CN111973619A - Nucleic acid, pharmaceutical composition containing nucleic acid, siRNA conjugate, preparation method and application - Google Patents

Nucleic acid, pharmaceutical composition containing nucleic acid, siRNA conjugate, preparation method and application Download PDF

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CN111973619A
CN111973619A CN202010427992.1A CN202010427992A CN111973619A CN 111973619 A CN111973619 A CN 111973619A CN 202010427992 A CN202010427992 A CN 202010427992A CN 111973619 A CN111973619 A CN 111973619A
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nucleotide sequence
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sirna
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CN111973619B (en
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张鸿雁
高山
康代武
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Suzhou Ruibo Biotechnology Co Ltd
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification

Abstract

The present disclosure provides an siRNA that inhibits transmembrane serine protease 6(TMPRSS6) gene expression, pharmaceutical compositions and siRNA conjugates. Each nucleotide in the siRNA is independently a modified or unmodified nucleotide, the siRNA comprises a sense strand and an antisense strand, the sense strand comprises a nucleotide sequence I, and the nucleotide sequence I is identical to the nucleotide sequence shown in SEQ ID NO: 1, and NO more than 3 nucleotide differences, and the antisense strand comprises a nucleotide sequence II that is identical to SEQ ID NO: 2 are equal in length and differ by no more than 3 nucleotides. The siRNA, the pharmaceutical composition thereof and the siRNA conjugate provided by the disclosure can effectively treat and/or prevent iron overload complications.

Description

Nucleic acid, pharmaceutical composition containing nucleic acid, siRNA conjugate, preparation method and application
Technical Field
The present disclosure relates to a nucleic acid capable of inhibiting transmembrane serine protease 6(TMPRSS6) gene expression and pharmaceutical compositions and siRNA conjugates containing the same. The disclosure also relates to methods of making and uses of the nucleic acids, pharmaceutical compositions, and siRNA conjugates.
Background
Aplastic Anemia (AA), myelodysplastic syndrome (MDS), thalassemia and other diseases often require blood transfusion treatment to maintain life, and long-term repeated blood transfusion treatment can cause iron content overload in the body, lead cells to take in iron and deposit in tissues and organs, cause injuries such as chronic toxicity and DNA damage, cause the problem of excessive iron absorption of body tissues, and cause the risk of serious iron overload complications.
Transmembrane serine protease 6(TMPRSS6) is one of the key targets for the treatment of iron overload complications. By inhibiting the expression of TMPRSS6 gene, the absorption of tissues to iron ions in blood can be effectively inhibited, thereby avoiding transfusional iron overload. Therefore, silencing gene expression from the gene level and blocking the production of TMPRSS6 would be clearly the most desirable therapeutic approach.
Small interfering RNA (siRNA) can inhibit or block the expression of a target gene of interest in a sequence-specific manner based on the mechanism of RNA interference (RNAi), thereby achieving the purpose of treating diseases.
One of the keys to the development of siRNA drugs that inhibit TMPRSS6 gene expression and treat iron overload complications is to find suitable sirnas and their modifications and effective delivery systems.
