CN118291459A - 3'UTR sequence for promoting mRNA translation and its use - Google Patents

Abstract

The present disclosure relates to the biotechnology field, in particular to a 3' utr sequence for promoting mRNA translation and uses thereof. The present disclosure provides a 3' utr sequence comprising one of the following groups: (1) the nucleotide sequence shown in any one of SEQ ID NOs 1 to 3; (2) A complement of the nucleotide sequence shown in any one of SEQ ID NOs 1 to 3; (3) A nucleotide sequence having at least 80% homology with the nucleotide sequence set forth in any one of SEQ ID NOs 1 to 3; or (4) a nucleotide sequence having at least 80% homology with the complementary sequence of the nucleotide sequence shown in any one of SEQ ID NOS: 1-3. The invention provides a brand new 3' UTR sequence, and the advantage of optimizing translation is realized by reasonably optimizing the nucleic acid sequence of a functional region, so that the nucleic acid molecule has wide application prospect in the fields of gene therapy, vaccine research and development and the like.

Description

3'UTR sequence for promoting mRNA translation and its use

Technical Field

The present disclosure relates to the field of biotechnology, and in particular to a 3'UTR sequence for promoting mRNA translation and a use thereof.

Background technique

In vitro transcribed (IVT) mRNA refers to a messenger RNA (mRNA) obtained by transcription using DNA as a template. It is similar to natural mRNA in structure and can transmit genetic information in cells in vivo. mRNA drugs have the advantages of being independent of cells during transcription, easy to adjust the sequence, theoretically no integration risk, low cost, high efficiency and modular production. Therefore, mRNA drugs have broad application prospects and unique advantages in the fields of treatment and vaccines. To achieve effective treatment and vaccine effects, the key is to ensure that mRNA can be fully expressed. The expression and regulation of mRNA is a complex, multi-level regulatory process, and the regulation of transcriptional and post-transcriptional levels is particularly critical. Among them, in the process of translation regulation, the interaction between multiple protein factors and mRNA is often involved, and the 3'UTR is located at the 3' end of mRNA, regulating mRNA stability, localization and expression. Therefore, the 3'UTR is a key node affecting translation efficiency.

At present, there have been reports of 3’UTRs screened from naturally occurring genomes to improve mRNA translation efficiency. However, 3’UTRs are usually related to mRNA stability, and the presence of microRNA binding sites may cause inhibition of translation/RNA degradation. Therefore, naturally occurring 3’UTR sequences may have certain limitations in terms of regulatory mechanisms and translation efficiency, and cannot meet the needs of high-level gene expression. In addition to using 3’UTRs from naturally occurring genomes, 3’UTRs can also be designed from scratch, based on design principles such as reducing secondary structures and reducing miRNA targeting effects. Although a large number of 3’UTR sequences with good effects have been screened in this field based on different design principles and methods, the development of new 3’UTR sequences that promote mRNA translation and have wide applicability, further reducing the unit dose of mRNA administration and reducing costs, so as to achieve better applications in mRNA therapy and mRNA vaccines, is still a research focus in this field.

In addition, in nature, the 3'UTR segments of many mRNA molecules contain microRNA binding sites, which allow microRNA to fine-tune mRNA, including inhibiting its translation and accelerating its degradation. MicroRNAs are considered to be highly specific biomarkers due to their differential expression in different tissue types and stages of disease progression. Accordingly, the introduction of microRNA binding sites into the 3'UTR segment has become a commonly used method to regulate the function of mRNA. In theory, the interaction between miRNA and target mRNA is mainly based on the complementarity of the seed sequence of miRNA (generally 2 to 8 nucleotides long) with the corresponding position in the 3'UTR of mRNA. This complementary pairing is sufficient to locate the RNA-induced silencing complex (RISC) to a specific mRNA, thereby blocking its translation or accelerating its degradation. Numerous studies have shown that in addition to the matching of the seed sequence, additional complementary pairing between the non-seed sequence portion of miRNA and mRNA can also improve the efficiency of miRNA-mediated gene regulation. This additional pairing outside the miRNA seed sequence can enhance the binding affinity between miRNA and its target, thereby improving the inhibitory effect of mRNA. For example, one of the strategies adopted by Moderna is to add the reverse complementary sequence of the full length of miRNA to the 3'UTR segment. This design is not limited to the pairing of the seed sequence, allowing miRNA to have a wider range of complementary pairing with its target mRNA, thereby enhancing the ability of miRNA to regulate mRNA.

Summary of the invention

In order to solve the above technical problems, the present disclosure provides a 3'UTR sequence for promoting mRNA translation and its use.

In one aspect, the present disclosure provides a 3'UTR sequence, wherein the 3'UTR sequence comprises one of the following groups (1)-(4):

(1) the nucleotide sequence shown in any one of SEQ ID NOs: 1-3;

(2) a complementary sequence of the nucleotide sequence shown in any one of SEQ ID NOs: 1-3;

(3) a nucleotide sequence having at least 80% homology to the nucleotide sequence shown in any one of SEQ ID NOs: 1-3; or

(4) A nucleotide sequence having at least 80% homology with the complementary sequence of the nucleotide sequence shown in any one of SEQ ID NOs: 1-3.

