WO2023109814A1 - miRNA-2911分子作为核酸稳定剂的应用 - Google Patents

miRNA-2911分子作为核酸稳定剂的应用 Download PDF

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WO2023109814A1
WO2023109814A1 PCT/CN2022/138695 CN2022138695W WO2023109814A1 WO 2023109814 A1 WO2023109814 A1 WO 2023109814A1 CN 2022138695 W CN2022138695 W CN 2022138695W WO 2023109814 A1 WO2023109814 A1 WO 2023109814A1
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nucleic acid
mirna
gene
rna
target nucleic
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张晓霞
曾秒
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成都凌泰氪生物技术有限公司
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    • 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
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    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
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    • 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
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2320/00—Applications; Uses
    • C12N2320/50—Methods for regulating/modulating their activity
    • C12N2320/51—Methods for regulating/modulating their activity modulating the chemical stability, e.g. nuclease-resistance

Definitions

  • the invention relates to the fields of biomedicine and biotechnology, in particular to the application of a miRNA-2911 molecule as a nucleic acid stabilizer.
  • nucleic acid tools or nucleic acid drugs By utilizing the ability of nucleic acid to bind to proteins or other targets, a variety of nucleic acid tools or nucleic acid drugs can be developed, including ASO drugs, mRNA drugs, RNA aptmers, and regulatory RNAs. Among them, mRNA is translated into protein to be developed as mRNA drug. Nucleic acid aptamers can bind to targets (proteins, small molecules, cells, etc.), play a role similar to "chemical antibodies", and are used in the field of nucleic acid drugs. However, these nucleic acid drugs have problems such as being easily degraded by nucleases, unstable affinity with the target, and difficult to locate and transport.
  • nucleic acid drugs In order to protect nucleic acid drugs from being degraded, a variety of methods have been developed to resist the degradation of nucleases, including chemical modification methods and non-chemical modification methods.
  • the methods widely used at present are mainly chemical modification methods, including phosphate backbone modification, sugar modification, phosphoric acid modification and base modification, etc., to enhance the stability of nucleic acid drugs.
  • chemically modified nucleic acid drugs have potential safety issues, such as causing immune response by activating toll-like receptors, causing cytotoxicity (such as inducing apoptosis and causing proteinuria), affecting blood coagulation function, and causing thrombocytopenia, etc.
  • thermodynamically stable hairpin structure hairpin (GCGAAAGC ) can protect the oligonucleotide from being degraded by the 3-terminal exonuclease, and optimize the hairpin to obtain the min-hairpin (GCGAAGC).
  • GCGAAAGC thermodynamically stable hairpin structure hairpin
  • the protective effect of this method is very limited.
  • a patent report (US7022832B2) designed the 5' end or/and 3' end of the oligonucleotide as a hairpin structure, which can play a protective role in 10% heat-inactivated serum, in animals Gene knockdown is achieved with the assistance of nanomaterials, but the protective effect is limited and has not been widely used at present.
  • a patent report (US6121434A) that continuous G bases (wherein the number of G is 0-10) can protect oligonucleotides, but this method is usually used in conjunction with thio-modification. Therefore, at present Nor has it been widely used.
  • mRNA also has the problem of being easily degraded by nucleases.
  • miRNA-2911 was discovered by Zhang Chenyu's research group in the honeysuckle boiling liquid, indicating that miRNA-2911 has good stability and found its antiviral activity. However, whether miRNA-2911 can protect nucleic acid from being degraded has not been reported so far.
  • the present invention is based on the discovery that miRNA-2911 and its derivatives can be used to protect nucleic acids and enhance their stability in serum.
  • the present disclosure provides a miRNA-2911 for improving the stability of a target nucleic acid and nucleic acid molecules derived therefrom, characterized in comprising the following formula:
  • X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 and X 8 are independently selected from A, C, T and U;
  • n 0 or 1
  • L represents a linker group not exceeding 10 nucleotides in length.
  • the linker group comprises G. In some embodiments, the linker group consists of G. In some embodiments, the linker group is 1-5 nucleotides in length. In some embodiments, the linker group is GGGGG.
  • the miRNA-2911 and derived nucleic acid molecules thereof comprise the formula:
  • X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 and X 8 are independently selected from A, C, T and U.
  • Xi , X2 , X4 , and X6 are the same.
  • X 1 , X 2 , X 4 and X 6 are the same and are all A, C or U.
  • X3 , X5 , and X8 are the same.
  • X3 , X5 and X8 are the same and are all A, C or U.
  • X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , and X 8 are independently selected from A, C, and U
  • X 7 is independently selected from A, C, T, and U .
  • X 1 , X 2 , X 3 , X 4 , X 5 , X 6 and X 8 when any one of X 1 , X 2 , X 3 , X 4 , X 5 , X 6 and X 8 is U, X 1 , X 2 , X 3 , X 4 , X 5 , The rest of the nucleotides in X6 and X8 are U. In some embodiments, X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , and X 8 are all A, C, or U.
  • X 7 when X 7 is T, X 1 , X 2 , X 4 , and X 6 are all C, and X 3 , X 5 , and X 8 are all A. In some embodiments, X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , and X 8 are all A.
  • the miRNA-2911 and derived nucleic acid molecules thereof comprise 1 or more (for example 2) of SEQ ID NO: 1 or SEQ ID NO: 3 connected directly or indirectly through a linker, or consist of composition.
  • the linker is no more than 10 nucleotides in length.
  • the linker comprises G.
  • the linker consists of G.
  • the linker is 1-5 nucleotides in length.
  • the linker is GGGGG.
  • the miRNA-2911 and derived nucleic acid molecules thereof comprise or are selected from the following sequences: SEQ ID NO: 4-10.
  • the nucleic acid of interest is DNA or RNA. In some embodiments, the nucleic acid of interest is modified or unmodified, eg, not thio-modified. In some embodiments, the miRNA-2911 and nucleic acid molecules derived therefrom are DNA or RNA.
  • the miRNA-2911 and nucleic acid molecules derived therefrom are modified or unmodified, eg, not thio-modified.
  • the target nucleic acid is 8-5000nt in length, such as 8-4000nt, 8-3000nt, 8-2000nt, 8-1000nt, 8-500nt, 8-200nt, 8-150nt, 8-100nt, 8 -80nt, 8-50nt, 8-40nt, 8-30nt, 8-20nt, 8-10nt, 30-180nt, 20-100nt, 30-50nt, etc.
  • the miRNA-2911 and nucleic acid molecules derived therefrom are other than the following RNA molecules: GGCCGGGGGACGGACUGGGA (SEQ ID NO: 2).
  • the present disclosure provides a protected target nucleic acid, characterized in that the target nucleic acid is composed of the miRNA- 2911 and its derived nucleic acid molecules or nucleic acid molecules with the sequence GGCCGGGGGACGGACUGGGA (SEQ ID NO: 2) are conjugated, or the miRNA-2911 of the first aspect and its derived nucleic acid molecules or with the sequence GGCCGGGGGGACGGACUGGGA (SEQ ID NO: 2) The nucleic acid molecule is inserted into the target nucleic acid, and the stability of the target nucleic acid is increased.
  • the target nucleic acid is composed of the miRNA- 2911 and its derived nucleic acid molecules or nucleic acid molecules with the sequence GGCCGGGGGACGGACUGGGA (SEQ ID NO: 2) are conjugated, or the miRNA-2911 of the first aspect and its derived nucleic acid molecules or with the sequence GGCCGGGGGGACGGACUGGGA (SEQ ID NO: 2)
  • the nucleic acid molecule is
  • the nucleic acid of interest is > 8 nt in length.
  • the target nucleic acid is 8-5000nt in length, such as 8-4000nt, 8-3000nt, 8-2000nt, 8-1000nt, 8-500nt, 8-200nt, 8-150nt, 8-100nt, 8 -80nt, 8-50nt, 8-40nt, 8-30nt, 8-20nt, 8-10nt, 30-180nt, 20-100nt, 30-50nt, etc.
  • the target nucleic acid is nucleic acid for gene knockdown, nucleic acid for gene knockout, nucleic acid for gene activation, nucleic acid for gene modification, nucleic acid for gene editing, nucleic acid for gene regulation, nucleic acid for protein regulation, protein expression Using nucleic acid, nucleic acid for biological detection or nucleic acid drug.
  • the nucleic acid of interest is an oligonucleotide, single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, mRNA or ncRNA (non-coding RNA).
  • the oligonucleotide is an antisense oligonucleotide ASO or an aptamer.
  • the ncRNA is miRNA (microRNA), siRNA (small interfering RNA), saRNA (small activating RNA), piRNA (RNA interacting with Piwi protein), lncRNA (long non-coding RNA), circRNA (circular RNAs), their fragments or other regulatory RNAs.
  • the nucleic acid of interest is DNA or RNA.
  • the nucleic acid of interest is modified or unmodified.
  • the miRNA-2911 and nucleic acid molecules derived therefrom are modified or unmodified.
  • the conjugation is a chemical covalent linkage, preferably via a phosphodiester linkage.
  • the method of constructing the protected target nucleic acid is: chemical synthesis, genetic engineering based on the principle of PCR or biosynthesis.
  • the present disclosure provides a pharmaceutical composition, which is characterized in that the pharmaceutical composition comprises the protected target nucleic acid of the second aspect and a pharmaceutically acceptable carrier.
  • the present disclosure provides a method for increasing the stability of a nucleic acid of interest, characterized in that the miRNA-2911 of the first aspect and its derived nucleic acid molecule or having the sequence GGCCGGGGGACGGACUGGGA (SEQ ID NO: 2 ) is conjugated to the 5' end, the 3' end, or both the 5' end and the 3 end of the nucleic acid of interest, or inserted into the nucleic acid of interest.
  • the target nucleic acid length is ⁇ 8nt, such as 8-5000nt such as 8-4000nt, 8-3000nt, 8-2000nt, 8-1000nt, 8-500nt, preferably 8-200nt, such as 8-150nt, 8-100nt, 8-80nt, 8-50nt, 8-40nt, 8-30nt, 8-20nt, 8-10nt, 30-180nt, 20-100nt, 30-50nt, etc.
  • 8nt such as 8-5000nt such as 8-4000nt, 8-3000nt, 8-2000nt, 8-1000nt, 8-500nt, preferably 8-200nt, such as 8-150nt, 8-100nt, 8-80nt, 8-50nt, 8-40nt, 8-30nt, 8-20nt, 8-10nt, 30-180nt, 20-100nt, 30-50nt, etc.
  • the target nucleic acid is nucleic acid for gene knockdown, nucleic acid for gene knockout, nucleic acid for gene activation, nucleic acid for gene modification, nucleic acid for gene editing, nucleic acid for gene regulation, nucleic acid for protein regulation, protein expression Using nucleic acid, nucleic acid for biological detection or nucleic acid drug.
  • the nucleic acid of interest is an oligonucleotide, single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, mRNA or ncRNA.
  • the oligonucleotide is an antisense oligonucleotide ASO or an aptamer.