Disclosure of Invention
The inventors of the present disclosure surprisingly found that the siRNA conjugates provided with the present disclosure can specifically inhibit the expression of TMPRSS6 gene and specifically target the liver, inhibit the expression of TMPRSS6 gene in the liver, and achieve the treatment or prevention of iron overload complications. In addition, the inventor also invented siRNA and pharmaceutical composition with higher activity. In some embodiments, the present disclosure provides an siRNA conjugate having a structure as shown in formula (308):
Figure BDA0002498737070000011
wherein: n1 is an integer selected from 1 to 3, n3 is an integer selected from 0 to 4; m1, m2 or m3 are independently an integer selected from 2 to 10; r10、R11、R12、R13、R14Or R15Each independently is H, or is selected from the group consisting of: c1-C10Alkyl radical, C1-C10Haloalkyl and C1-C10An alkoxy group;
R3a group of the structure shown in formula a 59:
Figure BDA0002498737070000021
wherein E is1Is OH, SH or BH2；
Nu is siRNA, the siRNA comprises a sense strand and an antisense strand, each nucleotide in the siRNA is a modified or unmodified nucleotide independently, wherein the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially complementary reversely to form a double-stranded region, and the nucleotide sequence I and the nucleotide sequence II are selected from the group consisting of the following I) to vi):
i) the nucleotide sequence I is similar to SEQ ID NO: 1 is equal in length and differs by NO more than 3 nucleotides, and the nucleotide sequence II is identical to SEQ ID NO: 2 are equal in length and differ by no more than 3 nucleotides:
5′-CCGCCAAAGCCCAGAAGAZl-3′(SEQ ID NO：1)；
5′-Z2UCUUCUGGGCUUUGGCGG-3′(SEQ ID NO：2)，
wherein Z is1Is U, Z2Is A, the nucleotide sequence I comprises a position corresponding to Z1Nucleotide Z of3The nucleotide sequence II comprises a position corresponding to Z2Nucleotide Z of4Z is the same as4Is the first nucleotide at the 5' end of the antisense strand;
ii) the nucleotide sequence I is identical to SEQ ID NO: 61 and NO more than 3 nucleotides different, and the nucleotide sequence II is identical to the nucleotide sequence shown in SEQ ID NO: 62 are equal in length and differ by no more than 3 nucleotides:
5′-AGGCACUGCUGGUGGAGGZ5-3′(SEQ ID NO：61)；
5′-Z6CCUCCACCAGCAGUGCCU-3′(SEQ ID NO：62)，
wherein Z is5Is A, Z6Is U, the nucleotide sequence I comprises a position corresponding to Z5Nucleotide Z of7The nucleotide sequence II comprises a position corresponding to Z6Nucleotide Z of8Z is the same as8Is the first nucleotide at the 5' end of the antisense strand;
iii) the nucleotide sequence I has the sequence shown in SEQ ID NO: 121 and NO more than 3 nucleotides different, and the nucleotide sequence II is identical to SEQ ID NO: 122 are equal in length and differ by no more than 3 nucleotides:
5′-GGAGGCAGAAGUAUGAUUZ9-3′(SEQ ID NO：121)；
5′-Z10AAUCAUACUUCUGCCUCC-3′(SEQ ID NO：122)，
wherein Z is9Is U, Z10Is A, the nucleotide sequence I comprises a position corresponding to Z9Nucleotide Z of11The nucleotide sequence II comprises a position corresponding to Z10Nucleotide Z of12Z is the same as12Is the first nucleotide at the 5' end of the antisense strand;
iv) the nucleotide sequence I has the sequence shown in SEQ ID NO: 181 and NO more than 3 nucleotides different, and the nucleotide sequence II is identical to SEQ ID NO: 182 are equal in length and differ by no more than 3 nucleotides:
5′-GAGUUCCUCUGUUCUGUGZ13-3′(SEQ ID NO：181)；
5′-Z14CACAGAACAGAGGAACUC-3′(SEQ ID NO：182)，
wherein Z is13Is A, Z14Is U, the nucleotide sequence I comprises a position corresponding to Z13Nucleotide Z of15The nucleotide sequence II comprises a position corresponding to Z14Nucleotide Z of16Z is the same as16Is the first nucleotide at the 5' end of the antisense strand;