In some embodiments, the aforementioned 3'UTR sequence further comprises a 3'UTR sequence or its complementary sequence inserted with an additional sequence; preferably, the additional sequence is a miRNA binding site; more preferably, the additional sequence is selected from the full-length microRNA reverse complementary sequence or the reverse complementary sequence of its seed sequence; preferably, the full-length microRNA reverse complementary sequence is 19-25nt in length; preferably, the reverse complementary sequence of the seed sequence is 7-8nt in length.

On the other hand, the present disclosure provides a DNA molecule comprising the aforementioned 3'UTR sequence.

In some embodiments, the DNA molecule further comprises an open reading frame encoding a polypeptide or protein of interest.

In some embodiments, the DNA molecule is circular DNA.

On the other hand, the present disclosure provides an mRNA comprising the aforementioned 3'UTR sequence.

In some embodiments, the aforementioned mRNA comprises in the 5'-3' direction:

(1) an open reading frame encoding a polypeptide or protein of interest; and

(2) The 3’UTR sequence described.

In another aspect, the present disclosure provides an expression vector, wherein the expression vector comprises the aforementioned DNA molecule.

On the other hand, the present disclosure provides a host cell, which comprises a 3'UTR sequence, the aforementioned DNA molecule, the aforementioned mRNA and/or the aforementioned expression vector.

In another aspect, the present disclosure provides a pharmaceutical composition comprising one of the following components (1)-(5), and a pharmaceutically acceptable carrier;

(1) the aforementioned 3’UTR sequence;

(2) the aforementioned DNA molecule;

(3) the aforementioned mRNA;

(4) the aforementioned expression vector; or

(5) The aforementioned cells.

On the other hand, the present disclosure provides a method for promoting the expression of a polypeptide or protein of interest, the method comprising introducing a 3'UTR sequence, the aforementioned DNA molecule, the aforementioned mRNA and/or the aforementioned vector into a host cell.

On the other hand, the present disclosure provides a use of a 3'UTR sequence, the aforementioned DNA molecule, the aforementioned mRNA, the aforementioned vector, the aforementioned cell, the aforementioned pharmaceutical composition and/or the aforementioned method in promoting the expression of a polypeptide or protein of interest.

The beneficial effects of the present disclosure are at least as follows:

1. Optimizing translation efficiency: In the 3'UTR of the coding transcript, the nucleic acid molecule of the present invention adopts specific sequence optimization, which helps to improve the scanning of ribosomes and the formation of translation initiation complexes, thereby enhancing the translation efficiency of proteins.

2. Wide applicability: The nucleic acid molecules of the present invention are flexibly designed and applicable to different genes and application scenarios. The sequences of their functional regions can be customized according to specific needs to meet the requirements of different experiments and applications.

In summary, the present invention provides a new nucleic acid molecule design scheme, which achieves the advantage of optimized translation by rationally optimizing the nucleic acid sequence of the functional region, making the nucleic acid molecule have broad application prospects in the fields of gene therapy, vaccine development, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a bar graph showing the relative light units of firefly luciferase in cell lysate detected by a multi-well plate reader (Agilent), indicating its expression level.

FIG2 is a fluorescence quantitative analysis of the relative intensity of green fluorescent protein after transfection of 293T cells with mRNA of the 3'UTR of the present invention containing three microRNA binding sites, and its changing trend within 24 hours.

Detailed ways

Definition and Description

In order to more easily understand the present invention, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. It should be understood that the present invention is not limited to specific methods, reagents, compounds, compositions or biosystems, and certainly the above can be changed. It should also be understood that the terms used in this application are only for the purpose of describing specific embodiments and are not intended to be limited.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

As used herein, the terms "include" and "have" and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product or device comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps that are not listed, or may optionally include other steps that are inherent to these processes, methods, products or devices. The "plurality" mentioned in this disclosure refers to two or more. "And/or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and/or B may represent: A exists alone, A and B exist at the same time, and B exists alone. The character "/" generally indicates that the previously associated objects are in an "or" relationship.

As used herein, the terms "nucleic acid", "nucleotide" and "polynucleotide" are used interchangeably to refer to deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and polymers thereof in single-stranded, double-stranded or multi-stranded form. The term includes, but is not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and/or pyrimidine bases or other natural, chemically modified, biochemically modified, non-natural, synthetic or derived nucleotide bases. In some embodiments, nucleic acids may include mixtures of DNA, RNA and their analogs. The term also encompasses nucleic acids containing known analogs of natural nucleotides, which have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. "Nucleic acid" is used interchangeably with "gene", "DNA" and "mRNA" encoded by a gene.

As used herein, the term "homology" refers to a region (site) in which two nucleic acids share at least partial complementarity. A region of homology can span only a portion of a sequence. For example, only a portion of a nucleotide can be homologous to a site in a genome. Different portions of a nucleotide can be homologous to several different sites in a genome, and a complete nucleotide can be homologous to another site in a genome. As with any partially complementary nucleic acid sequence, when two sequences are compared, a region of homology can contain one or more mismatches and gaps. A smaller nucleic acid chain (e.g., an oligonucleotide) can be homologous to a region (site) in a larger nucleic acid (e.g., a gene or genome). The term "degree of homology" between two sequences refers to the degree of identity between sequences. The degree of identity is generally expressed as the ratio of the total number of mismatched nucleotides to nucleotides in a homology region expressed as a percentage. For example, a 20 base oligonucleotide hybridized to a homology region (site) in a target genome with two mismatches is referred to as having 90% identity with the region. As used herein, homology is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

As used herein, the term "complementary sequence" refers to the ability of one nucleic acid to form hydrogen bonds with another nucleic acid sequence through traditional Watson-Crick or other non-traditional types. The percentage of complementarity represents the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Fully complementary" means that all consecutive residues of a nucleic acid sequence will hydrogen bond with the same number of consecutive residues in a second nucleic acid sequence, and "substantially complementary" means a degree of complementarity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides, or that two nucleic acids hybridize under stringent conditions.

