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WO2025167868A1 - Acide nucléique modifié et son utilisation - Google Patents

Acide nucléique modifié et son utilisation

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Publication number
WO2025167868A1
WO2025167868A1 PCT/CN2025/075700 CN2025075700W WO2025167868A1 WO 2025167868 A1 WO2025167868 A1 WO 2025167868A1 CN 2025075700 W CN2025075700 W CN 2025075700W WO 2025167868 A1 WO2025167868 A1 WO 2025167868A1
Authority
WO
WIPO (PCT)
Prior art keywords
mir
nucleic acid
natural nucleic
microrna
utr
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2025/075700
Other languages
English (en)
Chinese (zh)
Inventor
李林鲜
黄慧
金亮
杨柳
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Shenxin Biotechnology Co Ltd
Original Assignee
Shenzhen Shenxin Biotechnology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shenzhen Shenxin Biotechnology Co Ltd filed Critical Shenzhen Shenxin Biotechnology Co Ltd
Publication of WO2025167868A1 publication Critical patent/WO2025167868A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the present invention belongs to the field of biotechnology and relates to a modified nucleic acid and an application thereof.
  • cytokines When therapeutic protein drugs are used clinically to treat diseases, they often induce immune responses in the patient's body, such as the release of cytokines or the production of anti-drug antibodies (ADA).
  • ADA anti-drug antibodies
  • the production of cytokines and ADA can change the pharmacokinetics, pharmacodynamics, efficacy and safety of therapeutic protein drugs.
  • lipid-containing delivery vehicles including liposomes and lipid nanoparticles (LNPs), which contain protonable lipids, cholesterol, phospholipids, and PEG-lipids.
  • LNPs lipid nanoparticles
  • B cells e.g., B1a cells
  • B2 cells activated to initiate an adaptive anti-PEG IgM immune response against PEGylated lipids.
  • LNP-based mRNA therapy has great application prospects, but requires repeated administration through a delivery vehicle. Therefore, the problem of accelerated blood clearance caused by lipid-containing delivery vehicles needs to be solved.
  • DNA or mRNA containing microRNA binding sites can bind to microRNA expressed in immune cells, reducing or inhibiting the expression of DNA or mRNA containing microRNA binding sites in immune cells, reducing or inhibiting undesirable immune responses, and thus reducing or inhibiting anti-drug antibody responses and accelerated blood clearance.
  • microRNA binding sites are placed in the untranslated region (UTR) of mRNAs, such as the 3’-UTR and/or 5’-UTR.
  • UTR untranslated region
  • mRNA binding site downstream of the 3’-UTR of synthetic nucleic acids such as mRNA or DNA
  • placing the microRNA binding site downstream of the 3’-UTR of synthetic nucleic acids can also exert the corresponding effect.
  • the present disclosure provides a non-natural nucleic acid comprising a 3'-UTR and one or more microRNA binding sites, wherein the microRNA binding site is located downstream of the 3'-UTR.
  • the non-natural nucleic acid further comprises a ploy(A) tail, wherein the ploy(A) tail is located downstream of the 3'-UTR, and the one or more microRNA binding sites are located at one of the following positions:
  • the microRNA binding site located in the poly(A) tail is at the 5' end, between the 5' and 3' ends, and/or at the 3' end of the poly(A) tail.
  • the multiple microRNA binding sites are identical.
  • the multiple microRNA binding sites are different and bind to the same microRNA or different microRNAs.
  • the different microRNAs are from the same cell, tissue and/or organ, or the different microRNAs are from different cells, tissues and/or organs.
  • the one or more microRNA binding sites are capable of binding to microRNA expressed in target cells, target tissues and/or target organs to reduce or inhibit the expression of the non-natural nucleic acid in the target cells, target tissues and/or target organs.
  • the target cells, target tissues, and/or target organs include one or more of the following: immune cells, liver, lung, heart, nervous system, pancreas, kidney, muscle, endothelial cells, epithelial cells, embryonic stem cells, and abnormal cells;
  • the target cell is an immune cell.
  • the one or more microRNA binding sites are capable of binding to microRNAs that include one or more of the following: miR-122, miR-126, miR-142-3p, miR-142-5p, miR-144, miR-146-3p, miR-146-5p, miR-155, miR-16, miR-21, miR-223, miR-24, and miR-27.
  • the one or more microRNA binding sites are capable of binding to microRNAs comprising one or more of the following: miR-142-3p, miR-142-5p, miR-126, miR-146-3p, miR-146-5p, and miR-155.
  • the one or more microRNA binding sites are capable of binding to microRNAs comprising one or more of the following: miR-142-3p, miR-142-5p, and miR-126.
  • the one or more microRNA binding sites are capable of binding to a microRNA comprising miR-142-3p, miR-142-5p, miR-126, or miR-122.
  • the microRNA that the one or more microRNA binding sites are capable of binding is miR-142-3p.
  • the one or more microRNA binding sites are capable of binding to microRNAs including miR-142-3p and one or more of the following: miR-142-5p, miR-146-3p, miR-146-5p, miR-155, miR-126, miR-16, miR-21, miR-223, miR-24, and miR-27.
  • the one or more microRNA binding sites are capable of binding to microRNAs including miR-142-5p and one or more of the following: miR-142-3p, miR-146-3p, miR-146-5p, miR-155, miR-126, miR-16, miR-21, miR-223, miR-24, and miR-27.
  • the one or more microRNA binding sites are capable of binding to microRNAs including miR-126 and one or more of the following: miR-142-3p, miR-142-5p, miR-146-3p, miR-146-5p, miR-155, miR-16, miR-21, miR-223, miR-24, and miR-27.
  • the one or more microRNA binding sites are capable of binding to microRNAs including miR-122 and one or more of the following: miR-142-3p, miR-142-5p, miR-146-3p, miR-146-5p, miR-155, miR-126, miR-16, miR-21, miR-223, miR-24, and miR-27.
  • the non-natural nucleic acid comprises 1, 2, 3, or 4 of the microRNA binding sites.
  • the non-natural nucleic acid further comprises one or more of the following: a 5'-UTR and a coding region encoding a polypeptide or protein of interest.
  • the non-natural nucleic acid further comprises a coding region encoding a polypeptide or protein of interest.
  • the non-natural nucleic acid further comprises a 5'-UTR and a coding region encoding a polypeptide or protein of interest.
  • the 3'-UTR is heterologous to the coding region encoding the polypeptide or protein of interest.
  • the 5'-UTR is heterologous to the coding region encoding the polypeptide or protein of interest.
  • the 5'-UTR and 3'-UTR are heterologous to the coding region encoding the polypeptide or protein of interest.
  • the nucleotides comprising the ploy(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.
  • the nucleotides comprising the poly(A) tail include one or more nucleotides other than A nucleotides.
  • the non-natural nucleic acid further comprises at least one microRNA binding site located in the 3'-UTR and/or in the 5'-UTR.
  • the non-natural nucleic acid is mRNA.
  • nucleotide sequence of the DNA corresponding to the 3’-UTR is shown in SEQ ID NO: 2 or 3.
  • nucleotide sequence of the DNA corresponding to the 5’-UTR is shown in SEQ ID NO: 4.
  • nucleotide sequence of the DNA corresponding to the microRNA binding site is shown as ACACTAC, SEQ ID NO: 1 or 13.
  • the non-natural nucleic acid is an mRNA, and the mRNA comprises a cap structure.
  • the cap structure is selected from at least one of m 7 GpppG, m 2 7,3′-O GpppG, m 7 Gppp(5′)N1, and m 7 Gppp(m 2′-O )N1.
  • the non-natural nucleic acid contains modified nucleotides.
  • the non-natural nucleic acid contains modified nucleosides.
  • the modified nucleoside comprises at least one of a modified uridine, a modified cytidine, a modified adenosine, and a modified guanosine.
  • the uridine in the non-natural nucleic acid is 100% modified.
  • the non-natural nucleic acid is DNA.
  • the present disclosure provides a genetic engineering vector comprising the non-natural nucleic acid according to any one of the aforementioned embodiments, or the genetic engineering vector comprises a polynucleotide capable of being transcribed into the non-natural nucleic acid according to any one of the aforementioned embodiments.
  • the present disclosure provides a host cell comprising the genetic engineering vector according to any one of the above embodiments.
  • the present disclosure provides a delivery vector comprising the non-natural nucleic acid according to any of the aforementioned embodiments, the genetically engineered vector according to any of the aforementioned embodiments, or the host cell according to any of the aforementioned embodiments.
  • the delivery vehicle is a lipid nanoparticle, a cationic liposome, a cationic protein, or a lipid polymer.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the non-natural nucleic acid of any of the above embodiments, the genetic engineering vector of any of the above embodiments, the host cell of any of the above embodiments, or the delivery vector of any of the above embodiments, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition includes a plurality of said delivery vehicles
  • the pharmaceutical composition comprises a plurality of said mRNAs.
  • the present disclosure also provides a use of the non-natural nucleic acid of any of the above embodiments, the genetic engineering vector of any of the above embodiments, the host cell of any of the above embodiments, the delivery vector of any of the above embodiments, or the pharmaceutical composition of any of the above embodiments in the preparation of a drug.
  • the medicament is for gene therapy, genetic vaccination, protein replacement therapy, antisense therapy, or treatment by interfering RNA.
  • the medicament is for the treatment and/or prevention of a disease.
  • the medicament is used to treat and/or prevent one or more of the following diseases: rare diseases, cancer, infectious diseases, autoimmune diseases, metabolic diseases, neurological diseases, cardiovascular diseases, transplant rejection, inflammatory response, genetic diseases and musculoskeletal diseases.
  • the drug is a nucleic acid drug, wherein the nucleic acid comprises one or more of the following: RNA and DNA.
  • the DNA includes one or more of: a plasmid and an antisense oligonucleotide.
  • the RNA includes one or more of the following: antisense oligonucleotides, messenger RNA, ribosomal RNA, microRNA, transfer RNA, small inhibitory RNA, small nuclear RNA, small hairpin RNA, single-stranded guide RNA and Cas9 mRNA.
  • the present disclosure also provides a method for preventing or treating a disease, comprising administering to a subject the non-natural nucleic acid of any of the above embodiments, the genetically engineered vector of any of the above embodiments, the host cell of any of the above embodiments, the delivery vector of any of the above embodiments, or the pharmaceutical composition of any of the above embodiments.
  • the present disclosure also provides a method for reducing or inhibiting the expression of a non-natural nucleic acid in undesired cells, tissues and/or organs, comprising administering to a subject a non-natural nucleic acid according to any of the above embodiments, wherein the non-natural nucleic acid encodes a polypeptide or protein of interest and comprises one or more microRNA binding sites capable of binding to microRNA expressed in undesired cells, tissues and/or organs.
  • the undesired cells are immune cells.
  • the present disclosure provides a method for reducing or inhibiting undesirable immune cell activation, comprising administering to a subject a non-natural nucleic acid according to any of the above embodiments, a genetically engineered vector according to any of the above embodiments, or a delivery vector according to any of the above embodiments, wherein the non-natural nucleic acid encodes a polypeptide or protein of interest and comprises one or more microRNA binding sites capable of binding to microRNA expressed in immune cells.
  • the methods reduce or inhibit undesired immune cell activation by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% compared to a non-natural nucleic acid that does not contain a microRNA binding site.
  • the undesirably activated immune cells are selected from one or more of the following: T cells, B cells, plasma cells, and NK cells.
  • the present disclosure also provides a method for reducing or inhibiting the production of undesirable cytokines, comprising administering to a subject a non-natural nucleic acid according to any of the above embodiments, a genetically engineered vector according to any of the above embodiments, or a delivery vector according to any of the above embodiments, wherein the non-natural nucleic acid encodes a polypeptide or protein of interest and comprises one or more microRNA binding sites capable of binding to microRNA expressed in immune cells.
  • the methods reduce or inhibit the production of an undesired cytokine by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% compared to a non-natural nucleic acid that does not contain a microRNA binding site.
  • the present disclosure also provides a method for reducing or inhibiting an anti-drug antibody response in a subject who is repeatedly administered a drug, comprising administering to the subject a non-natural nucleic acid according to any of the above embodiments, a genetically engineered vector according to any of the above embodiments, or a delivery vector according to any of the above embodiments, wherein the non-natural nucleic acid encodes a polypeptide or protein of interest and comprises one or more microRNA binding sites capable of binding to microRNA expressed in immune cells, such that upon repeated administration, the anti-drug antibody response in the subject is reduced or inhibited.
  • the present disclosure also provides a method for reducing or inhibiting accelerated blood clearance in a subject who is repeatedly administered a drug, comprising administering to a subject a non-natural nucleic acid according to any of the above embodiments, wherein the non-natural nucleic acid is encapsulated in lipid nanoparticles, and the non-natural nucleic acid encodes a polypeptide or protein of interest and comprises one or more microRNA binding sites capable of binding to microRNA expressed in immune cells, such that upon repeated administration, accelerated blood clearance in the subject is reduced or inhibited.
  • the present disclosure also provides a method for reducing or inhibiting the production of polyethylene glycol-bound IgM molecules in a subject that is repeatedly administered, comprising administering to the subject a non-natural nucleic acid according to any of the above embodiments, wherein the non-natural nucleic acid is encapsulated in a lipid nanoparticle, and the non-natural nucleic acid encodes a polypeptide or protein of interest and comprises one or more microRNA binding sites capable of binding to microRNA expressed in immune cells, such that upon repeated administration, the production of polyethylene glycol-bound IgM molecules in the subject is reduced or inhibited.
  • the lipid nanoparticle encapsulating the non-natural nucleic acid comprises PEG-lipid.
  • the non-natural nucleic acid is mRNA.
  • the non-natural nucleic acid is DNA.
  • the genetically engineered vector is a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.
  • the non-natural nucleic acid comprises one or more microRNA binding sites that can bind to microRNAs including one or more of the following: miR-122, miR-126, miR-142-3p, miR-142-5p, miR-144, miR-146-3p, miR-146-5p, miR-155, miR-16, miR-21, miR-223, miR-24, and miR-27.
  • the one or more microRNA binding sites are capable of binding to microRNAs comprising one or more of the following: miR-142-3p, miR-142-5p, miR-126, miR-146-3p, miR-146-5p, and miR-155.
  • the one or more microRNA binding sites are capable of binding to a microRNA comprising miR-142-3p, miR-142-5p, miR-126, or miR-122.
  • the one or more microRNA binding sites are capable of binding to microRNAs including miR-142-3p and one or more of the following: miR-142-5p, miR-146-3p, miR-146-5p, miR-155, miR-126, miR-16, miR-21, miR-223, miR-24, and miR-27.
  • the one or more microRNA binding sites are capable of binding to microRNAs including miR-142-5p and one or more of the following: miR-142-3p, miR-146-3p, miR-146-5p, miR-155, miR-126, miR-16, miR-21, miR-223, miR-24, and miR-27.
  • the one or more microRNA binding sites are capable of binding to microRNAs including miR-126 and one or more of the following: miR-142-3p, miR-142-5p, miR-146-3p, miR-146-5p, miR-155, miR-16, miR-21, miR-223, miR-24, and miR-27.
  • the one or more microRNA binding sites are capable of binding to microRNAs including miR-122 and one or more of the following: miR-142-3p, miR-142-5p, miR-146-3p, miR-146-5p, miR-155, miR-126, miR-16, miR-21, miR-223, miR-24, and miR-27.
  • the one or more microRNA binding sites are capable of binding to microRNAs comprising one or more of the following: miR-142-3p, miR-142-5p, and miR-126.
  • the microRNA to which the one or more microRNA binding sites are capable of binding is miR-142-3p.
  • the non-natural nucleic acid comprises 2 to 6 microRNA binding sites capable of binding to miR-142-3p;
  • the non-natural nucleic acid comprises three microRNA binding sites capable of binding miR-142-3p.
  • the subject is a mammal; preferably, the mammal is a human.
  • the number of administrations is one, two, three, four, or more times.
  • the time interval between administrations is no more than 8 weeks, 7 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 2 weeks, or 1 week.
  • the route of administration is intravenous or intramuscular injection.
  • Figures 1A to 1C show the luciferase expression levels in mice after a single dose.
  • C-Fluc refers to a control group injected with lipid nanoparticles encapsulating mRNA encoding Fluc without microRNA binding sites (numbered C-hFluc), while the other groups were experimental groups injected with lipid nanoparticles encapsulating mRNA encoding Fluc with microRNA binding sites.
  • 437-Fluc refers to an experimental group injected with lipid nanoparticles encapsulating mRNA encoding Fluc with microRNA binding sites (numbered 437-Fluc), and the same applies to the others.
  • Figure 2 shows the hEPO expression level in the serum of rats after multiple administrations.
  • PBS refers to the control group injected with PBS
  • C-hEPO refers to the control group injected with lipid nanoparticles encapsulating mRNA encoding hEPO without microRNA binding sites (numbered C-hEPO)
  • the other groups are experimental groups injected with lipid nanoparticles encapsulating mRNA encoding hEPO containing microRNA binding sites.
  • 437-hEPO refers to the experimental group injected with lipid nanoparticles encapsulating mRNA encoding hEPO containing microRNA binding sites (numbered 437-hEPO), and the same applies to the others.
  • FIG. 3 shows the anti-PEG IgG antibody levels in the serum of rats after multiple administrations.
  • 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 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.”
  • the numerical ranges described herein should be understood to encompass any and all subranges contained therein.
  • 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.
  • the terms "and/or,” “any combination thereof,” and their grammatical equivalents are used interchangeably. These terms are intended to expressly refer to any combination.
  • the phrases “A, B, and/or C” or “A, B, C, or any combination thereof” refer to any of the following: "A alone; B alone; C alone; A and B; B and C; A and C; and A, B, and C.”
  • naturally occurring refers to the fact that a substance can be found in nature.
  • a peptide, amino acid, protein, or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and has not been experimentally modified by man is naturally occurring.
  • non-naturally occurring or “non-natural” when used in connection with nucleic acids herein is intended to mean that the nucleic acid is not found in nature.
  • a non-naturally occurring nucleic acid encoding a viral peptide or protein has at least one genetic alteration or chemical modification that is not normally found in wild-type strains of the virus in question.
  • Such genetic alterations include, for example, the introduction of an expressible nucleic acid sequence encoding a peptide or polypeptide that is heterologous to the virus in question, other nucleic acid additions, nucleic acid deletions, nucleic acid substitutions, and/or other functional disruptions to the viral genetic material.
  • Chemical modifications include, for example, one or more functional nucleotide analogs as described herein.
  • heterologous refers to a non-natural nucleic acid comprising two elements, such as a polynucleotide encoding a polypeptide and/or protein of interest and a 5'-UTR, that do not naturally exist in this combination (in nature), or a polynucleotide encoding a polypeptide and/or protein of interest and a 3'-UTR that do not naturally exist in this combination (in nature). They are typically recombinant.
