6.

[0301] The first nucleotide sequence may comprise two or more tandem polynucleotides encoding the 5'-UTR from the above-mentioned gene, polynucleotides encoding a fragment of the 5'-UTR from the above-mentioned gene, polynucleotides encoding a variant of the 5'-UTR from the above-mentioned gene, or polynucleotides encoding a variant of the fragment of the 5'-UTR from the above-mentioned gene.

[0302] In some embodiments, the first nucleotide sequence comprises at least one of the following polynucleotides: (a): a polynucleotide encoding the 5'-UTR of at least two genes selected from the group consisting of PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, HBB, MYSM1, LENG1, TMSB4X, CASP4, IFNA1, PGLYRP1, UCHL1, CPAMD8, TTR, APOA2, GH1, DTYMK, APOC2, and CDK7; (b): a polynucleotide encoding a fragment of the 5'-UTR of at least two of the genes described in (a); (c): a polynucleotide encoding a variant of the 5'-UTR of at least two of the genes described in (a); and (d): a polynucleotide encoding a variant of the fragment of the 5'-UTR of at least two of the genes described in (a). In some embodiments, the first nucleotide sequence comprises at least one of the following polynucleotides: (e): a polynucleotide encoding a 5'-UTR derived from at least two genes of genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, and HBB; (f): a polynucleotide encoding a fragment of the 5'-UTR described in (e); (g): a polynucleotide encoding a variant of the 5'-UTR described in (e); and (h): a polynucleotide encoding a variant of the fragment described in (f).

[0303] In some embodiments, the first nucleotide sequence comprises at least one of the following polynucleotides: a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 1 to 21, a fragment of a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 1 to 21, a variant of a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 1 to 21, and a variant of a fragment of a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 1 to 21. In some embodiments, the fragment, variant, or variant of the fragment of a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 1 to 21 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the polynucleotide having a sequence as set forth in one of SEQ ID NOs: 1 to 21. In some embodiments, a fragment, variant, or variant of a fragment of a polynucleotide whose sequence is as shown in one of SEQ ID NOs: 1 to 21 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide insertions, additions, deletions or substitutions compared to a polynucleotide whose sequence is as shown in one of SEQ ID NOs: 1 to 21.

[0304] In some embodiments, the first nucleotide sequence comprises at least one of the following polynucleotides: a polynucleotide having a sequence as shown in at least two of SEQ ID NOs: 9, 7, 18, 12, 8, 1 and 6, a fragment of a polynucleotide having a sequence as shown in at least two of SEQ ID NOs: 9, 7, 18, 12, 8, 1 and 6, a variant of a polynucleotide having a sequence as shown in at least two of SEQ ID NOs: 9, 7, 18, 12, 8, 1 and 6, and a variant of a fragment of a polynucleotide having a sequence as shown in at least two of SEQ ID NOs: 9, 7, 18, 12, 8, 1 and 6. In some embodiments, a fragment, variant, or variant of a fragment of a polynucleotide having a sequence as set forth in SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a polynucleotide having a sequence as set forth in SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6. In some embodiments, a fragment, variant, or variant of a fragment of a polynucleotide having a sequence as set forth in SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertions, additions, deletions, or substitutions of nucleotides compared to a polynucleotide having a sequence as set forth in SEQ ID NO: 9, 7, 18, 12, 8, 1, or 6.

[0305] In some embodiments, the first nucleotide sequence comprises at least one of the following polynucleotides: a): at least two copies of a polynucleotide encoding a 5'-UTR derived from at least one of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, HBB, MYSM1, LENG1, TMSB4X, CASP4, IFNA1, PGLYRP1, UCHL1, CPAMD8, TTR, APOA2, GH1, DTYMK, APOC2, and CDK7; b): at least two copies of a fragment of a polynucleotide encoding a 5'-UTR of at least one of the genes in a); c): at least two copies of a polynucleotide encoding a variant of a 5'-UTR of at least one of the genes in a); and d): at least two copies of a polynucleotide encoding a variant of a fragment of a 5'-UTR of at least one of the genes in a). In some embodiments, the first nucleotide sequence comprises at least one of the following polynucleotides: e) at least two copies of a polynucleotide encoding a 5'-UTR derived from at least one of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, and HBB; f) at least two copies of a polynucleotide encoding a fragment of a 5'-UTR derived from at least one of the genes in e); g) at least two copies of a polynucleotide encoding a variant of a 5'-UTR derived from at least one of the genes in e); h) at least two copies of a polynucleotide encoding a variant of a fragment of a 5'-UTR derived from at least one of the genes in e). Preferably, the at least two copies are two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, or nine copies.

[0306] In some embodiments, the first nucleotide sequence comprises at least one of the following polynucleotides: at least two copies of a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1-21, at least two copies of a fragment of a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1-21, at least two copies of a variant of a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1-21, and at least two copies of a variant of a fragment of a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1-21. Preferably, the at least two copies are two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, or nine copies. In some embodiments, the fragment, variant, or variant of the polynucleotide having a sequence as set forth in one of SEQ ID NOs: 1-21 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the polynucleotide having a sequence as set forth in one of SEQ ID NOs: 1-21. In some embodiments, a fragment, variant, or variant of a polynucleotide whose sequence is as shown in one of SEQ ID NOs: 1 to 21 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide insertions, additions, deletions, or substitutions compared to a polynucleotide whose sequence is as shown in one of SEQ ID NOs: 1 to 21.

[0307] In some embodiments, the first nucleotide sequence comprises at least one of the following polynucleotides: at least two copies of a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 9, 7, 18, 12, 8, 1, and 6, at least two copies of a fragment of a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 9, 7, 18, 12, 8, 1, and 6, at least two copies of a variant of a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 9, 7, 18, 12, 8, 1, and 6, and at least two copies of a variant of a fragment of a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 9, 7, 18, 12, 8, 1, and 6. Preferably, the at least two copies are two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, or nine copies. In some embodiments, the fragment, variant, or variant of a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 9, 7, 18, 12, 8, 1, and 6 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the polynucleotide having a sequence as set forth in SEQ ID NOs: 9, 7, 18, 12, 8, 1, or 6. In some embodiments, the homolog or variant has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertions, additions, deletions, or substitutions of nucleotides compared to SEQ ID NOs: 9, 7, 18, 12, 8, 1, or 6.

[0308] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: a polynucleotide with a sequence as set forth in at least one of SEQ ID NOs: 22 to 48, a fragment of a polynucleotide with a sequence as set forth in at least one of SEQ ID NOs: 22 to 48, a variant of a polynucleotide with a sequence as set forth in at least one of SEQ ID NOs: 22 to 48, and a variant of a fragment of a polynucleotide with a sequence as set forth in at least one of SEQ ID NOs: 22 to 48. In some embodiments, the fragment, variant, or variant of the fragment of a polynucleotide with a sequence as set forth in at least one of SEQ ID NOs: 22 to 48 is at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a polynucleotide with a sequence as set forth in one of SEQ ID NOs: 22 to 48. In some embodiments, a fragment, variant, or variant of a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 22 to 48 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide insertions, additions, deletions, or substitutions compared to a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 22 to 48.

[0309] In a preferred embodiment, the gene is selected from at least one of MPND, FBXW10, FBXW12, and PGLYRP1. In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: a polynucleotide with a sequence as set forth in SEQ ID NO: 24, 22, 23, or 25, a fragment of a polynucleotide with a sequence as set forth in SEQ ID NO: 24, 22, 23, or 25, a variant of a polynucleotide with a sequence as set forth in SEQ ID NO: 24, 22, 23, or 25, and a variant of a fragment of a polynucleotide with a sequence as set forth in SEQ ID NO: 24, 22, 23, or 25. In some embodiments, the fragment, variant, or variant of a fragment of a polynucleotide having a sequence as set forth in SEQ ID NO: 24, 22, 23, or 25 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the polynucleotide having a sequence as set forth in SEQ ID NO: 24, 22, 23, or 25. In some embodiments, the fragment, variant, or variant of a fragment of a polynucleotide having a sequence as set forth in SEQ ID NO: 24, 22, 23, or 25 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertions, additions, deletions, or substitutions of nucleotides compared to the polynucleotide having a sequence as set forth in SEQ ID NO: 24, 22, 23, or 25.