  • the ncRNA is miRNA, siRNA, saRNA, piRNA, lncRNA, circRNA, fragments thereof, or other regulatory RNA.
  • the nucleic acid of interest is modified or unmodified.
  • the miRNA-2911 and nucleic acid molecules derived therefrom are modified or unmodified.
  • the conjugation is a chemical covalent linkage, preferably via a phosphodiester linkage.
  • the method of constructing the protected target nucleic acid is: chemical synthesis, genetic engineering based on the principle of PCR or biosynthesis.
  • the nucleic acid of interest is DNA or RNA.
  • Figure 1 shows the stability of RNA drugs containing miRNA-2911 in serum
  • Figure 2 shows the stability in serum of RNA drugs containing RNA modified based on miRNA-2911
  • Figure 3 shows the stability in serum of nucleic acid drug chimeras containing RNA modified based on miRNA-2911;
  • Figure 4 shows the stability in serum of DNA drugs containing DNA modified based on miRNA-2911
  • Figure 5 shows the stability in serum of nucleic acid drug chimeras containing DNA modified based on miRNA-2911
  • Figure 6 shows the stability in serum of nucleic acid drugs containing R2911A with different sequences
  • Figure 7 shows the stability in serum of nucleic acid drugs of different lengths containing R2911A
  • Figure 8 shows the stability results of nucleic acid drugs containing R2911A in animals
  • Figure 9 shows the stability in serum of nucleic acid drugs containing R2911A at the 5' end or 3' end and inside;
  • Figure 10 evaluates the ability of siRNA containing R2911A to knock down target RNA in cells (Hep3B, 97H cell lines);
  • Figure 11 evaluates the effect of R2911A-containing mRNA on protein expression in cells.
  • the methods for protecting nucleic acid drugs from degradation in the prior art include chemical modification methods and non-chemical modification methods, but both have their limitations.
  • the inventors of the present application pioneered the use of miRNA-2911 and its derivatives to protect nucleic acid drugs, which significantly enhanced their stability in serum.
  • the present invention has the following beneficial effects:
  • nucleic acid molecules and methods of the present invention can enhance the stability of nucleic acid drugs in serum without any modification
  • nucleic acid drug of the present invention has the advantage of high stability in serum compared to the existing conventional chemically modified protection methods and non-chemically modified protection methods;
  • the protection method of the present invention can also be used to protect ncRNA and mRNA, greatly improving the stability of ncRNA and mRNA, and providing strong support for the development of ncRNA and mRNA drugs.
  • ranges are used as shorthand for describing each and every value that is within the range. Any value within a range, such as an integer value, is optional as the terminus of the range.
  • 10 nucleotides means not more than 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 nucleotides and any subranges formed by them such as 1-8nt, 2-5nt, 3-7nt etc.
  • nucleic acid refers to any DNA, RNA or DNA/RNA chimera, and may be an oligonucleotide or a polynucleotide, and may be is unmodified RNA or DNA or modified RNA or DNA.
  • the term includes, but is not limited to, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, including A hybrid molecule of DNA and RNA that is single- or double-stranded or a mixture of single- and double-stranded regions.
  • a nucleic acid may comprise one or more modified nucleotides, modified linkages, etc.
  • modified linkages or internucleotide linkages include phosphorothioate, phosphorodithioate, and the like.
  • the nucleotides include phosphorus derivatives.
  • Phosphorus derivatives (or modified phosphate groups) that can be attached to sugar or sugar analog moieties in the modified nucleotides of the present invention can be monophosphate, diphosphate, triphosphate, alkyl phosphate , alkane phosphate, phosphorothioate, etc.
  • the preparation of the abovementioned phosphate analogs, as well as their incorporation into nucleotides, modified nucleotides and oligonucleotides is also known per se and need not be described here.
  • unmodified or “natural” nucleotides include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U).
  • Modified nucleotides include nucleotides that occur only infrequently or transiently in natural nucleic acids, for example, hypoxanthine, 6-methyladenine, 5-Me pyrimidine, especially 5-methylcytosine (also known as 5-methyl-2'deoxycytosine (and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC, and gentiobiosyl HMC, and synthetic nuclei Nucleic acid, for example, 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalkylamino)adenine, or other heterosubstituted alkyladenine , 2-thiouracil,
  • nucleic acids of the invention comprise one or more modifications (e.g., base modifications, backbone modifications, etc.) to provide new or enhanced characteristics to the nucleic acid (e.g., improved stability).
  • modifications e.g., base modifications, backbone modifications, etc.
  • nucleosides are base-sugar combinations.
  • the base portion of a nucleoside is usually a heterocyclic base.
  • the two most common classes of such heterocyclic bases are purines and pyrimidines.
  • a nucleotide is a nucleoside that also includes a phosphate group covalently linked to the sugar moiety of the nucleoside.
  • the phosphate group can be attached to the 2', 3' or 5' hydroxyl moiety of the sugar.
  • phosphate groups covalently link adjacent nucleosides to each other to form a linear polymeric compound.
  • ends of linear polymeric compounds can be further linked to form cyclic compounds, however, linear compounds are generally suitable.
  • linear compounds may have internal nucleotide base complementarity and thus may fold in such a way as to produce fully or partially double-stranded compounds.
  • the phosphate groups are often referred to as forming the internucleoside backbone of the oligonucleotide. The normal bond or backbone of RNA and DNA is a 3' to 5' phosphodiester bond.
  • nucleic acids containing modifications include nucleic acids containing modified backbones or non-natural internucleoside linkages.
  • Nucleic acids (with modified backbones) include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
  • Suitable modified oligonucleotides in which a phosphorus atom is contained include, for example, phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl Phosphonates include 3'-alkylene phosphates, 5'-alkylene phosphates and chiral phosphates, phosphonates, phosphoramidates include 3'-aminophosphoramidates and aminoalkylphosphoramidates, Phosphorodiamidate, phosphorothioate, phosphorothioate, phosphorothioate, phosphoroselenoate and phosphoroborate with normal 3'-5' linkages, 2'- 5' linkage analogs as well as those of reverse polarity in which one or more of the internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage.
  • Suitable oligonucleotides with reverse polarity contain a single 3' to 3' bond at the most 3' internucleotide bond, i.e. a single reverse of an abasic (nucleobase missing or replaced by a hydroxyl).
  • Transnucleoside residues Also included are various salts (such as, for example, potassium or sodium), mixed salts and free acid forms.
  • nucleic acids with morpholino backbone structures include, for example, nucleic acids with morpholino backbone structures.
  • nucleic acids of the invention comprise a 6-membered morpholino ring in place of a ribose ring.
  • phosphorodiamidate or other non-phosphodiester internucleoside linkages replace phosphodiester linkages.
  • Suitable modified polynucleotide backbones that do not include phosphorus atoms have short-chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short-chain heteroatoms.
  • the backbone formed by bonds between atoms or heterocyclic nucleosides.
  • morpholino linkages formed in part from the sugar moieties of nucleosides
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formoacetyl and thioformyl acetyl backbones Methyleneformyl acetyl and thioformyl acetyl skeletons
  • riboacetyl (riboacetyl) skeletons olefin-containing skeletons; sulfamate skeletons; Sulfonate and sulfonamide backbones; amide backbones; and other backbones with mixed N, O, S, and CH2 moieties.
  • LNA locked nucleic acid
  • the chain can be a methylene group ( -CH2- ) (group bridging the 2' oxygen atom and the 4' carbon atom), where n is 1 or 2 (Singh et al., Chem. Commun., 1998, 4, 455-456) .
  • LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil together with their oligomerization and nucleic acid recognition properties have been described in the prior art (Koshkin et al., Tetrahedron , 1998, 54, 3607-3630).
  • Nucleic acids of the invention may also include one or more substituted sugar moieties.
  • Suitable polynucleotides comprise a sugar substituent selected from: OH; F; O-, S- or N-alkyl; O-, S- or N-alkenyl; O-, S- or N-alkynyl or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl groups may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl.
  • Particularly suitable are O((CH 2 ) n O) m CH 3 , O(CH 2 ) n OCH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O( CH 2 ) n ONH 2 and O(CH 2 ) n ON((CH 2 ) n CH 3 ) 2 , where n and m are 1 to about 10.
  • Suitable polynucleotides comprise sugar substituents selected from the group consisting of C1 to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O- Aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , Heterocycloalkyl, Heterocycle Alkaryl, aminoalkylamino, polyalkylamino, substituted silyl groups, RNA cleavage groups, reporter groups, intercalators, groups for modifying the pharmacokinetic properties of oligonucleotides or with Groups for improving the pharmacodynamic properties of oligonucleotides and other substituents with similar properties.
  • Suitable modifications include 2'-methoxyethoxy (2' - O- CH2CH2OCH3 , also known as 2'-O-(2-methoxyethyl) or 2'-MOE) ( Martin et al., Helv. Chim. Acta, 1995, 78, 486-504), ie alkoxyalkoxy.
  • Another suitable modification includes 2'-dimethylaminooxyethoxy, the O(CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2'-DMAOE, as in the examples below and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), namely 2'-O-CH 2 -O-CH 2 -N(CH 3 ) 2 .
  • the 2'-sugar substituent can be in the arabinose (upper) or ribose (lower) position.
  • a suitable 2'-arabinose modification is 2'-F.
  • Similar modifications can also be made at other positions on the oligomeric compound, specifically at the 3' position and the 5' end of the sugar on the 3' terminal nucleoside or in 2'-5' linked oligonucleotides. The 5' position of the nucleotide.
  • the oligomeric compound may also have a sugar mimetic such as a cyclobutyl moiety in place of the pentofuranose.
  • Nucleic acids of the invention may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobases include the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C) and uracil ( U).
  • Additional modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine Cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-cluster substituted phenoxazine cytidine (e.g.
  • Heterocyclic base moieties may also include those in which a purine or pyrimidine base is replaced by other heterocycles, such as 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone.
  • Additional nucleobases include those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pp. 858-859, Kroschwitz, J.I. Ed. John Wiley & Sons, 1990, those disclosed by Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Those published in Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pp. 289-302, Crooke, S.T. and Lebleu, B.
  • nucleobases are useful to increase the binding affinity of oligomeric compounds. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynyl base cytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6°C-1.2°C (Sanghvi et al., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and for example when Suitable base substitutions when combined with 2'-O-methoxyethyl sugar modification.
  • nucleic acids of the invention involves chemically linking to the polynucleotide one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide.
  • moieties or conjugates may include conjugate groups that covalently bind functional groups such as primary or secondary hydroxyl groups.
  • Conjugate groups include, but are not limited to, intercalators, reporters, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. group.
  • Suitable conjugate groups include, but are not limited to, cholesterol, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes.
  • Groups that enhance pharmacodynamic properties include groups that improve absorption, increase resistance to degradation, and/or enhance sequence-specific hybridization to target nucleic acids.
  • Groups that enhance pharmacokinetic properties include groups that improve the absorption, distribution, metabolism or excretion of nucleic acids of the invention.