v) the nucleotide sequence I has the sequence shown in SEQ ID NO: 241 are equal in length and differ by NO more than 3 nucleotides, and said nucleotide sequence II is identical to SEQ ID NO: 242 are equal in length and differ by no more than 3 nucleotides:
5′-GGAUGUGCAGUUGAUCCCZl7-3′(SEQ ID NO：241)；
5′-Z18GGGAUCAACUGCACAUCC-3′(SEQ ID NO：242)，
wherein Z is17Is A, Z18Is U, the nucleotide sequence I comprises a position corresponding to Z17Nucleotide Z of19The nucleotide sequence II comprises a position corresponding to Z18Nucleotide Z of20Z is the same as20Is the first nucleotide at the 5' end of the antisense strand; and the number of the first and second electrodes,
vi) the nucleotide sequence I is identical to SEQ ID NO: 301 is of equal length and differs by NO more than 3 nucleotides, and said nucleotide sequence II is identical to SEQ ID NO: 302 are equal in length and differ by no more than 3 nucleotides:
5′-UAACUUGGGAUCUGGGAAZ21-3′(SEQ ID NO：301)；
5′-Z22UUCCCAGAUCCCAAGUUA-3′(SEQ ID NO：302)，
wherein Z is21Is U, Z22Is A, the nucleotide sequence I comprises a position corresponding to Z21Nucleotide Z of23The nucleotide sequence II comprises a position corresponding to Z22Nucleotide Z of24Z is the same as24Is the first nucleotide at the 5' end of the antisense strand;
R2is a straight chain alkylene group of 1 to 20 carbon atoms in length, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: c (O), NH, O, S, CH-N, S (O)2、C2-C10Alkenylene radical, C2-C10Alkynylene, C6-C10Arylene radical, C3-C18Heterocyclylene and C5-C10A heteroarylene group; and wherein R2May optionally have a substituent of any one or more of the group consisting of: c1-C10Alkyl radical, C6-C10Aryl radical, C5-C10Heteroaryl group, C1-C10Haloalkyl, -OC1-C10Alkyl, -OC1-C10Alkylphenyl, -C1-C10alkyl-OH, -OC1-C10Haloalkyl, -SC1-C10Alkyl, -SC1-C10Alkylphenyl, -C1-C10alkyl-SH, -SC1-C10Haloalkyl, halogen substituents, -OH, -SH, -NH2、-C1-C10alkyl-NH2、-N(C1-C10Alkyl) (C1-C10Alkyl), -NH (C)1-C10Alkyl group), N (C)1-C10Alkyl) (C1-C10Alkylphenyl), NH (C)1-C10Alkylphenyl), cyano, nitro, -CO2H、-C(O)O(C1-C10Alkyl), -CON (C)1-C10Alkyl) (C1-C10Alkyl), -CONH (C)1-C10Alkyl), -CONH2，-NHC(O)(C1-C10Alkyl), -NHC (O) (phenyl), -N (C)1-C10Alkyl) C (O) (C)1-C10Alkyl), -N (C)1-C10Alkyl) C (O) (phenyl), -C (O) C1-C10Alkyl, -C (O) C1-C10Alkylphenyl, -C (O) C1-C10Haloalkyl, -OC (O) C1-C10Alkyl, -SO2(C1-C10Alkyl), -SO2(phenyl), -SO2(C1-C10Haloalkyl), -SO2NH2、-SO2NH(C1-C10Alkyl), -SO2NH (phenyl), -NHSO2(C1-C10Alkyl), -NHSO2(phenyl) and-NHSO2(C1-C10Haloalkyl);
each L1Is a straight chain alkylene group of 1 to 70 carbon atoms in length, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: c (O), NH, O, S, CH-N, S (O)2、C2-C10Alkenylene radical, C2-C10Alkynylene, C6-C10Arylene radical, C3-C18Heterocyclylene and C5-C10A heteroarylene group; and wherein L1May optionally have a substituent of any one or more of the group consisting of: c1-C10Alkyl radical, C6-C10Aryl radical, C5-C10Heteroaryl group, C1-C10Haloalkyl, -OC1-C10Alkyl, -OC1-C10Alkylphenyl, -C1-C10alkyl-OH, -OC1-C10Haloalkyl, -SC1-C10Alkyl, -SC1-C10Alkylphenyl, -C1-C10alkyl-SH, -SC1-C10Haloalkyl, halogen substituents, -OH, -SH, -NH2、-C1-C10alkyl-NH2、-N(C1-C10Alkyl) (C1-C10Alkyl), -NH (C)1-C10Alkyl group), N (C)1-C10Alkyl) (C1-C10Alkylphenyl), NH (C)1-C10Alkylphenyl), cyano, nitro, -CO2H、-C(O)O(C1-C10Alkyl), -CON (C)1-C10Alkyl) (C1-C10Alkyl), -CONH (C)1-C10Alkyl), -CONH2，-NHC(O)(C1-C10Alkyl), -NHC (O) (phenyl), -N (C)1-C10Alkyl) C (O) (C)1-C10Alkyl), -N (C)1-C10Alkyl) C (O) (phenyl), -C (O) C1-C10Alkyl, -C (O) C1-C10Alkylphenyl, -C (O) C1-C10Haloalkyl, -OC (O) C1-C10Alkyl, -SO2(C1-C10Alkyl), -SO2(phenyl), -SO2(C1-C10Haloalkyl), -SO2NH2、-SO2NH(C1-C10Alkyl), -SO2NH (phenyl), -NHSO2(C1-C10Alkyl), -NHSO2(phenyl) and-NHSO2(C1-C10Haloalkyl);
Figure BDA0002498737070000041
represents the site of covalent attachment of a group; m1Represents a targeting group.