As used herein, the term "5'UTR" generally refers to the sequence between the 5' end of the mRNA molecule and the translation start codon, which can recruit ribosome complexes and initiate the translation of mRNA. 5'UTR includes a 5'UTR region structure on the mRNA or the structure corresponds to a coding sequence on the DNA template. 5'UTR regulates post-transcriptional modification, the formation and stability of the translation start complex by interacting with transcription factors, ribosomes and other transcription regulatory proteins. The sequence design and optimization of this region are essential for improving the efficiency of post-transcriptional modification and protein expression. As used herein, the terms "5'UTR structure", "5'UTR", "5'UTR sequence" and "5'UTR element" etc. are used interchangeably, and 5'UTR has a length of 3-500 nucleotides, 5-150 nucleotides, 10-100 nucleotides, 15-90 nucleotides or 20-70 nucleotides. The 5'UTR element involved in the present disclosure comprises or consists of the following nucleic acid sequence: the nucleic acid sequence is derived from the 5'UTR of a eukaryotic protein-coding gene, preferably from the 5'UTR of a vertebrate protein-coding gene, more preferably from the 5'UTR of a mammalian protein-coding gene, even more preferably from the 5'UTR of a primate protein-coding gene, and in particular from the 5'UTR of a human or mouse protein-coding gene. The 5'UTR element involved in the present disclosure is derived from the 5'UTR of a 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, such as a collagen α1 (I) gene, or a variant of the 5'UTR of a 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, such as a collagen α1 (I) gene. The 5'UTR involved in the present disclosure comprises the 5'UTR of the TOP gene, preferably the 5'UTR of the human large ribosomal protein 32 lacking the 5'-terminal oligopyrimidine tract. The sequence involved in the present disclosure comprises one of the following groups (1)-(4): (1) the nucleotide sequence shown in GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC (SEQ ID NO: 4); (2) the complementary sequence of the nucleotide sequence shown in SEQ ID NO: 4; (3) a nucleotide sequence having at least 80% homology with the nucleotide sequence shown in SEQ ID NO: 4; or (4) a nucleotide sequence having at least 80% homology with the complementary sequence of the nucleotide sequence shown in SEQ ID NO: 4.

As used herein, the term "3'UTR" refers to the sequence between the stop codon of the polypeptide coding sequence in the mRNA and the poly (A) sequence. The 3'UTR can regulate the translation of the mRNA by interacting with mRNA binding proteins, miRNA, etc. The 3'UTR includes the 3'UTR region structure on the mRNA or the structure corresponds to the coding sequence on the DNA template. It is closely related to post-transcriptional modification and mRNA stability. The sequence and structural features of 3'UTR can affect the stability of mRNA, ribosome scanning and the formation of translation termination complex, etc., thereby affecting the expression level of protein. As used herein, the terms "3'UTR structure", "3'UTR", "3'UTR sequence" and "3'UTR element" are used interchangeably, all referring to the 3'UTR elements that can enhance the expression of the target gene obtained by the inventors after a large number of screenings. The 3'UTR sequence comprises one of the following groups (1)-(3): (1) the nucleotide sequence shown in any one of SEQ ID NOs: 1-3; (2) the complementary sequence of the nucleotide sequence shown in any one of SEQ ID NOs: 1-3; (3) a nucleotide sequence having at least 80% homology with the nucleotide sequence shown in any one of SEQ ID NOs: 1-3; or (4) a nucleotide sequence having at least 80% homology with the complementary sequence of the nucleotide sequence shown in any one of SEQ ID NOs: 1-3. The 3'UTR element of the present invention can be used for the design of mRNA molecular structure and DNA molecular template for mRNA therapy, mRNA vaccine and personalized immunotherapy to improve translation efficiency and enhance the expression amount of the target gene.

As used herein, the term "polyA tail" is a poly (A) sequence, which includes the structure of the poly A tail region on the mRNA or the structure corresponding to the coding sequence on the DNA template. The addition of the poly A sequence contributes to the stability and transport of the mRNA, prevents its degradation, and plays an important role in the post-transcriptional modification process. The poly (A) sequence can be a continuous chain of pure adenine nucleotides, or it can contain non-adenine nucleotides. In any form, as long as its function is equivalent to that of a traditional poly (A) sequence, that is, it can provide biological functions similar to those of a traditional poly (A) sequence, such as affecting the stability of mRNA, translation efficiency, or ribosome binding, the sequence is identified as a poly (A) sequence. This includes but is not limited to known variants such as the human growth hormone (hGH) poly (A) sequence and the simian virus 40 (SV40) poly (A) sequence, which may differ in nucleotide composition but are functionally identified as equivalent to traditional poly (A) sequences. In the present disclosure, the poly(A) sequence has a length of 20-500 adenine nucleotides, for example, a length of 25, 50, 100, 150, 175, 200, 300, 400 or 500 adenine nucleotides.