[0310] The second nucleotide sequence may comprise two or more tandem polynucleotides encoding the 3'-UTR from the above gene, polynucleotides encoding variants of the 3'-UTR from the above gene, or polynucleotides encoding variants of fragments of the 3'-UTR from the above gene.

[0311] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: (a): a polynucleotide encoding a 3'-UTR derived from at least two of the genes MPND, FBXW10, FBXW12, PGLYRP1, HPX, CDK7, APOC2, PFN1, RBP4, FTCD, NAAA, ALB, GSDMD, FBXL8, ORM1, CASP4, CHMP2A, LENG1, MYCBPAP, APOC1, GAPDH, HSPA8, APOA2, UCHL1, TSG101, NAE1, and NFKB2; (b): a polynucleotide encoding a fragment of the 3'-UTR of at least two of the genes described in (a); (c): a polynucleotide encoding a variant of the 3'-UTR of at least two of the genes described in (a); and (d): a polynucleotide encoding a variant of the fragment of the 3'-UTR of at least two of the genes described in (a). In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: (e): a polynucleotide encoding a 3'-UTR derived from at least two genes of the genes MPND, FBXW10, FBXW12, and PGLYRP1; (f): a polynucleotide encoding a fragment of the 3'-UTR in (e); (g): a polynucleotide encoding a variant of the 3'-UTR in (e); and (h): a polynucleotide encoding a variant of the fragment in (f).

[0312] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: a polynucleotide with a sequence as set forth in at least two of SEQ ID NOs: 22 to 48, a fragment of a polynucleotide with a sequence as set forth in at least two of SEQ ID NOs: 22 to 48, a variant of a polynucleotide with a sequence as set forth in at least two of SEQ ID NOs: 22 to 48, and a variant of a fragment of a polynucleotide with a sequence as set forth in at least two of SEQ ID NOs: 22 to 48. In some embodiments, the fragment, variant, or variant of the fragment of a polynucleotide with a sequence as set forth in one of SEQ ID NOs: 22 to 48 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the polynucleotide with a sequence as set forth in one of SEQ ID NOs: 22 to 48. In some embodiments, the fragment, variant, or variant of the fragment of a polynucleotide whose sequence is shown in one of SEQ ID NOs: 22 to 48 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide insertions, additions, deletions, or substitutions compared to the polynucleotide whose sequence is shown in one of SEQ ID NOs: 22 to 48.

[0313] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 24, 22, 23, and 25, a fragment of a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 24, 22, 23, and 25, a variant of a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 24, 22, 23, and 25, and a variant of a fragment of a polynucleotide having a sequence as set forth in at least two of SEQ ID NOs: 24, 22, 23, and 25. In some embodiments, the fragment, variant, or variant of the fragment of the polynucleotide having a sequence as set forth in SEQ ID NOs: 24, 22, 23, or 25 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the polynucleotide having a sequence as set forth in SEQ ID NOs: 24, 22, 23, or 25. In some embodiments, the sequence is such as a fragment, variant, or variant of a polynucleotide as shown in SEQ ID NO: 24, 22, 23 or 25, having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide insertions, additions, deletions or substitutions compared to the polynucleotide as shown in SEQ ID NO: 24, 22, 23 or 25.

[0314] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: a): at least two copies of a polynucleotide encoding a 3'-UTR derived from at least one of the genes MPND, FBXW10, FBXW12, PGLYRP1, HPX, CDK7, APOC2, PFN1, RBP4, FTCD, NAAA, ALB, GSDMD, FBXL8, ORM1, CASP4, CHMP2A, LENG1, MYCBPAP, APOC1, GAPDH, HSPA8, APOA2, UCHL1, TSG101, NAE1, and NFKB2; b): at least two copies of a polynucleotide encoding a fragment of the 3'-UTR of at least one of the genes in a); c): at least two copies of a polynucleotide encoding a variant of the 3'-UTR of at least one of the genes in a); and d): at least two copies of a polynucleotide encoding a variant of a fragment of the 3'-UTR of at least one of the genes in a). In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: e): at least two copies of a polynucleotide encoding a 3'-UTR derived from one of the genes MPND, FBXW10, FBXW12, and PGLYRP1; f): at least two copies of a polynucleotide encoding a fragment of the 3'-UTR of at least one of the genes in e); g): at least two copies of a polynucleotide encoding a variant of the 3'-UTR of at least one of the genes in e); and h): at least two copies of a polynucleotide encoding a variant of the 3'-UTR of at least one of the genes in e). Preferably, the at least two copies are two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, or nine copies.

[0315] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: at least two copies of a polynucleotide having a sequence as set forth in any one of SEQ ID NOs: 22 to 48, at least two copies of a fragment of a polynucleotide having a sequence as set forth in any one of SEQ ID NOs: 22 to 48, at least two copies of a variant of a polynucleotide having a sequence as set forth in any one of SEQ ID NOs: 22 to 48, and at least two copies of a variant of a fragment of a polynucleotide having a sequence as set forth in any one of SEQ ID NOs: 22 to 48. Preferably, the at least two copies are two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, or nine copies. In some embodiments, the fragment, variant, or variant of a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 22 to 48 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 22 to 48. In some embodiments, the fragment, variant, or variant of a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 22 to 48 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertions, additions, deletions, or substitutions of nucleotides compared to a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 22 to 48.

[0316] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: at least two copies of a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 24, 22, 23, and 25, at least two copies of a fragment of a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 24, 22, 23, and 25, at least two copies of a variant of a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 24, 22, 23, and 25, and at least two copies of a variant of a fragment of a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 24, 22, 23, and 25. Preferably, the at least two copies are two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, or nine copies. In some embodiments, the polynucleotide as set forth in one of SEQ ID NOs: 24, 22, 23, and 25 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the polynucleotide as set forth in SEQ ID NOs: 24, 22, 23, or 25. In some embodiments, the polynucleotide as set forth in one of SEQ ID NOs: 24, 22, 23, and 25 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertions, additions, deletions, or substitutions of nucleotides compared to the polynucleotide as set forth in SEQ ID NOs: 24, 22, 23, or 25.

[0317] In some embodiments, the vector is used to produce a recombinant RNA molecule, such as an mRNA molecule. In some embodiments, the vector comprises the first nucleotide sequence and the second nucleotide sequence. In some embodiments, the vector further comprises a third nucleotide sequence encoding a polypeptide and / or protein of interest between the first nucleotide sequence and the second nucleotide sequence. In some embodiments, the third nucleotide sequence encodes at least one polypeptide of interest. For example, one, two, three, four, five, six, seven, eight, nine, or ten polypeptides of interest. In some embodiments, the third nucleotide sequence encodes at least one protein of interest. For example, one, two, three, four, five, six, seven, eight, nine, or ten proteins of interest. In some embodiments, the third nucleotide sequence encodes at least one polypeptide of interest and at least one protein of interest. For example, one, two, three, four, five, six, seven, eight, nine, or ten polypeptides of interest and one, two, three, four, five, six, seven, eight, nine, or ten proteins of interest.