  • the 3'-UTR and/or 5'-UTR are derived from a different gene than the polynucleotide encoding the polypeptide and/or protein of interest, that is, the source gene of the 3'-UTR and/or 5'-UTR is different from the source gene of the polynucleotide encoding the polypeptide and/or protein of interest.
  • the source gene of the polynucleotide encoding the polypeptide and/or protein of interest and the source gene of the 3'-UTR and/or the 5'-UTR are genes that encode different proteins.
  • the source gene of the polynucleotide encoding the polypeptide and/or protein of interest and the source gene of the 3'-UTR and/or the 5'-UTR are genes that encode the same protein but belong to different species.
  • the term "wild type” means that the sequence is naturally occurring and has not been artificially modified, including naturally occurring mutants.
  • 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.
  • a fragment of a nucleic acid comprises at least 70% or 80% of the nucleotide residues from the nucleic acid.
  • at least 90%, 95%, 96%, 97%, 98% or 99% of the nucleotide residues are present. Typically, this can be a shorter portion of the full length of the nucleic acid.
  • variant nucleic acids refers to a nucleic acid variant that differs from a reference nucleic acid (or "parent") in at least one nucleotide.
  • a variant nucleic acid includes single or multiple nucleotide deletions, additions, mutations, and/or insertions, wherein: deletions include removal of 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 a 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 transitions)); insertions include adding at least one nucleotide.
  • a nucleic acid variant nucleic acid include, but are not limited to, substitutions (e.g.
  • nucleic acid variant includes naturally occurring variants and engineered variants. Therefore, " nucleic acid variant " as defined herein can be derived from a reference nucleic acid, separated, relevant, based on or homologous to a reference nucleic acid sequence.” nucleic acid variant " optionally has 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% of a corresponding naturally occurring (wild type) nucleic acid or its homologue, fragment or derivative.
  • At least 70%, at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity includes degenerate nucleic acid sequences, wherein the degenerate nucleic acid sequences according to the present invention are due to the degeneracy of genetic code and the nucleic acid different from the reference nucleic acid in codon sequence.
  • % 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.
  • % identity or “% identity” 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.
  • 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 to determine sequence identity can be performed using tools known in the art, preferably using optimal sequence alignment, for example, using Align using standard settings, preferably EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, Gap Extend 0.5.
  • a fragment, variant, or nucleic acid having a specific degree of identity to a specific nucleic acid 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.
  • nucleotide includes deoxyribonucleotides, ribonucleotides, deoxyribonucleotide derivatives, and ribonucleotide derivatives.
  • ribonucleotide is a constituent 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 ⁇ -D-ribofuranosyl group.
  • Deoxyribonucleotide is a constituent 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 ⁇ -D-ribofuranosyl group is replaced by a hydrogen atom. It is the main chemical component of chromosomes.
  • Nucleotides are generally referred to by single letters representing the bases: "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.
  • a nucleoside refers to adenine deoxyribonucleotide
  • 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.
  • nucleic acids herein also include derivatives of nucleic acids.
  • 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.
  • nucleic acids can be in the form of single-stranded or double-stranded linear or covalently closed circular molecules.
  • Polynucleotide sequence can be used interchangeably to refer to the order of nucleotides in a polynucleotide. It should be understood by those skilled in the art that the DNA coding strand (sense strand) and the RNA it encodes can be regarded as having the same nucleotide sequence, and the deoxythymidylate in the DNA coding strand sequence corresponds to the uridine nucleotide in the RNA sequence it encodes.
  • the RNA corresponding to the DNA refers to the polynucleotide after all T in the DNA is replaced by U.
  • the RNA corresponding to the DNA shown in the nucleotide sequence ACACTAC refers to the RNA formed by replacing all T in the DNA shown in the nucleotide sequence ACACTAC with U.
  • the DNA corresponding to the RNA refers to the polynucleotide after all U in the RNA is replaced with T.
  • the polynucleotide may comprise one or more segments (nucleic acid fragments) (e.g., 1, 2, 3, 4, 5, 6, 7, 8 segments).
  • the polynucleotide may comprise a segment encoding a polypeptide of interest (e.g., a polypeptide and polypeptide antigen described herein).
  • the polynucleotide may comprise a segment encoding a polypeptide of interest and a regulatory segment (including but not limited to segments for transcriptional regulation and translational regulation).
  • 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.
  • promoter refers to a polynucleotide located upstream of the 5' end of the coding region of a gene, which contains a conserved sequence required for the specific binding of RNA polymerase and the initiation of transcription, can activate RNA polymerase, enable RNA polymerase to accurately bind to the template DNA and have the specificity of transcription initiation. Promoters can be derived from viruses, bacteria, fungi, plants, insects and animals.
  • promoters include bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV 40 late promoter and CMV IE promoter.
  • the term "5' untranslated region" or "5'-UTR” can be an RNA sequence in an mRNA that is upstream of the coding sequence and is not translated into protein.
  • the 5'-UTR in a gene generally begins at the transcription start site and ends at the nucleotides upstream of the translation start codon of the coding sequence.
  • the 5'-UTR can contain elements that control gene expression, such as a ribosome binding site, a 5'-terminal oligopyrimidine tract, and a translation initiation signal such as a Kozak sequence.
  • mRNA can be post-transcriptionally modified by the addition of a 5' cap. Therefore, the 5'-UTR in a mature mRNA can also refer to the RNA sequence between the 5' cap and the start codon.
  • 3' untranslated region can be an RNA sequence in an mRNA that is located downstream of a coding sequence and is not translated into protein.
  • the 3'-UTR in an 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.
  • 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.
  • the partial 5’ or 3’-UTR of the mRNA of gene A includes a partial 5’-UTR formed by splicing together multiple fragments of the 5’-UTR of the mRNA of gene A, or a partial 3’-UTR formed by splicing together multiple fragments of the 3’-UTR of the mRNA of gene A.
  • poly(A),” “polyA,” “poly(A) sequence,” and “poly(A) tail” are used interchangeably.
  • Naturally occurring poly(A) sequences are typically composed of adenine ribonucleotides.
  • 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, for example, at the 3' end (downstream) of the 3'-UTR.
  • the term "5'-cap structure” refers to a 5'-cap structure typically located at the 5' end of a mature mRNA. In some embodiments, the 5'-cap structure is linked 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 (particularly, guanine nucleotide derivatives).
  • 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.
  • the 5'-cap structure includes, but is not limited to, cap 0, cap 1 (a cap structure formed by further methylating the 2'-OH of the first nucleotide sugar group of hnRNA on the basis of cap 0, or referred to as "cap 1”), cap 2 (a cap structure formed by further methylating the 2'-OH of the second nucleotide sugar group of hnRNA on the basis of cap 1, or referred to as "cap 2”), cap 4, cap 0 analogs, cap 1 analogs, cap 2 analogs, or cap 4 analogs.
  • the term "expression” includes transcription and/or translation of a nucleotide sequence. Thus, expression can involve the production of transcripts and/or polypeptides.
  • transcription refers to the process of transcribing the genetic code in a DNA sequence into RNA (transcript).
  • 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.
  • a vector that can be used to produce a transcript is also called a "transcription vector,” which contains the regulatory sequences required for transcription.
  • transcription encompasses "in vitro transcription.”
  • 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.
  • 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.
  • 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.
  • the host cell can be a cell expressing a polypeptide of the present invention therein.
  • recombinant or “recombinant” means "produced by genetic engineering.”
  • "recombinant material” such as a recombinant RNA molecule, is non-naturally occurring.
  • 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.
  • 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. In some embodiments, the DNA plasmid is a medium copy or high copy plasmid.
  • the DNA plasmid is a high copy plasmid.
  • 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.
  • an antigen generally refers to a substance that can be recognized by the immune system, preferably recognized by the adaptive immune system, and can trigger an antigen-specific immune response (e.g., forming antibodies and/or antigen-specific T cells).
  • an antigen can be or can comprise a peptide or protein that can be presented to a T cell by MHC.
  • an antigen can be a translation product of a provided nucleic acid (e.g., RNA, RNA molecules, DNA herein).
  • fragments, variants, and derivatives of peptides or proteins derived from peptides or proteins comprising at least one epitope or antigen can be understood as antigens.
  • vaccine is typically understood to mean a prophylactic or therapeutic material that provides at least one antigen or antigenic function that can stimulate the body's adaptive immune system to provide an adaptive immune response.
  • the term "treatment” etc. is used in this article to generally mean obtaining the pharmacological and/or physiological effect of expectation. Therefore, the treatment of the present application may relate to the treatment of the state of a certain disease, but may also relate to prophylactic treatment for preventing a disease or its symptoms completely or in part. In some embodiments, the term “treatment” should be understood as being therapeutic in terms of partially or completely curing a disease and/or the adverse effects and/or symptoms owing to the disease. Treatment may also be prophylactic or preventive treatment, i.e., measures taken to prevent a disease, such as, for example, to prevent infection and/or the onset of a disease.
  • the terms “subject” and “patient” can be used interchangeably.
  • the subject is a mammal, such as a human, non-human primate (e.g., ape, chimpanzee, monkey, and orangutan), domesticated animal (including dog and cat and livestock (e.g., horse, cattle, pig, sheep, and goat)) or other mammal.
  • Other mammals include, but are not limited to, mice, rats, guinea pigs, rabbits, hamsters, etc.
  • the subject is a human.
  • the subject is a mammal (e.g., a human) suffering from a genetic disease, a rare disease, or an infectious disease.
  • the subject is a mammal (e.g., a human) at risk of developing a genetic disease, a rare disease, or an infectious disease.
  • administer refers to providing or administering a medicament to a subject by any effective route.
  • routes of administration include, but are not limited to, one or more of the following: injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, intrathecal, intracerebroventricular, or intravenous), oral, intracavitary, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation.
  • injection e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, intrathecal, intracerebroventricular, or intravenous
  • oral intracavitary, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation.
  • administration of the substance is typically performed after the onset of the disease, disorder, condition, or symptom.
  • administration of the substance is typically performed before the onset of the disease, disorder, condition, or symptom.
  • ABS accelerated blood clearance
  • ADA anti-drug antibody
  • ADA anti-drug antibody
  • nucleic acid therapeutics e.g., mRNA therapeutics
  • ADA responses include antibody responses observed in animal studies described herein, such as the production of antibodies in animals that bind to therapeutic proteins encoded by mRNA therapeutics.
  • This antibody response against therapeutic proteins encoded by mRNA therapeutics is also referred to as an anti-protein antibody (APA) response, a term used interchangeably herein with ADA responses.
  • APA anti-protein antibody
  • microRNA refers to a small non-coding RNA. Generally, microRNAs are numbered according to the order in which they were discovered. MicroRNAs with the same number indicate that they are derived from the same pre-miRNA. For example, miRNA-142-3p refers to the mature miRNA-142 derived from the 3' end arm of pre-miRNA-142, and miRNA-142-5p refers to the mature miRNA-142 derived from the 5' end arm of pre-miRNA-142. As used herein, unless otherwise specified, if a microRNA is not specified as “5p” or "3p", it refers to "5p” and/or “3p”. For example, unless otherwise specified, miR-142 refers to miRNA-142-5p and/or miRNA-142-3p.
  • microRNA binding site refers to a polynucleotide, such as DNA or RNA, that has sufficient complementarity with all or part of a region of a miRNA to enable interaction, association, or binding with the microRNA.
  • MicroRNAs bind to microRNA binding sites on mRNAs, triggering microRNA-mediated mRNA regulation, such as microRNA-mediated mRNA degradation or inhibition of mRNA translation, thereby reducing expression of the protein encoded by the mRNA.
  • the non-natural nucleic acid comprises a plurality of microRNA binding sites located in the poly (A) tail; in another embodiment, the microRNA binding site located downstream of the 3'-UTR is capable of binding to one or more of the following microRNAs: miR-122, miR-126, miR-142-3p, miR-142-5p, miR-144, miR-146-3p, miR-146-5p, miR-155, miR-16, miR-21, miR-223, miR-24 and miR-26.
  • the non-natural nucleic acid contains multiple microRNA binding sites located in the poly (A) tail, and the multiple microRNA binding sites can bind to one or more of the following microRNAs: miR-122, miR-126, miR-142-3p, miR-142-5p, miR-144, miR-146-3p, miR-146-5p, miR-155, miR-16, miR-21, miR-223, miR-24 and miR-27.
  • the present disclosure provides a non-natural nucleic acid comprising a 3'-UTR and one or more microRNA binding sites, wherein the one or more microRNA binding sites are located downstream of the 3'-UTR.
  • the non-natural nucleic acid comprises a microRNA binding site downstream of the 3'-UTR.
  • the non-natural nucleic acid comprises a plurality of microRNA binding sites downstream of the 3'-UTR.
  • the non-natural nucleic acid comprises 2 to 6 microRNA binding sites located downstream of the 3'-UTR, for example 2, 3, 4, 5 or 6. In some embodiments, the non-natural nucleic acid comprises 3 microRNA binding sites located downstream of the 3'-UTR.
  • the non-natural nucleic acid comprises a plurality of microRNA binding sites downstream of the 3’-UTR, and the plurality of microRNA binding sites downstream of the 3’-UTR are identical.
  • the non-natural nucleic acid comprises three identical microRNA binding sites downstream of the 3’-UTR that are capable of binding to miR-142-3p.
  • the non-natural nucleic acid comprises a plurality of microRNA binding sites downstream of the 3’-UTR, and the nucleotide sequences of the plurality of microRNA binding sites downstream of the 3’-UTR or their corresponding DNAs are as shown in ACACTAC or SEQ ID NO: 1.
  • the non-natural nucleic acid comprises three microRNA binding sites downstream of the 3’-UTR, and the nucleotide sequences of the three microRNA binding sites downstream of the 3’-UTR or their corresponding DNAs are as shown in ACACTAC or SEQ ID NO: 1.
  • the non-natural nucleic acid comprises a plurality of microRNA binding sites located downstream of the 3'-UTR, the plurality of microRNA binding sites located downstream of the 3'-UTR are different, and the plurality of microRNA binding sites located downstream of the 3'-UTR bind to the same microRNA or different microRNAs.
  • the non-natural nucleic acid comprises a plurality of microRNA binding sites located downstream of the 3'-UTR, the plurality of microRNA binding sites located downstream of the 3'-UTR are different, and the plurality of microRNA binding sites located downstream of the 3'-UTR are capable of binding to the same microRNA.
  • the plurality of microRNA binding sites located downstream of the 3'-UTR can reduce or inhibit the expression of the non-natural nucleic acid in one or more specific cells, tissues and/or organs.
  • the non-natural nucleic acid comprises three microRNA binding sites located downstream of the 3'-UTR and capable of binding to miR-142-3p, wherein the first microRNA binding site is capable of binding to the 5' end of miR-142-3p, and the second and third microRNA binding sites are capable of binding to the 3' end of miR-142-3p.
  • the above-mentioned non-natural nucleic acid contains three microRNA binding sites located downstream of the 3’-UTR and capable of binding to miR-142-3p, wherein the nucleotide sequence of the first microRNA binding site or its corresponding DNA is shown as SEQ ID NO: 1, and the nucleotide sequences of the second and third microRNA binding sites or their corresponding DNA are shown as ACACTAC.
  • the non-natural nucleic acid comprises multiple microRNA binding sites downstream of the 3'-UTR, the multiple microRNA binding sites downstream of the 3'-UTR are different, the multiple microRNA binding sites downstream of the 3'-UTR bind to different microRNAs, the different microRNAs are from the same cell, tissue and/or organ, or the different microRNAs are from different cells, tissues and/or organs.
  • the multiple microRNA binding sites downstream of the 3'-UTR can reduce or inhibit the expression of the non-natural nucleic acid in one or more specific cells, tissues and/or organs.
  • the non-natural nucleic acid comprises multiple microRNA binding sites downstream of the 3'-UTR, wherein the multiple microRNA binding sites downstream of the 3'-UTR are different, and the multiple microRNA binding sites downstream of the 3'-UTR are capable of binding to different microRNAs, and the different microRNAs are derived from the same cell, tissue, and/or organ.
  • the non-natural nucleic acid comprises three microRNA binding sites downstream of the 3'-UTR, wherein the first microRNA binding site is capable of binding to miR-142 expressed in immune cells, the second microRNA binding site is capable of binding to miR-155 expressed in immune cells, and the third microRNA binding site is capable of binding to miR-223 expressed in immune cells.
  • the non-natural nucleic acid comprises multiple microRNA binding sites downstream of the 3'-UTR, wherein the multiple microRNA binding sites downstream of the 3'-UTR are different and can bind to different microRNAs, each of which originates from different cells, tissues, and/or organs.
  • the non-natural nucleic acid comprises three microRNA binding sites downstream of the 3'-UTR, wherein the first microRNA binding site can bind to miR-122 expressed in the liver, and the second and third microRNA binding sites can bind to miR-142 expressed in immune cells.
  • the microRNA binding site located downstream of the 3'-UTR is capable of binding to a microRNA expressed in a target cell, target tissue, and/or target organ to reduce or inhibit the expression of the non-natural nucleic acid in the target cell, target tissue, and/or target organ.
  • the target cells, target tissues, and/or target organs include one or more of the following: immune cells, liver, lung, heart, nervous system, pancreas, kidney, muscle, endothelial cells, epithelial cells, embryonic stem cells, and abnormal cells.
  • the target cell is an immune cell.
  • the microRNA binding site located downstream of the 3'-UTR can bind to a microRNA including a microRNA expressed in an immune cell, so as to reduce or inhibit the expression of the non-natural nucleic acid in the immune cell.
  • the immune cells are myeloid cells and/or lymphocytes.
  • the myeloid cells are selected from one or more of the following: dendritic cells, macrophages, monocytes, neutrophils, basophils, eosinophils, megakaryocytes, and platelets.
  • the lymphocytes are selected from one or more of the following: T cells, B cells, plasma cells, and NK cells.
  • the non-natural nucleic acid comprises one or more microRNA binding sites located downstream of the 3'-UTR, and the microRNA binding sites located downstream of the 3'-UTR can bind to microRNAs including microRNAs expressed in immune cells.
  • MicroRNAs expressed in immune cells include, but are not limited to, one or more of the following: hsa-let-7a-2-3p, hsa-let-7a-3p, hsa-7a-5p, hsa-let-7c, hsa-let-7e-3p, hsa-let-7e-5p, hsa-let-7g-3p, hsa-let-7g-5p, hsa-let-7i-3p, hsa-let-7i-5p, miR -10a-3p,miR-10a-5p,miR-1184,hsa-let-7f-l-3p,hsa-let-7f-2 ⁇ 5p,hsa-let-7f-5p,miR-125b-l
  • the microRNA expressed in immune cells is selected from the microRNA of Jima DD et al, Blood, 2010, 116:e118-e127, the microRNA of Vaz C et al, BMC Genomics, 2010, 11, 288, or a combination thereof with the aforementioned microRNAs expressed in immune cells.