  • Conjugate moieties include, but are not limited to: lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994,4,1053-1060), thioethers such as hexyl-S-trityl mercaptan (Manoharan et al, Ann.N.Y.Acad.Sci., 1992,660,306-309; Manoharan et al, Bioorg.Med. Chem.
  • lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994,4,1053-1060), thi
  • nucleic acid molecules of the invention comprise or consist of SEQ ID NO: 1 or 3.
  • the nucleic acid molecule of the present invention comprises two or more of SEQ ID NO: 1 or 3 (i.e. a dimer, trimer, etc. of SEQ ID NO: 1 or 3) directly or indirectly connected by a linker , or consist of it.
  • the terms “linker group” and “linker” are used interchangeably to refer to oligonucleotide sequences used to join different parts of sequences.
  • the oligonucleotide sequence may comprise unmodified or modified natural or non-natural nucleotides.
  • the linker group or linker comprises A, T, C, G and/or U.
  • the linker group or linker comprises G.
  • the linker group or linker consists of G.
  • the linker group or linker is 1-10 nucleotides, such as 1-5 nucleotides, such as 3-5 nucleotides in length.
  • the linker group or linker is GGGGG. In some embodiments, the presence of the linker group or linker does not affect the protection of the nucleic acid molecules of the invention.
  • conjugate and “link” are used interchangeably to refer to linking by a chemical bond.
  • the stability of the nucleic acid in the serum of the present invention refers to the stability of the nucleic acid in various concentrations of serum such as 50% serum.
  • the improvement of stability refers to that the stability of the target nucleic acid, for example in serum, is improved compared with the target nucleic acid not protected by the nucleic acid molecule of the present invention or protected by other methods.
  • the activity of the nucleic acid of interest is equivalent or similar to the activity of the nucleic acid of interest that is not protected by the nucleic acid molecule of the present invention or protected by other methods, but its stability is improved.
  • the activity of the nucleic acid of interest remains unchanged.
  • the term "pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. The use of such media and reagents is well known in the art. Unless any conventional media or agents are incompatible with the materials provided herein, they are contemplated for use in the compositions.
  • the materials used in the examples herein are commercially available, and the various specific experimental methods used to carry out the experiments are routine experimental methods in the art or according to the steps and conditions suggested by the manufacturer, and can be obtained by those skilled in the art. Personnel are routinely identified as needed.
  • the serum used in the examples of this specification is Gibco special grade fetal bovine serum.
  • the degradation system contains: oligonucleotide drug, 1 ⁇ PBS, 50% serum and water.
  • the electrophoresis conditions used are: 180V, 20min; the loading amount is: 2 ⁇ g-5 ⁇ g.
  • the miRNA-2911 sequence and its derivatives in the examples of this specification are all added to both ends of the protected sequence.
  • Example 1 The stability of RNA drugs containing miRNA-2911 is improved in serum
  • miRNA-2911 Specific sequences (miRNA-2911) were added to the 5' end and 3' end of the RNA (R1) to be protected to form miRNA2911-R1 (shown as mi2911-R1 in Figure 1), and random sequence RRs were selected as negative controls ( The reason for choosing this random sequence is that the length of the oligonucleotide itself is short and it is easy to degrade. Therefore, a random control sequence that is basically the same length as the protected sequence is selected, so that the electrophoresis results are almost in a straight line, which is convenient for comparison) . Each sequence was incubated in serum for different times: 6h, 12h and 24h, and the results were analyzed by 3% agarose gel electrophoresis.
  • Example 1 the sequence information of the nucleic acid fragments used is as follows:
  • miRNA-2911 GGCCGGGGGACGGACUGGGA (SEQ ID NO: 2)
  • RNA modified based on miRNA-2911 improves the stability of RNA drugs in serum
  • the above sequence was incubated in serum for different time: 6h, 12h and 24h, and then the results were analyzed by 3% agarose gel electrophoresis.
  • the results are shown in Figure 2-(1), Figure 2-(2) and Figure 2-(3), in which the degrees of degradation of different modified nucleic acid drugs in serum are different, but there are intact RNAs that have not been degraded, All have protective capabilities.
  • RNA drug containing RNA engineered based on miRNA-2911 was incubated in serum for up to 36 hours, and the results are shown in Figure 2-(4).
  • the RNA drug containing RNA modified based on miRNA-2911 still has a complete sequence that has not been degraded at 36 hours, indicating that the RNA drug containing RNA modified based on miRNA-2911 has high stability in serum.
  • Example 2 the sequence information of the nucleic acid fragment used is as follows:
  • R2911A GGAAGGGGGAAGGAAAGGGA (SEQ ID NO: 4)
  • R2911C GGCCGGGGGCCGGCCCGGGC (SEQ ID NO: 5)
  • R2911U GGUUGGGGGUUGGUUUGGGU (SEQ ID NO: 6)
  • R2911A-5G GGAAGGGGGGGGGGAAGGAAAGGGA (SEQ ID NO: 7)
  • R2911A-D GGAAGGGGGAAGGAAAGGGAGGAAGGGGGAAGGAAAGGGA (SEQ ID NO: 8)
  • Example 3 Improving the serum stability of nucleic acid drug chimeras after transforming miRNA-2911 into DNA
  • Example 3 the sequence information of D1 and DRs is as follows:
  • Example 4 Transforming miRNA-2911 into DNA can improve the stability of DNA drugs in serum
  • the inventors transformed the miRNA-2911 sequence into DNA (D2911 and D2911A), added the transformed sequences to both ends of D1 to form D2911-D1 and D2911A-D1, and evaluated the ability of the transformed sequences to protect DNA.
  • D1 and DDs were used as negative controls.
  • the above sequences were incubated in serum for different time: 12h, 24h and 36h, and then the results were analyzed by 3% agarose gel electrophoresis.
  • Figure 4-(1) and 4-(2) wherein the stability of D2911-D1 and D2911A-D1 in serum is significantly enhanced, and Figure 4-(2) shows that the enhancement of nucleic acid drug stability is caused by D2911A brings, not the sequence itself.
  • sequence information of the nucleic acid fragment used is as follows:
  • D2911 GGCCGGGGGACGGACTGGGA (SEQ ID NO:9)
  • D2911A GGAAGGGGGAAGGAAAGGGA (SEQ ID NO: 10)
  • Example 5 Transforming miRNA-2911 into DNA can improve the serum stability of nucleic acid drugs
  • D2911A sequence was added to both ends of R1 to form D2911A-R1, and random sequence DRs were selected as negative controls. Each sequence was incubated in serum for 1 h and the results were analyzed by 3% agarose gel electrophoresis. The results are shown in Figure 5, the nucleic acid drug chimera containing D2911A still has certain stability in serum.
  • Example 5 the sequence information of the nucleic acid fragments used is as follows:
  • Example 6 Evaluation of the stability in serum of nucleic acid drugs containing different sequences of R2911A
  • nucleic acid drugs containing different sequences of R2911A in serum There are differences in the degree of degradation of nucleic acid drugs containing different sequences of R2911A in serum, but there are complete DNA sequences that have not been degraded. Combined with the protection ability of R1 in Example 1, it shows that the nucleic acid drugs containing R2911A Nucleic acid drugs with different sequences are all stable in serum, indicating that the method of the present invention has universal applicability for protecting nucleic acid sequences.
  • Example 6 the sequence information of the nucleic acid fragments used is as follows:
  • R2 ACGGGGUCAUUAGUUCAUAG (SEQ ID NO: 17)
  • R3 UAAGAUACACCUGCAAAGGC (SEQ ID NO: 18)
  • R6 UGACCCUGAAGUUCAUCUGC (SEQ ID NO: 21)
  • Example 7 Evaluation of the stability in serum of nucleic acid drugs of different lengths containing R2911A
  • RNA of 2000nt was used as their negative controls (R80-RC and R2K-RC respectively); the RNA of 2000nt represented the protective ability of the method of the present invention to long-chain RNA (including mRNA and lncRNA).
  • Example 7 the sequence information of the nucleic acid fragment used is as follows:
  • Example 8 Evaluation of the stability of nucleic acid drugs containing R2911A in animals
  • the inventors used cy5.5 to label a nucleic acid drug containing R2911A (R2911A-R1, formed by adding R2911A sequences to both ends of R1 respectively), to form Cy5.5-R2911A-R1. Before injection, this Cy5.5-R2911A-R1 was diluted with physiological saline. The mice were weighed and administered according to the injection dose of 2 mg/kg in mice undergoing abdominal depilation. Mice were injected subcutaneously. Grab the mouse and fix it, inject it in the skin between the two hind limbs, draw out the drug with a defined volume according to the body weight with a syringe, gently pierce the skin with the needle bevel facing upward, and inject slowly along the subcutaneous needle about 1-2cm.
  • Example 9 Evaluate the stability in serum of nucleic acid drugs containing R2911A at the 5' end, 3' end and inside
  • Example 9 the sequence information of the nucleic acid fragment used is as follows:
  • R2911A-L GGAAGGGGGAAGGAAAGGGAUGAAUGUAGAGAUGCGGUGG (SEQ ID NO: 27)
  • R2911A-R UGAAUGUAGAGAUGCGGUGGGGAAGGGGGAAGGAAAGGGA (SEQ ID NO: 28)
  • RRs ACCCGACCUCUUCUAUCUGGACCCGACCGUCUCUUUUUUG (SEQ ID NO: 29).
  • Example 10 Evaluation of the knockdown effect of siRNA drugs containing R2911A on target RNA in cells
  • inclisiran-R2911A Using FDA-approved inclisiran as a positive control (Shenzhen ginseng is a chemically modified siRNA), by adding R2911A at the 3' end of its RNA sequence to form an unmodified siRNA drug containing R2911A (inclisiran-R2911A), using Lipofectamine 2000 for siRNA transfection: add an appropriate amount of inclisiran-2911A to 50 ⁇ L of Opti-MEM for dilution, and at the same time dilute 4.8 ⁇ L of Lipofectamine 2000 with 50 ⁇ L of Opti-MEM, gently pipette to mix and then place at room temperature for 5 minutes.
  • the reverse transcription system Take out 1 ⁇ g of total RNA for reverse transcription, the reverse transcription system is 20 ⁇ L, after the reverse transcription is completed, it will be cDNA.
  • the PCR reaction conditions are: 95°C for 3min; 95°C for 10s, 60°C for 10s, 72°C for 20s (this program is 40 cycles); 95°C for 15s; 60°C for 60s; One fluorescent signal; 95°C for 10s.
  • the target RNA of the siRNA (inclisiran or inclisiran connected with 2911) used in this example is PCSK9, and its knockdown ability is tested at the cellular level.
  • the tested cell lines are 97H cells and Hep3B cells;
  • the PBS group was used as a negative control (Mock), and inclisiran was set as a positive control.