[0318] In some embodiments, the nucleotides constituting the poly(A) tail comprise at least 20, at least 40, at least 80, at least 100, or at least 120 A nucleotides. Preferably, the nucleotides constituting the poly(A) tail comprise at least 20, at least 40, at least 80, at least 100, or at least 120 consecutive A nucleotides. In some embodiments, the poly(A) tail comprises one or more nucleotides other than A nucleotides. In some embodiments, the poly(A) tail comprises two or more consecutive nucleotides other than A nucleotides, wherein the first and last nucleotides in the sequence of two or more consecutive nucleotides are nucleotides other than A nucleotides. Preferably, the poly(A) tail is truncated, i.e., m consecutive A nucleotides and n consecutive A nucleotides are reconnected by a linker sequence consisting of p non-A nucleotides, wherein m, n, and p are positive integers. Preferably, m is 30, n is 70, and p is 10.

[0319] In some embodiments, in the vector, the DNA sequence corresponding to the poly(A) tail is shown as SEQ ID NO:53.

[0320] In addition, the vector may further comprise expression control elements for proper expression of the vector in the host. Such control elements are known to those skilled in the art and may include promoters, splice cassettes, translation initiation codons, translation and insertion sites for introducing inserts into the vector.

[0321] In some embodiments, the vector of the present invention may be, for example, a plasmid, a cosmid, a virus, a phage or another vector conventionally used in genetic engineering, and may contain additional genes, such as a marker gene that allows selection of the vector in a suitable host cell and under suitable conditions.

[0322] The recombinant RNA molecules and vectors of the present invention can be introduced into cells directly or via liposomes, viral vectors (eg, adenovirus, retrovirus), electroporation, ballistics (eg, gene gun), or other delivery systems.

[0323] In addition, the present invention also provides a method for preparing mRNA, comprising contacting the vector of the present invention with RNA polymerase.

[0324] In some embodiments, the methods of the present invention further comprise the step of linearizing the plasmid. In some embodiments, prior to linearization, the supercoiled rate of the plasmid is at least about 90%. In some embodiments, the methods of the present invention further comprise the step of purifying the linearized plasmid. In some embodiments, the methods of the present invention further comprise the step of purifying the mRNA.

[0325] In some embodiments, the method of the present invention further comprises the steps of capping and optionally purifying the capped product. In some embodiments, the cap is a Cap1 type cap. The Cap1 type cap structure is as shown in formula (I): cap G1G2=m7G-5'-ppp-5'-Gm2'-3'-p-[m7=7-CH3;m2'=2'-O-CH3;-ppp-=- PO2H-O-PO2H-O-PO2H)-;-p-=-PO2H-].

[0326] The capping reaction is as follows: pppN1(p)Nx-OH(3')→ppN1(pN)x-OH(3')+Pi ppN1(pN)x-OH(3')+GTP→G(5')ppp(5')N1(pN)x-OH(3')+PPi G(5')ppp(5')N1(pN)x-OH(3')+AdoMet→m7G(5')ppp(5')N1(pN)x-OH(3')+AdoHyc m7GpppN1(pN)x-OH(3')+AdoMet→m7Gppp[m2'-O]N1(pN)x-OH(3')+AdoHyc.

[0327] In addition, the present invention also provides a host cell, such as a bacterial cell, comprising the recombinant RNA molecule, DNA molecule, and / or vector of the present invention. The vector of the present invention, such as a plasmid, is stored and / or amplified in the host cell.

[0328] Host cells of the present invention can be prepared by transforming competent host cells with the vector of the present invention. Competent host cells are cells with the ability of free extracellular genetic material (such as DNA plasmids) that do not rely on sequence uptake. Various bacterial cells well known to those skilled in the art can naturally uptake exogenous DNA from the environment, and therefore can serve as bacterial host cells according to the present invention. In addition, it is known to those skilled in the art that competent bacterial host cells can be obtained from natural non-competent bacterial cells using, for example, electroporation or chemicals (such as with calcium ion treatment and with high temperature exposure). After uptake, the DNA plasmid is preferably neither degraded nor integrated in the genomic information of the bacterial host cell. Bacterial host cells include Escherichia coli (E.coli) cells well known to those skilled in the art.

[0329] VI. Lipid Nanoparticles Containing Recombinant RNA Molecules of the Present Invention's 5' and / or 3'-UTR, Pharmaceutical Compositions Containing Recombinant RNA Molecules of the Present Invention's 5' and / or 3'-UTR, and Methods for Treating / Preventing Diseases

[0330] The present invention provides a lipid nanoparticle comprising the recombinant RNA molecule, DNA molecule or vector of the present invention.

[0331] In some embodiments, the lipid nanoparticles contain protonatable cationic lipids.

[0332] In some embodiments, the lipid nanoparticles further contain one or more of a helper lipid, a structural lipid, and a PEG-lipid (polyethylene glycol-lipid). In some further embodiments, the lipid nanoparticles further contain the helper lipid, the structural lipid, and the PEG-lipid.

[0333] In some embodiments as described above, the helper lipid is a phospholipid. The phospholipid is generally semi-synthetic, but may also be of natural origin or chemically modified. The phospholipid includes, but is not limited to, DSPC (distearoylphosphatidylcholine), DOPE (dioleoylphosphatidylethanolamine), DOPC (dioleoylphosphatidylcholine), DOPS (dioleoylphosphatidylserine), DSPG (1,2-dioctadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol)), DPPG (dipalmitoylphosphatidylglycerol), DPPC (dipalmitoylphosphatidylcholine), DGTS (1,2-dipalmitoyl-sn-glycero-3-O-4'-(N,N,N-trimethyl)homoserine), lysophospholipids, and the like. Preferably, the helper lipid is one or more selected from DSPC, DOPE, DOPC, and DOPS. In some embodiments, the helper lipid is DSPC and / or DOPE.

[0334] In some embodiments, the structured lipid is a sterol substance, including but not limited to cholesterol, cholesterol ester, sterol hormones, sterol vitamins, bile acid, cholesterol, ergosterol, β-sitosterol and oxidized cholesterol derivatives. Preferably, the structured lipid is at least one selected from cholesterol, cholesterol ester, sterol hormones, sterol vitamins and bile acids. In some embodiments, the structured lipid is cholesterol, preferably high-purity cholesterol, especially injection-grade high-purity cholesterol, such as CHO-HP (produced by AVT).

[0335] As used herein, the term PEG-lipid (polyethylene glycol-lipid) is a conjugate of polyethylene glycol and a lipid structure. Preferably, the PEG-lipid is selected from PEG-DMG and PEG-distearoylphosphatidylethanolamine (PEG-DSPE), preferably PEG-DMG. Preferably, the PEG-DMG is a polyethylene glycol (PEG) derivative of 1,2-dimyristyl glycerol. Preferably, the average molecular weight of the PEG is about 2000 to 5000, preferably about 2000.

[0336] In some further embodiments, the lipid nanoparticle further contains the helper lipid, the structural lipid, and the PEG-lipid. In some embodiments, the lipid nanoparticles comprise the protonatable cationic lipid in an amount (molar percentage) of about 25.0%-75.0%, such as about 25.0%-28.0%, 28.0%-32.0%, 32.0%-35.0%, 35.0%-40.0%, 40.0%-42.0%, 42.0%-45.0%, 45.0%-46.3%, 46.3%-48.0%, 48.0%-49.5%, 49.5%-50.0%, 50.0%-55.0%, 55.0%-60.0%, 60.0%-65.0%, or 65.0%-75.0%, based on the total amount of the protonatable cationic lipid, the helper lipid, the structural lipid, and the PEG-lipid.

[0337] The present invention provides a cationic liposome comprising the recombinant RNA molecule, DNA molecule or vector of the present invention.

[0338] The present invention provides a cationic protein comprising the recombinant RNA molecule, DNA molecule or vector of the present invention.

[0339] The present invention provides a cationic polymer comprising the recombinant RNA molecule, DNA molecule or vector of the present invention.