  • the microRNA expressed in immune cells is a microRNA that is highly abundantly expressed or specifically expressed in immune cells.
  • the microRNA that is highly abundantly expressed or specifically expressed in immune cells is miR-142.
  • the microRNA that is highly abundantly expressed or specifically expressed in immune cells is miR-142-3p.
  • the microRNA binding site located downstream of the 3'-UTR can bind to a microRNA that includes a microRNA expressed in the liver.
  • MicroRNAs expressed in the liver include, but are not limited to, one or more of the following: miR-107, miR-122-3p, miR-122-5p, miR-1228-3p, miR-1228-5p, miR-1249, miR-129-5p, miR-1303, miR-151a-3p, miR-151a-5p, miR-152, miR-194-3p, miR-194-5p, miR-199a-3p, miR-199a-5p, miR-199b-3p, miR-199b-5p, miR-296-5p, miR-557, miR-581, miR-939-3p, and miR-939-5p.
  • the microRNA expressed in the liver is a microRNA that is highly expressed or specifically expressed in normal liver cells and is low in abundance or not expressed in abnormal liver cells (e.g., liver cancer cells).
  • the microRNA that is highly expressed or specifically expressed in normal liver cells and is low in abundance or not expressed in abnormal liver cells (e.g., liver cancer cells) is miR-122.
  • the microRNA binding site located downstream of the 3'-UTR can bind to microRNAs that are expressed in the lung.
  • MicroRNAs expressed in the lung include, but are not limited to, one or more of the following: let-7a-2-3p, let-7a-3p, let-7a-5p, miR-126-3p, miR-126-5p, miR-127-3p, miR-127-5p, miR-130a-3p, miR-130a-5p, miR-130b-3p, miR-130b-5p, miR-133a, miR-133b, miR-133b-5p ...
  • the microRNA to which the microRNA binding site located downstream of the 3'-UTR can bind includes a microRNA expressed in the heart.
  • the microRNA expressed in the heart includes, but is not limited to, one or more of the following: miR-1, miR-133a, miR-133b, miR-149-3p, miR-149-5p, miR-186-3p, miR-186-5p, miR-208a, miR-208b, miR-210, miR-296-3p, miR-320, miR-451a, miR-451b, miR-499a-3p, miR-499a-5p, miR-499b-3p, miR-499b-5p, miR-744-3p, miR-744-5p, miR-92b-3p, and miR-92b-5p.
  • the microRNA binding site located downstream of the 3'-UTR can bind to a microRNA including a microRNA expressed in the nervous system.
  • the microRNA expressed in the nervous system includes, but is not limited to, one or more of the following: miR-124-5p, miR-125a-3p, miR-125a-5p, miR-125b-1-3p, miR-125b-2-3p, miR-125b-5p, miR-1271-3p, miR-1271-5p, miR-128, miR-132-5p, miR-135a-3p, miR-135a-5p, miR-135b -3p,miR-135b-5p,miR-137,miR-139-5p,miR-139-3p,miR-149-3p,miR-149-5p,miR-153,miR-181c-3p,miR -181c-5p,miR-183-3p,miR-183-5p,miR-190a,miR-
  • the microRNA binding site located downstream of the 3'-UTR can bind to microRNAs that include microRNAs expressed in neurons and/or microRNAs expressed in glial cells.
  • MicroRNAs expressed in neurons include, but are not limited to, one or more of the following: miR-132-3p, miR-132-3p, miR-148b-3p, miR-148b-5p, miR-151a-3p, miR-151a-5p, miR-212-3p, miR-212-5p, miR-320b, miR-320e, miR-323a-3p, miR-323a-5p, miR-324-5p, miR-325, miR-326, miR-328, and miR-922.
  • MicroRNAs expressed in glial cells include, but are not limited to, one or more of the following: miR-1250, miR-219-1-3p, miR-219-2-3p, miR-219-5p, miR-23a-3p, miR-23a-5p, miR-3065-3p, miR-3065-5p, miR-30e-3p, miR-30e-5p, miR-32-5p, miR-338-5p, and miR-657.
  • the microRNA that the microRNA binding site located downstream of the 3'-UTR can bind to includes a microRNA expressed in the pancreas.
  • the microRNA expressed in the pancreas includes, but is not limited to, one or more of the following: miR-105-3p, miR-105-5p, miR-184, miR-195-3p, miR-195-5p, miR-196a-3p, miR-196a-5p, miR-214-3p, miR-214-5p, miR-216a-3p, miR-216a-5p, miR-30a-3p, miR-33a-3p, miR-33a-5p, miR-375, miR-7-1-3p, miR-7-2-3p, miR-493-3p, miR-493-5p, and miR-944.
  • the microRNA binding site located downstream of the 3'-UTR can bind to microRNAs that include microRNAs expressed in the kidney.
  • MicroRNAs expressed in the kidney include, but are not limited to, one or more of the following: miR-122-3p, miR-145-5p, miR-17-5p, miR-192-3p, miR-192-5p, miR-194-3p, miR-194-5p, miR-20a-3p, miR-20a-5p, miR-204-3p, miR-204-5p, miR-210, miR-216a-3p, miR-217a-5p, miR-218a-3p, miR-219a-5p, miR-220a-3p, miR-221a-5p, miR-222a-3p, miR-222a-5p, miR-223a-3p, miR-223a-5p, miR-224a-3p, miR-224a-5p, miR-226a-3p, miR-227a-5p, miR-228a-3
  • the microRNA binding site located downstream of the 3'-UTR can bind to a microRNA that includes a microRNA expressed in muscle.
  • the microRNA expressed in muscle includes, but is not limited to, one or more of the following: let-7g-3p, let-7g-5p, miR-1, miR-1286, miR-133a, miR-133b, miR-140-3p, miR-143-3p, miR-143-5p, miR-145-3p, miR-145-5p, miR-188-3p, miR-188-5p, miR-206, miR-208a, miR-208b, miR-25-3p, and miR-25-5p.
  • the microRNA binding site located downstream of the 3'-UTR can bind to microRNAs that include microRNAs expressed in endothelial cells.
  • the microRNAs expressed in endothelial cells include, but are not limited to, one or more of the following: let-7b-3p, let-7b-5p, miR-100-3p, miR-100-5p, miR-101-3p, miR-101-5p, miR-126-3p, miR-126-5p, miR-1236-3p, miR-1236-5p, miR-130a -3p,miR-130a-5p,miR-17-5p,miR-17-3p,miR-18a-3p,miR-18a-5p,miR-19a-3p,miR-19 a-5p,miR-19b-l-5p,miR-19b-2-5p,miR-19b-3p,miR-20a-3p,miR-20a-5p,miR-217,miR -210,m
  • the microRNA binding site located downstream of the 3'-UTR can bind to a microRNA that includes a microRNA expressed in epithelial cells.
  • the microRNA expressed in epithelial cells includes, but is not limited to, one or more of the following: let-7b-3p, let-7b-5p, miR-1246, miR-200a-3p, miR-200a-5p, miR-200b-3p, miR-200b-5p, miR-200c-3p, miR-200c-5p, miR-338-3p, miR-429, miR-451a, miR-451b, miR-494, and miR-802.
  • microRNAs expressed in respiratory ciliated epithelial cells include, but are not limited to, one or more of the following: miR-34a, miR-34b-5p, miR-34c-5p, miR-449a, miR-449b-3p, and miR-449b-5p.
  • MicroRNAs expressed in lung epithelial cells include, but are not limited to, one or more of the following: the let-7 family, miR-133a, miR-133b, and miR-126.
  • MicroRNAs expressed in renal tubular epithelial cells include, but are not limited to, one or more of the following: miR-382-3p and miR-382-5p.
  • MicroRNAs expressed in corneal epithelial cells include, but are not limited to, miR-762.
  • the microRNA binding site located downstream of the 3'-UTR can bind to a microRNA that includes a microRNA expressed in embryonic stem cells.
  • the microRNA expressed in embryonic stem cells includes, but is not limited to, one or more of the following: let-7a-2-3p, let-a-3p, let-7a-5p, let7d-3p, let-7d-5p, miR-103a-2-3p, miR-103a-5p, miR-106b-3p, miR-106b-5p, miR-1246, miR-1275, miR-138-1-3p, miR-138-2-3p, miR-138-5p, miR-154 -3p,miR-154-5p,miR-200c-3p,miR-200c-5p,miR-290,miR-301a-3p,miR-301a-5p,miR-302a-3p,miR-302a-5p,mi R-302b-3p,miR-302b-5p,m,m
  • the microRNA expressed in embryonic stem cells includes but is not limited to one or more of those mentioned in Morin RD et al, Genome Res, 2008, 18, 610-621, Goff LA et al, PLoS One, 2009, 4: e7192, and Bar M et al, Stem cells, 2008, 26, 2496-2505.
  • the microRNA binding site located downstream of the 3'-UTR can bind to microRNAs including microRNAs expressed in abnormal cells.
  • Some microRNAs are abnormally overexpressed in certain abnormal cells (e.g., cancer cells), while other microRNAs are underexpressed in certain abnormal cells.
  • cells, tissues, or diseases in which microRNAs are differentially expressed include: cancer cells (WO2008/154098, US2013/0059015, US2013/0042333, WO2011/157294), cancer stem cells (US2012/0053224), pancreatic cancer and diseases (US2009/0131348, US2011/0171646, US2010/0286232, US8 389210), asthma and inflammation (US8415096), prostate cancer (US2013/0053264), hepatocellular carcinoma (WO2012/151212, US2012/0329672, WO2008/054828, US8252538), lung cancer cells (WO2011/076143, WO2013/033640, WO2009/070653, US2010/0323357), skin T-cell lymphoma (WO2013/011378), colorectal cancer cells (WO2011/0281756, WO2011/076142), cancer-positive lymph nodes (WO2009/100430, US2009/0263803), nasopharyngeal carcinoma (EP21
  • the microRNA binding site located downstream of the 3'-UTR can bind to one or more of the following microRNAs:
  • the non-natural nucleic acid comprises two or more (e.g., two, three, four or more) microRNA binding sites located downstream of the 3'-UTR, wherein:
  • microRNA binding site capable of binding to a microRNA in hematopoietic cells (e.g., miR-142, miR-144, miR-150, miR-155, or miR-223), and at least one microRNA binding site capable of binding to a microRNA in plasmacytoid dendritic cells, platelets, or endothelial cells (e.g., miR-126);
  • microRNA binding site capable of binding to a microRNA in B cells (e.g., miR-142, miR-150, miR-16, or miR-223), and at least one microRNA binding site capable of binding to a microRNA in plasmacytoid dendritic cells, platelets, or endothelial cells (e.g., miR-126);
  • B cells e.g., miR-142, miR-150, miR-16, or miR-223
  • microRNA binding site capable of binding to a microRNA in plasmacytoid dendritic cells, platelets, or endothelial cells (e.g., miR-126);
  • microRNA binding site capable of binding to a microRNA in progenitor hematopoietic cells (e.g., miR-223, miR-451, miR-26a, or miR-16), and at least one microRNA binding site capable of binding to a microRNA in plasmacytoid dendritic cells, platelets, or endothelial cells (e.g., miR-126);
  • progenitor hematopoietic cells e.g., miR-223, miR-451, miR-26a, or miR-16
  • microRNA binding site capable of binding to a microRNA in plasmacytoid dendritic cells, platelets, or endothelial cells (e.g., miR-126);
  • (4) at least one microRNA binding site that binds to a microRNA of hematopoietic lineage cells e.g., miR-142, miR-144, miR-150, miR-155, or miR-223
  • a microRNA of B cells e.g., miR-142, miR-150, miR-16, or miR-223
  • at least one microRNA binding site that binds to plasmacytoid dendritic cells, platelets, or endothelial cells e.g., miR-126
  • microRNA binding sites i.e., binding to hematopoietic lineage cells, binding to B cells, binding to progenitor hematopoietic cells, and/or binding to plasmacytoid dendritic cells/platelets/endothelial cells.
  • the non-natural nucleic acid comprises one or more microRNA binding sites located downstream of the 3'-UTR, and the microRNA binding sites located downstream of the 3'-UTR can bind to one or more of the following microRNAs: miR-122, miR-126, miR-142-3p, miR-142-5p, miR-144, miR-146-3p, miR-146-5p, miR-155, miR-16, miR-21, miR-223, miR-24, and miR-27.
  • the microRNA that the microRNA binding site is capable of binding is miR-142-3p.
  • the microRNA that the microRNA binding site is capable of binding is miR-122.
  • the microRNA that the microRNA binding site is capable of binding is miR-126.
  • the microRNA binding site located downstream of the 3'-UTR can bind to a microRNA that includes or is: miR-142-3p and one or more of the following: miR-142-5p, miR-146-3p, miR-146-5p, miR-155, miR-126, miR-16, miR-21, miR-223, miR-24 and miR-27.
  • the microRNA binding site located downstream of the 3'-UTR can bind to a microRNA that includes or is: miR-142-5p and one or more of the following: miR-142-3p, miR-146-3p, miR-146-5p, miR-155, miR-126, miR-16, miR-21, miR-223, miR-24 and miR-27.
  • the microRNA binding site located downstream of the 3’-UTR can bind to microRNAs including or including miR-126 and one or more of the following: miR-142-3p, miR-142-5p, miR-146-3p, miR-146-5p, miR-155, miR-16, miR-21, miR-223, miR-24 and miR-27.
  • the microRNA binding site located downstream of the 3'-UTR can bind to microRNAs including or including miR-122 and one or more of the following: miR-142-3p, miR-142-5p, miR-146-3p, miR-146-5p, miR-155, miR-126, miR-16, miR-21, miR-223, miR-24and miR-27.
  • MiR-142, miR-126, miR-146 and miR-155 are expressed in large quantities in immune cells.
  • These microRNA sequences are known in the art, and therefore, one of ordinary skill in the art can easily design binding sequences or target sequences to which these microRNAs will bind based on Watson-Crick complementarity.
  • the non-natural nucleic acid comprises at least two microRNA binding sites located downstream of the 3'-UTR and capable of binding to a microRNA expressed in immune cells, wherein at least one microRNA binding site is capable of binding to miR-142-3p.
  • the non-natural nucleic acid comprises at least two microRNA binding sites located downstream of the 3'-UTR and capable of binding to a microRNA expressed in an immune cell, wherein:
  • At least one microRNA binding site capable of binding to miR-142-3p, and at least one microRNA binding site capable of binding to miR-155 e.g., miR-155-3p or miR-155-5p
  • At least one microRNA binding site is capable of binding to miR-142-3p, and at least one microRNA binding site is capable of binding to miR-146 (e.g., miR-146-3 or miR-146-5p); or
  • At least one microRNA binding site is capable of binding to miR-142-3p, and at least one microRNA binding site is capable of binding to miR-126 (e.g., miR-126-3p or miR-126-5p).
  • miR-126 e.g., miR-126-3p or miR-126-5p.
  • the non-natural nucleic acid comprises at least two microRNA binding sites located downstream of the 3'-UTR and capable of binding to a microRNA expressed in immune cells, wherein at least one microRNA binding site is capable of binding to miR-126-3p.
  • the non-natural nucleic acid comprises at least two microRNA binding sites located downstream of the 3'-UTR and capable of binding to a microRNA expressed in an immune cell, wherein:
  • At least one microRNA binding site is capable of binding to miR-126-3p, and at least one microRNA binding site is capable of binding to miR-155 (e.g., miR-155-3p or miR-155-5p);
  • At least one microRNA binding site is capable of binding to miR-126-3p, and at least one microRNA binding site is capable of binding to miR-146 (e.g., miR-146-3p or miR-146-5p); or
  • At least one microRNA binding site is capable of binding to miR-126-3p, and at least one microRNA binding site is capable of binding to miR-142 (e.g., miR-142-3p or miR-142-5p).
  • miR-142 e.g., miR-142-3p or miR-142-5p.
  • the non-natural nucleic acid comprises at least two microRNA binding sites located downstream of the 3'-UTR and capable of binding to a microRNA expressed in immune cells, wherein at least one microRNA binding site is capable of binding to miR-142-5p.
  • the non-natural nucleic acid comprises at least two microRNA binding sites located downstream of the 3'-UTR and capable of binding to a microRNA expressed in an immune cell, wherein:
  • At least one microRNA binding site capable of binding to miR-142-5p, and at least one microRNA binding site capable of binding to miR-155 e.g., miR-155-3p or miR-155-5p
  • At least one microRNA binding site is capable of binding to miR-142-5p, and at least one microRNA binding site is capable of binding to miR-146 (e.g., miR-146-3 or miR-146-5p); or
  • At least one microRNA binding site is capable of binding to miR-142-5p, and at least one microRNA binding site is capable of binding to miR-126 (e.g., miR-126-3p or miR-126-5p).
  • miR-126 e.g., miR-126-3p or miR-126-5p.
  • the non-natural nucleic acid comprises at least two microRNA binding sites located downstream of the 3'-UTR and capable of binding to a microRNA expressed in immune cells, wherein at least one microRNA binding site is capable of binding to miR-155-5p.
  • the non-natural nucleic acid comprises at least two microRNA binding sites located downstream of the 3'-UTR and capable of binding to microRNA expressed in immune cells, wherein:
  • At least one microRNA binding site capable of binding to miR-155-5p at least one microRNA binding site capable of binding to miR-142 (e.g., miR-142-3p or miR-142-5p);
  • At least one microRNA binding site is capable of binding to miR-155-5p, and at least one microRNA binding site is capable of binding to miR-146 (e.g., miR-146-3 or miR-146-5p); or
  • At least one microRNA binding site is capable of binding to miR-155-5p, and at least one microRNA binding site is capable of binding to miR-126 (e.g., miR-126-3p or miR-126-5p).
  • miR-126 e.g., miR-126-3p or miR-126-5p.
  • the non-natural nucleic acid comprises one or more microRNA binding sites located downstream of the 3'-UTR and capable of binding to miR-142 (e.g., miR-142-3p or miR-142-5p).
  • miR-142 e.g., miR-142-3p or miR-142-5p.
  • the non-natural nucleic acid comprises one or more microRNA binding sites located downstream of the 3'-UTR and capable of binding to miR-122 (e.g., miR-122-3p or miR-122-5p).
  • miR-122 e.g., miR-122-3p or miR-122-5p.
  • the non-natural nucleic acid comprises one or more microRNA binding sites located downstream of the 3'-UTR and capable of binding to miR-126 (e.g., miR-126-3p or miR-126-5p).
  • miR-126 e.g., miR-126-3p or miR-126-5p.
  • the microRNA binding site located downstream of the 3'-UTR is capable of binding to a microRNA that is expressed more highly in one cell, tissue, or organ than in another cell, tissue, or organ.