In some embodiments, the present disclosure provides an siRNA capable of inhibiting expression of TMPRSS6 gene, the siRNA comprising a sense strand and an antisense strand, each nucleotide in the sense strand and the antisense strand being independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide; the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially complementary reversely to form a double-stranded region, the fluorinated modified nucleotide is positioned in the nucleotide sequence I and the nucleotide sequence II, and according to the direction from 5 'end to 3' end, in the sense strand, the nucleotides at the 7 th, 8 th and 9 th positions of the nucleotide sequence I are fluorinated modified nucleotides, and the nucleotides at the rest positions in the sense strand are non-fluorinated modified nucleotides; in the direction from the 5 'end to the 3' end, in the antisense strand, the nucleotides at positions 2, 6, 14, 16 of the nucleotide sequence II are fluorine-modified nucleotides, the nucleotides at the remaining positions in the antisense strand are non-fluorine-modified nucleotides, and the nucleotide sequence I and the nucleotide sequence II are selected from the group consisting of I) to vi) as follows:
i) the nucleotide sequence I is similar to SEQ ID NO: 1 is equal in length and differs by NO more than 3 nucleotides, and the nucleotide sequence II is identical to SEQ ID NO: 2 are equal in length and differ by no more than 3 nucleotides:
5′-CCGCCAAAGCCCAGAAGAZ1-3′(SEQ ID NO：1)；
5′-Z2UCUUCUGGGCUUUGGCGG-3′(SEQ ID NO：2)，
wherein Z is1Is U, Z2Is A, the nucleotide sequence I comprises a position corresponding to Z1Nucleotide Z of3The nucleotide sequence II comprises a position corresponding to Z2Nucleotide Z of4Z is the same as4Is the first nucleotide at the 5' end of the antisense strand;
ii) the nucleotide sequence I is identical to SEQ ID NO: 61 and NO more than 3 nucleotides different, and the nucleotide sequence II is identical to the nucleotide sequence shown in SEQ ID NO: 62 are equal in length and differ by no more than 3 nucleotides:
5′-AGGCACUGCUGGUGGAGGZ5-3′(SEQ ID NO：61)；
5′-Z6CCUCCACCAGCAGUGCCU-3′(SEQ ID NO：62)，
wherein Z is5Is A, Z6Is U, the nucleotide sequence I comprises a position corresponding to Z5Nucleotide Z of7The nucleotide sequence II comprises a position corresponding to Z6Nucleotide Z of8Z is the same as8Is the first nucleotide at the 5' end of the antisense strand;
iii) the nucleotide sequence I has the sequence shown in SEQ ID NO: 121 and NO more than 3 nucleotides different, and the nucleotide sequence II is identical to SEQ ID NO: 122 are equal in length and differ by no more than 3 nucleotides:
5′-GGAGGCAGAAGUAUGAUUZ9-3′(SEQ ID NO：121)；
5′-Z10AAUCAUACUUCUGCCUCC-3′(SEQ ID NO：122)，
wherein Z is9Is U, Z10Is A, the nucleotide sequence I comprises a position corresponding to Z9Nucleotide Z of11The nucleotide sequence II comprises a position corresponding to Z10Nucleotide Z of12Z is the same as12Is the first nucleotide at the 5' end of the antisense strand;
iv) the nucleotide sequence I has the sequence shown in SEQ ID NO: 181 and NO more than 3 nucleotides different, and the nucleotide sequence II is identical to SEQ ID NO: 182 are equal in length and differ by no more than 3 nucleotides:
5′-GAGUUCCUCUGUUCUGUGZ13-3′(SEQ ID NO：181)；
5′-Z14CACAGAACAGAGGAACUC-3′(SEQ ID NO：182)，
wherein Z is13Is A, Z14Is U, the nucleotide sequence I comprises a position corresponding to Z13Nucleotide Z of15The nucleotide sequence II comprises a position corresponding to Z14Nucleotide Z of16Z is the same as16Is the first nucleotide at the 5' end of the antisense strand;
v) the nucleotide sequence I has the sequence shown in SEQ ID NO: 241 are equal in length and differ by NO more than 3 nucleotides, and said nucleotide sequence II is identical to SEQ ID NO: 242 are equal in length and differ by no more than 3 nucleotides:
5′-GGAUGUGCAGUUGAUCCCZ17-3′(SEQ ID NO：241)；
5′-Z18GGGAUCAACUGCACAUCC-3′(SEQ ID NO：242)，
wherein Z is17Is A, Z18Is U, the nucleotide sequence I comprises a position corresponding to Z17Nucleotide Z of19The nucleotide sequence II comprises a position corresponding to Z18Nucleotide Z of20Z is the same as20Is the first nucleotide at the 5' end of the antisense strand; and the number of the first and second electrodes,
vi) the nucleotide sequence I is identical to SEQ ID NO: 301 is of equal length and differs by NO more than 3 nucleotides, and said nucleotide sequence II is identical to SEQ ID NO: 302 are equal in length and differ by no more than 3 nucleotides:
5′-UAACUUGGGAUCUGGGAAZ21-3′(SEQ ID NO：301)；
5′-Z22UUCCCAGAUCCCAAGUUA-3′(SEQ ID NO：302)，
wherein Z is21Is U, Z22Is A, the nucleotide sequence I comprises a position corresponding to Z21Nucleotide Z of23The nucleotide sequence II comprises a position corresponding to Z22Nucleotide Z of24Z is the same as24Is the first nucleotide at the 5' end of the antisense strand.