[0340] The present invention provides a pharmaceutical composition comprising the recombinant RNA molecule, DNA molecule, vector, host cell, cationic liposome, cationic protein, cationic polymer or lipid nanoparticle of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.

[0341] In addition, the present invention also provides a use of the recombinant RNA molecule, DNA molecule, vector, lipid nanoparticle or pharmaceutical composition of the present invention in preparing a drug.

[0342] In some embodiments, the medicament is for gene therapy, genetic vaccination, protein replacement therapy, antisense therapy, or treatment by interfering RNA.

[0343] In some embodiments, the drug is a nucleic acid drug, wherein the nucleic acid comprises at least one of the following: RNA, messenger RNA (mRNA), antisense oligonucleotide, DNA, plasmid, ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small inhibitory RNA (siRNA), small nuclear RNA (snRNA), small hairpin RNA (shRNA), tRNA, single-stranded guide RNA (sgRNA), and Cas9 mRNA.

[0344] In some embodiments, the drug is used to treat and / or prevent a disease. Preferably, the disease is selected from the group consisting of rare diseases, infectious diseases, cancers, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases, renal vascular diseases, and metabolic diseases; preferably, the cancer comprises one or more of lung cancer, gastric cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, blood cancer, or prostate cancer; and the genetic disease comprises one or more of hemophilia, thalassemia, and Gaucher's disease.

[0345] In some embodiments, the medicament is a vaccine.In some embodiments, the recombinant molecule is used to produce an antigen or portion thereof of a pathogen.

[0346] In some embodiments, the drug is a gene therapy agent.In some embodiments, the recombinant molecule is used to produce a protein associated with a genetic disease.

[0347] In some embodiments, the recombinant molecules are used to generate antibodies, such as scFVs or nanobodies.

[0348] In addition, the present invention also provides a method for preventing or treating a disease, comprising administering the recombinant RNA molecule or pharmaceutical composition of the present invention to a subject in need thereof.

[0349] In some embodiments, the disease or condition is selected from the group consisting of rare diseases, infectious diseases, cancer, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases, renal vascular diseases, and metabolic diseases. Preferably, the cancer comprises one or more of lung cancer, gastric cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, blood cancer, or prostate cancer; and the genetic disease comprises one or more of hemophilia, thalassemia, and Gaucher's disease.

[0350] In some embodiments, the recombinant RNA molecule or pharmaceutical composition is used as a vaccine to prevent a disease.In some embodiments, the recombinant RNA molecule is used to produce an antigen or part thereof of a pathogen.

[0351] In some embodiments, the recombinant RNA molecule is used to produce the protein associated with the genetic disease.

[0352] In some embodiments, the recombinant RNA molecules are used to generate antibodies, such as scFVs or nanobodies.

[0353] 7. Beneficial effects of the present invention:

[0354] The present invention obtains optimized UTRs through extensive screening. These UTRs can improve the efficiency and / or stability of mRNA translation and increase the expression level of polypeptides and / or proteins, and have very important application value in the development of mRNA vaccines. Example

[0355] Example 1 Construction of Plasmid D

[0356] The construction method is as follows:

[0357] Construction of plasmid B:

[0358] Using the Luciferase-pcDNA3 plasmid (purchased from Addgene, plasmid catalog #18964) as the backbone, new restriction sites HindIII and BamHI were added before the Kozack sequence, and new restriction sites KpnI and ApaI were added after the luciferase stop codon to generate plasmid B. The nucleotide sequence of the Luciferase-pcDNA3 plasmid is shown in SEQ ID NO: 50, and the plasmid map of luciferase-pcDNA3 is shown in Figure 2. The nucleotide sequence of plasmid B is shown in SEQ ID NO: 51, and the plasmid map of plasmid B is shown in Figure 3.

[0359] Construction of plasmid C:

[0360] The Amp (ampicillin) resistance gene in plasmid B was replaced with the Kana (kanamycin) resistance gene, and the neo / KanR sequence from 3746 to 4540 was removed to obtain plasmid C. The plasmid map of plasmid C is shown in Figure 4, and the nucleotide sequence of plasmid C is shown in SEQ ID NO:52.

[0361] Construction of plasmid D:

[0362] Plasmid C was inserted with a poly(A) tail as shown in SEQ ID NO:53. Specifically, plasmid C was digested with ApaI, the product was purified, and the purified digestion product and the poly(A) tail as shown in SEQ ID NO:53 were recombined by homologous recombination. The recombinant product was transformed into DH5α, and clones were screened on LB plates containing 50 μg / mL kanamycin. Correctly sequenced clones were selected and plasmids were extracted to obtain plasmid D. A plasmid map of plasmid D is shown in Figure 5, and the nucleotide sequence of plasmid D is shown in SEQ ID NO:54.

[0363] The construction flowchart of plasmid D is shown in Figure 1A , and the schematic diagram of the construction and transformation is shown in Figure 1B .

[0364] Example 2 Construction of plasmids containing different 5'-UTRs

[0365] Design and synthesize the 5'-UTR as shown in Table 1:

[0366] The 5'-UTR sequence was synthesized by commissioning Sangon Biotech (Shanghai) Co., Ltd. According to the company's quality analysis report, the 5'-UTR sequence was consistent with the theoretical design sequence.

[0367] Construction of plasmids containing different 5'UTRs:

[0368] Plasmid D was double-digested with HindIII and BamHI, and fragment A with a molecular weight of approximately 6 kb was recovered from the gel (Axygen). Different 5'UTR sequences were introduced into the homology arm sequences by PCR, and homologous recombination with fragment A was performed using Takara's homologous recombination enzyme system at 50°C for 15 minutes. DH5α (Takara) competent cells were transformed with this reaction system and plated (LB plates with 50 μg / mL kanamycin). After 16 hours of culture, 3-4 single colonies were selected and cultured in medium containing 50 μg / mL kanamycin for 8 hours. The plasmids were then extracted and sequenced by Sangon Biotech (Shanghai) Co., Ltd. to obtain plasmids containing different 5'UTRs.

[0369] Example 3 Construction of plasmids containing different 3'UTRs

[0370] The 3'-UTRs shown in Table 2 were synthesized and designed:

[0371] The 3'-UTR sequence was synthesized by Shanghai Sangon Biotechnology Co., Ltd. According to the company's quality analysis report, the 3'-UTR sequence was consistent with the theoretical design sequence.

[0372] Plasmid construction with different 3'-UTR sequences:

[0373] Plasmid D was double-digested with KpnI and ApaI, and fragment A with a molecular weight of approximately 6 kb was recovered from the gel (Axygen). Different 3'-UTR sequences were introduced into the homology arm sequences by PCR, and homologous recombination with fragment A was performed using Takara's homologous recombination enzyme system at 50°C for 15 minutes. DH5α (Takara) competent cells were transformed with this reaction system and plated (LB plates with 50 μg / mL kanamycin). After 16 hours of culture, 3-4 single clones were selected and cultured in medium containing 50 μg / mL kanamycin for 8 hours. The plasmids were extracted and sequenced by Sangon Biotech (Shanghai) Co., Ltd. to obtain plasmids containing different 3'-UTRs.

[0374] Example 4 Synthesis of mRNA

[0375] 1. Extract the plasmid template and ensure that the supercoil rate of the plasmid is above 90%.

[0376] 2. Plasmid Linearization

[0377] (1) Enzyme linearization: Take 20 μg of plasmid and use the corresponding enzyme (BsaI) to linearize the plasmid.

[0378] Note: Adjust the reaction system according to actual conditions. Due to the influence of the purification kit column, the plasmid in each reaction system should not exceed 20μg.

[0379] (2) Prepare the system in a 0.2 ml tube, mix well, and place in a 37°C incubator overnight or for 2 hours for enzyme digestion reaction.