  • the microRNA binding site located downstream of the 3'-UTR is capable of binding to a microRNA that is expressed at a lower level in cancer cells than in non-cancerous cells of the same tissue.
  • the non-natural nucleic acid is present in cancer cells expressing such a low level of microRNA, the polypeptide or protein encoded by the non-natural nucleic acid will typically show increased expression. If the polypeptide or protein is capable of inducing apoptosis, this may result in cancer cells being killed more easily than normal cells. For example, liver cancer cells typically express low levels of miR-122 compared to normal liver cells.
  • a non-natural nucleic acid e.g., mRNA
  • a polypeptide or protein comprising at least one miR-122 binding site (e.g., in the poly(A) tail of the mRNA) will typically express relatively low levels of the polypeptide or protein in normal liver cells, and relatively high levels of the polypeptide in liver cancer cells. If the polypeptide or protein is capable of inducing apoptosis, this will result in liver cancer cells being killed preferentially compared to normal cells.
  • the microRNA binding site is a polynucleotide or a variant thereof that is complementary to the full-length microRNA or a portion of the microRNA.
  • the variant retains the ability of the microRNA binding site to bind to the microRNA and can achieve the binding of the microRNA to the mRNA where the microRNA binding site is located.
  • the microRNA binding site can be completely complementary to the full-length microRNA or a portion of the microRNA, or it can be incompletely complementary to the full-length microRNA or a portion of the microRNA. Therefore, in some embodiments, the microRNA binding site located downstream of the 3'-UTR can bind to the miroRNA with complete complementary pairing.
  • the microRNA binding site located downstream of the 3'-UTR can bind to the partial miroRNA with complete complementary pairing. In some embodiments, the microRNA binding site located downstream of the 3'-UTR can bind to the miroRNA with incomplete complementary pairing. In some embodiments, the microRNA binding site located downstream of the 3'-UTR can bind to the partial miroRNA with incomplete complementary pairing. In some embodiments, the microRNA binding site located downstream of the 3'-UTR can bind to the partial miroRNA with incomplete complementary pairing.
  • the length of the microRNA binding site located downstream of the 3'-UTR is 19 nt to 25 nt. In some embodiments, the length of the microRNA binding site located downstream of the 3'-UTR is 20 nt to 24 nt.
  • the non-natural nucleic acid comprises multiple microRNA binding sites downstream of the 3'-UTR, and each of the multiple microRNA binding sites downstream of the 3'-UTR is independently 19 nt to 25 nt in length. In some embodiments, the non-natural nucleic acid comprises multiple microRNA binding sites downstream of the 3'-UTR, and each of the multiple microRNA binding sites downstream of the 3'-UTR is independently 20 nt to 24 nt in length.
  • the non-natural nucleic acid comprises a plurality of microRNA binding sites located downstream of the 3'-UTR, and a spacer sequence is present between the plurality of microRNA binding sites located downstream of the 3'-UTR.
  • a plurality of microRNA binding sites located downstream of the 3'-UTR are separated by spacer sequences. In other embodiments, a plurality of microRNA binding sites located downstream of the 3'-UTR are separated by spacer sequences, while the remaining microRNA binding sites are not separated by spacer sequences.
  • the non-natural nucleic acid comprises three microRNA binding sites located downstream of the 3'-UTR, two of which are separated by spacer sequences, and the remaining microRNA binding site is directly linked to one of the aforementioned microRNA binding sites without a spacer sequence.
  • the non-natural nucleic acid comprises multiple microRNA binding sites downstream of the 3'-UTR, and the multiple microRNA binding sites downstream of the 3'-UTR are directly linked without any spacer sequence.
  • the non-natural nucleic acid comprises three microRNA binding sites downstream of the 3'-UTR, and the three microRNA binding sites are directly linked to form the DNA sequence shown in SEQ ID NO: 5.
  • the non-natural nucleic acid comprises a 3'-UTR, a ploy (A) tail located downstream of the 3'-UTR, and one or more microRNA binding sites, wherein the one or more microRNA binding sites are located at one of the following positions:
  • one or more microRNA binding sites are located in the poly(A) tail.
  • the microRNA binding site located in the poly(A) tail is located at the 5' end, between the 5' end and the 3' end, and/or at the 3' end of the poly(A) tail.
  • the microRNA binding site located in the poly(A) tail is located at the 5' end of the poly(A) tail.
  • the non-natural nucleic acid comprises a 3'-UTR, a poly(A) tail located downstream of the 3'-UTR, and a plurality of microRNA binding sites located after the 3'-UTR and before the poly(A) tail.
  • the non-natural nucleic acid comprises a 3'-UTR, a poly(A) tail located downstream of the 3'-UTR, and a plurality of microRNA binding sites located after the 3'-UTR and before the poly(A) tail, wherein at least two of the plurality of microRNA binding sites have a spacer sequence between them.
  • the non-natural nucleic acid comprises a 3'-UTR, a poly(A) tail located downstream of the 3'-UTR, and one or more microRNA binding sites located in the poly(A) tail. In some embodiments, the non-natural nucleic acid comprises a 3'-UTR, a poly(A) tail located downstream of the 3'-UTR, and multiple microRNA binding sites located in the poly(A) tail. In some embodiments, the non-natural nucleic acid comprises a 3'-UTR, a poly(A) tail located downstream of the 3'-UTR, and multiple microRNA binding sites located in the poly(A) tail, and at least two of the multiple microRNA binding sites have a spacer sequence between them.
  • the non-natural nucleic acid comprises a 3'-UTR, a poly(A) tail located downstream of the 3'-UTR, and a plurality of microRNA binding sites located in the poly(A) tail, wherein in the 5' to 3' direction, there are 2 to 20 nucleotides (e.g., A nucleotides) between the first microRNA binding site in the poly(A) tail and the 3'-UTR.
  • the non-natural nucleic acid comprises a 3'-UTR, a poly(A) tail downstream of the 3'-UTR, and a plurality of microRNA binding sites in the poly(A) tail, wherein in the 5' to 3' direction, there are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides between the first microRNA binding site in the poly(A) tail and the 3'-UTR.
  • the non-natural nucleic acid comprises a 3'-UTR, a poly(A) tail located downstream of the 3'-UTR, and a plurality of microRNA binding sites located in the poly(A) tail, wherein in the 5' to 3' direction, there are 2 to 20 A nucleotides between the first microRNA binding site located in the poly(A) tail and the 3'-UTR.
  • the non-natural nucleic acid comprises a 3'-UTR, a poly(A) tail located downstream of the 3'-UTR, and a plurality of microRNA binding sites located in the poly(A) tail, and in the 5' to 3' direction, there are 3 to 17 nucleotides (e.g., A nucleotides) between the first microRNA binding site in the poly(A) tail and the 3'-UTR.
  • the non-natural nucleic acid comprises a 3'-UTR, a poly(A) tail located downstream of the 3'-UTR, and a plurality of microRNA binding sites located in the poly(A) tail, and in the 5' to 3' direction, there are 9 to 17 nucleotides (e.g., A nucleotides) between the first microRNA binding site in the poly(A) tail and the 3'-UTR.
  • the non-natural nucleic acid comprises a 3'-UTR, a poly(A) tail located downstream of the 3'-UTR, and a plurality of microRNA binding sites located in the poly(A) tail, wherein there is a spacer sequence between at least two of the plurality of microRNA binding sites, and in the 5' to 3' direction, there are 3 to 17 nucleotides (e.g., A nucleotides) between the first microRNA binding site in the poly(A) tail and the 3'-UTR.
  • nucleotides e.g., A nucleotides
  • the non-natural nucleic acid comprises a 3'-UTR, a poly(A) tail located downstream of the 3'-UTR, and a plurality of microRNA binding sites located in the poly(A) tail, wherein at least two of the plurality of microRNA binding sites are separated by a spacer sequence, and in the 5' to 3' direction, there are 9 to 17 nucleotides (e.g., A nucleotides) between the first microRNA binding site in the poly(A) tail and the 3'-UTR.
  • nucleotides e.g., A nucleotides
  • the non-natural nucleic acid comprises a 3'-UTR, a poly(A) tail downstream of the 3'-UTR, and a plurality of microRNA binding sites downstream of the 3'-UTR, wherein at least one microRNA binding site is located in the poly(A) tail, and at least one microRNA binding site is located after the 3'-UTR and before the poly(A) tail.
  • the non-natural nucleic acid comprises a 3'-UTR, a poly(A) tail downstream of the 3'-UTR, and a plurality of microRNA binding sites downstream of the 3'-UTR, wherein at least two microRNA binding sites are located in the poly(A) tail, and at least two microRNA binding sites are located after the 3'-UTR and before the poly(A) tail.
  • there is a spacer sequence between the plurality of microRNA binding sites located in the poly(A) tail and/or there is a spacer sequence between the plurality of microRNA binding sites located after the 3'-UTR and before the poly(A) tail.
  • the microRNA binding site located downstream of the 3'-UTR is codon-optimized.
  • the microRNA binding site located downstream of the 3'-UTR or its corresponding DNA sequence comprises a nucleotide sequence as shown in ACACTAC, SEQ ID NO: 1 or 13. In some embodiments, the microRNA binding site located downstream of the 3'-UTR or its corresponding DNA sequence comprises a nucleotide sequence as shown in ACACTAC, SEQ ID NO: 1 or 13.
  • the 3'-UTR comprised by the non-natural nucleic acid comprises a 3'-UTR derived from one or more of the following: a ⁇ -globin gene (e.g., Karikó, Katalin, et al.
  • human growth hormone (hGH) gene e.g., US20140206753, WO2013185069, WO2014089486, WO2014144196, WO2014152659, WO2014152940, WO2014152774, WO2014153052
  • ribosomal rps9 protein gene e.g., WO2015101414
  • FIG4 gene e.g., WO2015101415
  • human albumin 7 gene e.g., WO2015101415, WO2015101414, WO201506273, WO2015024667, WO2105062737
  • viruses e.g., WO2015101415, WO2015101414, WO201506273, WO2015024667, WO2105062737
  • the 3'-UTR derived from a virus includes: the 3'-UTR of Venezuelan equine encephalitis virus (VEEV) (e.g., Andries, Oliwia, et al. "N1-methylpseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice.” Journal of Controlled Release 217(2015):337-344.
  • VEEV Venezuelan equine encephalitis virus
  • the 3’-UTR or its corresponding DNA sequence is as shown in SEQ ID NO: 2 or 3. It should be noted that the “DNA sequence corresponding to the 3’-UTR” refers to the 3’-UTR in DNA form, and the same applies to the “DNA sequence of the 5’-UTR” and the “DNA corresponding to the poly(A) tail” below. It is understood that in other embodiments, the 3’-UTR is not limited to the above, and can also be others, such as the 3’-UTR described in patents such as WO2017059902, WO2013143700, and WO2017001554.
  • 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. 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 consecutively.
  • the nucleotides comprising the poly(A) tail comprise one or more nucleotides other than A nucleotides.
  • poly(A) tail or its corresponding DNA sequence is shown as SEQ ID NO: 6.
  • poly (A) tail contained in the non-natural nucleic acid (e.g., RNA) disclosed herein is not limited to the above, and may also be other poly (A) tails, such as the poly (A) tails described in patents such as US20170166905 and WO2020074642.
  • the non-natural nucleic acid further comprises one or more of the following: a coding region encoding a polypeptide or protein of interest, a 5'-UTR, an internal ribosome entry site (IRES), and a coding region encoding a 2A peptide (2A self-cleaving peptides).
  • the non-natural nucleic acid further comprises a coding region encoding a polypeptide or protein of interest, and the coding region encoding the polypeptide or protein of interest is located upstream of the 3'-UTR.
  • the non-natural nucleic acid comprises a 3'-UTR and a coding region encoding a polypeptide or protein of interest that are heterologous.
  • the coding region encodes a polypeptide or protein of interest that is ⁇ -galactosidase
  • the 3'-UTR is a 3'-UTR from a ⁇ -globin gene.
  • the coding region comprising the non-natural nucleic acid encoding the polypeptide or protein of interest is codon-optimized.
  • a polypeptide or protein of interest refers to a therapeutically or pharmaceutically active polypeptide or protein having a therapeutic or prophylactic effect, whose function in or near a cell is desirable or beneficial.
  • a protein whose absence or defective form leads to a disease, and whose provision can modulate or prevent the disease, or a protein whose presence in or near a cell is beneficial to the body.
  • a polypeptide or protein of interest can comprise the entire protein or a functional variant thereof.
  • the polypeptide and/or protein expressed by the above-mentioned non-natural nucleic acid containing the coding region encoding the polypeptide and/or protein of interest comprises or is one or more of the following: (a) an antigen; (b) a therapeutic polypeptide or protein, a fragment, fragment or variant thereof; and (c) other polypeptides or proteins.
  • the peptide and/or protein expressed by the non-natural nucleic acid comprising a coding region encoding a polypeptide or protein of interest comprises or is an antigen.
  • the antigen expressed by the above-mentioned non-natural nucleic acid containing the coding region encoding the polypeptide or protein of interest is derived from one or more of the following: (1) pathogenic antigens, fragments, variants or variants of fragments thereof, (2) tumor antigens, fragments, variants or variants of fragments thereof, (3) allergic antigens, fragments, variants or variants of fragments thereof, (4) autoimmune self-antigens, fragments, variants or variants of fragments thereof.
  • 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).
  • the pathogenic organisms include or are one or more of the following: bacteria, viruses, fungi, and protozoa (e.g., single-cell organisms, multicellular organisms).
  • 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.
  • the pathogenic antigen comprises or is derived from a polypeptide or protein of a pathogen associated with an infectious disease.
  • 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.
  • the tumor antigen is selected from but not limited to the group consisting of the tumor antigens described in WO2018/078053A1, pages 47-51.
  • the antigens expressed by the non-natural nucleic acid comprising a coding region encoding a polypeptide or protein of interest include or are allergic antigens and autoimmune self-antigens.
  • 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.
  • the antigen expressed by the above-mentioned non-natural nucleic acid containing a coding region encoding a polypeptide or protein of interest is listed on pages 48 to 51 of WO2018/078053A1.
  • the polypeptide and/or protein expressed by the non-natural nucleic acid comprising a coding region encoding a polypeptide or protein of interest comprises or is a therapeutic polypeptide or protein.
  • the therapeutic polypeptide or protein includes or is one or more of the following:
  • Enzyme replacement therapy for the treatment of metabolic, endocrine or amino acid disorders or therapeutic polypeptides or proteins for replacing missing, defective or mutated proteins (2) Therapeutic polypeptides or proteins for the treatment of blood diseases, circulatory system diseases, respiratory system diseases, infectious diseases or immune deficiencies; (3) Therapeutic polypeptides or proteins for the treatment of cancer or tumor diseases; (4) Therapeutic polypeptides or proteins for hormone replacement therapy; (5) Therapeutic polypeptides or proteins for reprogramming somatic cells into pluripotent stem cells or totipotent stem cells; (6) Therapeutic polypeptides or proteins used as adjuvants or immunostimulants; (7) Therapeutic polypeptides or proteins as therapeutic antibodies; (8) Therapeutic polypeptides or proteins as gene editing agents; (9) Therapeutic polypeptides or proteins for the treatment or prevention of liver diseases selected from the group consisting of liver fibrosis, cirrhosis and liver cancer; and (10) Therapeutic polypeptides or proteins for the treatment or prevention of rare diseases.
  • the polypeptide or protein of interest is one or more of the following: a therapeutic protein, a cytokine, a growth factor, an antibody, or a fusion protein.
  • the coding region encoding a polypeptide or protein of interest comprises non-coding sequences (e.g., introns). It is understood that non-coding sequences can be removed through post-transcriptional modification. In other embodiments, the coding region encoding a polypeptide or protein of interest does not contain non-coding sequences.
  • the non-natural nucleic acid comprises one or more coding regions encoding a polypeptide or protein of interest.
  • the non-natural nucleic acid comprises multiple coding regions encoding polypeptides or proteins of interest, and the multiple coding regions encoding polypeptides or proteins of interest encode the same polypeptide or protein.
  • the non-natural nucleic acid comprises multiple coding regions encoding polypeptides or proteins of interest, and the multiple coding regions encoding polypeptides or proteins of interest encode different polypeptides or proteins.
  • the non-natural nucleic acid comprises a plurality of coding regions encoding polypeptides or proteins of interest, wherein the plurality of coding regions encoding polypeptides or proteins of interest encode different polypeptides or proteins, and a coding region encoding a 2A peptide is located between the coding regions encoding different polypeptides or proteins.
  • the non-natural nucleic acid further comprises a 5'-UTR, and the 5'-UTR is located upstream of the 3'-UTR.
  • the 5'-UTR contained in the non-natural nucleic acid includes a 5'-UTR derived from one or more of the following: ⁇ -globin gene (e.g., Kariko et al. (2008) Mol. Therap. 16: 1833-1840, US 8,278,063, US9012219, etc.), ⁇ -globin gene (e.g., US 9012219), human cytochrome b-245a polypeptide gene (CYBA) (e.g., Ferizi, Mehrije, et al.
  • CYBA human cytochrome b-245a polypeptide gene
  • TOP gene for example, WO2015101414, WO2015101415, WO2015062738, WO2015024667, etc.
  • L32 ribosomal protein large 32
  • ATP5A1 gene for example, WO2015024667
  • the 5'-UTR derived from a virus includes one or more 5'-UTRs of the following: a 5'-UTR from tobacco erosion virus (TEV) (e.g., Karikó, Katalin, et al. "Increased erythropoiesis in mice injected with submicrogram quantities of pseudouridine-containing mRNA encoding erythropoietin.” Molecular Therapy 20.5(2012):948-953, US8278063, US9012219, etc.), a 5'-UTR from Venezuelan equine encephalitis virus (VEEV) (e.g., Andries, Oliwia, et al.
  • TEV tobacco erosion virus
  • VEEV Venezuelan equine encephalitis virus
  • N1-methylpseudouridine-in Corporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice.” Journal of Controlled Release, 217(2015):337-344) and cytomegalovirus immediate early 1 (IE1) gene (e.g., US20140206753, WO2014089486, WO2013185069, WO2014144196, WO2014152659, WO2014152940, WO2014152774, WO2014153052, etc.).
  • IE1 cytomegalovirus immediate early 1
  • the 5'-UTR or its corresponding DNA sequence is as shown in SEQ ID NO: 4. It is understood that in other embodiments, the 5'-UTR is not limited to the above, and can also be other, such as the 5'-UTR described in patents such as WO2017059902, WO2013143700, and WO2017001554.
  • the non-natural nucleic acid further comprises a coding region encoding a polypeptide or protein of interest and a 5'-UTR, the coding region encoding the polypeptide or protein of interest is located upstream of the 3'-UTR, and the 5'-UTR is located upstream of the coding region encoding the polypeptide or protein of interest.