  • nucleic acid fragment information used is as follows:
  • nucleotides are modified with methoxy at the 2' position; nucleotides marked in italics are modified with fluorine (fluorine is the replacement of the hydroxyl group at the 2' position with a fluorine atom); * indicates sulfur modification (indicating the replacement of a non-bridging oxygen atom in a phosphate bond with a sulfur atom).
  • R2911A was added to both ends of the firefly luciferase gene (mRNA1) to form R-mRNA1; mRNA1 without any protection was used as a negative control (mRRs).
  • mRNA1 firefly luciferase gene

Abstract

本发明涉及一种miRNA-2911分子作为核酸稳定剂应用。具体而言，所述的用于提高目的核酸的稳定性的miRNA-2911分子的结构如下式所示： GGX 1X 2GGGGG-(L) n-X 3X 4GGX 5X 6X 7GGGX 8(SEQ ID NO:1) 其中： X 1、X 2、X 3、X 4、X 5、X 6、X 7和X 8独立地选自A、C、T和U； n为0或1； L表示长度不超过10个核苷酸的接头基团。

Description

miRNA-2911分子作为核酸稳定剂的应用 技术领域
本发明涉及生物医药和生物技术领域，特别是涉及一种miRNA-2911分子作为核酸稳定剂应用。
背景技术
通过利用核酸与蛋白质或其它靶标结合的能力，可以开发多种核酸工具或核酸药物，包括了ASO药物、mRNA药物、RNA核酸适配体(apatmer)及调控性RNA等。其中，mRNA通过蛋白翻译，被开发为mRNA药物。核酸适配体可以与靶标(蛋白质、小分子、细胞等)结合，发挥类似“化学抗体”的作用，被应用于核酸药物领域。但是，这类核酸药物均存在易被核酸酶降解、与靶标亲和力不稳定、定位及运输困难的问题等。
为保护核酸药物不被降解，已开发了多种方法来对抗核酸酶的降解，包括化学修饰方法和非化学修饰方法两类。目前被广泛应用的方法主要是化学修饰方法，包括磷酸盐骨架修饰、糖修饰、磷酸修饰和碱基修饰等，用于增强核酸药物的稳定性。但是化学修饰的核酸药物存在潜在的安全性问题，比如通过激活toll样受体引起免疫应答、导致细胞毒性(如诱导凋亡而引起蛋白尿)、影响凝血功能和导致血小板减少等。
目前报道的非化学修饰的方法甚少，在1989年报道了能够形成极稳定的二级结构的短链寡核苷酸，1993年Coulson课题组报道了一种热力学稳定的发夹结构hairpin(GCGAAAGC)可以保护寡核苷酸不被3端核酸外切酶降解，并对该hairpin进行优化，得到min-hairpin(GCGAAGC)。但是该方法的保护效果十分有限。在2002年有专利报道(US7022832B2)将寡核苷酸的5'端或/和3'端设计为发夹结构，在10％的热失活的血清中的可以起到保护效果，在动物中利用纳米材料的辅助实现基因敲低，但保护效果有限，目前并没有得到广泛应用。在2002年有专利报道(US6121434A)连续G碱基(其中G的个数为0-10)可以保护寡核苷酸，但该方法通常情况下是要和硫代修饰进行联用，因此，目前也没有得到广泛应用。另外，类似地，mRNA也存在易被核酸酶降解的问题。
miRNA-2911是张辰宇课题组在金银花熬煮液中发现的，说明miRNA-2911具有很好稳定性，并发现其抗病毒的活性。但miRNA-2911是否能保护核酸不被降解的性质至今还没有报道。
因此，对提高核酸稳定性和/或保护核酸免受降解的物质和方法存在未被满足的需求。
发明内容
本发明基于以下发现：miRNA-2911及其衍生物能够用于保护核酸，增强其在血清中的稳定性。
在第一方面，本公开内容提供了一种用于提高目的核酸的稳定性的miRNA-2911及其衍生的核酸分子，其特征在于包含下式：
GGX 1X 2GGGGG-(L) n-X 3X 4GGX 5X 6X 7GGGX 8(SEQ ID NO:1)
其中：
X 1、X 2、X 3、X 4、X 5、X 6、X 7和X 8独立地选自A、C、T和U；
n为0或1；
L表示长度不超过10个核苷酸的接头基团。
在一些实施方案中，所述接头基团包含G。在一些实施方案中，所述接头基团由G组成。在一些实施方案中，所述接头基团长度为1-5个核苷酸。在一些实施方案中，所述接头基团为GGGGG。
在一些实施方案中，所述miRNA-2911及其衍生的核酸分子包含下式：
GGX 1X 2GGGGGX 3X 4GGX 5X 6X 7GGGX 8(SEQ ID NO:3)
其中：
X 1、X 2、X 3、X 4、X 5、X 6、X 7和X 8独立地选自A、C、T和U。
在一些实施方案中，X 1、X 2、X 4和X 6相同。在一些实施方案中，X 1、X 2、X 4和X 6相同，且均为A、C或U。在一些实施方案中，X 3、X 5和X 8相同。在一些实施方案中，X 3、X 5和X 8相同，且均为A、C或U。在一些实施方案中，X 1、X 2、X 3、X 4、X 5、X 6和X 8独立地选自A、C和U，以及X 7独立地选自A、C、T和U。在一些实施方案中，当X 1、X 2、X 3、X 4、X 5、X 6和X 8中的任一个为U时，X 1、X 2、X 3、X 4、X 5、X 6和X 8中的其余核苷酸均为U。在一些实施方案中，X 1、X 2、X 3、X 4、X 5、X 6、X 7和X 8均为A、C或U。在一些实施方案中，当X 7为T时，X 1、X 2、X 4和X 6均为C，且X 3、X 5和X 8均为A。在一些实施方案中，X 1、X 2、X 3、X 4、X 5、X 6、X 7和X 8均为A。
在一些实施方案中，所述miRNA-2911及其衍生的核酸分子包含1个或多个(例如2个)直接或通过接头间接连接的SEQ ID NO:1或SEQ ID NO:3，或者由其组成。在一些实施方案中，所述接头长不超过10个核苷酸。在一些实施方案中，所述接头包含G。在一些实施方案中，所述接头由G组成。在一些实施方案中，所述接头长度为1-5个核苷酸。在一些实施方案中，所述接头为GGGGG。
在一些实施方案中，所述miRNA-2911及其衍生的核酸分子包含或选自下列序列：SEQ ID NO:4-10。
在一些实施方案中，所述目的核酸为DNA或RNA。在一些实施方案中，所述目的核酸为修饰的或未经修饰的例如未经硫代修饰的。在一些实施方案中，所述miRNA-2911及其衍生的核酸分子为DNA或RNA。
在一些实施方案中，所述miRNA-2911及其衍生的核酸分子为修饰的或未经修饰的例如未经硫代修饰的。
在一些实施方案中，所述目的核酸长度为8-5000nt，例如8-4000nt、8-3000nt、8-2000nt、8-1000nt、8-500nt、8-200nt、8-150nt、8-100nt、8-80nt、8-50nt、8-40nt、8-30nt、8-20nt、8-10nt、30-180nt、20-100nt、30-50nt等。
在一些实施方案中，所述miRNA-2911及其衍生的核酸分子不为以下RNA分子： GGCCGGGGGACGGACUGGGA(SEQ ID NO:2)。
在第二方面，本公开内容提供了一种经保护的目的核酸，其特征在于所述目的核酸在其5'端、3'端或5'端和3端两者由第一方面的miRNA-2911及其衍生的核酸分子或具有序列GGCCGGGGGACGGACUGGGA(SEQ ID NO:2)的核酸分子缀合，或者将第一方面的miRNA-2911及其衍生的核酸分子或具有序列GGCCGGGGGACGGACUGGGA(SEQ ID NO:2)的核酸分子插入所述目的核酸中，并且所述目的核酸的稳定性得到增加。
在一些实施方案中，所述目的核酸长度为≥8nt。在一些实施方案中，所述目的核酸长度为8-5000nt，例如8-4000nt、8-3000nt、8-2000nt、8-1000nt、8-500nt、8-200nt、8-150nt、8-100nt、8-80nt、8-50nt、8-40nt、8-30nt、8-20nt、8-10nt、30-180nt、20-100nt、30-50nt等。
在一些实施方案中，所述目的核酸为基因敲低用核酸、基因敲除用核酸、基因活化用核酸、基因修饰用核酸、基因编辑用核酸、基因调控用核酸、蛋白调控用核酸、蛋白表达用核酸、生物检测用核酸或核酸药物。
在一些实施方案中，所述目的核酸为寡核苷酸、单链DNA、双链DNA、单链RNA、双链RNA、mRNA或ncRNA(非编码RNA)。
在一些实施方案中，所述寡核苷酸为反义寡核苷酸ASO或核酸适配体。在一些实施方案中，所述ncRNA为miRNA(微小RNA)、siRNA(小干扰RNA)、saRNA(小激活RNA)、piRNA(与Piwi蛋白相作用的RNA)、lncRNA(长非编码RNA)、circRNA(环状RNA)、它们的片段或其它调控性RNA。
在一些实施方案中，所述目的核酸为DNA或RNA。
在一些实施方案中，所述目的核酸为修饰的或未经修饰的。在一些实施方案中，所述miRNA-2911及其衍生的核酸分子为修饰的或未经修饰的。在一些实施方案中，所述的缀合为化学共价连接，优选为通过磷酸二酯键连接。
在一些实施方案中，构建所述经保护的目的核酸的方式为：化学合成法、基于PCR原理的基因工程方法或生物合成方法。
在第三方面，本公开内容提供了一种药物组合物，其特征在于所述药物组合物包含第二方面的经保护的目的核酸和药学上可接受的载体。
在第四方面，本公开内容提供了一种用于增加目的核酸的稳定性的方法，其特征在于将第一方面的miRNA-2911及其衍生的核酸分子或具有序列GGCCGGGGGACGGACUGGGA(SEQ ID NO:2)的核酸分子缀合至所述目的核酸的5'端、3'端或5'端和3端两者，或者插入所述目的核酸中。