[0380] (3) Take 1 μL of the digested product and run electrophoresis together with the original plasmid to check whether the digestion is complete.

[0381] 3. Linearized plasmid purification: Use Takara recovery kit (Cat. No. 9761)

[0382] (1) Add 3 times the amount of Buffer DC to the PCR reaction solution (or other enzymatic reaction solution) (if the amount of Buffer DC to be added is less than 100 μL, add 100 μL) and mix evenly.

[0383] (2) Place the Spin Column in the kit on the Collection Tube.

[0384] (3) Transfer the solution from step (1) above to a spin column, centrifuge at 12,000 rpm for 1 minute at room temperature, and discard the filtrate.

[0385] (4) Add 700 μL of Buffer WB to the spin column, centrifuge at 12,000 rpm for 30 seconds at room temperature, and discard the filtrate.

[0386] 4. Phenol-chloroform extraction (to remove RNAse, protein, etc.)

[0387] (1) Dilute the plasmid to be extracted to 300 μL or 500 μL, add an equal volume of phenol chloroform, and incubate at 12,000 rpm for 10 min.

[0388] (2) After centrifugation, aspirate the upper layer of liquid as much as possible, then add an equal volume of phenol-chloroform, and centrifuge at 12,000 rpm for 10 min.

[0389] (3) After centrifugation, aspirate the upper liquid as much as possible, add 0.1 times 5M NaCl and 0.7 times isopropanol, and then ice-bathe for 15 minutes. Incubate at 12000 rpm for 10 minutes and discard the supernatant.

[0390] (4) Wash once with 200 μL of 70% ethanol (pre-cooled) at 12,000 rpm for 1 min, discard the supernatant, and then centrifuge again at 12,000 rpm for 1 min, and thoroughly aspirate with a small pipette tip.

[0391] (5) After drying at room temperature, add an appropriate amount of UltraPure DNase / RNase-Free-Distilled Water and mix well.

[0392] (6) Use OneDrop to determine the concentration of the purified sample.

[0393] Identification: Take 100 ng of the purified plasmid and perform electrophoresis together with the original plasmid. After confirming that the plasmid is completely linearized and has no impurity bands, it can be used for mRNA synthesis.

[0394] 5. In vitro transcription of mRNA (refer to Novozymes IVT reaction kit)

[0395] (1) Cleaning the experimental area: First, sterilize the biosafety cabinet with UV light for 0.5 h. Then, wipe the cabinet clean with an alcohol swab and spray it with RNase inhibitor. After 5 min, wipe the inhibitor clean with an alcohol swab and begin the RNA experiment.

[0396] (2) System configuration: Before preparing the system, take out each component reagent, vortex mix on an oscillator, centrifuge, and place in an ice box for later use.

[0397] Note: During the preparation of the system, the entire process was performed on ice, and the plasmid template and IVT system were prepared separately.

[0398] (3) Preparation of IVT system: All components except the plasmid template were added below the liquid level of the system, and finally T7 enzyme was added and vortexed to mix. Uracil (U) was replaced with N1-methyl pseudouridine.

[0399] (4) Take out the required amount of plasmid into a new PCR tube and incubate it with the IVT system for 5 minutes. Finally, mix the two systems together, vortex to mix, centrifuge and incubate at 37°C for 2 hours.

[0400] (5) Prepare the system in a 0.2 mL flat-top thin-walled tube, mix well, and place in a PCR instrument for reaction.

[0401] The reaction conditions are 37°C for 2 hours. Note: The reaction system can also be scaled up according to the required production volume.

[0402] (6) Use DNase to remove the linearized plasmid template. Add 1 μL (20 μL system) or 5 μL (100 μL system) DNase to the reaction system and incubate at 37°C for 15 min.

[0403] 6. Purification method: (refer to Thermo MEGAclear Kit)

[0404] Column purification

[0405] (1) Transfer each reaction sample to a 1.5 ml EP tube (if the volume is less than 100 μL, add Elution Solution to make it up to 100 μL) and mix gently.

[0406] (2) Add 350 μL of Binding Solution Concentrate and mix gently with a pipette;

[0407] (3) Add 250 μL of anhydrous ethanol and mix gently with a pipette;

[0408] (4) Insert the filter cartridge provided with the kit into the collection tube, add 700 μL of the mixture to the filter cartridge, combine at room temperature for 10 min, centrifuge at 12,000 g for 1 min, discard the filtrate, reuse the collection tube, and then wash the RNA;

[0409] (5) Add 500 μL of Wash Solution and filter through a filter (centrifuge at 12000 g for 1 min);

[0410] (6) Repeat step (5);

[0411] (7) Remove the wash solution and continue centrifugation at the highest speed for 1 min to remove the residual wash solution;

[0412] (8) Add an appropriate amount (80-100 μl) of preheated RNase-Free Distilled Water to the filter cartridge, close the lid, incubate at 70°C for 10 min, and centrifuge at 12,000 g for 1 min. You can increase the number of elutions as needed.

[0413] (9) Concentration determination: use onedrop or Qubit to determine the concentration (measure after 10-fold dilution);

[0414] 7. Identification: Take 5 μL of diluted purified sample and NorthernMax TM Mix the Formaldehyde Load Dye and incubate at 75°C for 10 minutes, then store on ice. Perform gel electrophoresis on a 1% agarose gel to confirm that the mRNA is of the correct size and that the bands are not diffuse. This allows for the capping reaction to proceed.

[0415] 8. Capping reaction (refer to cellscript kit)

[0416] mRNACap1 capping reaction:

[0417] The structure and reaction principle of Cap1 type cap are as follows: pppN1(p)Nx-OH(3')→ppN1(pN)x-OH(3')+Pi ppN1(pN)x-OH(3')+GTP→G(5')ppp(5')N1(pN)x-OH(3')+PPi G(5')ppp(5')N1(pN)x-OH(3')+AdoMet→m7G(5')ppp(5')N1(pN)x-OH(3')+AdoHyc m7GpppN1(pN)x-OH(3')+AdoMet→m7Gppp[m2'-O]N1(pN)x-OH(3'+AdoHyc

[0418] 5'-Cap1 type cap structure:

[0419] cap G 1 G 2 =m 7 G + -5'-ppp-5'-Gm 2 '-3'-p-[m 7 =7-CH3;m 2' =2'-O-CH3; -ppp-=-PO2H-O-PO2H-O-PO2H)-; -p-=-PO2H-], 37°C for 5 min or 65°C for 5 min (CELL SCRIPT, the reaction system is shown in Table 4 below).

[0420] Table 3: Cap1 type capping reaction system

[0421] Note: Due to the influence of the system, the amount of capped mRNA per single addition (100ul system) should not exceed 60ug.

[0422] Capping (100ul system) (refer to CELL SCRIPT Capping Reaction Kit) is shown in Table 5 below.

[0423] Table 4: 100 μL capping system

[0424] The pre-heated mRNA was mixed with the above system and incubated at 37°C for 1 h.

[0425] 9. Purification method: the same as the purification method of in vitro transcription products

[0426] Characterization data: Determine concentration using onedrop or Qubit.

[0427] Experimental results: An mRNA final product containing a Cap1-type cap and different UTRs was obtained, and all uridine (U) nucleosides in the mRNA final product were replaced by N1-methyl pseudouridine.

[0428] Example 5 Screening of 5'-UTR

[0429] The effects of 5'-UTR on luciferase expression levels were compared using a cellular luciferase reporter assay.

[0430] HEK293 cells were transfected with mRNA containing different 5'-UTRs. Specifically, HEK293 cells were plated in a 96-well plate at a density of 40,000 cells per well one day in advance, and transfection was performed the next day when the cell confluence reached 70% to 90%.