As used herein, the terms "5' cap element" and "5' cap structure" are used interchangeably, including 5' cap elements present on natural mRNA and their analogs. The 5' cap element on natural mRNA refers to the methylated guanylate connected to the 5' terminal nucleotide of RNA via pyrophosphate to form a 5', 5'-triphosphate linkage (5', 5'-triphosphate linkage). There are usually three types of 5' cap elements (m7G5'ppp5'Np, m7G5'ppp5'NmpNp, m7G5'ppp5'NmpNmpNp), respectively referred to as Cap0, Cap1 and Cap2. Cap0 refers to the unmethylated ribose of the terminal nucleotide, Cap1 refers to the methylation of the ribose of one terminal nucleotide, and Cap2 refers to the methylation of the ribose of both terminal two nucleotides. Methods for capping mRNA molecules are known in the art. The 5' cap structure of the aforementioned mRNA molecule can be added by enzymatic reaction after obtaining the mRNA molecule by chemical synthesis or in vitro transcription (for example, by a commercial kit containing vaccinia capping enzyme and mRNA cap structure 2'-O-methyltransferase). However, mRNA with a cap structure can also be produced by directly incorporating a nucleotide analog with a cap structure as the first nucleotide into the transcript during in vitro transcription.

As used herein, the terms "promoter" and "promoter element" are used interchangeably in the present invention and are a specific nucleic acid sequence that a transcriptase can recognize and bind to to start the transcription process. The promoter is located near the 5' end of a nucleic acid molecule and provides the necessary regulatory sequences for subsequent transcription and translation. The promoter involved in the present invention is a T7 RNA polymerase promoter, a T6 viral RNA polymerase promoter, an SP6 viral RNA polymerase promoter, a T3 viral RNA polymerase promoter, or a T4 viral RNA polymerase promoter.

As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably in the present invention to refer to polymers of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The terms "polypeptide", "peptide", "amino acid sequence" and "protein" may also include modified forms, including but not limited to glycosylation, lipid linkage, sulfation, gamma carboxylation, hydroxylation and ADP-ribosylation of glutamic acid residues. The polypeptide may be a polypeptide of eukaryotic, prokaryotic or viral origin. In certain embodiments, the polypeptide may be any polypeptide for therapeutic, preventive or diagnostic purposes. For example, the polypeptide may be an antigen, an antibody, a gene editing enzyme such as a CRISPR nuclease, etc. The polypeptide may also be a chimeric antigen receptor, an immunomodulatory protein, a transcription factor, etc. Examples of polypeptides include, but are not limited to: luciferase, red/green fluorescent protein, human erythropoietin, β-galactosidase.

As used herein, the term "open reading frame" (ORF) is a normal nucleotide sequence of a structural gene, which has the potential to encode a protein or polypeptide, starting from the start codon and ending at the stop codon, without a stop codon that interrupts translation. On an mRNA chain, the ribosome starts translating from the start codon, synthesizes the polypeptide chain along the RNA sequence and continues to extend, and when the stop codon is encountered, the extension reaction of the polypeptide chain is terminated.

As used herein, the term "vector" refers to a section of DNA extracted from a virus, plasmid or a cell of a higher organism, into which a foreign DNA fragment can be inserted or has been inserted for cloning and/or expression purposes. In certain embodiments, the vector can be stably maintained in an organism. The vector may include, for example, an origin of replication, a selective marker or a reporter gene, such as antibiotic resistance or GFP, and/or a multiple cloning site (MCS). The term includes linear DNA fragments (e.g., PCR products, linear plasmid fragments), plasmid vectors, viral vectors, cosmids, bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), etc.

As used herein, the terms "cell" and "host cell" are used interchangeably in the present invention and refer to cells that express or are capable of expressing a sequence to be expressed. The host cells of the present invention express polynucleotides encoding polypeptides or RNAs with a variety of uses, including biotechnology, molecular biology, and clinical applications. Host cells include prokaryotic cells or eukaryotic cells, and examples of suitable host cells in the present invention include, but are not limited to, bacteria, yeast cells, insect cells, animal cells, and mammalian cells.

As used herein, the term "pharmaceutically acceptable carrier" refers to one or more compatible solid, semisolid, liquid or gel fillers that are suitable for human or animal use and must have sufficient purity and sufficiently low toxicity. "Compatibility" refers to the components in the pharmaceutical composition and the active ingredients of the drug and their mutual blending without significantly reducing the efficacy. In the present invention, the aforementioned pharmaceutically acceptable carrier includes but is not limited to a buffer, an excipient, a stabilizer or a preservative. Examples of pharmaceutically acceptable carriers are physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, such as salts, buffers, sugars, antioxidants, aqueous or non-aqueous carriers, preservatives, wetting agents, surfactants or emulsifiers or combinations thereof. The amount of a pharmaceutically acceptable carrier in a pharmaceutical composition can be determined experimentally based on the activity of the carrier and the desired characteristics of the formulation, such as stability and/or minimal oxidation.

Detailed description of the specific implementation method

In one aspect, the present disclosure provides a 3'UTR sequence, wherein the 3'UTR sequence comprises one of the following groups (1)-(4):

(1) the nucleotide sequence shown in any one of SEQ ID NOs: 1-3;

(2) a complementary sequence of the nucleotide sequence shown in any one of SEQ ID NOs: 1-3;

(3) a nucleotide sequence having at least 80% homology to the nucleotide sequence shown in any one of SEQ ID NOs: 1-3; or

(4) A nucleotide sequence having at least 80% homology with the complementary sequence of the nucleotide sequence shown in any one of SEQ ID NOs: 1-3.