In some embodiments, each non-fluorinated modified nucleotide is independently selected from one of a nucleotide or a nucleotide analog in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with a non-fluorinated group.
In some embodiments, the nucleotide formed by substituting the hydroxyl group at the 2 '-position of the ribosyl group of the nucleotide with a non-fluorine group is selected from one of a 2' -alkoxy-modified nucleotide, a 2 '-substituted alkoxy-modified nucleotide, a 2' -alkyl-modified nucleotide, a 2 '-substituted alkyl-modified nucleotide, a 2' -amino-modified nucleotide, a 2 '-substituted amino-modified nucleotide, and a 2' -deoxynucleotide; the nucleotide analog is selected from one of isonucleotides, LNA, ENA, cET, UNA and GNA.
In some embodiments, each non-fluorinated modified nucleotide is a methoxy modified nucleotide.
In some embodiments, the present disclosure provides a pharmaceutical composition comprising the siRNA of the present disclosure described above and a pharmaceutically acceptable carrier.
In some embodiments, the present disclosure provides an siRNA conjugate comprising an siRNA provided by the present disclosure and a conjugate group conjugated to the siRNA.
In some embodiments, the present disclosure provides the use of an siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure in the manufacture of a medicament for the treatment and/or prevention of iron overload complications.
In some embodiments, the present disclosure provides a method of treating and/or preventing iron overload complications, the method comprising administering an effective amount of an siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure to a subject having iron overload complications.
In some embodiments, the present disclosure provides a method of inhibiting TMPRSS6 gene expression in a hepatocyte, the method comprising contacting the hepatocyte with an effective amount of an siRNA and/or a pharmaceutical composition and/or an siRNA conjugate of the present disclosure.
In some embodiments, the present disclosure provides a kit comprising an siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure.
Advantageous effects
The siRNA, pharmaceutical compositions and siRNA conjugates provided by the present disclosure have good stability, higher TMPRSS6mRNA inhibitory activity, lower off-target effects, and/or can significantly treat or alleviate iron overload complications symptoms.
In some embodiments, the siRNA, pharmaceutical composition or siRNA conjugate provided by the present disclosure exhibits excellent target mRNA inhibitory activity in an in vitro cell assay. In some embodiments, the siRNA, pharmaceutical composition or siRNA conjugate provided by the present disclosure exhibits a target mRNA inhibition rate of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in hepatocytes.
In some embodiments, the sirnas provided by the present disclosure exhibit higher in vitro inhibitory activity in HEK293A cells, particularly sirnas of the present disclosure have target sequence inhibition rates of up to 70.98% at siRNA concentrations of 0.1 nM. In some embodiments, the siRNA conjugates provided by the present disclosure exhibit high TMPRSS6mRNA inhibitory activity in human primary hepatocytes at an inhibition rate of at least 53.63% and even up to 67.07% at an siRNA concentration of 50 nM. In some embodiments, the siRNA conjugates of the present disclosure with fluorescent labels are injected subcutaneously into C57 mice, the mice are sacrificed for 48h for organ dissection, real-time fluorescence imaging is performed on the mice, and the distribution of fluorescence in organs is observed to find that the siRNA conjugates almost all gather in the liver, which indicates that the siRNA conjugates provided by the present disclosure can effectively deliver siRNA specifically to the liver, indicating that the siRNA conjugates can specifically inhibit the expression of target genes in the liver.