  • the non-natural nucleic acid comprises a 5'-UTR, a coding region encoding a polypeptide or protein of interest, a 3'-UTR, a poly(A) tail, and one or more microRNA binding sites located in the poly(A) tail.
  • the non-natural nucleic acid comprises a 5'-UTR that is heterologous to the coding region encoding the polypeptide or protein of interest.
  • the coding region encodes an ⁇ -galactosidase polypeptide or protein of interest
  • the 5'-UTR is the 5'-UTR from the ⁇ -globin gene.
  • the non-natural nucleic acid comprises a 5'-UTR and a 3'-UTR that are heterologous to the coding region encoding the polypeptide or protein of interest.
  • the non-natural nucleic acid comprises a 5'-UTR, a coding region encoding a polypeptide or protein of interest, a 3'-UTR, a poly(A) tail, and one or more microRNA binding sites, wherein the one or more microRNA binding sites are located downstream of the 3'-UTR, and at least one of the 5'-UTR and the 3'-UTR is heterologous to the coding region encoding the polypeptide or protein of interest.
  • the non-natural nucleic acid comprises a 5'-UTR, a coding region encoding a polypeptide or protein of interest, a 3'-UTR, a poly(A) tail, and one or more microRNA binding sites, wherein the one or more microRNA binding sites are located in the poly(A) tail, and at least one of the 5'-UTR and the 3'-UTR is heterologous to the coding region encoding the polypeptide or protein of interest.
  • the non-natural nucleic acid comprises a 5'-UTR, a coding region encoding a polypeptide or protein of interest, a 3'-UTR, a poly(A) tail, and one or more microRNA binding sites, wherein the one or more microRNA binding sites are located downstream of the 3'-UTR, and both the 5'-UTR and the 3'-UTR are heterologous to the coding region encoding the polypeptide or protein of interest.
  • the non-natural nucleic acid comprises a 5'-UTR, a coding region encoding a polypeptide or protein of interest, a 3'-UTR, a poly(A) tail, and one or more microRNA binding sites, wherein the one or more microRNA binding sites are located after the 3'-UTR and before the poly(A), and/or in the poly(A) tail, and the 5'-UTR and the 3'-UTR are heterologous to the coding region encoding the polypeptide or protein of interest.
  • the non-natural nucleic acid comprises a 5'-UTR, a coding region encoding a polypeptide or protein of interest, a 3'-UTR, a poly(A) tail, and one or more microRNA binding sites, wherein the one or more microRNA binding sites are located in the poly(A) tail, and the 5'-UTR and the 3'-UTR are heterologous to the coding region encoding the polypeptide or protein of interest.
  • the non-natural nucleic acid comprises a 5'-UTR, a coding region encoding a polypeptide or protein of interest, a 3'-UTR, a poly(A) tail, and one or more microRNA binding sites, wherein the one or more microRNA binding sites are located after the 3'-UTR and before the poly(A), and/or in the poly(A) tail, wherein:
  • nucleotide sequence of the microRNA binding site or its corresponding DNA is represented by ACACTAC, SEQ ID NO: 1 or 13;
  • nucleotide sequence of the 3'-UTR or its corresponding DNA is shown in SEQ ID NO: 2 or 3;
  • nucleotide sequence of 5’-UTR or its corresponding DNA is shown in SEQ ID NO: 4.
  • the non-natural nucleic acid further comprises at least one microRNA binding site located in the 3'-UTR and/or the 5'-UTR.
  • the non-natural nucleic acid further comprises one or more microRNA binding sites located in the 5'-UTR.
  • the non-natural nucleic acid further comprises one or more microRNA binding sites located in the 3'-UTR.
  • the non-natural nucleic acid further comprises one or more microRNA binding sites located in the 5'-UTR and one or more microRNA binding sites located in the 3'-UTR.
  • the specific position, length, number, source of the bound microRNA, and degree of complementarity with the bound microRNA of the microRNA binding site located in the 3'-UTR and/or the 5'-UTR in the UTR are not particularly limited.
  • the position of the microRNA binding site located in the 3'-UTR and/or the 5'-UTR in the UTR is as described in WO2017062513A1.
  • the length of the microRNA binding site located in the 3'-UTR and/or the 5'-UTR is 2nt to 25nt.
  • the number of microRNA binding sites located in the 3'-UTR and/or the 5'-UTR is one or more (e.g., 2, 3, 4, 5, or 6).
  • the number of microRNA binding sites located in the 3'-UTR and/or the 5'-UTR is 2 to 4.
  • the source of the microRNA that can be bound by the microRNA binding site located in the 3'-UTR and/or the 5'-UTR can be, but is not limited to, the description above.
  • the microRNA binding site located in the 3'-UTR and/or the 5'-UTR is partially complementary or fully complementary to the microRNA to which it can bind.
  • the non-natural nucleic acid further comprises a microRNA binding site located downstream of the 3'-UTR, and a microRNA binding site located in the 3'-UTR and/or the 5'-UTR, wherein the microRNA binding site located in the 3'-UTR and/or the 5'-UTR is the same as the microRNA binding site located downstream of the 3'-UTR.
  • the microRNA binding site located in the 3'-UTR and/or the 5'-UTR and the microRNA binding site located downstream of the 3'-UTR are both RNAs corresponding to the DNA shown in ACACTAC or SEQ ID NO: 1.
  • the microRNA binding site located in the 3'-UTR and/or in the 5'-UTR is different from the microRNA binding site located downstream of the 3'-UTR.
  • the microRNA binding site located in the 3'-UTR and/or the 5'-UTR is different from the microRNA binding site located downstream of the 3'-UTR, but the microRNA binding site located in the 3'-UTR and/or the 5'-UTR binds to the same microRNA as the microRNA binding site located downstream of the 3'-UTR.
  • the microRNA binding site located in the 3'-UTR and/or the 5'-UTR is different from the microRNA binding site located downstream of the 3'-UTR, but the microRNA that can be bound is miR-142-3p, wherein the microRNA binding site located in the 3'-UTR and/or the 5'-UTR is the RNA corresponding to the DNA shown as ACACTAC, and the microRNA binding site located downstream of the 3'-UTR is the RNA corresponding to the DNA shown as SEQ ID NO: 1.
  • the microRNA binding site located in the 3'-UTR and/or the 5'-UTR is different from the microRNA binding site located downstream of the 3'-UTR, and the microRNA binding site located in the 3'-UTR and/or the 5'-UTR binds to a different microRNA than the microRNA binding site located downstream of the 3'-UTR.
  • the microRNA binding site located in the 3'-UTR and/or the 5'-UTR is different from the microRNA binding site located downstream of the 3'-UTR, and the microRNA binding site located in the 3'-UTR and/or the 5'-UTR is different from the microRNA binding site located downstream of the 3'-UTR.
  • the microRNAs that the microRNA binding site located in the 3'-UTR and/or the 5'-UTR is different from the microRNA binding site located downstream of the 3'-UTR.
  • the microRNAs that the microRNA binding site located in the 3'-UTR and/or the 5'-UTR is different from the microRNA binding site located downstream of the 3'-UTR.
  • the non-natural nucleic acid further comprises a microRNA binding site located downstream of the 3'-UTR, and a microRNA binding site located in the 3'-UTR and/or the 5'-UTR, and the number of microRNA binding sites located in the 3'-UTR and/or the 5'-UTR is the same as the number of microRNA binding sites located downstream of the 3'-UTR.
  • the non-natural nucleic acid comprises three microRNA binding sites located in the 3'-UTR and/or the 5'-UTR and three microRNA binding sites located downstream of the 3'-UTR.
  • the number of microRNA binding sites located in the 3'-UTR and/or the 5'-UTR is different from the number of microRNA binding sites located downstream of the 3'-UTR.
  • the non-natural nucleic acid contains one or two microRNA binding sites located in the 3'-UTR and/or the 5'-UTR and three microRNA binding sites located downstream of the 3'-UTR.
  • the non-natural nucleic acid comprises a 5'-UTR, a coding region encoding a polypeptide or protein of interest, a 3'-UTR, a poly(A) tail, one or more microRNA binding sites located in the poly(A) tail, and one or more microRNA binding sites located after the 3'-UTR and before the poly(A).
  • the non-natural nucleic acid comprises a 5'-UTR, a coding region encoding a polypeptide or protein of interest, a 3'-UTR, a poly(A) tail, one or more microRNA binding sites in the poly(A) tail, and one or more microRNA binding sites in the 5'-UTR.
  • the non-natural nucleic acid comprises a 5'-UTR, a coding region encoding a polypeptide or protein of interest, a 3'-UTR, a poly(A) tail, one or more microRNA binding sites in the poly(A) tail, and one or more microRNA binding sites in the 3'-UTR.
  • the non-natural nucleic acid comprises a 5'-UTR, a coding region encoding a polypeptide or protein of interest, a 3'-UTR, a poly(A) tail, one or more microRNA binding sites located after the 3'-UTR and before the poly(A), and one or more microRNA binding sites located in the 5'-UTR.
  • the non-natural nucleic acid comprises a 5'-UTR, a coding region encoding a polypeptide or protein of interest, a 3'-UTR, a poly(A) tail, one or more microRNA binding sites located after the 3'-UTR and before the poly(A), and one or more microRNA binding sites located in the 3'-UTR.
  • the non-natural nucleic acid comprises a 5'-UTR, a coding region encoding a polypeptide or protein of interest, a 3'-UTR, a poly(A) tail, one or more microRNA binding sites in the poly(A) tail, one or more microRNA binding sites after the 3'-UTR and before the poly(A), and one or more microRNA binding sites in the 5'-UTR.
  • the non-natural nucleic acid comprises a 5'-UTR, a coding region encoding a polypeptide or protein of interest, a 3'-UTR, a poly(A) tail, one or more microRNA binding sites located in the poly(A) tail, one or more microRNA binding sites located after the 3'-UTR and before the poly(A), and one or more microRNA binding sites located in the 3'-UTR.
  • the non-natural nucleic acid comprises a 5'-UTR, a coding region encoding a polypeptide or protein of interest, a 3'-UTR, a poly(A) tail, one or more microRNA binding sites in the poly(A) tail, one or more microRNA binding sites after the 3'-UTR and before the poly(A), one or more microRNA binding sites in the 5'-UTR, and one or more microRNA binding sites in the 3'-UTR.
  • the non-natural nucleic acid does not comprise a microRNA binding site in the 3'-UTR and/or the 5'-UTR.
  • the non-natural nucleic acid is an artificially synthesized nucleic acid.
  • the non-natural nucleic acid is an artificially synthesized isolated nucleic acid.
  • the non-natural nucleic acid is RNA.
  • the non-natural nucleic acid is mRNA.
  • the microRNA binding site contained in the mRNA binds to the microRNA, triggering microRNA-mediated mRNA regulation, such as mRNA degradation or inhibition of mRNA translation, thereby reducing the expression of the protein encoded by the mRNA.
  • the microRNA contained in the mRNA is highly abundantly expressed or specific in cells, tissues and/or organs where expression is not desired, thereby reducing the expression of the protein encoded by the mRNA in cells, tissues and/or organs where expression is not desired.
  • the non-natural nucleic acid is mRNA, which contains a 3’-UTR, and the nucleotide sequence of the DNA corresponding to the 3’-UTR is shown in SEQ ID NO: 2 or 3.
  • the non-natural nucleic acid is mRNA, which further comprises a 5’-UTR, and the nucleotide sequence of the DNA corresponding to the 5’-UTR is shown in SEQ ID NO: 4.
  • the mRNA comprises a cap structure.
  • the cap structure is located at the 5' end of the 5'-UTR, also known as a "5'-cap structure”.
  • the cap structure is selected from at least one of m 7 GpppG, m 2 7,3′-O GpppG, m 7 Gppp(5′)N1, and m 7 Gppp(m 2′-O )N1; wherein “m 7 G” represents a 7-methylguanosine cap nucleoside, “ppp” represents a 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 a guanine nucleoside, "7” represents a methyl group at the 7-position of guanine, and "m 2′-O " represents a methyl group at the 2′-O position of the nucleotide.
  • the cap structure is m 7 Gppp(5′)N1 or m 7 Gppp(m 2′-O )N1. It will be understood that in other embodiments, the cap
  • the non-natural nucleic acid does not contain modified nucleotides.
  • the non-natural nucleic acid contains modified nucleotides.
  • the non-natural nucleic acids described above contain modified nucleosides.
  • one or more of the following regions in the non-natural nucleic acid contain modified nucleosides: 5'-UTR, coding region encoding a polypeptide or protein of interest, 3'-UTR, poly(A) tail, and microRNA site.
  • the modified nucleosides contained in the non-natural nucleic acid include at least one of modified uridine, modified cytidine, modified adenosine, and modified guanosine.
  • the modified nucleoside of the non-natural nucleic acid comprises or is modified uridine.
  • 0.1% to 100% of the uridine in the non-natural nucleic acid is modified.
  • 80% to 100% of the uridine is modified. In some embodiments, 100% of the uridine is modified.
  • Exemplary modified uridines include pseudouridine ( ⁇ ), 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-oxyacetic acid (cmo5U), uridine-5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine ( ⁇ ),
  • the modified uridine in the non-natural nucleic acid is a single species.
  • the non-natural nucleic acid comprises a plurality of modified uridines, each of which is N1-methylpseudouridine.
  • the modified uridine in the non-natural nucleic acid is a plurality of species.
  • the non-natural nucleic acid comprises a plurality of modified uridines, each of which is selected from at least two of the exemplary modified uridines (e.g., pseudouridine and N1-methylpseudouridine).
  • the modified nucleosides in the non-natural nucleic acids comprise or are modified cytidines.
  • 0.1% to 100% of the cytidines in the non-natural nucleic acids are modified.
  • at least 0.1%, at least 0.5%, at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% of the cytidines in the non-natural nucleic acids are modified.
  • 80% to 100% of the cytidines are modified. In some embodiments, 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-pseu
  • the modified cytidine in the non-natural nucleic acid is one.
  • the non-natural nucleic acid comprises a plurality of modified cytidines, and the plurality of modified cytidines are all 5-aza-cytidine.
  • the modified cytidine in the non-natural nucleic acid is multiple.
  • the non-natural nucleic acid comprises a plurality of modified cytidines, and the plurality of modified cytidines are selected from at least two of the exemplary modified cytidines (e.g., 5-aza-cytidine and 6-aza-cytidine).
  • the modified nucleosides in the non-natural nucleic acids comprise or are modified adenosines.
  • 0.1% to 100% of the adenosines in the non-natural nucleic acids are modified.
  • at least 0.1%, at least 0.5%, at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% of the adenosines in the non-natural nucleic acids are modified.
  • 80% to 100% of the adenosines are modified. In some embodiments, 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-methyl-
  • the modified adenosine in the non-natural nucleic acid is a single species.
  • the non-natural nucleic acid comprises a plurality of modified adenosines, each of which is 2-amino-purine.
  • the modified adenosine in the non-natural nucleic acid is a plurality of species.
  • the non-natural nucleic acid comprises a plurality of modified adenosines, each of which is selected from at least two of the exemplary modified adenosines (e.g., 2-amino-purine and 2,6-diaminopurine).
  • the modified nucleoside in the non-natural nucleic acid is a modified guanosine.
  • 0.1% to 100% of the guanosine in the non-natural nucleic acid is modified.
  • guanosine In some embodiments, 80% to 100% of the guanosine is modified. In some embodiments, 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), wyosine (yW), peroxy wyosine (o2yW), hydroxy wyosine (OHyW), undermodified hydroxy wyosine (OHyW*), 7-deaza-guanosine, queuosine (Q), epoxy queuosine (o Q), galactosyl-braided guanosine (galQ), mannosyl-braided guanosine (manQ), 7-cyano-7-deaza-guanosine (preQ0
  • the non-natural nucleic acid contains a single modified guanosine.
  • the non-natural nucleic acid contains multiple modified guanosines, each of which is inosine.
  • the non-natural nucleic acid contains multiple modified guanosines.
  • the non-natural nucleic acid contains multiple modified guanosines, each of which is selected from at least two of the exemplary modified guanosines (e.g., inosine and 1-methyl-inosine).
  • the modified nucleotides of the non-natural nucleic acid comprise nucleotides containing isotopes.
  • the non-natural nucleic acid comprises nucleotides containing isotopes of hydrogen. Hydrogen isotopes are not limited to deuterium and tritium. Furthermore, in some embodiments, the non-natural nucleic acid further comprises or contains nucleotides containing isotopes of elements other than hydrogen, including but not limited to carbon, oxygen, nitrogen, and phosphorus.
  • the non-natural nucleic acid comprises a 5'-UTR, a coding region encoding a polypeptide or protein of interest, a 3'-UTR, a poly(A) tail, and one or more microRNA binding sites, wherein the one or more microRNA binding sites are located downstream of the 3'-UTR, and the non-natural nucleic acid is mRNA and contains modified nucleotides.
  • the non-natural nucleic acid comprises a 5'-UTR, a coding region encoding a polypeptide or protein of interest, a 3'-UTR, a poly(A) tail, and one or more microRNA binding sites, wherein the one or more microRNA binding sites are located after the 3'-UTR and before the poly(A), and/or in the poly(A) tail, and the non-natural nucleic acid is mRNA and contains modified nucleotides.
  • the non-natural nucleic acid comprises a 5'-UTR, a coding region encoding a polypeptide or protein of interest, a 3'-UTR, a poly(A) tail, and one or more microRNA binding sites, wherein the one or more microRNA binding sites are located downstream of the 3'-UTR, at least one of the 5'-UTR and the 3'-UTR is heterologous to the coding region encoding the polypeptide or protein of interest, and the non-natural nucleic acid is mRNA and contains modified nucleotides.
  • the non-natural nucleic acid comprises a 5'-UTR, a coding region encoding a polypeptide or protein of interest, a 3'-UTR, a poly(A) tail, and one or more microRNA binding sites, wherein the one or more microRNA binding sites are located downstream of the 3'-UTR, at least one of the 5'-UTR and the 3'-UTR is heterologous to the coding region encoding the polypeptide or protein of interest, and the non-natural nucleic acid is mRNA and contains modified uridine.
  • At least 0.1%, at least 0.5%, at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% of the uridines in the non-natural nucleic acid are modified.
  • the modified uridines in the non-natural nucleic acid are N1-methylpseudouridines.
  • 100% of the uridines in the non-natural nucleic acid are modified and the modified uridines are N1-methylpseudouridines.
  • the non-natural nucleic acid comprises a terminator codon. It is understood that in other embodiments, the non-natural nucleic acid does not contain a terminator codon. When using the non-natural nucleic acid that does not contain a terminator codon, one of ordinary skill in the art will appreciate that a terminator codon (e.g., UGA or TGA) should be added at the appropriate location. It is understood that the non-natural nucleic acid may comprise one or more terminator codons.