In some embodiments, the aforementioned 3'UTR sequence includes a nucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the nucleotide sequence shown in any one of SEQ ID NOs: 1-3 or its complementary sequence.

In some embodiments, the aforementioned 3'UTR sequence further comprises a 3'UTR sequence or its complementary sequence inserted with an additional sequence; preferably, the additional sequence is a miRNA binding site; more preferably, the additional sequence is selected from the full-length microRNA reverse complementary sequence or the reverse complementary sequence of its seed sequence; preferably, the full-length microRNA reverse complementary sequence is 19-25nt in length; preferably, the reverse complementary sequence of the seed sequence is 7-8nt in length.

In some embodiments, the aforementioned 3’UTR sequence further comprises a 3’UTR sequence or its complementary sequence into which at least one additional sequence is inserted; preferably, the aforementioned 3’UTR sequence further comprises a 3’UTR sequence or its complementary sequence into which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional sequences are inserted.

In some embodiments, the aforementioned 3'UTR sequence comprises a nucleotide sequence shown in any one of SEQ ID NO: 1-3 or its complementary sequence with an inserted additional sequence; preferably, the additional sequence is a miRNA binding site; more preferably, the additional sequence is selected from the full-length microRNA reverse complementary sequence or the reverse complementary sequence of its seed sequence; preferably, the full-length microRNA reverse complementary sequence is 19-25nt in length; and the reverse complementary sequence of the seed sequence is 7-8nt in length. Inserting a microRNA binding site into a candidate 3'UTR does not affect the translational regulatory function of the 3'UTR, nor does it affect the regulatory function of the 3'UTR on the stability of mRNA. Therefore, inserting a microRNA binding site into a candidate 3'UTR does not affect its function.

In some embodiments, the aforementioned 3'UTR sequence comprises a nucleotide sequence shown in any one of SEQ ID NOs: 1-3 or a complementary sequence thereof with at least one additional sequence inserted therein; preferably, the aforementioned 3'UTR sequence further comprises a nucleotide sequence shown in any one of SEQ ID NOs: 1-3 or a complementary sequence thereof with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional sequences inserted therein.

In some embodiments, the reverse complementary sequence of the full-length microRNA includes the nucleotide sequence shown in SEQ ID NO:8.

On the other hand, the present disclosure provides a DNA molecule comprising the aforementioned 3'UTR sequence.

In some embodiments, the aforementioned DNA molecule further comprises an open reading frame encoding a polypeptide or protein of interest.

In some embodiments, the DNA molecule is circular DNA.

On the other hand, the present disclosure provides an mRNA comprising the aforementioned 3'UTR sequence.

In some embodiments, the aforementioned mRNA comprises in the 5'-3' direction:

(1) an open reading frame encoding a polypeptide or protein of interest; and

(2) The aforementioned 3’UTR sequence.

In some preferred embodiments, the mRNA comprises in the 5'-3' direction:

(1) No or 5' cap element;

(2) no 5′UTR sequence or internal ribosome entry site (IRES) sequence;

(3) an open reading frame encoding a target polypeptide or target protein;

(4) the aforementioned 3'UTR sequence; and

(5) No or poly(A) sequence.

In some preferred embodiments, the aforementioned 5' cap element is selected from Cap0 cap structure, Cap1 cap structure or Cap2 cap structure. In some more preferred embodiments, the aforementioned 5' cap element is Cap1 cap structure.

In some embodiments, the sequence of the aforementioned 5'UTR element comprises one of the following groups (1)-(4):

(1) the nucleotide sequence shown in SEQ ID NO:4;

(2) a complementary sequence to the nucleotide sequence shown in SEQ ID NO: 4;

(3) a nucleotide sequence having at least 80% homology to the nucleotide sequence shown in SEQ ID NO: 4; or

(4) A nucleotide sequence having at least 80% homology with the complementary sequence of the nucleotide sequence shown in SEQ ID NO:4.

In some embodiments, the aforementioned poly A sequence has a length of 20-500 adenine nucleotides. In some more preferred embodiments, the aforementioned poly A sequence has a length of 25, 50, 100, 150, 175, 200, 300, 400 or 500 adenine nucleotides.

In some preferred embodiments, the aforementioned DNA molecule further comprises at least one nucleotide modification. At least one nucleotide modification includes but is not limited to cytidine modification, uridine modification or adenosine modification. In some more preferred embodiments, at least one nucleoside modification includes but is not limited to 5-methylcytosine (m5C), N6-methyladenosine (m6A), pseudouridine (ψ), N1-methylpseudouridine (m1ψ) and 5-methoxyuridine (5moU).

In another aspect, the present disclosure provides an expression vector, wherein the expression vector comprises the aforementioned DNA molecule.

In some embodiments, the aforementioned vector also comprises an RNA polymerase promoter sequence operably connected to the coding sequence of the DNA molecule. The operably connected promoter allows in vivo and/or in vitro transcription of the DNA molecule. The aforementioned promoter is a T7 RNA polymerase promoter, a T6 viral RNA polymerase promoter, an SP6 viral RNA polymerase promoter, a T3 viral RNA polymerase promoter, or a T4 viral RNA polymerase promoter.