在一些实施方案中，所述目的核酸长度为≥8nt，例如8-5000nt如8-4000nt、8-3000nt、8-2000nt、8-1000nt、8-500nt，优选8-200nt，例如8-150nt、8-100nt、8-80nt、8-50nt、8-40nt、8-30nt、8-20nt、8-10nt、30-180nt、20-100nt、30-50nt等。
在一些实施方案中，所述目的核酸为基因敲低用核酸、基因敲除用核酸、基因活化用核酸、基因修饰用核酸、基因编辑用核酸、基因调控用核酸、蛋白调控用核酸、蛋白表达用核 酸、生物检测用核酸或核酸药物。
在一些实施方案中，所述目的核酸为寡核苷酸、单链DNA、双链DNA、单链RNA、双链RNA、mRNA或ncRNA。
在一些实施方案中，所述寡核苷酸为反义寡核苷酸ASO或核酸适配体。
在一些实施方案中，所述ncRNA为miRNA、siRNA、saRNA、piRNA、lncRNA、circRNA、它们的片段或其它调控性RNA。
在一些实施方案中，所述目的核酸为修饰的或未经修饰的。在一些实施方案中，所述miRNA-2911及其衍生的核酸分子为修饰的或未经修饰的。
在一些实施方案中，所述的缀合为化学共价连接，优选为通过磷酸二酯键连接。
在一些实施方案中，构建所述经保护的目的核酸的方式为：化学合成法、基于PCR原理的基因工程方法或生物合成方法。在一些实施方案中，所述目的核酸为DNA或RNA。
下列描述和实施例详细阐述了本发明的实施方案。要理解的是，本发明不限于本文所述的具体实施方案并因此可改动。本领域技术人员将认识的是，存在本发明的许多变动和修改，所述变动和修改均包含在其范围之内。
附图说明
上述仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，以下结合附图与具体实施方式对本发明作进一步的详细说明。
图1显示了含有miRNA-2911的RNA药物在血清中的稳定性；
图2显示了含有基于miRNA-2911改造的RNA的RNA药物在血清中的稳定性；
图3显示了含有基于miRNA-2911改造的RNA的核酸药物嵌合体在血清中的稳定性；
图4显示了含有基于miRNA-2911改造的DNA的DNA药物在血清中的稳定性；
图5显示了含有基于miRNA-2911改造的DNA的核酸药物嵌合体在血清中的稳定性；
图6显示了含有R2911A的具有不同序列的核酸药物在血清中的稳定性；
图7显示了含有R2911A的不同长度的核酸药物在血清中的稳定性；
图8显示了含有R2911A的核酸药物在动物体内的稳定性结果；
图9显示了5'端或者3'端及内部含有R2911A的核酸药物在血清中稳定性；
图10评估含有R2911A的siRNA在细胞内对靶标RNA的敲低能力(Hep3B、97H细胞系)；
图11评估含有R2911A的mRNA在细胞内表达蛋白的效果。
具体实施方式
参考用于说明的示例应用在下文中描述本发明的数个方面。应当理解的是，陈述许多具体细节、关系和方法来提供对本发明的充分理解。然而，在相关领域的普通技术人员将容易地认识到，可在不含一个或多个具体细节的情况下实施本发明或者可用其他方法来实施本发明。
现有技术的保护核酸药物不被降解的方法包括化学修饰方法和非化学修饰方法两类，但均有其局限性。本申请的发明人首创性地利用miRNA-2911及其衍生物对核酸药物进行保护，显著地增强其了在血清中的稳定性。
相比于现有技术，本发明具有如下有益效果：
1.本发明核酸分子和方法能够在无需任何修饰的情况下增强核酸药物在血清中的稳定性；
2.经验证，相对于现有常规的化学修饰的保护方法和非化学修饰的保护方法，本发明的核酸药物在血清中具有稳定性高的优点；和
3.本发明的保护方法除了应用于寡核苷酸药物，还可用于保护ncRNA和mRNA，大大提高了ncRNA和mRNA的稳定性，为ncRNA和mRNA类药物的开发提供了有力的支持。
本文所用的术语仅以描述具体的实施方案为目的而不意图限制本发明。除非上下文另有明确指示，否则本文所用的单数形式“一”、“一个”和“所述”也意图包括复数形式。此外，开放式的表述“包括”和“包含”解释为还可以含有没有述及的结构组成部分或方法步骤，但需要注意的是，该开放式的表述也涵盖仅由所述的组分和方法步骤组成的情形(即涵盖了封闭式表述“由……组成”的情形)。
如全文所用，范围用作描述该范围内的每个数值和所有数值的简写形式。范围内的任何数值例如整数值都可选做该范围的终点。例如不超过10个核苷酸表示不超过1、2、3、4、5、6、7、8、9和10个核苷酸及它们形成的任何子范围例如1-8nt、2-5nt、3-7nt等。
本说明书中提及的所有科学技术术语具有与本领域技术人员通常理解的含义相同的含义，在冲突的情况下以本说明书中的定义为准。为了使本发明的描述更易于理解，以下对一些术语进行解释说明。
在本发明中，术语“核酸”、“核酸分子”和“核酸药物”可互换使用，指任何DNA、RNA或DNA/RNA嵌合体，并且可以为寡核苷酸或多核苷酸，并且可以是未修饰的RNA或DNA或者修饰的RNA或DNA。该术语包括但不限于单链和双链的DNA、为单链区和双链区的混合物的DNA、单链和双链的RNA以及为单链区和双链区的混合物的RNA、包含可以是单链或双链或者是单链区和双链区的混合物的DNA和RNA的杂合分子。
在一些实施方案中，核酸可包含一个或多个经修饰的核苷酸、经修饰的键等。经修饰的键或核苷酸间键的实例包括硫代磷酸酯、二硫代磷酸酯等。在一些实施方案中，所述核苷酸包括磷衍生物。可连接到本发明的修饰的核苷酸中的糖或糖类似物部分的磷衍生物(或经修饰的磷酸基)，可为单磷酸酯、二磷酸酯、三磷酸酯、烷基磷酸酯、链烷磷酸酯、硫代磷酸酯等。上述磷酸酯类似物的制备，以及它们掺入到核苷酸、修饰核苷酸和寡核苷酸中本身也为已知的且无需在此描述。
本文所用的“未修饰的”或“天然的”核苷酸包括腺嘌呤(A)、鸟嘌呤(G)、胸腺嘧啶(T)、胞嘧啶(C)和尿嘧啶(U)。修饰核苷酸包括在天然核酸中仅稀少或短暂地存在的核苷酸，例如，次黄嘌呤、6-甲基腺嘌呤、5-Me嘧啶、特别地5-甲基胞嘧啶(也称为5-甲基-2’脱氧胞嘧啶且常常在本领域中称为5-Me-C)、5-羟甲基胞嘧啶(HMC)、糖基HMC和龙胆二糖基HMC，以 及合成核苷酸，例如，2-氨基腺嘌呤、2-(甲氨基)腺嘌呤、2-(咪唑基烷基)腺嘌呤、2-(氨烷基氨基)腺嘌呤或其他杂取代的烷基腺嘌呤、2-硫尿嘧啶、2-硫胸腺嘧啶、5-溴尿嘧啶、5-羟基甲基尿嘧啶、8-氮杂鸟嘌呤、7-脱氮杂鸟嘌呤、N6(6-氨基己基)腺嘌呤和2,6-二氨基嘌呤。可包括本领域已知的“通用的”碱基，例如，肌苷。
核酸修饰
(1)核酸结构
在一些实施方案中，本发明核酸(例如，靶向DNA的RNA)包含一个或多个修饰(例如，碱基修饰、骨架修饰等)以对核酸提供新的或增强的特征(例如，改进的稳定性)。如本领域中所已知，核苷为碱基-糖组合。核苷的碱基部分通常为杂环碱基。此类杂环碱基的两个最常见类别为嘌呤和嘧啶。核苷酸为还包括共价连接至核苷的糖部分的磷酸酯基团的核苷。对于包括呋喃戊糖的那些核苷，磷酸酯基团可连接至糖的2'、3'或5'羟基部分。在形成寡核苷酸中，磷酸酯基团共价连接彼此相邻的核苷以形成线性聚合化合物。反过来，线性聚合化合物的各端可进一步连接以形成环状化合物，然而，线性化合物通常为适合的。另外，线性化合物可具有内部核苷酸碱基互补性并且因此可以为了产生完全或部分双链化合物的方式折叠。在寡核苷酸内，磷酸酯基团通常称为形成寡核苷酸的核苷间骨架。RNA和DNA的正常键或骨架为3'至5'的磷酸二酯键。
(2)修饰的骨架和修饰的核苷间键
含有修饰的适合的核酸的实例包括含有修饰的骨架或非天然的核苷间键的核酸。核酸(具有修饰的骨架)包括在骨架中保留磷原子的那些和在骨架中不具有磷原子的那些。
其中含有磷原子的适合的修饰的寡核苷酸包括例如，硫代磷酸酯、手性硫代磷酸酯、二硫代磷酸酯、磷酸三酯、氨基烷基磷酸三酯、甲基和其它烷基磷酸酯包括3'-亚烷基磷酸酯、5'-亚烷基磷酸酯和手性磷酸酯、膦酸盐、氨基磷酸酯包括3'-氨基氨基磷酸酯和氨基烷基氨基磷酸酯、二氨基磷酸酯、硫羰氨基磷酸酯、硫羰烷基磷酸酯、硫羰烷基磷酸三酯、具有正常3'-5'键的硒代磷酸酯和硼代磷酸酯、这些的2'-5'连接类似物以及具有反极性的那些，其中一个或多个核苷酸间键为3'至3'、5'至5'或2'至2'键。具有反极性的适合的寡核苷酸在最3'核苷酸间键处包含单个3'至3'键，即可为无碱基(核碱基丢失或其被羟基替代)的单个反转核苷残基。还包括各种盐(例如像钾或钠)、混合盐和游离酸形式。
在一些实施方案中，本发明核酸包含一个或多个硫代磷酸酯键和/或杂原子核苷间键，具体为-CH 2-NH-O-CH 2-、-CH 2-N(CH 3)-O-CH 2-(称为亚甲基(甲基亚氨基)或MMI骨架)、-CH 2-O-N(CH 3)-CH 2-、-CH 2-N(CH 3)-N(CH 3)-CH 2-以及-O-N(CH 3)-CH 2-CH 2-(其中天然磷酸二酯核苷间键表示为-O-P(＝O)(OH)-O-CH 2-)。
其他的骨架修饰还包括例如吗啉代骨架结构的核酸。例如，在一些实施方案中，本发明核酸包含替代核糖环的6元吗啉代环。在这些实施方案的一些中，二氨基磷酸酯或其它非磷酸二酯核苷间键替代磷酸二酯键。
不包括磷原子的适合的修饰的多核苷酸骨架具有通过短链烷基或环烷基核苷间键、混合杂原子和烷基或环烷基核苷间键或一个或多个短链杂原子或杂环核苷间键形成的骨架。这些 包括具有以下的那些：吗啉代键(部分地从核苷的糖部分中形成)；硅氧烷骨架；硫化物、亚砜和砜骨架；甲酰乙酰基和硫代甲酰乙酰基骨架；亚甲基甲酰乙酰基和硫代甲酰乙酰基骨架；核糖乙酰基(riboacetyl)骨架；含烯烃的骨架；氨基磺酸酯骨架；亚甲基亚胺基和亚甲基肼基骨架；磺酸酯和磺酰胺骨架；酰氨骨架；以及具有混合N、O、S和CH 2组成部分的其它骨架。
另一种骨架修饰包括锁定核酸(LNA)，其中2'-羟基连接至糖环的4'碳原子从而形成2'-C、4'-C-氧基亚甲基键，从而形成双环糖部分。链可为亚甲基(-CH 2-)(桥联2'氧原子和4'碳原子的基团)，其中n为1或2(Singh等,Chem.Commun.,1998,4,455-456)。LNA和LNA类似物显现出与互补DNA和RNA具有非常高的双链体热稳定性(T m＝+3℃至+10℃)、朝向3'-核酸外切降解的稳定性和良好的溶解性质。
现有技术中已描述了LNA单体腺嘌呤、胞嘧啶、鸟嘌呤、5-甲基-胞嘧啶、胸腺嘧啶和尿嘧啶的合成和制备连同其低聚化以及核酸识别性质(Koshkin等,Tetrahedron,1998,54,3607-3630)。
(3)修饰的糖部分