[0431] Using the transfection reagent lipoMAX (Invitrogen, follow the instructions for lipoMAX), mRNAs containing different 5'-UTRs were transfected into HEK293 cells, with 100 ng of mRNA per well. After incubation at 37°C in a CO2 incubator for 16 h, luciferase reporter assay was performed (according to the instructions of the Promega kit).

[0432] The experimental results are shown in Figure 6. Compared with mRNA without UTR, mRNA containing 5UTR-NO2, 5UTR-NO4, 5UTR-NO12, 5UTR-NO13, 5UTR-NO14, 5UTR-NO17, 5UTR-NO20, 5UTR-NO22, 5UTR-NO26, 5UTR-NO29, 5UTR-NO34, and 5UTR-NO37 can significantly increase protein expression levels.

[0433] Example 6 Screening of 3'-UTR

[0434] The effects of 3'-UTR on luciferase expression levels were compared using a cellular luciferase reporter assay.

[0435] HEK293 cells were transfected with mRNA containing different 3'-UTRs. Specifically, HEK293 cells were plated in a 96-well plate at 40,000 cells per well one day in advance, and transfection was performed the next day when the cell confluence reached 70% to 90%.

[0436] Using the transfection reagent lipoMAX (Invitrogen, follow the instructions for lipoMAX), mRNAs containing different 3'-UTRs were transfected into HEK293 cells, with 100 ng of mRNA per well. After incubation at 37°C in a CO2 incubator for 16 h, luciferase reporter assay was performed (according to the instructions of the Promega kit).

[0437] The experimental results are shown in Figure 7. Compared with mRNA without UTR, mRNA containing 3UTR-NO4, 3UTR-NO5, 3UTR-NO6, 3UTR-NO8, 3UTR-NO10, 3UTR-NO11, 3UTR-NO13, 3UTR-NO14, 3UTR-NO16, 3UTR-NO22, 3UTR-NO23, 3UTR-NO24, 3UTR-NO25, 3UTR-NO32, 3UTR-NO36, 3UTR-NO41, 3UTR-NO46, 3UTR-NO48, and 3UTR-NO50 can increase the expression level of protein.

[0438] Example 7 Screening of 5'-UTR and 3'-UTR Combinations

[0439] 1. Compare the expression of a series of mRNAs containing the 5'-UTR-NO54 and one of the different 3'-UTRs in mice using in vivo imaging (IVIS). The specific procedures include:

[0440] 1. 5UTR-NO54 (DNA sequence shown in SEQ ID NO: 55, commissioned to Sangon Biotech (Shanghai) Co., Ltd. for synthesis) was inserted between HindIII and BamHI of blank plasmid D (for the construction method of blank plasmid D, see Example 1). After fixing the 5'UTR sequence, 3UTR-NO6 and 3UTR-NO8 were respectively inserted between ApaI and KpnI of the plasmid containing 5UTR-NO54 to obtain a series of plasmids containing 5UTR-NO54 and one of the different 3'-UTRs.

[0441] 2. Using the plasmids from step 1, a series of mRNAs containing 5'UTR-NO54 and one of the different 3'UTRs were prepared with reference to Example 4, and then respective LNP-mRNA preparations were prepared. The steps for preparing the LNP-mRNA preparations included:

[0442] (1) LNP encapsulation: The volume of the preparation solution (the total volume of the aqueous phase and its alcohol phase of each system) is 1.5 mL, wherein the mass ratio of mRNA to lipid is 1:10, and the concentration of HAc-NaOAc buffer (0.2 M, pH 5.0) in the final aqueous phase is 0.025 M. Using a microfluidic device (MPE-L2) and a chip (SN.000035), each sample was prepared at a flow rate of aqueous phase:alcohol phase = 9 mL / min:3 mL / min. The two phases were injected into the microfluidic chip according to the interface requirements for mixing. The aqueous phase contained 0.3 mg of mRNA with a total volume of 1125 μL; the alcohol phase contained lipids (SM-102) dissolved in ethanol: DSPC:CHO-HP:DMG-PEG2000 (Mol%) = 50:10:38.5:1.5, with a total volume of 375 μL. The chemical structure of SM-102 is as follows:

[0443] SM-102 is commercially available or can be prepared according to techniques known in the art.

[0444] (2) Dialysis and fluid exchange to prepare LNP-mRNA preparation:

[0445] a. Prepare dialysis fluid:

[0446] 1×PBS + 8% (m / V) sucrose solution: Take 2 packets of 1×PBS pre-made powder into a beaker, dissolve and mix with 2L of DEPC water, then add 160g of sucrose and mix to obtain 1×PBS + 8% (m / V) sucrose solution.

[0447] b. Dialysis: Each drug solution was placed in a 100KD dialysis bag and immersed in a beaker containing 1L of dialysis solution. The beaker was wrapped with aluminum foil and dialyzed at 100 rpm and room temperature for 1 hour. The dialysate was then replaced and dialysis was continued for 1 hour to obtain different LNP-mRNA preparations.

[0448] 3. The different LNP-mRNA formulations obtained in step 2 were subjected to in vivo live imaging experiments. The experiments used female Balb / c mice, 6 weeks old; 3 mice per group; each mouse was injected with 12 μg of LNP-mRNA formulation via the tail vein. After 12 hours, the mice were anesthetized by isoflurane inhalation and injected with the luciferase substrate D-luciferin (150 mg / kg). The animals were then placed in the supine position, and the luciferin signal distribution and intensity in the mice were observed using an IVIS live imaging system.

[0449] The results of luciferase expression in mice using various LNP-mRNA formulations are shown in Figure 8 . The unit of average photon number in Figure 8 is p / s / cm 2 / sr. The only difference between Control 1 and the other groups in Figure 8 is that Control 1 does not contain a 3'-UTR. The LNP-mRNA preparation method for Control 1 refers to the above. The results showed that compared with Control 1, mRNA containing 5'-UTR-NO54 and one of the 3'-UTRs selected from 3'-UTR-NO6 and 3'-UTR-NO8 achieved higher luciferase expression in mice.

[0450] Second, the expression of a series of mRNAs containing 3UTR-NO3 and one of the different 5'-UTRs in mice was compared by in vivo imaging. The specific steps included:

[0451] 1. 3UTR-NO3 (DNA sequence as shown in SEQ ID NO: 56, commissioned to Sangon Biotech (Shanghai) Co., Ltd. for synthesis) was inserted between ApaI and KpnI of blank plasmid D (for the construction method of blank plasmid D, see Example 1). After fixing the 3'-UTR sequence, 5UTR-NO12, 5UTR-NO2, 5UTR-NO11, 5UTR-NO13, 5UTR-NO14, 5UTR-NO18 and 5UTR-NO29 were respectively inserted between HindIII and BamHI of the plasmid containing 3UTR-NO3 to obtain a series of plasmids containing 3UTR-NO3 and one of different 5'-UTRs.

[0452] 2. Using the plasmids from step 1, prepare a series of mRNAs containing 3UTR-NO3 and one of the different 5'-UTRs as described above, and further prepare the corresponding LNP-mRNA preparations.

[0453] 3. Perform mouse imaging experiments using the different LNP-mRNA formulations obtained in Step 2. Six-week-old female Balb / c mice were used in this experiment; each group consisted of three mice. Each mouse received a tail vein injection of 12 μg of the LNP-mRNA formulation. After 12 hours, the mice were anesthetized by isoflurane inhalation and injected with 150 mg / kg of the luciferase substrate, D-luciferin. The animals were then placed in a supine position, and the luciferin signal distribution and intensity were observed using an IVIS in vivo imaging system.