In some embodiments, the aforementioned vector includes but is not limited to linear DNA fragments (e.g., PCR products, linear plasmid fragments), plasmid vectors, viral vectors, cosmids, bacterial artificial chromosomes (BAC) or yeast artificial chromosomes (YAC) etc. In some preferred embodiments, the aforementioned vector includes a plasmid vector or a viral vector. In some more preferred embodiments, the aforementioned vector comprises a restriction endonuclease site, such as an IIS type restriction endonuclease site, at the 3' flank of the coding sequence of the aforementioned mRNA molecule. Suitable restriction endonucleases include but are not limited to BsmBI, BsaI or SapI etc. The aforementioned restriction endonuclease site can be used to linearize the vector for in vitro transcription.

On the other hand, the present disclosure provides a host cell, which comprises a 3'UTR sequence, the aforementioned DNA molecule and/or the aforementioned expression vector.

In some embodiments, the aforementioned cells include prokaryotic cells or eukaryotic cells. In some preferred embodiments, the aforementioned cells are selected from the group consisting of Escherichia coli, yeast cells, or mammalian cells. In some more preferred embodiments, the cells are mammalian cells, including but not limited to rodent cells such as mouse cells or rat cells, etc.; primate cells such as monkey cells or human cells, etc. In some more preferred embodiments, the aforementioned cells are human cells. Furthermore, the aforementioned cells are 293T cells.

In another aspect, the present disclosure provides a pharmaceutical composition comprising one of the following components (1)-(5), and a pharmaceutically acceptable carrier;

(1) the aforementioned 3’UTR sequence;

(2) the aforementioned DNA molecule;

(3) the aforementioned mRNA;

(4) the aforementioned expression vector; or

(5) The aforementioned cells.

In some embodiments, the mRNA itself in the aforementioned pharmaceutical composition can also serve as an adjuvant.

In some embodiments, the dosage form of the aforementioned pharmaceutical composition is selected from an injection or a lyophilized preparation.

On the other hand, the present disclosure provides a method for promoting the expression of a polypeptide or protein of interest, the method comprising introducing a 3'UTR sequence, the aforementioned DNA molecule, the aforementioned mRNA molecule and/or the aforementioned vector into a host cell. The method of the present invention can make the aforementioned host cell contain the aforementioned nucleic acid molecule or the aforementioned vector, or integrate the aforementioned 3'UTR sequence or nucleic acid molecule into its genome, thereby improving the translation of mRNA or vector in the host cell.

In some embodiments, introduction can be performed by methods known in the art, such as microinjection or liposome-mediated transfection or electroporation.

In some embodiments, the aforementioned mRNA is sequentially coupled and cloned into the multiple cloning site of the vector in a "head-to-tail relationship" order, and an IIS type restriction endonuclease site is added after the poly A tail to guide the IIS type restriction endonuclease to cut the DNA template. The multiple cloning site of the vector refers to a nucleic acid region containing a restriction endonuclease, any of which can be used to cut the vector and insert a sequence. The restriction endonuclease can recognize a specific sequence (binding site) on a double-stranded DNA molecule and cut the phosphodiester bond. The vector will be cut under the action of the IIS type restriction endonuclease, and the cutting site is located at a distance determined from the DNA binding site. The product is recovered to obtain a linearized plasmid. The IIS type restriction endonuclease involved in the present disclosure includes, but is not limited to, BsmBI, BsaI or SapI.

By using T7 RNA polymerase to perform in vitro transcription on the linearized plasmid template, mRNA molecules can be produced and a 5' cap structure can be added at the same time. The 5' cap structure can be capped by co-transcription, where the cap analog is incorporated into the transcript as the first nucleotide during in vitro transcription to directly produce mRNA with a cap1 structure. The cap1 structure can also be capped post-transcriptionally, where the cap0 structure is added using vaccinia capping enzyme after in vitro transcription, and the cap1 structure is further added using mRNA cap structure 2'-O-methyltransferase. The mRNA obtained in this way is purified and resuspended in water. Subsequent transfection and detection work is then performed.

The mRNA molecule containing the generated 5'UTR sequence element and the ORF of the firefly luciferase sequence was transfected into mammalian 293T cells. After 24 hours of transfection, the cell samples were collected and chemiluminescence intensity analysis was performed to compare the effects of different optimized 5'UTRs on the expression level of firefly luciferase protein. This experiment was used to demonstrate the significant effect of the optimized 5'UTR on the translation efficiency of the protein.

On the other hand, the present disclosure provides a use of a 3'UTR sequence, the aforementioned DNA molecule, the aforementioned mRNA molecule, the aforementioned vector, the aforementioned cell, the aforementioned pharmaceutical composition and/or the aforementioned method in promoting the expression of a polypeptide or protein of interest.

For purposes of clarity and concise description, features are described herein as part of the same or separate embodiments, however, it will be understood that the scope of the present disclosure may include embodiments having a combination of all or some of the described features.

Example

Example 1: Construction of a vector containing a 3'UTR sequence

Using human cell Ribo-seq data and RNA-seq data published in public databases, a deep learning prediction model was trained, which can predict protein translation efficiency based on mRNA 3’UTR sequences. Subsequently, artificial intelligence analysis strategies, including adversarial generative networks (GANs) and long short-term memory recurrent neural networks (LSTMs), were used to train 3’UTR sequences in the human genome to construct a generative learning model, from which non-natural, new 3’UTR sequences were generated. The generative model was combined with the prediction model to evaluate the generated non-natural 3’UTR sequences, predict the translation efficiency of RNA composed of these 3’UTRs and protein-coding ORFs, and screen out a group of 3’UTR sequences that may achieve higher protein translation efficiency. Then, the sequences with low protein translation efficiency in actual expression were excluded through wet experiments, and the candidate 3’UTR sequences finally obtained are shown in Table 1.