本发明核酸还可包括一个或多个取代的糖部分。适合的多核苷酸包含选自以下的糖取代基：OH；F；O-、S-或N-烷基；O-、S-或N-烯基；O-、S-或N-炔基；或O-烷基-O-烷基，其中烷基、烯基和炔基可为取代或未取代的C1至C10烷基或C2至C10烯基和炔基。特别适合的为O((CH 2) nO) mCH 3、O(CH 2) nOCH 3、O(CH 2) nNH 2、O(CH 2) nCH 3、O(CH 2) nONH 2和O(CH 2) nON((CH 2) nCH 3) 2，其中n和m为1至约10。其它适合的多核苷酸包含选自以下的糖取代基：C1至C10低级烷基、取代的低级烷基、烯基、炔基、烷芳基、芳烷基、O-烷芳基或O-芳烷基、SH、SCH 3、OCN、Cl、Br、CN、CF 3、OCF 3、SOCH 3、SO 2CH 3、ONO 2、NO 2、N 3、NH 2、杂环烷基、杂环烷芳基、氨基烷基氨基、多烷基氨基、取代的甲硅烷基、RNA裂解基团、报道基团、嵌入剂、用于改进寡核苷酸的药物代谢动力学性质的基团或用于改进寡核苷酸的药效性质的基团以及具有类似性质的其它取代基。适合的修饰包括2'-甲氧基乙氧基(2'-O-CH 2CH 2OCH 3，又称为2'-O-(2-甲氧基乙基)或2'-MOE)(Martin等,Helv.Chim.Acta,1995,78,486-504)，即烷氧基烷氧基。另一适合的修饰包括2'-二甲基氨基氧基乙氧基，即O(CH 2) 2ON(CH 3) 2基团，又称为2'-DMAOE，如在下文的实施例中所述；和2'-二甲基氨基乙氧基乙氧基(在本领域中又称为2'-O-二甲基-氨基-乙氧基-乙基或2'-DMAEOE)，即2'-O-CH 2-O-CH 2-N(CH 3) 2。
其它适合的糖取代基包括甲氧基(-O-CH 3)、氨基丙氧基(--OCH 2CH 2CH 2NH 2)、烯丙基(-CH 2-CH＝CH 2)、-O-烯丙基(--O--CH 2—CH＝CH 2)以及氟(F)。2'-糖取代基可处于阿拉伯糖(上)位或核糖(下)位。适合的2'-阿拉伯糖修饰为2'-F。还可在低聚化合物上的其它位置上做出类似的修饰，具体地在3'末端核苷上或在2'-5'连接的寡核苷酸中的糖的3'位置和5'末端核苷酸的5'位置。低聚化合物还可具有替代呋喃戊糖的糖模拟物如环丁基部分。
(4)碱基修饰和取代
本发明核酸还可包括核碱基(在本领域中常常简称为“碱基”)修饰或取代。如本文所使用，“未修饰”或“天然”核碱基包括嘌呤碱基腺嘌呤(A)和鸟嘌呤(G)以及嘧啶碱基胸腺嘧啶 (T)、胞嘧啶(C)和尿嘧啶(U)。修饰的核碱基包括其它合成和天然的核碱基如5-甲基胞嘧啶(5-me-C)、5-羟甲基胞嘧啶、黄嘌呤、次黄嘌呤、2-氨基腺嘌呤、腺嘌呤和鸟嘌呤的6-甲基衍生物和其它烷基衍生物、腺嘌呤和鸟嘌呤的2-丙基衍生物和其它烷基衍生物、2-硫尿嘧啶、2-硫胸腺嘧啶和2-硫胞嘧啶、5-卤代尿嘧啶和胞嘧啶、5-丙炔基(-C＝C-CH 3)尿嘧啶和胞嘧啶以及嘧啶碱基的其它炔基衍生物、6-偶氮基尿嘧啶、胞嘧啶和胸腺嘧啶、5-尿嘧啶(假尿嘧啶)、4-硫尿嘧啶、8-卤代、8-氨基、8-巯基、8-硫烷基、8-羟基和其它8-取代的腺嘌呤和鸟嘌呤、5-卤代(具体为5-溴)、5-三氟甲基和其它5-取代的尿嘧啶和胞嘧啶、7-甲基鸟嘌呤和7-甲基腺嘌呤、2-F-腺嘌呤、2-氨基-腺嘌呤、8-氮杂鸟嘌呤和8-氮杂腺嘌呤、7-脱氮鸟嘌呤和7-脱氮腺嘌呤以及3-脱氮鸟嘌呤和3-脱氮腺嘌呤。另外的修饰的核碱基包括三环嘧啶如吩噁嗪胞苷(1H-嘧啶并(5,4-b)(1,4)苯并噁嗪-2(3H)-酮)、吩噻嗪胞苷(1H-嘧啶并(5,4-b)(1,4)苯并噻嗪-2(3H)-酮)、G-夹如取代的吩噁嗪胞苷(例如9-(2-氨基乙氧基)-H-嘧啶并(5,4-(b)(1,4)苯并噁嗪-2(3H)-酮)、咔唑胞苷(2H-嘧啶并(4,5-b)吲哚-2-酮)、吡啶并吲哚胞苷(H-吡啶并(3',2':4,5)吡咯并(2,3-d)嘧啶-2-酮)。
杂环碱基部分还可包括其中嘌呤或嘧啶碱基被其它杂环替代的那些，例如7-脱氮腺嘌呤、7-脱氮鸟苷、2-氨基吡啶和2-吡啶酮。另外的核碱基包括公开于The Concise Encyclopedia Of Polymer Science And Engineering,第858-859页,Kroschwitz,J.I.编著John Wiley&Sons,1990中的那些、由Angewandte Chemie,International Edition,1991,30,613公开的那些以及由Sanghvi,Y.S.,第15章,Antisense Research and Applications,第289-302页,Crooke,S.T.和Lebleu,B.编著,CRC Press,1993公开的那些。这些核碱基中的某些有用于增加低聚化合物的结合亲和力。这些包括5-取代的嘧啶、6-氮杂嘧啶以及N-2、N-6和O-6取代的嘌呤，包括2-氨基丙基腺嘌呤、5-丙炔基尿嘧啶和5-丙炔基胞嘧啶。5-甲基胞嘧啶取代已显示出使核酸双链体稳定性增加0.6℃-1.2℃(Sanghvi等编著,Antisense Research and Applications,CRC Press,Boca Raton,1993,第276-278页)并且例如当与2'-O-甲氧基乙基糖修饰组合时为适合的碱基取代。
(5)缀合物
本发明核酸的另一种可能的修饰涉及将增强寡核苷酸的活性、细胞分布或细胞吸收的一个或多个部分或缀合物化学连接至多核苷酸。这些部分或缀合物可包括共价结合如伯羟基或仲羟基的官能团的缀合物基团。缀合物基团包括但不限于嵌入剂、报道分子、多胺、聚酰胺、聚乙二醇、聚醚、增强低聚物药效性质的基团以及增强低聚物药物代谢动力学性质的基团。适合的缀合物基团包括但不限于胆固醇、脂质、磷脂、生物素、吩嗪、叶酸酯、菲啶、蒽醌、吖啶、荧光素、罗丹明、香豆素以及染料。增强药效性质的基团包括改进吸收、增强对降解的抗性和/或加强与靶核酸的序列特异性杂交的基团。增强药物代谢动力学性质的基团包括改进本发明核酸的吸收、分布、代谢或排泄的基团。
缀合物部分包括但不限于：脂质部分如胆固醇部分(Letsinger等,Proc.Natl.Acad.Sci.USA,1989,86,6553-6556)、胆酸(Manoharan等,Bioorg.Med.Chem.Let.,1994,4,1053-1060)、硫醚例如己基-S-三苯甲基硫醇(Manoharan等,Ann.N.Y.Acad.Sci.,1992,660,306-309；Manoharan 等,Bioorg.Med.Chem.Let.,1993,3,2765-2770)、硫代胆固醇(Oberhauser等,Nucl.Acids Res.,1992,20,533-538)、脂肪族链例如十二烷二醇或十一烷基残基(Saison-Behmoaras等,EMBO J.,1991,10,1111-1118；Kabanov等,FEBS Lett.,1990,259,327-330；Svinarchuk等,Biochimie,1993,75,49-54)、磷脂例如二-十六烷基-外消旋甘油或1,2-二-O-十六烷基-外消旋甘油-3-H-磷酸三乙基铵盐(Manoharan等,Tetrahedron Lett.,1995,36,3651-3654；Shea等,Nucl.Acids Res.,1990,18,3777-3783)、多胺或或聚乙二醇链(Manoharan等,Nucleosides&Nucleotides,1995,14,969-973)、或金刚烷乙酸(Manoharan等,Tetrahedron Lett.,1995,36,3651-3654)、棕榈基部分(Mishra等,Biochim.Biophys.Acta,1995,1264,229-237)、或十八烷基胺或己基氨基-羰基-氧基胆固醇部分(Crooke等,J.Pharmacol.Exp.Ther.,1996,277,923-937。
在一些实施方案中，本发明核酸分子包含SEQ ID NO:1或3或由其组成。在一些实施方案中，本发明核酸分子包含两个或更多个直接或通过接头间接连接的SEQ ID NO:1或3(即SEQ ID NO:1或3的二聚体、三聚体等)，或者由其组成。
在本发明中，术语“接头基团”和“接头”可互换使用，是指用于连接序列的不同部分的寡核苷酸序列。所述寡核苷酸序列可包含未修饰的或修饰的天然或非天然核苷酸。在一些实施方案中，所述接头基团或接头包含A、T、C、G和/或U。在一些实施方案中，所述接头基团或接头包含G。在一些实施方案中，所述接头基团或接头由G组成。在一些实施方案中，所述接头基团或接头长度为1-10个核苷酸，例如1-5个核苷酸，例如3-5个核苷酸。在一些实施方案中，所述接头基团或接头为GGGGG。在一些实施方案中，所述接头基团或接头的存在不影响本发明核酸分子的保护作用。
在本发明中，术语“缀合”和“连接”可互换使用，指通过化学键连接。
在本文中，本发明所述的核酸在血清中的稳定性，是指核酸在各种浓度的血清例如50％的血清中的稳定性。在本文中，稳定性的提高是指与未经本发明核酸分子保护或用其它方法保护的目的核酸相比，所述目的核酸的稳定性例如在血清中的稳定性得到提高。在一些实施方案中，目的核酸的活性等同或类似于未经本发明核酸分子保护或用其它方法保护的目的核酸的活性，但其稳定性得到提高。在一些实施方案中，目的核酸的活性保持不变。
本文使用的术语“药学上可接受的载体”旨在包括与药物给予相容的任何和所有溶剂、分散介质、包衣剂、抗细菌和抗真菌剂、等渗剂和吸收延迟剂等。合适的载体描述于最新版本的Remington's Pharmaceutical Sciences(本领域的标准参考文本)，其通过引用结合于本文中。这种载体或稀释剂的优选实例包括但不限于水、盐水、林格氏溶液、右旋糖溶液和5％人血清白蛋白。这种介质和试剂的使用为本领域熟知的。除非任何常规介质或试剂与本文提供的物质不相容，否则考虑在组合物中使用它们。
以下以具体实施例来说明本发明的方案，但并非是对本发明的技术方案的限制，本领域技术人员应该理解，依然可以对发明进行修改或等同替换，而不脱离本发明的精神和范围的任何修改或局部替换，其均应涵盖在本发明的保护范围之中。
具体实施例