[0454] The results of luciferase expression in mice using various LNP-mRNA formulations are shown in Figure 9 . The unit of average photon number in Figure 9 is p / s / cm 2 / sr. Control 2 in Figure 9 differed from the other groups only in that it lacked a 5'-UTR. The LNP-mRNA preparation method for Control 2 was described above. The results showed that compared to Control 2, mRNA containing 3UTR-NO3 and a 5'UTR selected from 5UTR-NO12, 5UTR-NO2, 5UTR-NO11, 5UTR-NO13, 5UTR-NO14, 5UTR-NO18, and 5UTR-NO29 achieved higher luciferase expression in mice.

Claims

1. A recombinant RNA molecule comprising: (1) a first nucleotide sequence encoding a polypeptide and / or protein of interest; and (2) a second nucleotide sequence containing a 5'-untranslated region (5'-UTR); The 5'-UTR comprises at least one selected from the following polynucleotides: (a): 5'-UTR derived from at least one of the following genes: PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, HBB, MYSM1, LENG1, TMSB4X, CASP4, IFNA1, PGLYRP1, UCHL1, CPAMD8, TTR, APOA2, GH1, DTYMK, APOC2, and CDK7; (b): a fragment of the 5'-UTR described in (a); (c): a variant of the 5'-UTR described in (a); and (d): a variant of the fragment described in (b); The first nucleotide sequence and the second nucleotide sequence do not naturally occur in the same RNA molecule.

2. The recombinant RNA molecule of claim 1, wherein the gene is a human gene.

3. The recombinant RNA molecule of claim 1 or 2, wherein the second nucleotide sequence comprises at least one of the following polynucleotides: (a): 5'-UTR derived from at least one of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1 and HBB; (b): a fragment of the 5'-UTR described in (a); (c): a variant of the 5'-UTR described in (a); and (d): A variant of the fragment described in (b).

4. The recombinant RNA molecule of any one of claims 1 to 3, wherein the second nucleotide sequence comprises at least one of the following polynucleotides: RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 1 to 21, a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 1 to 21, a variant of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 1 to 21, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 1 to 21; Preferably, variants of the RNA encoded by the polynucleotides having a sequence as shown in at least one of SEQ ID NOs: 1 to 21, fragments of the RNA encoded by the polynucleotides having a sequence as shown in at least one of SEQ ID NOs: 1 to 21, and variants of fragments of the RNA encoded by the polynucleotides having a sequence as shown in at least one of SEQ ID NOs: 1 to 21 have at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the RNA encoded by the polynucleotides having a sequence as shown in at least one of SEQ ID NOs: 1 to 21.

5. The recombinant RNA molecule of any one of claims 1 to 4, wherein the second nucleotide sequence comprises: At least one of the 5'-UTRs of at least two of the genes described in (a), fragments of the 5'-UTRs of at least two of the genes described in (a), variants of the 5'-UTRs of at least two of the genes described in (a), and variants of the fragments of the 5'-UTRs of at least two of the genes described in (a).

6. The recombinant RNA molecule of any one of claims 1 to 4, wherein the second nucleotide sequence comprises at least one of: at least two copies of the 5'-UTR in (a), at least two copies of a fragment of the 5'-UTR in (a), at least two copies of a variant of the 5'-UTR in (a), and at least two copies of a variant of a fragment of the 5'-UTR in (a). 7 . The recombinant RNA molecule according to claim 1 , further comprising at least one of a promoter, a 5′-cap structure, a 3′-UTR, and a poly(A) tail.

8. The recombinant RNA molecule of claim 7, wherein the 5'-cap structure comprises m 7 GpppG、m2 7,3′-O GpppG、m 7 Gppp(5')N1 and m 7 Gppp(m 2′-O ) At least one of N1.

9. The recombinant RNA molecule of claim 7 or 8, wherein the 3'-UTR comprises: i) a 3'-UTR derived from at least one gene selected from the group consisting of an albumin gene, an α-globin gene, a β-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen α gene; ii) a variant of the 3'-UTR in i); iii) at least one selected from the 3'-UTR of at least one of the genes MPND, FBXW10, FBXW12, PGLYRP1, HPX, CDK7, APOC2, PFN1, RBP4, FTCD, NAAA, ALB, GSDMD, FBXL8, ORM1, CASP4, CHMP2A, LENG1, MYCBPAP, APOC1, GAPDH, HSPA8, APOA2, UCHL1, TSG101, NAE1, NFKB2, and GH1, fragments, variants, and variants of fragments thereof; preferably, at least one selected from the group consisting of RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 22 to 49, fragments of RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 22 to 49, variants of RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 22 to 49, and variants of a fragment of RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 22 to 49; Preferably, the variants of the RNA encoded by the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 22 to 49, the fragments of the RNA encoded by the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 22 to 49, and the variants of the fragments of the RNA encoded by the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 22 to 49 are at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the RNA encoded by the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 22 to 49; iv) at least two copies of a polynucleotide selected from i), ii) or iii); or v) at least two polynucleotides selected from the group consisting of the polynucleotides described in i) to iii).

10. The recombinant RNA molecule of any one of claims 7 to 9, wherein the nucleotides constituting the poly(A) tail comprise at least 20, at least 40, at least 80, at least 100 or at least 120 A nucleotides; preferably, the nucleotides constituting the poly(A) tail comprise at least 20, at least 40, at least 80, at least 100 or at least 120 A nucleotides consecutively.

11. The recombinant RNA molecule according to any one of claims 7 to 10, wherein the nucleotides constituting the poly(A) tail include one or more nucleotides other than A nucleotides; Optionally, the nucleotides constituting the poly(A) include two or more consecutive nucleotides other than A nucleotides.

12. A recombinant RNA molecule comprising: (1) a first nucleotide sequence encoding a polypeptide and / or protein of interest; and (2) a second nucleotide sequence containing a 3'-untranslated region (3'-UTR); The 3'-UTR comprises at least one selected from the following polynucleotides: (a): 3'-UTR derived from at least one of the genes MPND, FBXW10, FBXW12, PGLYRP1, HPX, CDK7, APOC2, PFN1, RBP4, FTCD, NAAA, ALB, GSDMD, FBXL8, ORM1, CASP4, CHMP2A, LENG1, MYCBPAP, APOC1, GAPDH, HSPA8, APOA2, UCHL1, TSG101, NAE1, and NFKB2; (b): a fragment of the 3'-UTR described in (a); (c): a variant of the 3'-UTR described in (a); and (d): a variant of the fragment described in (b); The first nucleotide sequence and the second nucleotide sequence do not naturally occur in the same RNA molecule.

13. The recombinant RNA molecule of claim 12, wherein the gene is a human gene.

14. The recombinant RNA molecule of claim 12 or 13, wherein the second nucleotide sequence comprises at least one of the following polynucleotides: (a): 3'-UTR derived from at least one of the genes MPND, FBXW10, FBXW12, and PGLYRP1; (b): a fragment of the 3'-UTR described in (a); (c): a variant of the 3'-UTR described in (a); and (d): A variant of the fragment described in (b).

15. The recombinant RNA molecule of any one of claims 12 to 14, wherein the second nucleotide sequence comprises at least one of the following polynucleotides: RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 22 to 48, a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 22 to 48, a variant of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 22 to 48, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 22 to 48; Preferably, variants of the RNA encoded by the polynucleotides having a sequence as shown in at least one of SEQ ID NOs: 22 to 48, fragments of the RNA encoded by the polynucleotides having a sequence as shown in at least one of SEQ ID NOs: 22 to 48, and variants of fragments of the RNA encoded by the polynucleotides having a sequence as shown in at least one of SEQ ID NOs: 22 to 48 have at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the RNA encoded by the polynucleotides having a sequence as shown in at least one of SEQ ID NOs: 22 to 48.

16. The recombinant RNA molecule of any one of claims 12 to 15, wherein the second nucleotide sequence comprises at least one of: 3'-UTRs of at least two of the genes described in (a), fragments of 3'-UTRs of at least two of the genes described in (a), variants of 3'-UTRs of at least two of the genes described in (a), and variants of fragments of 3'-UTRs of at least two of the genes described in (a).