Table 1 Candidate 3'UTR sequences

To test candidate 3'UTRs, a nucleic acid fragment containing a T7 promoter, a 3'UTR (Table 1), a sequence encoding firefly luciferase (Table 2), a 5'UTR (SEQ ID NO:4), a poly(A) sequence containing 120 A nucleotide residues, and a type IIS restriction endonuclease cleavage site was synthesized in vitro and cloned into an in vitro transcription vector (pIVTRup, Addgene plasmid #101362).

Table 2 Sequence information

Example 2: mRNA molecule length and integrity detection

The vector obtained in Example 1 was linearized, and T7-RNA polymerase was used to perform in vitro transcription to produce mRNA molecules, and a 5' cap structure was added at the same time. The 5' cap structure was added by co-transcriptional capping, and the cap analog was incorporated into the transcript as the first nucleotide during in vitro transcription, directly producing mRNA molecules with Cap1 structure.

The mRNA molecules thus obtained were purified and resuspended in water. The mRNA molecules were quality checked using a 5200 fragment analyzer (Agilent) to detect the length and integrity of the mRNA molecules (the values were derived from the area under the curve of the expected length fragments). If they met the requirements, they could be used for subsequent testing of different candidate 3'UTR sequence elements.

Example 3: Verification of translation efficiency of 3'UTR sequence elements

In this example, the luciferase reporter gene system was used to identify the translation efficiency regulation effect of 3'UTR sequence elements.

The experimental test of whether the 3'UTR sequence element has a positive regulatory effect on translation was performed by the following method:

Mammalian cells 293T were transfected with an RNA molecule containing a 3'UTR sequence element (the protein sequence encoding firefly luciferase). The chemiluminescent light absorption value of luciferase was detected at a specific time point (24 hours) after transfection, representing its protein expression level. The light absorption value reading containing the 3'UTR sequence element was subtracted from the light absorption value of the background group to obtain the relative light unit of the group. Each 3'UTR sequence element was processed to the corresponding relative light unit after detection in this experiment, which can indicate the translation regulation ability of the 3'UTR sequence element.

The specific experimental procedure for determining the luciferase chemiluminescence absorbance value of translation efficiency is as follows:

293T human embryonic kidney cells were seeded in 48-well plates at a density of 5×10 ⁴ cells/well. The next day, the cells were washed in Opti-MEM and then transfected with 200ng/well of Lipofectamine2000-complexed mRNA encoding firefly luciferase containing 3'UTR sequence elements in Opti-MEM. Cells without any RNA molecules were used as background groups. After 6 hours of transfection, the mixed culture medium was aspirated and replaced with complete culture medium. After 24 hours of transfection, the culture medium was aspirated, 100μL of lysis buffer (Promega) was added, and lysis was performed at room temperature for 5 minutes.

Luciferase activity was measured in relative light units (RLU) in a multiwell plate reader (Agilent). The activity of firefly luciferase was measured sequentially from individual samples in the luciferase assay. 20 μL of lysate was pipetted, 50 μL of buffer containing firefly luciferase substrate was added, the plate was shaken to mix, and the light absorbance was measured, see Table 3.

Table 3 contains the raw absorbance readings of 3'UTR sequence elements

The absorbance readings of the background wells were subtracted from the absorbance readings containing the 3’UTR sequence elements to obtain the relative light units of each group (see Table 4).

Table 4 Relative light units containing 3'UTR sequence elements

The data in Statistical Table 4 are made into a bar graph 1. As can be seen from Figure 1, the activation strength of each group of 3'UTRs for the luciferase reporter gene is different, but all can be effectively activated, indicating that the non-natural 3'UTRs provided by the present disclosure all have the ability to guide protein expression similar to the natural 3'UTR.

Example 4: Insertion of a microRNA binding site into a candidate 3'UTR does not affect its function

In this example, a fluorescent protein (d1EGFP) that is rapidly degraded in cells is used to test whether the function of the candidate 3'UTR sequence element is changed after modification, confirming that it has a positive regulatory function on translation and a regulatory effect on the stability of mRNA molecules.

Methods as below:

1) Insert the full-length hsa-microRNA-1-3p reverse complementary sequence into three random sites in the candidate 3’UTR to obtain a new 3’UTR.

2) Transfect mammalian cell lines such as 293T, Hela, raw264.7, etc. with RNA molecules containing the new 3’UTR and the original 3’UTR sequence elements (both encoding 1-hour degraded green fluorescent protein, d1EGFP).

3) Within 24 hours after transfection, the cells were photographed every hour to detect the intensity of the fluorescent protein, which represents the protein expression level;

4) Divide the intensity of green fluorescent protein by the internal reference (intensity of red fluorescent protein) to obtain the relative intensity of the experimental group or control group. The relative intensity can indicate the translational regulation ability of the 3'UTR sequence, and the change in fluorescence intensity within 24 hours can indicate the effect of the 3'UTR sequence on the stability of the mRNA molecule.