除非另外说明，否则本文实施例所用的材料均市购获得，用于进行实验的各种具体实验方法均为本领域常规的实验方法或者按照制造商所建议的步骤和条件，并能由本领域技术人员根据需要常规地确定。此外，本说明书实施例中所用血清均为Gibco特级胎牛血清。降解体系包含：寡核苷酸药物、1×PBS、50％血清和水。所用电泳条件均为：180V，20min；上样量为：2μg-5μg。除非另外说明，否则本说明书实施例中的miRNA-2911序列及其衍生物均添加至受保护的序列的两端。
实施例1：含有miRNA-2911的RNA药物在血清中稳定性提升
在需要保护的RNA(R1)的5'端和3'端分别添加特定序列(miRNA-2911)，构成miRNA2911-R1(图1中显示为mi2911-R1)，其中选取随机序列RRs作为阴性对照(选取该随机序列的原因是因为寡核苷酸本身长度较短，很容易降解，因此选择了与保护后的序列长度基本一致的随机对照序列，使其电泳结果图几乎在一条直线，方便对照)。将各序列在血清中孵育不同时间：6h，12h和24h，然后通过3％琼脂糖凝胶电泳分析结果。结果如图1显示，其中不添加本发明的特定序列(miRNA-2911)的随机序列RRs作为阴性对照(NC)，在6h时完全降解，而miRNA-2911-R1在12h依然存在没被降解的完整的序列，说明两端的miRNA-2911极大地提高了R1对抗血清中核酸酶的降解能力，提高了R1在血清中的稳定性。
在实施例1中，使用的核酸片段的序列信息如下：
miRNA-2911：GGCCGGGGGACGGACUGGGA(SEQ ID NO:2)
R1：UGAAUGUAGAGAUGCGGUGG(SEQ ID NO:11)
RRs:
Figure PCTCN2022138695-appb-000001
实施例2：基于miRNA-2911改造的RNA提高RNA药物在血清中的稳定性
发明人选择对miRNA-2911序列进行不同改造，将改造后的序列分别添加在R1(SEQ ID NO:11)的两端，评估改造后的序列对RNA的保护能力，其中选取随机序列RRs作为阴性对照。将上述序列在血清中孵育不同时间：6h，12h和24h，然后通过3％琼脂糖凝胶电泳分析结果。结果如图2-(1)、图2-(2)和图2-(3)所示，其中不同改造的核酸药物在血清中降解程度存在差异，但均存在没被降解的完整的RNA，均具备保护能力。另外，将含有基于miRNA-2911改造的RNA的RNA药物在血清中孵育长达36h，其结果如图2-(4)所示。含有基于miRNA-2911改造的RNA的RNA药物在36h依然存在没被降解的完整的序列，说明含有基于miRNA-2911改造的RNA的RNA药物在血清中具有较高稳定性。
在实施例2中，使用的核酸片段的序列信息如下：
R2911A:GGAAGGGGGAAGGAAAGGGA(SEQ ID NO:4)
R2911C:GGCCGGGGGCCGGCCCGGGC(SEQ ID NO:5)
R2911U:GGUUGGGGGUUGGUUUGGGU(SEQ ID NO:6)
R2911A-5G：GGAAGGGGGGGGGGAAGGAAAGGGA(SEQ ID NO:7)
R2911A-D:GGAAGGGGGAAGGAAAGGGAGGAAGGGGGAAGGAAAGGGA(SEQ ID NO:8)
RRs:
Figure PCTCN2022138695-appb-000002
实施例3：将miRNA-2911改造为DNA后提升核酸药物嵌合体的血清稳定性
测试了改造后的序列对DNA的保护能力，在需要保护的DNA(D1)的5'端和3'端分别添加特定序列R2911A，构成R2911A-D1，其中选取随机序列RDs作为阴性对照。将各序列在血清中孵育不同时间：4h，6h和12h，然后通过3％琼脂糖凝胶电泳分析结果。结果如图3显示，其中R2911A-D1在12h依然存在没被降解的完整的序列，说明含有R2911A的核酸药物嵌合体在血清中具有较高稳定性。
在实施例3中，D1及DRs的序列信息如下：
D1：TGAATGTAGAGATGCGGTGG(SEQ ID NO:13)
RDs：
Figure PCTCN2022138695-appb-000003
实施例4：将miRNA-2911改造为DNA后能提升DNA药物在血清稳定性
发明人将miRNA-2911序列改造为DNA(D2911和D2911A)，将改造后的序列分别添加在D1的两端，构成D2911-D1和D2911A-D1，并评估改造后的序列对DNA的保护能力。同时以D1和DDs作为阴性对照。将上述序列分别在血清中孵育不同时间：12h，24h和36h，然后通过3％琼脂糖凝胶电泳分析结果。结果如图4-(1)和4-(2)所示，其中D2911-D1和D2911A-D1在血清中的稳定性显著增强，而图4-(2)表明核酸药物稳定性的增强是由D2911A带来的，而非序列本身。
在实施例4中，使用的核酸片段的序列信息如下：
D1：TGAATGTAGAGATGCGGTGG(SEQ ID NO:13)
D2911:GGCCGGGGGACGGACTGGGA(SEQ ID NO:9)
D2911A：GGAAGGGGGAAGGAAAGGGA(SEQ ID NO:10)
DDs：
Figure PCTCN2022138695-appb-000004
实施例5：将miRNA-2911改造为DNA后能提升核酸药物的血清稳定性
测试了D2911A对RNA的保护能力，将D2911A序列分别添加在R1的两端，构成D2911A-R1，其中选取随机序列DRs作为阴性对照。将各序列在血清中孵育1h，然后通过3％琼脂糖凝胶电泳分析结果。结果如图5所示，其中含有D2911A的核酸药物嵌合体在血清中依然具有一定的稳定性。
在实施例5中，使用的核酸片段的序列信息如下：
D2911A-R1：
Figure PCTCN2022138695-appb-000005
DRs:
Figure PCTCN2022138695-appb-000006
实施例6：评估含有R2911A的不同序列的核酸药物在血清中的稳定性
发明人测试了R2911A对不同序列的核酸药物的保护能力。具体而言，选择不同序列的核酸R2-R6，将R2911A分别添加在R2-R6的两端，分别构成R2911A-R2、R2911A-R3、R2911A-R4、R2911A-R5和R2911A-R6，其中选取随机序列RRs作为不使用本发明方法进行保护的阴性对照。将各序列在血清中孵育6h，然后通过3％琼脂糖凝胶电泳分析结果。结果如图6所示，含有R2911A的不同序列的核酸药物在血清中降解程度存在差异，但均存在没被降解的完整的DNA序列，结合实施例1中对R1的保护能力，说明含有R2911A的不同序列的核酸药物在血清中均具有稳定性，说明本发明方法对保护核酸序列具有普遍适用性。
在实施例6中，使用的核酸片段的序列信息如下：
R2:ACGGGGUCAUUAGUUCAUAG(SEQ ID NO:17)
R3:UAAGAUACACCUGCAAAGGC(SEQ ID NO:18)
R4:GCUCUCCUCAAGCGUAUUCA(SEQ ID NO:19)
R5:GUCGAGCUGGACGGCGACGU(SEQ ID NO:20)
R6:UGACCCUGAAGUUCAUCUGC(SEQ ID NO:21)
RRs:
Figure PCTCN2022138695-appb-000007
实施例7：评估含有R2911A的不同长度的核酸药物在血清中的稳定性
发明了测试了R2911A对不同长度的核酸药物的保护能力。具体而言，选择不同长度的核酸，以80nt(R80)及2000nt(R2K)长度的RNA作为代表，将R2911A分别添加它们的两端，分别构成R2911A-R80和R2911A-R1K，其中采用没有任何保护的RNA分别作为它们的阴性对照(分别为R80-RC和R2K-RC)；2000nt的RNA代表本发明方法对长链RNA(包括mRNA和lncRNA)的保护能力。将各序列在10％血清中分别孵育1h和20min，然后通过3％琼脂糖凝胶电泳分析结果。结果如图7-(1)和图7-(2)所示，其中含有R2911A的不同长度的核酸在血清中的降解程度存在差异，但均存在没被降解的完整的RNA序列，结合实施例6中对20nt核酸的保护能力，说明含有RNA R2911A的不同长度核酸药物均具有稳定性。另外，也测试了R2911A对长度为5000nt的RNA的保护能力，结果显示R2911A对该长度的RNA具有保护能力(结果未显示)。
在实施例7中，使用的核酸片段的序列信息如下：
R2911A-R80:
Figure PCTCN2022138695-appb-000008
R80-RC：
Figure PCTCN2022138695-appb-000009
R2911A-R2K:
Figure PCTCN2022138695-appb-000010
Figure PCTCN2022138695-appb-000011
R2K-RC：
Figure PCTCN2022138695-appb-000012
Figure PCTCN2022138695-appb-000013
实施例8：评估含有R2911A的核酸药物在动物体内的稳定性
发明人采用cy5.5对含有R2911A的核酸药物(R2911A-R1，将R2911A序列分别添加在R1的两端而形成)进行标记，形成Cy5.5-R2911A-R1。注射前，将此Cy5.5-R2911A-R1用生理盐水稀释。将小鼠称重并按照2mg/kg的注射剂量进行腹部脱毛处理的小鼠给药。采用皮下注射方式对小鼠进行注射。抓取小鼠并固定，在两后肢之间的皮肤进行注射，用注射器抽取根据体重定义体积的药物，针头斜面朝上轻轻刺入皮肤，沿皮下行针约1-2cm，缓慢注射。成功皮下注射后，注射位点的小鼠皮肤会隆起鼓包，沿进针反方向平稳抽出针，完成注射。分别在注射后的0d、1d、2d和3d进行活体成像，观察Cy5.5-R2911A-R1皮下注射后的分布以及在小鼠体内的降解情况。初步考察了R2911A-R1核酸药物在动物体内的稳定性。结果如图8显示，其中注射R2911A-R1核酸药物，在3d依然可以检测到明显的荧光，而未含有R2911A的Cy5.5标记的Cy5.5-RRs(图8中显示为Cy5.5-RS)在3d后基本检测不到荧光。本实施例表明含有R2911A的核酸药物在动物体内的依然具有很好的稳定性。
实施例9：评估在5'端、3'端和内部含有R2911A的核酸药物在血清中稳定性