  • a terminator codon e.g., UGA or TGA
  • the non-natural nucleic acid is DNA.
  • the non-natural nucleic acid is DNA, which can be transcribed into RNA in vitro.
  • the non-natural nucleic acid is DNA, which further comprises a 3’-UTR, and the nucleotide sequence of the 3’-UTR is as shown in SEQ ID NO: 2 or 3.
  • the non-natural nucleic acid is DNA, which further comprises a 5’-UTR, and the nucleotide sequence of the 5’-UTR is shown in SEQ ID NO: 4.
  • the non-natural nucleic acids (e.g., mRNA) disclosed herein containing one or more microRNA binding sites located downstream of the 3'-UTR can reduce the activation of unwanted immune cells and the corresponding immune response in the subject, thereby reducing or inhibiting the anti-drug antibody response.
  • mRNA containing one or more microRNA binding sites located downstream of the 3'-UTR that can bind to microRNAs expressed in immune cells e.g., miR142, miR126, or miR-155
  • the non-natural nucleic acids (such as mRNA) disclosed herein containing one or more microRNA binding sites located downstream of the 3'-UTR can reduce the activation of unwanted immune cells and the corresponding immune response of the subject, thereby reducing or inhibiting the production of anti-PEG IgM, and reducing or inhibiting accelerated blood clearance.
  • mRNA containing one or more microRNA binding sites located downstream of the 3'-UTR that can bind to microRNA expressed in immune cells (such as miR142, miR126 or miR-155) can reduce or inhibit the production of anti-PEG IgM, and reduce or inhibit accelerated blood clearance.
  • the non-natural nucleic acids (such as mRNA) disclosed herein containing one or more microRNA binding sites located downstream of the 3'-UTR can reduce the activation of undesirable immune cells and the corresponding immune response of the subject, thereby reducing or inhibiting the production of cytokines (such as IL-6, TNF- ⁇ and INF- ⁇ ).
  • cytokines such as IL-6, TNF- ⁇ and INF- ⁇ .
  • mRNA containing one or more microRNA binding sites located downstream of the 3'-UTR that bind to microRNAs expressed in immune cells can reduce or inhibit the production of cytokines.
  • the production of undesirable cytokines can be reduced or inhibited by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%.
  • the present disclosure also provides a genetic engineering vector comprising the DNA according to any one of the above embodiments.
  • the genetic engineering vector comprising the DNA of any of the above embodiments is a plasmid, cosmid, virus, phage or other vectors conventionally used in genetic engineering.
  • the genetic engineering vector comprising the DNA of any of the above embodiments is a plasmid.
  • the genetically engineered vector comprising the DNA of any of the above embodiments is an adenovirus, an adeno-associated virus, a lentivirus, or a retrovirus.
  • the genetic engineering vector comprises one or more of the following: an origin of replication (ORI), a marker gene or a fragment thereof, a reporter gene or a fragment thereof, and a restriction site allowing insertion of DNA elements.
  • the restriction site is a multiple cloning site (MCS).
  • the genetic engineering vector is an expression vector.
  • the genetic engineering vector comprises a promoter, a 5'-UTR, a coding region encoding a polypeptide or protein of interest, a 3'-UTR, a poly(A) tail, and one or more microRNA binding sites located after the 3'-UTR and before or within the poly(A) tail.
  • the genetic engineering vector is a cloning vector.
  • the present disclosure also provides a host cell, which comprises the RNA of any one of the above embodiments, the DNA of any one of the above embodiments, or the genetic engineering vector of any one of the above embodiments.
  • the host cell is an isolated cell.
  • the host cells are used to store and/or amplify the DNA.
  • the host cell is a bacterial cell.
  • Bacterial host cells include Escherichia coli (E. coli) cells well known to those skilled in the art.
  • Host cells of the present disclosure can be prepared by transforming competent host cells using the genetically engineered vectors of any of the above-mentioned embodiments.
  • Competent host cells are cells with the ability of free extracellular genetic material (such as DNA plasmids) that does not rely on sequence uptake.
  • Various bacterial cells well known to those skilled in the art are naturally able to take in exogenous DNA from the environment, and therefore can serve as bacterial host cells according to the present disclosure.
  • competent bacterial host cells can be obtained from natural non-competent bacterial cells using, for example, electroporation or chemicals (such as, for example, calcium ion treatment and accompanying high temperature exposure). After uptake, exogenous DNA is preferably neither degraded nor integrated in the genome of the bacterial host cell.
  • the present disclosure also provides a method for preparing RNA, which comprises the step of using the genetic engineering vector of any of the above embodiments to perform transcription.
  • the method for preparing RNA is an in vitro method.
  • the method for preparing RNA comprises contacting the genetic engineering vector (e.g., plasmid) of any of the above embodiments with an RNA polymerase.
  • the method for preparing RNA further comprises the step of linearizing the genetic engineering vector (e.g., plasmid).
  • the supercoil rate of the genetic engineering vector e.g., plasmid
  • the method for preparing RNA further comprises the step of purifying the linearized genetic engineering vector.
  • the method for preparing RNA further comprises the step of purifying RNA.
  • the present disclosure also provides another method for preparing RNA, comprising the step of preparing the RNA by chemical synthesis based on the nucleotide sequence of the RNA or DNA corresponding to any of the above embodiments. It is understood that the specific method of the chemical synthesis method can be a method known in the art, such as the solid-phase phosphoramidite method.
  • the non-natural nucleic acid is mRNA.
  • any of the above methods for preparing RNA further comprises the steps of capping and optionally purifying the capped product.
  • the cap is a Cap1 cap.
  • the Cap1 cap structure is as follows:
  • the capping reaction is as follows:
  • the method for preparing RNA is a partially in vitro method.
  • the RNA preparation method includes the following steps: preparing a genetically engineered vector according to any of the above embodiments in vitro; and introducing the genetically engineered vector into the body (e.g., in the form of a plasmid).
  • the genetically engineered vector is encapsulated in a delivery vehicle. In this case, the genetically engineered vector is delivered into the body via the delivery vehicle.
  • the RNA prepared in the method for preparing RNA according to any of the above embodiments comprises modified nucleosides or modified nucleotides.
  • the raw materials for preparing the RNA include one or more modified nucleosides or nucleotides. It is understood that the amount and type of the modified nucleosides or nucleotides correspond to the RNA to be prepared.
  • the present disclosure also provides an RNA, which is prepared by the method for preparing RNA according to any of the above embodiments.
  • the RNA is mRNA.
  • the present disclosure also provides a delivery vector comprising the RNA according to any one of the above embodiments, the DNA according to any one of the above embodiments, the genetic engineering vector according to any one of the above embodiments, or the host cell according to any one of the above embodiments.
  • the delivery vector comprises an RNA, DNA, genetically engineered vector, or host cell.
  • the delivery vector comprises multiple RNAs, multiple DNAs, multiple genetically engineered vectors, or multiple host cells.
  • the delivery vector comprises multiple RNAs encoding different polypeptides or proteins.
  • the delivery vector comprises two RNAs encoding different polypeptides or proteins.
  • the RNA in the delivery vector is mRNA. In some embodiments, the delivery vector comprises multiple mRNAs, each of which encodes a different polypeptide or protein.
  • the delivery vehicle is selected from a combination or one of the following: lipid nanoparticles (LNPs), liposomes, cationic proteins, vesicles, microparticles, polymers, and micelles.
  • LNPs lipid nanoparticles
  • the delivery vehicle is selected from a combination or one of the following: lipid nanoparticles, liposomes, cationic proteins, vesicles, microparticles, polymers, and micelles.
  • the delivery vector is a lipid nanoparticle (LNPs) comprising the RNA of any of the above embodiments, the DNA of any of the above embodiments, the genetic engineering vector of any of the above embodiments, or the host cell of any of the above embodiments.
  • LNPs lipid nanoparticle
  • lipid nanoparticles refer to particles having a nanometer scale (eg, 1 nm to 1000 nm) that include one or more lipids.
  • the average diameter of the lipid nanoparticles is 20 nm to 800 nm, 20 nm to 500 nm, 20 nm to 400 nm, 20 nm to 300 nm, 20 nm to 200 nm, 20 nm to 100 nm, 30 nm to 700 nm, 30 nm to 500 nm, 30 nm to 300 nm, 30 nm to 200 nm, 30 nm to 100 nm, 40 nm to 800 nm, 40 nm to 600 nm, 40 nm to 500 nm, 40 nm to 300 nm, 40 nm to 200 nm, 40nm ⁇ 100nm, 50nm ⁇ 800nm, 50nm ⁇ 600nm, 50nm ⁇ 500nm, 50nm ⁇ 500nm, 50nm ⁇ 400nm, 50nm ⁇ 500nm, 50nm ⁇ 400nm, 50nm ⁇ 300nm, 50nm ⁇ 200n
  • the average diameter of the lipid nanoparticles is 26 nm, 31 nm, 36 nm, 41 nm, 46 nm, 51 nm, 56 nm, 61 nm, 66 nm, 71 nm, 76 nm, 81 nm, 86 nm, 91 nm, 96 nm, 101 nm, 106 nm, 111 nm, 116 nm, 121 nm, 126 nm, 131 nm, 136 nm, 141 nm, 146 nm, 151 nm, 156 nm, 161 nm, 166 nm, 171 nm, 176 nm, 181 nm, 186 nm, 191 nm, 196 nm, 201 nm, 206 nm, 211 nm, 216 nm, 221 nm, 226 nm
  • the lipid nanoparticles include one of the following: cationic lipid nanoparticles, solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC), and nonlamellar lipid nanoparticles.
  • the lipid nanoparticles are cationic lipid nanoparticles.
  • lipid nanoparticles contain one or more of the following substances: cationic lipids, auxiliary lipids, structural lipids, and polymer-lipids.
  • cationic lipid refers to a lipid that becomes positively charged when the pH drops below the pKa of the ionizable group of the lipid, but gradually becomes neutral at higher pH values. At pH values below the pKa, the positively charged lipid is able to bind to negatively charged nucleic acids.
  • the cationic lipid comprises a zwitterionic lipid.
  • the cationic lipid comprises the following compound (I), an N-oxide thereof, a salt thereof, or an isomer thereof:
  • R1 is selected from the group consisting of C5 - C30 alkyl, C5 - C20 alkenyl, -R*YR", -YR", and -R"'M'R';
  • R 2 and R 3 are independently selected from the group consisting of H, C 1 -C 14 alkyl, C 2 -C 14 alkenyl, -R*YR", -YR", and -R*OR", or R 2 and R 3 together with the atoms to which they are attached form a heterocyclic or carbocyclic ring;
  • R4 is selected from the group consisting of hydrogen, C3 - C6 carbocycle, -( CH2 ) nQ , -( CH2 ) nCHQR , -( CH2 ) oC ( R10 ) 2 ( CH2 ) noQ , -CHQR, -CQ(R) 2 , and unsubstituted C1 - C6 alkyl, wherein Q is selected from carbocycle, heterocycle, -OR, -O( CH2 ) nN (R) 2 , -C(O)OR, -OC(O)R, -CX3 , -CX2H , -CXH2, -CN, -N(R) 2 , -C(O)N(R) 2 , -N(R) C (O)R, -N(R)S(O) 2R , -N(R)C(O)N(R) 2 , -N(R)C(
  • each R 5 is independently selected from the group consisting of OH, C 1 -C 3 alkyl, C 2 -C 3 alkenyl, and H;
  • Each R 6 is independently selected from the group consisting of OH, C 1 -C 3 alkyl, C 2 -C 3 alkenyl, and H;
  • M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M"-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O) 2- , -SS-, an aryl group, and a heteroaryl group, wherein M" is a bond, a C1 - C13 alkylene group, or a C2 -- C13 alkenylene group;
  • R7 is selected from the group consisting of C1-3 alkyl, C2 - C3 alkenyl and H;
  • R 8 is selected from the group consisting of C 3-6 carbocyclic and heterocyclic rings
  • R 9 is selected from the group consisting of H, CN, NO 2 , C 1 -C 6 alkyl, -OR, -S(O) 2 R, -S(O) 2 N(R) 2 , C 2 -C 6 alkenyl, C 3 -C 6 carbocycle, and heterocycle;
  • R 10 is selected from the group consisting of H, C 1 -C 3 alkyl and C 2 -C 3 alkenyl;
  • Each R is independently selected from the group consisting of C 1 -C 3 alkyl, C 2 -C 3 alkenyl, (CH 2 ) q OR** and H,
  • each q is independently selected from 1, 2, and 3;
  • Each R' is independently selected from the group consisting of: C1 - C18 alkyl, C2 - C18 alkenyl, -R*YR", -YR", H and and R 11 is selected from the group consisting of C 1 -C 12 alkylene and C 2 -C 12 alkenylene, and R 12 and R 13 are each independently selected from the group consisting of C 1 -C 12 alkyl and C 2 -C 12 alkenyl;
  • each R" is independently selected from the group consisting of C 3 -C 15 alkyl and C 3 -C 15 alkenyl;
  • each R'' is independently selected from the group consisting of C 3 -C 15 alkylene and C 3 -C 15 alkenylene;
  • each R* is independently selected from the group consisting of absent, C 1 -C 12 alkylene, and C 2 -C 12 alkenylene;
  • each R** is independently selected from the group consisting of absent, C 1 -C 12 alkyl, and C 2 -C 12 alkenyl;
  • Each Y is independently a C 3 -C 6 carbocycle
  • each X is independently selected from the group consisting of: F, Cl, Br, and I;
  • n is selected from 5, 6, 7, 8, 9, 10, 11, 12 and 13; and wherein when R 4 is -(CH 2 ) n Q, -(CH 2 ) n CHQR, -CHQR or -CQ(R) 2 , then (i) when n is 1, 2, 3, 4 or 5, Q is not -N(R) 2 ; or (ii) when n is 1 or 2, Q is not 5-, 6- or 7-membered heterocycloalkyl.
  • the cationic lipid is the following compound (I), its N-oxide, its salt or its isomer:
  • R 1 -R 7 , M and m are as defined above.
  • the cationic lipid comprises the following compound (II), an N-oxide thereof, a salt thereof, or an isomer thereof:
  • R 1 , R 2 , R 3 , R 5 , R 6 , M and R 7 are as described above,
  • RN is H or C1 - C3 alkyl
  • Xa and Xb are each independently O or S;
  • R 14 is selected from H, halogen, -OH, R b , -N(R b ) 2 , -CN, -N 3 , -C(O)OH, -C(O)OR b , -OC(O)R b , -OR b , -SR b , -S(O)R b , -S(O)OR b , -S(O) 2 OR b , -NO 2 , -S(O) 2 N(R b ) 2 , -N(R b )S(O) 2 R b , -NH(CH 2 ) t1 N(R b ) 2 , -NH(CH 2 ) p1 O(CH 2 ) q1 N(R b ) 2 , -NH(CH 2 ) s1 OR b , -N((CH 2 ) S OR b ) 2 , -N(R b
  • Each R b is independently selected from the group consisting of C 1 -C 3 alkyl, C 2 -C 3 alkenyl, and H;
  • u is 5, 6, 7, 8, 9, 10, 11, 12, or 13;
  • w is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
  • r is 0 or 1;
  • t1 is 1, 2, 3, 4, or 5;
  • p1 is 1, 2, 5, 4, or 5;
  • q 1 is 1, 2, 5, 4, or 5;
  • s1 is 1, 2, 3, 4, or 5.
  • the cationic lipid is the following compound (II), its N-oxide, its salt or its isomer:
  • R 1 -R 3 , R 5 -R 7 , R 14 , X a , X b , RN , M, u, w and r are as defined above.
  • the cationic lipid comprises the following compound (III), an N-oxide thereof, a salt thereof, or an isomer thereof:
  • G1 and G2 are each independently an unsubstituted C1 - C12 alkylene group or a C2 - C12 alkenylene group;
  • G 3 is C 1 -C 24 alkylene, C 2 -C 24 alkenylene, C 3 -C 8 cycloalkylene, or C 3 -C 8 cycloalkenylene;
  • R a is H or a C 1 -C 12 hydrocarbon group
  • R 15 and R 16 are each independently C 6 -C 24 alkyl or C 6 -C 24 alkenyl
  • R 19 is a C 1 -C 12 hydrocarbon group
  • R 18 is H or C 1 -C 6 hydrocarbon group
  • x 0, 1, or 2.
  • the cationic lipid is the following compound (III), its N-oxide, its salt or its isomer:
  • R 15 -R 17 , G 1 -G 3 , L 1 -L 2 are as defined above.
  • the cationic lipid is Compound 2-5, a salt thereof, or an isomer thereof:
  • the helper lipids of the lipid nanoparticles include phospholipids.
  • Phospholipids are typically semi-synthetic, but can also be naturally derived or chemically modified.
  • the helper lipids of the lipid nanoparticles are phospholipids.
  • the phospholipids of the lipid nanoparticles include one or more of the following: 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), and lysophospholipids.
  • DSPC disearoylphosphatidylcholine
  • DOPE dioleoylphosphatid
  • the helper lipids of the lipid nanoparticles are selected from one or more of the following: DSPC, DOPE, DOPC, and DOPS. In some embodiments, the helper lipid of the lipid nanoparticle is DSPC and/or DOPE.
  • the structural lipids of the lipid nanoparticles include sterols.
  • the structural lipids of the lipid nanoparticles are sterols.
  • the sterols of the lipid nanoparticles include one or more of the following: 20 ⁇ -hydroxycholesterol, cholesterol, cholesterol esters, sterol hormones, sterol vitamins, bile acids, ergosterol, ⁇ -sitosterol, and oxidized cholesterol derivatives.
  • the structural lipids of the lipid nanoparticles include at least one of cholesterol, cholesterol esters, sterol hormones, sterol vitamins, and bile acids.
  • the structural lipid of the lipid nanoparticles is cholesterol.
  • the structural lipid of the lipid nanoparticles is high-purity cholesterol, particularly injection-grade high-purity cholesterol, such as CHO-HP (produced by AVT).
  • the structural lipid is 20 ⁇ -hydroxycholesterol.
  • Polymer-lipid refers to a conjugate comprising a polymer and a lipid coupled to the polymer.
  • Polymer-lipid e.g., polyethylene glycol-lipid
  • lipid nanoparticles can improve the stability of lipid nanoparticles in vivo.
  • the lipids of the polymer-lipid used to form lipid nanoparticles include one or more of the following: 1,2-dimyristoyl-sn-glycerol (DMG), distearoyl-phosphatidyl-ethanolamine (DSPE), diacylglycerol (DAG), dialkyloxypropyl (DAA), phospholipids, ceramide (Cer), 1,2-distearoyl-rac-glycerol (DSG) and 1,2-dipalmitoyl-rac-glycero (DPG).