The sequence of the full-length hsa-microRNA-1-3p is shown below (SEQ ID NO: 7):

TGGAATGTAAAGAAGTATGTAT.

The reverse complementary sequence of the full-length hsa-microRNA-1-3p is shown below (SEQ ID NO: 8):

ATACATACTTCTTTACATTCCA.

The sequence of the new 3'UTR obtained by inserting the reverse complementary sequence of the full-length hsa-microRNA-1-3p into three random sites in 3'UTR-seq1 is as follows (SEQ ID NO: 9):

ATACATACTTCTTTACATTCCAAGACGCTGGCTGGACTGTTACCAGTAGGGATGTAT GTAGCATACATACTTCTTTACATTCCATCGTGCAGACACTTCTTAGAAAATATGACATAC ATACTTCTTTACATTCCAAGTTATGTAATGTATATTCCCTTTTCATAGC.

The amino acid sequence of 1 hour degraded green fluorescent protein (d1EGFP) is as follows (SEQ ID NO: 10):

MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKKLSHGFPPAVAAQDDGTLPMSCAQESGMDRHPAACASARINV*.

Among them, "*" means termination.

The sequence of the mRNA containing the green fluorescent protein degraded in 1 hour and the new 3'UTR sequence obtained by inserting the reverse complementary sequence of the full-length hsa-microRNA-1-3p into three random sites in 3'UTR-seq1 is as follows (SEQ ID NO: 11):

GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAG CCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGC ATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGAAGCTTAGCCATGGCTTCCCGCCGGCGGTGG CGGCGCAGGATGATGGCACGCTGCCCATGTCTTGTGCCCAGGAGAGCGGGATGGACCGTCACCCTGCAGCCTGTGCTTCTGCTAGGATCAATGTGTAGATACATACTTCTTTACATTCCAAGACGCTGGCTGGACTGTTACCAGTAGGGATGTATGTAGCATACATACTTCTTTACATTCCATCGTGCAGACACTTCTTAGAAAATATGACATACATACTTCTTTACATTCCAAGTTATGTAATGTATATTCCCTTTTCATAGC.

The experimental results show that compared with the control group without microRNA binding site, the relative light intensity difference of the microRNA binding site inserted into the candidate 3'UTR is not significant, that is, it does not affect the translation regulation function of the 3'UTR; the curve trend of the relative light intensity within 24 hours is not significantly different, that is, it does not affect the regulation function of the 3'UTR on the stability of mRNA. In summary, the insertion of microRNA binding sites into the candidate 3'UTR does not affect its function (Figure 2).

Claims (11)

1. A 3'UTR sequence, wherein the 3'UTR sequence comprises one of the following groups (1)-(4): (1) the nucleotide sequence shown in any one of SEQ ID NOs: 1-3; (2) a complementary sequence of the nucleotide sequence shown in any one of SEQ ID NOs: 1-3; (3) a nucleotide sequence having at least 80% homology to the nucleotide sequence shown in any one of SEQ ID NOs: 1-3; or (4) A nucleotide sequence having at least 80% homology with the complementary sequence of the nucleotide sequence shown in any one of SEQ ID NOs: 1-3. 2. A 3'UTR sequence according to claim 1, wherein the 3'UTR sequence further comprises a 3'UTR sequence or its complementary sequence inserted with an additional sequence; preferably, the additional sequence is a miRNA binding site; more preferably, the additional sequence is selected from a full-length microRNA reverse complementary sequence or a reverse complementary sequence of its seed sequence; preferably, the full-length microRNA reverse complementary sequence is 19-25 nt in length; preferably, the reverse complementary sequence of the seed sequence is 7-8 nt in length. 3. A DNA molecule comprising the 3'UTR sequence of claim 1 or 2. 4. A DNA molecule according to claim 3, further comprising an open reading frame encoding a polypeptide or protein of interest. The DNA molecule according to claim 3 , wherein the DNA molecule is a circular DNA. 6. An mRNA molecule, the mRNA comprising the 3'UTR sequence of claim 1 or 2, preferably the mRNA comprises in the 5'-3' direction: (1) an open reading frame encoding a polypeptide or protein of interest; and (2) The 3’UTR sequence of claim 1. 7. An expression vector comprising the DNA molecule according to any one of claims 3 to 5. 8. A host cell, comprising the 3'UTR sequence of claim 1 or 2, the DNA molecule of any one of claims 3-5, the mRNA molecule of claim 6 and/or the expression vector of claim 6. 9. A pharmaceutical composition comprising one of the following components (1) to (5), and a pharmaceutically acceptable carrier; (1) The 3'UTR sequence of claim 1 or 2; (2) The DNA molecule according to any one of claims 3 to 5; (3) the mRNA according to claim 6; (4) the expression vector according to claim 7; or (5) The host cell according to claim 8. 10. A method for promoting the expression of a polypeptide or protein of interest, the method comprising introducing into a host cell the 3'UTR sequence of claim 1 or 2, the DNA molecule of any one of claims 3-5, the mRNA molecule of claim 6 and/or the vector of claim 7. 11. Use of the 3'UTR sequence of claim 1 or 2, the DNA molecule of any one of claims 3-5, the mRNA molecule of claim 6, the vector of claim 7, the cell of claim 8, the pharmaceutical composition of claim 9 and/or the method of claim 10 in promoting the expression of a polypeptide or protein of interest.