为了评估在5'端、3'端和内部含有R2911A的核酸药物在血清中的稳定性，发明人选择了在R1的5'端、3'端或内部添加R2911A，其中采用没有任何保护的RNA作为阴性对照(RRs”)。将这些序列分别在血清中孵育12h、24h、1h，然后通过3％琼脂糖凝胶电泳分析结果。结果如图9-(1)和9-(2)所示，其中当在内部添加R2911A时，保护效果不理想(图9-(2))，而在5'端或者3'端添加R2911A时，在24h依然存在没被降解的完整的序列(图9-(1))，说明在5'端或者3'端的R2911A提高了RNA在血清中的稳定性。结合实施例2中对R1的保护能力，说明当在R1两端都存在本发明保护核酸分子时，在血清中的稳定性得到更加显著的增加。
在实施例9中，使用的核酸片段的序列信息如下：
R2911A-M:
Figure PCTCN2022138695-appb-000014
R2911A-L:GGAAGGGGGAAGGAAAGGGAUGAAUGUAGAGAUGCGGUGG(SEQ ID NO:27)
R2911A-R:UGAAUGUAGAGAUGCGGUGGGGAAGGGGGAAGGAAAGGGA(SEQ ID NO:28)
RRs”:ACCCGACCUCUUCUAUCUGGACCCGACCGUCUCUUUUUUG(SEQ ID NO:29)。
实施例10：评估含有R2911A的siRNA药物在细胞内对靶标RNA的敲低效果
1.采用FDA批准的inclisiran为阳性对照(阳参为经化学修饰的siRNA)，通过在其RNA序列的3’端添加R2911A，形成未修饰的含有R2911A的siRNA药物(inclisiran-R2911A)，采用Lipofectamine 2000转染siRNA：将适量的inclisiran-2911A加入50μL的Opti-MEM进 行稀释，同时将4.8μL的Lipofectamine 2000用50μL的Opti-MEM进行稀释，轻轻吹打混匀后室温放置5min。然后将Lipofectamine 2000稀释液加入到inclisiran-2911A稀释液当中形成混合液，室温放置孵育20min。同时将12孔板的细胞(97H细胞和Hep3B细胞50-60％细胞密度)换成无抗生素(青霉素-链霉素)的培养基(DMEM)，将混合液滴加到细胞中，轻柔混匀，5～6h后更换成含血清和抗生素的完全培养基。待转染时间达到48h、72h、96h后即可收取细胞提取总RNA。由于转染96h后细胞的密度已较高不适合在12孔板中继续培养，因此需要将其传代至6孔板中，待达到120h的转染时间后再收取细胞提取总RNA。
2.细胞总RNA的提取：
(1)弃掉培养基，然后用预冷的1×PBS润洗细胞一次。
(2)向12孔板的细胞中加入0.5mL的Trizol，6孔板的细胞加1mL，用移液器吹打混匀，彻底裂解细胞。
(3)向完全裂解后的Trizol中加入总体积20％的氯仿，盖上管盖，用力摇晃使之充分混匀，并在室温下静置2min，然后4℃，12000×g离心15min。
(4)吸取大约200μL(12孔)、400μL(6孔)的水相层至一个新的EP管中，加入等体积的异丙醇混匀后，在-80℃低温冰箱中放置半小时，然后4℃，12000×g离心15min。
(5)离心后可看见管底有白色沉淀，此为RNA。弃掉上清，加入1mL 75％的乙醇并重悬沉淀，然后4℃，7500×g离心10min。
(6)吸去乙醇，小心不要吸到沉淀。管子在室温下静置，待乙醇挥发完后，加入50μL无RNA酶H 2O，并于42℃孵育2min，使得RNA充分溶解。然后取出少量RNA测其浓度，并用TBE电泳检测RNA的完整性。
(7)取出1μg的总RNA进行反转录，反转录体系为20μL，反转录完成后即为cDNA。
3.实时荧光定量PCR：
(1)从-20℃冰箱中取出cDNA，室温溶解，然后加入180μL的ddH 2O稀释10倍。
(2)qPCR反应体系如下：
Figure PCTCN2022138695-appb-000015
PCR反应条件为：95℃3min；95℃10s，60℃10s，72℃20s(此程序为40个循环)；95℃15s；60℃60s；溶解曲线60℃～95℃，每上升0.5℃吸收一次荧光信号；95℃10s。
(3)利用ABI step one plus定量仪进行PCR，根据结果运用△△Ct算法分析实验数据。
本实施例所使用的siRNA(inclisiran或与2911连接的inclisiran)针对的靶标RNA是PCSK9，在细胞水平测试其敲低能力，测试的细胞系为97H细胞和Hep3B细胞；同时设置转染等体积的PBS组作为阴性对照(Mock)，设置inclisiran为阳性对照。
结果(图10)显示， 含有R2911A的siRNA药物(inclisiran-R2911A)在细胞内具有良 好的靶基因敲低能力。
在本实施例中，使用的核酸片段信息如下：
Inclisiran(全修饰序列): A*C* AAAA GC AA AACA GG UC UAG* A* A
带下划线标记的核苷酸进行2’位的甲氧基修饰；斜体字标记的核苷酸进行氟代修饰(氟代是将2’位上的羟基替换为氟原子)；*表示硫代修饰(表示将磷酸键中的一个非桥连的氧原子用硫原子来置换)。
Inclisiran-R2911A:
Figure PCTCN2022138695-appb-000016
实施例10：评估含有R2911A的mRNA在细胞内表达蛋白的效果
为了评估含有R2911A的mRNA药物在细胞内表达蛋白的效果，选择在萤火虫荧光素酶基因(mRNA1)两端添加R2911A，形成R-mRNA1；采用没有任何保护的mRNA1作为阴性对照(mRRs)。
将这些序列经体外转录后分别转染293U细胞，24h后加入与培养基等体积的检测试剂(Promega，ONE-GloUM萤光素酶检测系统，E6110)通过酶标仪(Thermo，Scientific VARioskan LUX)检测发光信号。结果如图11所示，当mRNA1两端添加R2911A时，R-mRNA1在细胞内蛋白表达量显著高于mRRs，说明含有R2911A显著提高了mRNA药物在细胞内表达蛋白的能力。
其中序列信息：
mRNA1：
Figure PCTCN2022138695-appb-000017
Figure PCTCN2022138695-appb-000018
R-mRNA1：
Figure PCTCN2022138695-appb-000019
Figure PCTCN2022138695-appb-000020
以上所述，仅是本发明的较佳实施例而已，并非对本发明作任何形式上的限制，本领域技术人员利用上述揭示的技术内容做出些许简单修改、等同变化或修饰，均落在本发明的保护范围内。

Claims (10)

  1. 一种用于提高目的核酸的稳定性的miRNA-2911及其衍生的核酸分子，其特征在于，所述的miRNA-2911及其衍生的核酸分子的核苷酸序列，包含如下式所示的核酸序列：
    GGX 1X 2GGGGG-(L) n-X 3X 4GGX 5X 6X 7GGGX 8(SEQ ID NO:1)
    其中：
    X 1、X 2、X 3、X 4、X 5、X 6、X 7和X 8独立地选自A、C、T和U；
    n为0或1；
    L表示长度不超过10个核苷酸的接头基团。
  2. 根据权利要求1所述的miRNA-2911及其衍生的核酸分子，其特征在于，L表示GGGGG。
  3. 根据权利要求1所述的miRNA-2911及其衍生的核酸分子，其特征在于，所述的miRNA-2911及其衍生的核酸分子包含如下式所示的核酸序列：
    GGX 1X 2GGGGGX 3X 4GGX 5X 6X 7GGGX 8(SEQ ID NO:3)
    其中：
    X 1、X 2、X 3、X 4、X 5、X 6、X 7和X 8独立地选自A、C、T和U。
  4. 根据权利要求1-3中任一项所述的miRNA-2911及其衍生的核酸分子，其特征在于，
    X 1、X 2、X 4和X 6相同，任选地为A、C或U；和/或
    X 3、X 5和X 8相同，任选地为A、C或U。
  5. 根据权利要求1-4中任一项所述的miRNA-2911及其衍生的核酸分子，其特征在于，所述miRNA-2911及其衍生的核酸分子的序列如SEQ ID NO:2、4-10任一所示。
  6. 一种经保护的目的核酸，其特征在于，
    所述目的核酸在其5'端和/或3'端，由权利要求1-5中任一项所述的miRNA-2911及其衍生的核酸分子缀合，或者，
    将权利要求1-5中任一项所述的miRNA-2911及其衍生的核酸分子插入所述目的核酸中；
    并且，所述经保护的目的核酸的稳定性得到增加。
  7. 根据权利要求6所述的经保护的目的核酸，其特征在于，
    所述目的核酸长度为≥8nt，优选为8-5000nt；
    优选地，所述目的核酸为基因敲低用核酸、基因敲除用核酸、基因活化用核酸、基因修饰用核酸、基因编辑用核酸、基因调控用核酸、蛋白调控用核酸、蛋白表达用核酸、生物检测用核酸或核酸药物；
    更优选地，所述目的核酸为：
    (1)寡核苷酸，包括但不限于反义寡核苷酸ASO或核酸适配体；
    (2)单链DNA、双链DNA、单链RNA、双链RNA、mRNA或ncRNA，所述的ncRNA包括但不限于miRNA、siRNA、saRNA、piRNA、lncRNA、circRNA、它们的片段或其它调控性RNA。
  8. 一种药物组合物，其特征在于，所述药物组合物包含：
    (1)治疗有效量的权利要求6-7中任一项所述的经保护的目的核酸，和
    (2)药学上可接受的载体。
  9. 一种用于增加目的核酸的稳定性的方法，其特征在于，
    将权利要求1-5中任一项所述的miRNA-2911及其衍生的核酸分子缀合至所述目的核酸的5'端和/或3'端，或者插入所述目的核酸中。
  10. 根据权利要求9的方法，其特征在于，
    所述目的核酸长度为≥8nt，优选为8-5000nt；
    优选地，所述目的核酸为基因敲低用核酸、基因敲除用核酸、基因活化用核酸、基因修饰用核酸、基因编辑用核酸、基因调控用核酸、蛋白调控用核酸、蛋白表达用核酸、生物检测用核酸或核酸药物；
    更优选地，所述目的核酸为：
    (1)寡核苷酸，包括但不限于反义寡核苷酸ASO或核酸适配体；
    (2)单链DNA、双链DNA、单链RNA、双链RNA、mRNA或ncRNA，所述的ncRNA包括但不限于miRNA、siRNA、saRNA、piRNA、lncRNA、circRNA、它们的片段或其它调控性RNA。
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