  • DMG 1,2-dimyristoyl-sn-glycerol
  • DSPE distearoyl-phosphatidyl-ethanolamine
  • DAG diacylglycerol
  • DAA dialkyloxypropyl
  • phospholipids ceramide
  • Cer 1,2-distearoyl-rac-glycerol
  • DPG 1,2-dipalmitoyl-rac-glycero
  • the polymer of the polymer-lipid used to form the lipid nanoparticles includes one or both of the following: a hydrophilic polymer and an amphiphilic polymer.
  • the polymer of the polymer-lipid used to form the lipid nanoparticles is a hydrophilic polymer. In other embodiments, the polymer of the polymer-lipid used to form the lipid nanoparticles is an amphoteric polymer.
  • the hydrophilic polymer includes one or more of the following: polyethylene glycol (PEG), polyoxazolines (POX), polyglycerols (PGs), polyhydroxypropyl methacrylate (PHPMA), poly(2-hydroxyethyl methacrylate), poly(N-(2-hydroxypropyl)methacrylamide (HPMA), polyvinyl pyrrolidone (PVP ...
  • PVP poly(N,N-dimethyl acrylamide)
  • PDMA poly(N-acryloyl morpholine)
  • PAcM poly(N-acryloyl morpholine)
  • GAGs glycosaminoglycans
  • HA heparin
  • hyaluronic acid HA
  • PSA polysialic acid
  • ELPs elastin-like polypeptide
  • polymer-lipid includes one or more of the following: polyethylene glycol-lipid (PEG-lipid), polyoxazoline-lipid, polyglycerol-lipid, polyhydroxypropyl methacrylate-lipid, polymethacrylate-2-hydroxyethyl ester-lipid, poly N-(2-hydroxypropyl) methacrylamide-lipid, polyvinyl pyrrolidone-lipid, poly N, N-dimethylacrylamide-lipid, poly N acryloylmorpholine-lipid, glycosaminoglycan-lipid, heparin-lipid, hyaluronic acid-lipid, polysialic acid-lipid, elastin-lipid, serum albumin-lipid and CD47-lipid.
  • PEG-lipid polyethylene glycol-lipid
  • polyoxazoline-lipid polyglycerol-lipid
  • polyhydroxypropyl methacrylate-lipid polymethacrylate-2-hydroxyethyl ester-lipid
  • PEG-lipid is a conjugate of polyethylene glycol and lipid
  • polyoxazoline-lipid refers to a conjugate formed by coupling polyoxazoline with lipid
  • polyglycerol-lipid refers to a conjugate formed by coupling polyglycerol with lipid
  • the hydrophilic polymer includes polyethylene glycol.
  • the polymer-lipid comprises a PEG-lipid.
  • the polymer-lipid is a PEG-lipid.
  • the PEG-lipid comprises one or more of the following: myristoyl diglycerol-PEG (DMG-PEG), distearoylphosphatidylethanolamine-PEG (DSPE-PEG), diacylglycerol-PEG (DAG-PEG), dialkyloxypropyl-PEG (DAA-PEG), phospholipid-PEG, ceramide-PEG (Cer-PEG), 1,2-distearoyl-rac-glycerol-PEG (DSG-PEG), and 1,2-dipalmitoyl-rac-glycerol-PEG (PEG-DPG).
  • the PEG-lipid is preferably DMG-PEG, DSG-PEG, or DPG-PEG.
  • DMG-PEG is a polyethylene glycol derivative of 1,2-dimyristoylglycerol.
  • the average molecular weight of the PEG in the PEG-lipid is approximately 2000 to 5000. In an alternative embodiment, the average molecular weight of PEG in the PEG-lipid is about 2000.
  • the amphoteric polymer comprises one or more of the following: poly(carboxybetaine) (pCB), poly(sulfobetaine) (pSB), phosphobetaine-based polymers, and phosphorylcholine polymers.
  • the amphoteric polymer includes one or more of the following: poly(carboxybetaine acrylamide) (pCBAA), poly(carboxybetaine methacrylate), poly(sulfobetaine methacrylate), poly(methacryloyloxyethyl phosphorylcholine), poly(vinyl-pyridinio propanesulfonate), poly(carboxybetaine) based on vinylimidazole, poly(sulfobetaine) based on vinylimidazole, and poly(sulfobetaine) based on vinylpyridine.
  • poly(carboxybetaine acrylamide) pCBAA
  • poly(carboxybetaine methacrylate) poly(sulfobetaine methacrylate)
  • poly(vinyl-pyridinio propanesulfonate) poly(carboxybetaine) based on vinylimidazole
  • the polymer-lipid comprises one or more of the following: polyhydroxybetaine-lipid, polysulfobetaine-lipid, phosphobetaine-based polymer-lipid and phosphorylcholine polymer-lipid.
  • the polymer-lipid comprises one or more of the following: poly(carboxybetaine acrylamide)-lipid, poly(carboxybetaine methacrylate)-lipid, poly(sulfobetaine methacrylate)-lipid, poly(methacryloyloxyethylphosphorylcholine)-lipid, poly(vinylpyridylpropanesulfonate)-lipid, polyvinylimidazolylbetaine-lipid, polyvinylimidazolylsulfobetaine-lipid, polyvinylpyridylsulfobetaine-lipid.
  • polymers used in nanoparticles in Hoang Thi, Thai Thanh et al. “The Importance of Poly (ethylene glycol) Alternatives for Overcoming PEG Immunogenicity in Drug Delivery and Bioconjugation.” Polymers vol. 12, 2298 are also introduced herein.
  • the lipid nanoparticles contain cationic lipids, helper lipids, structural lipids, and polymer-lipids (e.g., PEG-lipids). In some embodiments, the lipid nanoparticles contain the following amount (molar percentage) of cationic lipids based on the total amount of cationic lipids, helper lipids, structural lipids, and polymer-lipids: 25% to 75%.
  • the lipid nanoparticles comprise the following amount (molar percentage) of helper lipids based on the total amount of cationic lipids, helper lipids, structural lipids and polymer-lipids (e.g., PEG-lipids): 5% to 45%, for example, 5% to 9%, 9% to 9.4%, 9.4% to 10%, 10% to 10.5%, 10.5% to 11%, 11% to 15%, 15% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 33.5%, 33.5% to 37%, 37% to 40%, 40% to 42%, or 42% to 45%.
  • 5% to 45% for example, 5% to 9%, 9% to 9.4%, 9.4% to 10%, 10% to 10.5%, 10.5% to 11%, 11% to 15%, 15% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 33.5%, 33.5% to 37%, 37% to 40%, 40% to 42%, or 42% to 45%.
  • the lipid nanoparticles comprise the following amounts (in molar percentages) of structural lipids, based on the total amount of cationic lipids, helper lipids, and structural lipid polymers-lipids (e.g., PEG-lipids): 0% to 50%. For example, 0% to 10%, 10% to 15.5%, 15.5% to 18.5%, 18.5% to 22.5%, 22.5% to 23.5%, 23.5% to 28.5%, 28.5% to 33.5%, 33.5% to 35%, 35% to 36.5%, 36.5% to 38%, 38% to 38.5%, 38.5% to 39.5%.
  • structural lipid polymers-lipids e.g., PEG-lipids
  • the lipid nanoparticles comprise the following amount (molar percentage) of polymer-lipid: 0.5% to 5%, based on the total amount of cationic lipids, helper lipids, structural lipids and polymer-lipids (e.g., PEG-lipids), such as 0.5% to 1%, 1% to 1.5%, 1.5% to 1.6%, 1.6% to 2%, 2% to 2.5%, 2.5% to 3%, 3% to 3.5%, 3.5% to 4%, 4% to 4.5%, or 4.5% to 5%.
  • polymer-lipid e.g., PEG-lipids
  • the molar ratio of cationic lipid:helper lipid:structural lipid:PEG lipid is 45:10:42.5:2.5, 45:11:41.5:2.5, 42:10.5:45:2.5, 42:16:39.5:2.5, 40:16:41.5:2.5, 40:18:39.5:2.5, 35:16:46.5:2.5, 35:25:36.5:3.5, 28:33.5:35:3.5, 32:37:40.5:0.5, 35:40:22.5:2.5, 40:42:15.5:2.5, 40:20:38.5:1.5, 45: 15:38.5:1.5, 55:5:38.5:1.5, 60:5:33.5:1.5, 45:20:33.5:1.5, 50:20:28.5:1.5, 55:20:23.5:1.5, 60:20:18.5:1.5, 40:15:43.5:1.5, 50:15:33.
  • the helper lipid is DOPE and the
  • the molar ratio of cationic lipid:helper lipid:structural lipid:PEG lipid is about 50:10:38.5:1.5, 50:9:38:3, 49.5:10:39:1.5, 48:10:40.5:1.5, 46.3:9.4:42.7:1.6, 45:9:43:3, 45:11:41.5:2.5, 42:10.5:45:2.5, 42:16:39.5:2.5, 40:16:41.5:2.5, 40:18:39.5:2.5, 35:40:22.5:2.5, 40:20:38.5:1.5, 45:15 : 38.5:1.5, 55:5:38.5:1.5, 60:5:33.5:1.5, 45:20:33.5:1.5, 50:20:28.5:1.5, 55:20:23.5:1.5, 60:20:18.5:1.5, 40:15:43.5:1.5, 50:15:33.5 : 1.5, 55:15:28.5:1.5, 60:15:15:1.5, 60:15:43.5:1.5, 50:
  • the non-lamellar lipid nanoparticles are selected from one of the following: ethosomes and echogenic liposomes.
  • the delivery vector is a liposome comprising the non-natural nucleic acid (e.g., DNA or mRNA) of any of the above embodiments, the genetically engineered vector of any of the above embodiments, or the host cell of any of the above embodiments.
  • the liposome utilizes a vesicle formed by a phospholipid bilayer membrane to encapsulate the non-natural nucleic acid (e.g., DNA or mRNA), genetically engineered vector, or host cell of any of the above embodiments.
  • the components of the liposome include phospholipids and cholesterol.
  • the delivery vector is a cationic protein loaded with a non-natural nucleic acid (e.g., DNA or mRNA) according to any of the above embodiments, a genetically engineered vector according to any of the above embodiments, or a host cell according to any of the above embodiments.
  • cationic proteins include but are not limited to protamine.
  • the delivery vector is a polymer comprising the non-natural nucleic acid (such as DNA or mRNA) of any of the above-mentioned embodiments, the genetic engineering vector of any of the above-mentioned embodiments, or the host cell of any of the above-mentioned embodiments.
  • the polymer is a lipid polymer (lipopolyplex, LPP) and/or a hyaluronic acid polymer (such as hyaluronic acid gel) comprising the non-natural nucleic acid (such as DNA or mRNA) of any of the above-mentioned embodiments, the genetic engineering vector or the host cell of any of the above-mentioned embodiments.
  • the polymer is a lipid polymer or a hyaluronic acid gel.
  • Lipid polymer is a double-layer structure in which a polymer-encapsulated non-natural nucleic acid (such as mRNA) is a core and a lipid (such as phospholipid) is wrapped as an outer shell.
  • the delivery vectors applicable to the present disclosure are not limited to the above, and may also be other substances capable of delivering the non-natural nucleic acid (e.g., DNA or mRNA) of any of the above embodiments, the genetically engineered vector of any of the above embodiments, or the host cell of any of the above embodiments into the body, such as vesicles (e.g., exosomes).
  • vesicles e.g., exosomes
  • the present disclosure also provides a pharmaceutical composition, which comprises a non-natural nucleic acid (such as DNA or mRNA) according to any of the above embodiments, a genetically engineered vector according to any of the above embodiments, a host cell according to any of the above embodiments, or a delivery vector according to any of the above embodiments, and a pharmaceutically acceptable carrier.
  • a non-natural nucleic acid such as DNA or mRNA
  • the pharmaceutical composition comprises a plurality of non-natural nucleic acids (such as DNA or mRNA) according to any of the above embodiments, a plurality of genetically engineered vectors according to any of the above embodiments, a plurality of host cells according to any of the above embodiments, or a plurality of delivery vectors according to any of the above embodiments.
  • a plurality of non-natural nucleic acids such as DNA or mRNA
  • a plurality of genetically engineered vectors according to any of the above embodiments
  • a plurality of host cells according to any of the above embodiments
  • a plurality of delivery vectors according to any of the above embodiments.
  • the pharmaceutical composition comprises a plurality of non-natural nucleic acids (eg, DNA or mRNA) according to any of the above embodiments, and the polypeptides or proteins encoded by the plurality of non-natural nucleic acids are different or partially identical.
  • a plurality of non-natural nucleic acids eg, DNA or mRNA
  • the polypeptides or proteins encoded by the plurality of non-natural nucleic acids are different or partially identical.
  • the pharmaceutical composition comprises a plurality of delivery vectors, and the polypeptides or proteins encoded by the non-natural nucleic acids contained in the plurality of delivery vectors are different or partially identical.
  • the present disclosure also provides a use of a non-natural nucleic acid (such as DNA or mRNA) according to any of the above embodiments, a genetically engineered vector according to any of the above embodiments, a host cell according to any of the above embodiments, a delivery vector according to any of the above embodiments, or a pharmaceutical composition according to any of the above embodiments in the preparation of a drug;
  • a non-natural nucleic acid such as DNA or mRNA
  • the above-mentioned medicament is used for the treatment and/or prevention of a disease.
  • the above-mentioned medicament is used for gene therapy, gene vaccination, protein replacement therapy, antisense therapy or treatment by interfering RNA.
  • the above-mentioned drug is used to treat a disease corresponding to the above-mentioned polypeptide or protein of interest.
  • the above-mentioned drugs are used to treat and/or prevent one or more of the following diseases: rare diseases, cancer, infectious diseases, autoimmune diseases, metabolic diseases, neurological diseases, cardiovascular diseases, transplant rejection, inflammatory response, genetic diseases and musculoskeletal diseases.
  • the rare disease comprises one or more of the following: Brittle Bone Disease, Wilson Disease, Spinal Muscular Atrophy (SMA), Huntington's Disease, Rett Syndrome, Amyotrophic Lateral Sclerosis (ALS), Duchenne Type Muscular dystrophy, Friedrichs Ataxia, Methylmalonic Acidemia (MMA), Cystic Fibrosis (CF), Glycogen Storage Disease 1a (GSD1a), Glycogen Storage Disease III (GSDIII), Crigler-Najjar Syndrome, Ornithine Transcarbamylase Deficiency, Deficiency (OTCD), propionic acidemia (PA), phenylketonuria (PKU), hemophilia A, hemophilia B, ⁇ -thalassemia, Lafora disease, Dravet syndrome (DS), Alexander disease, Leber's congenital amaurosis (LCA), myelodysplastic syndrome (MDS), and homocystinuria due to CBS deficiency.
  • the cancer comprises one or more of the following: hematological malignancies, lung cancer, liver cancer, kidney cancer, head and neck cancer, esophageal cancer, gastric cancer, colorectal cancer, pancreatic cancer, brain cancer, prostate cancer, gallbladder cancer, ovarian cancer, breast cancer, cervical cancer, endometrial cancer, bladder cancer, melanoma.
  • infectious diseases include diseases caused by one or more infections selected from the group consisting of viruses, fungi, and bacteria.
  • the autoimmune disease comprises one or more of the following: acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, systemic lupus erythematosus, rheumatoid arthritis, psoriasis, inflammatory bowel disease, multiple sclerosis, celiac disease, type 1 diabetes mellitus, diffuse toxic goiter.
  • the genetic disease comprises one or more of the following: hemophilia, thalassemia, and Gaucher disease.
  • the neurological disease comprises one or more of the following: amyotrophic lateral sclerosis, Alzheimer's disease, and glioma.
  • the above-mentioned drug is a vaccine.
  • the vaccine is a multivalent vaccine or a combination vaccine.
  • the vaccine does not include an adjuvant.
  • the vaccine further comprises an adjuvant.
  • the dosage form of the vaccine is not particularly limited.
  • the above-mentioned drug is a nucleic acid drug, wherein the nucleic acid comprises at least one of the following: RNA and DNA.
  • the DNA includes one or more of: a plasmid and an antisense oligonucleotide.
  • the RNA includes one or more of the following: antisense oligonucleotides, messenger RNA (mRNA), ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small hairpin RNA (shRNA), single-stranded guide RNA (sgRNA) and Cas9 mRNA.
  • mRNA messenger RNA
  • rRNA ribosomal RNA
  • miRNA microRNA
  • tRNA transfer RNA
  • siRNA small interfering RNA
  • snRNA small nuclear RNA
  • shRNA small hairpin RNA
  • sgRNA single-stranded guide RNA
  • Cas9 mRNA Cas9 mRNA.
  • the vaccine is an mRNA vaccine.
  • the present disclosure also provides a drug comprising the non-natural nucleic acid (such as DNA or mRNA) of any of the above embodiments, the genetic engineering vector of any of the above embodiments, the host cell of any of the above embodiments, the delivery vector of any of the above embodiments, or the pharmaceutical composition of any of the above embodiments.
  • non-natural nucleic acid such as DNA or mRNA
  • the disease or condition that the drug prevents or treats is as described herein above.
  • the present disclosure also provides a drug prepared from the pharmaceutical composition according to any of the above embodiments.
  • the disease or condition that the drug prevents or treats is as described herein above.
  • the present disclosure also provides a method for preventing or treating a disease, comprising the step of administering to a subject the non-natural nucleic acid of any of the above embodiments, the genetically engineered vector of any of the above embodiments, the host cell of any of the above embodiments, the delivery vector (e.g., lipid nanoparticles) of any of the above embodiments, the pharmaceutical composition of any of the above embodiments, or the drug of any of the above embodiments.
  • the disease or condition to be prevented or treated is as described above in the present disclosure.
  • the present disclosure also provides a method for reducing or inhibiting the expression of a non-natural nucleic acid in an undesirable cell, tissue and/or organ, comprising administering to a subject a non-natural nucleic acid of the present disclosure encoding a polypeptide or protein of interest, wherein the non-natural nucleic acid comprises one or more microRNA binding sites that bind to a microRNA expressed in an undesirable cell, tissue and/or organ.
  • the microRNA expressed in the undesirable cell, tissue and/or organ is a microRNA that is highly abundantly expressed or specifically expressed in the undesirable cell, tissue and/or organ.
  • the undesired cells are normal hepatocytes, and the microRNA highly expressed or specifically expressed in the undesired cells is miR-122.
  • the methods described above reduce or inhibit expression of the non-naturally occurring nucleic acid in undesired cells, tissues, and/or organs by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% compared to the non-naturally occurring nucleic acid without the microRNA binding site.
  • the present disclosure also provides a method for treating or preventing liver disease, comprising administering to a subject a non-natural nucleic acid according to any of the above embodiments, wherein the non-natural nucleic acid comprises one or more microRNA binding sites that bind to a microRNA that is expressed in high abundance or specifically expressed in normal liver cells and is expressed in low abundance or not expressed in abnormal liver cells (e.g., liver cancer cells).