17. The recombinant RNA molecule of any one of claims 12 to 15, wherein the second nucleotide sequence comprises at least one of: at least two copies of the 3'-UTR in (a), at least two copies of a fragment of the 3'-UTR in (a), at least two copies of a variant of the 3'-UTR in (a), and at least two copies of a variant of a fragment of the 3'-UTR in (a). 18 . The recombinant RNA molecule according to claim 12 , further comprising at least one of a promoter, a 5′-cap structure, a 5′-UTR, and a poly(A) tail.

19. The recombinant RNA molecule of claim 17, wherein the 5'-cap structure comprises m 7 GpppG、m2 7,3′-O GpppG、m 7 Gppp(5')N1 or m 7 Gppp(m 2′-O ) At least one of N1.

20. The recombinant RNA molecule of claim 18 or 19, wherein the 5'-UTR comprises: i) at least one of the 5'-UTR of at least one of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, HBB, MYSM1, LENG1, TMSB4X, CASP4, IFNA1, PGLYRP1, UCHL1, CPAMD8, TTR, APOA2, GH1, DTYMK, APOC2, and CDK7, fragments, variants, and variants of fragments thereof; preferably, an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21, a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21, a variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21; Preferably, variants of the RNA encoded by the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21, fragments of the RNA encoded by the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21, and variants of fragments of the RNA encoded by the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21 are at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the RNA encoded by the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21; ii) at least two copies of one of the polynucleotides described in i); or iii) at least two polynucleotides according to i).

21. The recombinant RNA molecule of any one of claims 18 to 20, wherein the nucleotides constituting the poly(A) tail comprise at least 20, at least 40, at least 80, at least 100 or at least 120 A nucleotides; preferably, the nucleotides constituting the poly(A) tail comprise at least 20, at least 40, at least 80, at least 100 or at least 120 A nucleotides consecutively.

22. The recombinant RNA molecule according to any one of claims 18 to 21, wherein the nucleotides constituting the poly(A) tail comprise one or more nucleotides other than A nucleotides.

23. A DNA molecule encoding the recombinant RNA molecule according to any one of claims 1 to 22.

24. A vector comprising the DNA molecule of claim 23.

25. A host cell comprising the recombinant RNA molecule according to any one of claims 1 to 22, the DNA molecule according to claim 23, or the vector according to claim 24.

26. A lipid nanoparticle comprising the recombinant RNA molecule of any one of claims 1 to 22.

27. A pharmaceutical composition comprising the recombinant RNA molecule of any one of claims 1 to 22, the DNA of claim 23, the vector of claim 24, the host cell of claim 25 or the lipid nanoparticle of claim 26, and a pharmaceutically acceptable carrier.

28. A vector comprising a first nucleotide sequence encoding a 5'-UTR and / or a second nucleotide sequence encoding a 3'-UTR, wherein: The first nucleotide sequence comprises at least one of the following polynucleotides: (a): a polynucleotide encoding a 5'-UTR derived from at least one of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, HBB, MYSM1, LENG1, TMSB4X, CASP4, IFNA1, PGLYRP1, UCHL1, CPAMD8, TTR, APOA2, GH1, DTYMK, APOC2, and CDK7; (b): a polynucleotide encoding a fragment of the 5'-UTR in (a); (c): a polynucleotide encoding a variant of the 5'-UTR in (a); and (d): a polynucleotide encoding a variant of the fragment in (b); The second nucleotide sequence comprises at least one of the following polynucleotides: (e): a polynucleotide encoding a 3'-UTR derived from at least one of the genes MPND, FBXW10, FBXW12, PGLYRP1, HPX, CDK7, APOC2, PFN1, RBP4, FTCD, NAAA, ALB, GSDMD, FBXL8, ORM1, CASP4, CHMP2A, LENG1, MYCBPAP, APOC1, GAPDH, HSPA8, APOA2, UCHL1, TSG101, NAE1 and NFKB2; (f): a polynucleotide encoding a fragment of the 3'-UTR described in (e); (g): a polynucleotide encoding a variant of the 3'-UTR described in (e); and (h): a polynucleotide encoding a variant of the fragment described in (f).

29. The vector of claim 28, wherein the gene is a human gene.

30. The vector of claim 28 or 29, wherein the first nucleotide sequence comprises a 5'-UTR derived from at least one of the genes PPIA, HPX, FTCD, CDK5RAP3, HSPA8, HBA1, and HBB, or a variant thereof.

31. The vector of any one of claims 28 to 30, wherein the second nucleotide sequence comprises a 3'-UTR derived from at least one of the genes MPND, FBXW10, FBXW12, and PGLYRP1, or a variant thereof.

32. The vector according to any one of claims 28 to 31, comprising the first nucleotide sequence and the second nucleotide sequence.

33. The vector of any one of claims 28 to 32, wherein the first nucleotide sequence comprises: i) a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21, a fragment of a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21, a variant of a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21, and a variant of a fragment of a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21; Preferably, the variants of the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21, the fragments of the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21, and the variants of the fragments of the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21 have at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 1 to 21; ii) at least two copies of a polynucleotide according to i); or iii) at least two polynucleotides according to i).

34. The vector of any one of claims 28 to 33, wherein the second nucleotide sequence comprises: (1): a polynucleotide encoding a 3′-UTR derived from at least one of the albumin gene, the α-globin gene, the β-globin gene, the tyrosine hydroxylase gene, the lipoxygenase gene, and the collagen α gene; (2): a polynucleotide encoding a variant of the 3'-UTR in (1); (3): A polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 22 to 48, or a sequence as shown in SEQ ID NOs: at least one of a fragment of a polynucleotide as set forth in at least one of SEQ ID NOs: 22 to 48, a variant of a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 22 to 48, and a variant of a fragment of a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 22 to 48; Preferably, the RNA encoded by the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 22 to 48, the fragments encoded by the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 22 to 48, and the variants of the fragments encoded by the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 22 to 48 are 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 22 to 48; (4): at least two copies of a polynucleotide of (1), (2) or (3); or (5): At least two polynucleotides from the group consisting of the polynucleotides of (1) to (3).

35. The vector of any one of claims 28 to 34, further comprising a polynucleotide encoding a poly(A) tail.

36. The vector of claim 35, wherein the nucleotides constituting the poly(A) tail comprise at least 20, at least 40, at least 80, at least 100 or at least 120 A nucleotides; preferably, the nucleotides constituting the poly(A) tail comprise at least 20, at least 40, at least 80, at least 100 or at least 120 A nucleotides consecutively. The vector of claim 35 , wherein the nucleotides constituting the poly(A) tail comprise one or more nucleotides other than A nucleotides.

38. Use of the recombinant RNA molecule according to any one of claims 1 to 22, the DNA molecule according to claim 23, the vector according to claim 24 or 28, the host cell according to claim 25, the lipid nanoparticle according to claim 26, or the pharmaceutical composition according to claim 27 in the preparation of a medicament; Preferably, the medicament is used for gene therapy, gene vaccination or protein replacement therapy.

39. The use of claim 38, wherein the drug is a nucleic acid drug, wherein the nucleic acid comprises at least one of the following: RNA, messenger RNA (mRNA), DNA, plasmid, ribosomal RNA (rRNA), single-stranded guide RNA (sgRNA) and Cas9 mRNA.

40. The use according to any one of claims 38 to 39, wherein the drug is used for the treatment and / or prevention of a disease; Preferably, the disease is selected from the group consisting of rare diseases, infectious diseases, cancer, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases, renal vascular diseases, and metabolic diseases; Preferably, the cancer includes one or more of lung cancer, gastric cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, blood cancer or prostate cancer; the genetic disease includes one or more of hemophilia, thalassemia and Gaucher disease.

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