  • the non-natural nucleic acid comprises one or more microRNA binding sites that bind to a microRNA that is expressed in high abundance or specifically expressed in normal liver cells and is expressed in low abundance or not expressed in abnormal liver cells (e.g., liver cancer cells).
  • the microRNA that is highly expressed or specifically expressed in normal liver cells and is low in abundance or not expressed in abnormal liver cells is miR-122.
  • the liver disease is liver cancer.
  • the non-natural nucleic acid further comprises a coding region for a polypeptide or protein for treating or preventing liver disease.
  • one or more miR-122 binding sites are located in the poly(A) tail.
  • the methods described above reduce or inhibit expression of the non-natural nucleic acid in the liver by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% compared to the non-natural nucleic acid that does not contain the microRNA binding site.
  • the present disclosure also provides a method for reducing or inhibiting undesirable immune cell activation, comprising administering to a subject a non-natural nucleic acid (e.g., mRNA or DNA) of the present disclosure, a genetically engineered vector comprising the non-natural nucleic acid of the present disclosure, or a delivery vehicle (e.g., lipid nanoparticle) comprising the non-natural nucleic acid of the present disclosure, wherein the non-natural nucleic acid encodes a polypeptide or protein of interest and comprises one or more microRNA binding sites capable of binding to microRNA expressed in immune cells.
  • a non-natural nucleic acid e.g., mRNA or DNA
  • a genetically engineered vector comprising the non-natural nucleic acid of the present disclosure
  • a delivery vehicle e.g., lipid nanoparticle
  • the methods described above reduce or inhibit undesired immune cell activation by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% compared to a non-natural nucleic acid that does not contain a microRNA binding site.
  • the undesirably activated immune cells are myeloid cells and/or lymphocytes.
  • the undesirably activated myeloid cells are selected from one or more of the following: dendritic cells, macrophages, monocytes, neutrophils, basophils, eosinophils, megakaryocytes, and platelets.
  • the undesirably activated lymphocytes are selected from one or more of the following: T cells, B cells, plasma cells, and NK cells.
  • the undesirably activated immune cell is a B cell.
  • the undesirably activated immune cell is a B1a cell.
  • the present disclosure also provides a method for reducing or inhibiting the production of undesirable cytokines, comprising administering to a subject a non-natural nucleic acid (e.g., mRNA or DNA) of the present disclosure, a genetically engineered vector comprising the non-natural nucleic acid of the present disclosure, or a delivery vehicle (e.g., lipid nanoparticle) comprising the non-natural nucleic acid of the present disclosure, wherein the non-natural nucleic acid encodes a polypeptide or protein of interest and comprises one or more microRNA binding sites capable of binding to microRNA expressed in immune cells.
  • a non-natural nucleic acid e.g., mRNA or DNA
  • a genetically engineered vector comprising the non-natural nucleic acid of the present disclosure
  • a delivery vehicle e.g., lipid nanoparticle
  • the undesirable cytokine is a pro-inflammatory cytokine and/or chemokine.
  • the undesirable cytokine is one or more of the following: IL-6, TNF- ⁇ , and INF- ⁇ .
  • the methods described above reduce or inhibit the production of an undesired cytokine by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% compared to a non-natural nucleic acid that does not contain a microRNA binding site.
  • the present disclosure also provides a method for reducing or inhibiting an anti-drug antibody response (ADA) in a subject who is repeatedly administered a drug, comprising administering to the subject a non-natural nucleic acid (e.g., mRNA or DNA) of the present disclosure, a genetically engineered vector comprising the non-natural nucleic acid of the present disclosure, or a delivery vector (e.g., lipid nanoparticle) comprising the non-natural nucleic acid of the present disclosure, wherein the non-natural nucleic acid encodes a polypeptide or protein of interest and comprises one or more microRNA binding sites capable of binding to microRNA expressed in immune cells, such that upon repeated administration, the anti-drug antibody response of the subject is reduced or inhibited.
  • a non-natural nucleic acid e.g., mRNA or DNA
  • a genetically engineered vector comprising the non-natural nucleic acid of the present disclosure
  • a delivery vector e.g., lipid nanoparticle
  • the method of reducing or inhibiting an anti-drug antibody response in a subject to repeated administration comprises:
  • a non-natural nucleic acid e.g., mRNA or DNA
  • a genetically engineered vector comprising a non-natural nucleic acid of the present disclosure
  • a delivery vehicle e.g., lipid nanoparticle
  • the non-natural nucleic acid encodes a polypeptide or protein of interest and comprises one or more microRNA binding sites capable of binding to microRNA expressed in immune cells
  • non-natural nucleic acid of the present invention e.g., mRNA or DNA
  • a genetically engineered vector comprising the non-natural nucleic acid of the present invention
  • a delivery vehicle e.g., lipid nanoparticle
  • the non-natural nucleic acid encodes a polypeptide or protein of interest and comprises one or more microRNA binding sites capable of binding to microRNA expressed in immune cells, so as to reduce or inhibit the anti-drug antibody response of the subject caused by repeated administration of the non-natural nucleic acid.
  • the present disclosure further provides a method for reducing or inhibiting an anti-drug antibody response in a subject to repeated drug administration, comprising:
  • a non-natural nucleic acid e.g., mRNA or DNA
  • a genetically engineered vector comprising a non-natural nucleic acid of the present disclosure
  • a delivery vehicle e.g., lipid nanoparticle
  • the non-natural nucleic acid encodes a polypeptide or protein of interest and comprises one or more microRNA binding sites capable of binding to a microRNA expressed in an immune cell
  • a non-natural nucleic acid e.g., mRNA or DNA
  • a genetically engineered vector comprising a non-natural nucleic acid of the present disclosure, or a delivery vehicle (e.g., lipid nanoparticle) comprising a non-natural nucleic acid of the present disclosure has been administered; and
  • the non-natural nucleic acid e.g., mRNA or DNA
  • a genetically engineered vector comprising the non-natural nucleic acid of the present disclosure
  • a delivery vehicle e.g., lipid nanoparticle
  • the non-natural nucleic acid encodes a polypeptide or protein of interest and comprises one or more microRNA binding sites capable of binding to microRNA expressed in immune cells, to reduce or inhibit the subject's anti-drug antibody response caused by repeated administration of the non-natural nucleic acid.
  • the methods described above reduce or inhibit an anti-drug antibody response by at least 10%, at least 20%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% compared to a non-natural nucleic acid that does not contain a microRNA binding site.
  • the present disclosure provides a method for reducing or inhibiting accelerated blood clearance (ABC) in a subject subjected to repeated administration, comprising administering to the subject a non-natural nucleic acid of the present disclosure encapsulated in lipid nanoparticles (LNPs), wherein the non-natural nucleic acid encodes a polypeptide or protein of interest and comprises one or more microRNA binding sites capable of binding to microRNA expressed in immune cells, such that upon repeated administration, accelerated blood clearance in the subject is reduced or inhibited.
  • lipid nanoparticles LNPs
  • the above method of reducing or inhibiting accelerated blood clearance in a subject to repeated administration comprises:
  • a non-natural nucleic acid of the present disclosure encapsulated in lipid nanoparticles, wherein the non-natural nucleic acid encodes a polypeptide or protein of interest and comprises one or more microRNA binding sites capable of binding to a microRNA expressed in an immune cell;
  • non-natural nucleic acid of the present disclosure encapsulated in lipid nanoparticles, wherein the non-natural nucleic acid encodes a polypeptide or protein of interest and comprises one or more microRNA binding sites capable of binding to microRNA expressed in immune cells to reduce or inhibit accelerated blood clearance.
  • the lipid nanoparticles encapsulating the non-natural nucleic acids of the present disclosure comprise PEG-lipid.
  • the microRNA expressed in the immune cells in any of the above embodiments is a microRNA that is highly abundantly expressed or specifically expressed in the immune cells.
  • the microRNA expressed abundantly or specifically in immune cells in any of the above embodiments is miR-142-3p.
  • the one or more miR-142 binding sites in any of the above embodiments are located in the poly(A) tail.
  • one or more miR-142-3p binding sites in any of the above embodiments are located in a poly(A) tail.
  • the lipid nanoparticles encapsulating the non-natural nucleic acids of the present disclosure comprise PEG-lipid.
  • the present disclosure provides a method for reducing or inhibiting the production of IgG antibodies that bind to polyethylene glycol (PEG) (anti-PEG IgG antibodies, anti-PEG IgG antibodies) in a subject who is repeatedly administered the drug, comprising administering to the subject a non-natural nucleic acid of the present disclosure encapsulated in lipid nanoparticles (LNPs), wherein the non-natural nucleic acid encodes a polypeptide or protein of interest and contains one or more microRNA binding sites that can bind to microRNA expressed in immune cells, so that upon repeated administration, the production of anti-PEG IgG antibodies in the subject is reduced or inhibited.
  • PEG polyethylene glycol
  • LNPs lipid nanoparticles
  • the lipid nanoparticles encapsulating the non-natural nucleic acids of the present disclosure comprise PEG-lipid.
  • the non-natural nucleic acid administered by any of the above methods is mRNA.
  • the non-natural nucleic acid administered in any of the above methods is DNA.
  • the genetically engineered vector administered by any of the above methods is a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.
  • the non-natural nucleic acid administered by any of the above methods comprises one or more microRNA binding sites that bind to microRNAs including one or more of the following: miR-142-3p, miR-142-5p, miR-126, miR-146-3p, miR-146-5p, and miR-155.
  • the non-natural nucleic acid administered by any of the above methods comprises one or more microRNA binding sites that can bind to microRNAs including one or more of the following: miR-142-3p, miR-142-5p, and miR-126.
  • the non-natural nucleic acid administered by any of the above methods comprises one or more microRNA binding sites that are capable of binding to the microRNA miR-142-3p.
  • the non-natural nucleic acid administered by any of the above methods comprises 2-6 microRNA binding sites capable of binding to miR-142-3p.
  • the non-natural nucleic acid administered by any of the above methods comprises three microRNA binding sites capable of binding to miR-142-3p.
  • the subject administered by any of the above methods is a mammal, such as a human, a non-human primate (e.g., ape, chimpanzee, monkey, and orangutan), etc.
  • a mammal such as a human, a non-human primate (e.g., ape, chimpanzee, monkey, and orangutan), etc.
  • the subject is a human.
  • any of the above methods is administered once, twice, three times, four times or more.
  • the time intervals between multiple administrations of any of the above methods is no more than 8 weeks, 7 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 2 weeks, or 1 week.
  • the route of administration of any of the above methods is nasal, intratracheal, or by injection (eg, intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardial, intraperitoneal, and subcutaneous).
  • any of the above methods is administered by intravenous injection or intramuscular injection.
  • the 5'-UTR, nucleotide sequence encoding luciferase (Fluc), 3'-UTR, and poly(A) tail of each mRNA were introduced into the pCDNA3.0-Kana plasmid (GENEWIZ, Suzhou Jinweizhi Biotechnology Co., Ltd.) using subcloning technology (e.g., PCR-based subcloning technology and restriction endonuclease digestion or in-fusion technology), thereby constructing plasmids corresponding to different mRNAs encoding Fluc.
  • subcloning technology e.g., PCR-based subcloning technology and restriction endonuclease digestion or in-fusion technology
  • the mRNAs numbered 437-Fluc, 439-Fluc, 440-Fluc, 443-Fluc, 444-Fluc, and 445-Fluc in Table 1 all contain microRNA binding sites, and the microRNA binding sites are all located exclusively in the poly(A) tail.
  • the DNA sequence corresponding to its 5'-UTR is shown in SEQ ID NO: 4
  • the DNA sequence corresponding to its 3'-UTR is shown in SEQ ID NO: 3
  • the DNA sequence corresponding to the poly(A) tail is shown in SEQ ID NO: 6.
  • the plasmid was linearized by enzyme digestion, and then purified using a DNA fragment purification and recovery kit (Takara, 9761) and DNA magnetic beads (Rebecil, SP703) (to remove RNases and proteins, etc.).
  • RNA Cleaner kit (YEASEN, 12602ES56) to obtain different mRNAs encoding Fluc, which contained a Cap1-type cap structure, a 5'-UTR, a 3'-UTR, a poly(A) tail, and a microRNA binding site, in which all uridines were replaced by N1-methylpseudouridine, and an mRNA encoding Fluc, which contained a Cap1-type cap structure, a 5'-UTR, a 3'-UTR, and a poly(A) tail but did not contain a microRNA binding site, in which all uridines were replaced by N1-methylpseudouridine.
  • Example 2 The mRNA prepared in Example 2 was encapsulated using a microfluidic device and a microfluidic chip (SN.000038) to prepare crude lipid nanoparticles encapsulating the corresponding mRNA.
  • the aqueous phase consisted of an acetic acid-sodium acetate buffer (pH 5.0) containing the corresponding mRNA, and the alcohol phase contained the cationic lipid compound 2-5, DSPC, cholesterol, DMG-PEG2000, and ethanol.
  • the molar ratio of compound 2-5, DSPC, cholesterol, and DMG-PEG2000 was 48:10:40.5:1.5.
  • the crude product obtained by encapsulation was placed in dialysis bags, immersed in a beaker containing 1 L of dialysis fluid, wrapped in aluminum foil and dialyzed at 100 rpm for 1 hour at room temperature, and then the dialysis fluid was replaced and the dialysis was continued for 1 hour.
  • the dialyzed sample was sterile-filtered using a 0.22 ⁇ m PES membrane to obtain lipid nanoparticles encapsulating the corresponding mRNA. Testing showed that the lipid nanoparticles ranged in size from 80 nm to 100 nm, with an encapsulation efficiency exceeding 80%.
  • mice Female BALB/c mice aged 6 to 8 weeks were administered the drug via tail vein injection (IV). Each mouse was injected with 10 ⁇ g of the corresponding lipid nanoparticles encapsulating mRNA encoding luciferase (Fluc mRNA) prepared in Example 3. Three mice were injected with each lipid nanoparticle.
  • IV tail vein injection
  • mice After 6 hours, 200 ⁇ L of D-Luciferin luciferase development substrate (Cat. No.: 122799; Manufacturer: Perkin Elmer) was injected into the mice. After the substrate injection, the mice were anesthetized by isoflurane inhalation anesthesia. Ten minutes after the substrate injection, the animals were placed in a supine position, and the signal distribution and intensity of luciferase in the mice and various organs were observed under an in vivo imaging system (IVIS).
  • IVIS in vivo imaging system
  • mice administered with Fluc mRNA containing a miR-142-3p binding site in the poly(A) tail showed no significant change in total liver Fluc flux (Total Flux), but a significant decrease in total spleen Fluc flux (Total Flux) was observed.
  • miR-142-3p is a microRNA highly expressed in immune cells (immune organs).
  • mice administered with Fluc mRNA containing a miR-122 binding site within its poly(A) tail showed no significant change in total spleen Fluc flux (Total Flux), but a significant decrease in liver Fluc flux (Total Flux).
  • miR-122 is a microRNA highly expressed in hepatocytes, and these data suggest that the addition of a miR-122 binding site to the mRNA poly(A) tail significantly reduces its expression in the liver.
  • Plasmids corresponding to different mRNAs encoding hEPO were constructed with reference to Example 1, wherein the nucleotide sequence encoding luciferase (Fluc) was replaced with a nucleotide sequence encoding hEPO.
  • the corresponding mRNAs were numbered C-hEPO, 437-hEPO, 439-hEPO, 440-hEPO, 443-hEPO, 444-hEPO, and 445-hEPO.
  • Lipid nanoparticles encapsulating different mRNAs encoding hEPO (hEPO mRNA) were further prepared with reference to Examples 2-3.
  • the drug was administered to female SD rats weighing approximately 180 g via tail vein injection (IV). Each rat was injected with the corresponding lipid nanoparticles encapsulating the mRNA encoding hEPO prepared in step 1 of this example at a dose of 1 mg/kg mRNA. Three rats were injected with each lipid nanoparticle. The drug was administered once a week for multiple times.
  • Enzyme-linked immunosorbent assay was used to detect the hEPO concentration in the serum of rats 6 hours after administration, wherein the coated antibody was a recombinant Anti-EPO antibody (Cat. No.: ab272358, Manufacturer: Abcam).
  • ELISA was used to detect the anti-PEG IgG antibody titer in the serum of rats 120 hours after administration obtained in Example 5, where the coating antigen was DMG-PEG2000.

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Abstract

La présente invention concerne un acide nucléique modifié et son utilisation. L'acide nucléique modifié est un acide nucléique non naturel, et comporte une 3'-UTR et un ou plusieurs sites de liaison de microARN situés en aval de la 3'-UTR.
PCT/CN2025/075700 2024-02-05 2025-02-05 Acide nucléique modifié et son utilisation Pending WO2025167868A1 (fr)

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Citations (6)

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Publication number Priority date Publication date Assignee Title
WO2022233880A1 (fr) * 2021-05-03 2022-11-10 Curevac Ag Séquence d'acide nucléique améliorée pour l'expression spécifique de type cellulaire
CN115869332A (zh) * 2022-10-27 2023-03-31 北京新合睿恩生物医疗科技有限公司 递送至体内后在肝脏少表达的mRNA药物及其制备方法
EP4253544A2 (fr) * 2017-05-18 2023-10-04 ModernaTX, Inc. Arn messager modifié comprenant des éléments d'arn fonctionnels
CN117159495A (zh) * 2023-08-16 2023-12-05 北京大学 脂质纳米颗粒及其应用
WO2024026257A2 (fr) * 2022-07-25 2024-02-01 Modernatx, Inc. Polynucléotides modifiés pour l'expression sélective de cellules
CN119101681A (zh) * 2023-06-09 2024-12-10 仁景(苏州)生物科技有限公司 表达可调控的工程化rna分子

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4253544A2 (fr) * 2017-05-18 2023-10-04 ModernaTX, Inc. Arn messager modifié comprenant des éléments d'arn fonctionnels
WO2022233880A1 (fr) * 2021-05-03 2022-11-10 Curevac Ag Séquence d'acide nucléique améliorée pour l'expression spécifique de type cellulaire
WO2024026257A2 (fr) * 2022-07-25 2024-02-01 Modernatx, Inc. Polynucléotides modifiés pour l'expression sélective de cellules
CN115869332A (zh) * 2022-10-27 2023-03-31 北京新合睿恩生物医疗科技有限公司 递送至体内后在肝脏少表达的mRNA药物及其制备方法
CN119101681A (zh) * 2023-06-09 2024-12-10 仁景(苏州)生物科技有限公司 表达可调控的工程化rna分子
CN117159495A (zh) * 2023-08-16 2023-12-05 北京大学 脂质纳米颗粒及其应用

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