In some embodiments, the siRNA, pharmaceutical composition or siRNA conjugate provided by the present disclosure may have greater stability and/or greater activity in vivo. In some embodiments, the siRNA, pharmaceutical composition or siRNA conjugate provided by the present disclosure exhibits a rate of inhibition of expression of the target gene of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in vivo. In some embodiments, the siRNA, pharmaceutical composition or siRNA conjugate provided by the present disclosure exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% inhibition of TMPRSS6 gene expression in vivo. In some embodiments, the siRNA, pharmaceutical composition or siRNA conjugate provided by the present disclosure exhibits an inhibition rate of intrahepatic TMPRSS6 gene expression of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in vivo. In some embodiments, the siRNA, pharmaceutical composition or siRNA conjugate provided by the present disclosure exhibits an inhibition rate of intrahepatic TMPRSS6 gene expression in vivo in at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the animal models. In some embodiments, the siRNA, pharmaceutical composition or siRNA conjugate provided by the present disclosure exhibits an inhibition rate of TMPRSS6 gene expression in the liver in at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of human subjects in vivo. In some embodiments, the siRNA, pharmaceutical composition or siRNA conjugate provided by the present disclosure does not exhibit significant off-target effects. The off-target effect can be, for example, inhibition of normal expression of a gene other than the target gene. It is believed that off-target effects are not significant if the binding/inhibition of off-target gene expression is less than 50%, 40%, 30%, 20% or 10% compared to the effect on the target gene.
Therefore, the siRNA provided by the disclosure can inhibit the expression of TMPRSS6 gene, effectively treat and/or prevent iron overload complication symptoms caused by TMPRSS6 gene overexpression, and has good application prospect.
Additional features and advantages of the disclosure will be set forth in the detailed description which follows.
Drawings
FIG. 1 is a bar graph of the residual activity of the Renilla reporter gene in the psiCHECK system after transfection with different siRNAs.
Figure 2 is a bar graph of the relative expression levels of TMPRSS6mRNA in human primary hepatocytes after transfection of different siRNA conjugates and a reference siranc.
FIG. 3A is a photograph of fluorescence images of organs in a C57 mouse administered with 5ml/kg of 1 XPBS, 3mg/kg of Cy5-siRNA 1 or 3mg/kg of Cy 5-conjugate for 148 h.
FIG. 3B is a photograph of fluorescence images of organs in a C57 mouse administered with 5ml/kg of 1 XPBS, 3mg/kg of Cy5-siRNA 2 or 3mg/kg of Cy 5-conjugate for 248 hours.
Detailed Description
The following describes in detail specific embodiments of the present disclosure. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.
In the present disclosure, TMPRSS6mRNA refers to mRNA having a sequence as shown in Genbank accession No. NM — 001289001.1. Further, unless otherwise specified, the term "target gene" used in the present disclosure refers to a gene that transcribes the above-mentioned TMPRSS6mRNA, and the term "target mRNA" refers to the above-mentioned TMPRSS6 mRNA.
Definition of
In the above and below, capital C, G, U, A represents the base composition of nucleotides, unless otherwise specified; the lower case letter m indicates that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide; the lower case letter f indicates that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide; the lower case letter s indicates a phosphorothioate-based linkage between two nucleotides adjacent to the left and right of the letter s; p1 indicates that the nucleotide adjacent to the right of the P1 is a 5 ' -phosphate nucleotide or a 5 ' -phosphate analog modified nucleotide, the letter combination VP indicates that the nucleotide adjacent to the right of the letter combination VP is a vinyl phosphate modified nucleotide, the letter combination Ps indicates that the nucleotide adjacent to the right of the letter combination Ps is a phosphorothioate modified nucleotide, and the capital letter P indicates that the nucleotide adjacent to the right of the letter P is a 5 ' -phosphate nucleotide.
In the above and below, the "fluorine-modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2 '-position of the ribosyl group of the nucleotide is substituted with fluorine, and the "non-fluorine-modified nucleotide" refers to a nucleotide or a nucleotide analog in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with a non-fluorine group. "nucleotide analog" refers to a group that can replace a nucleotide in a nucleic acid, but that differs in structure from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. Such as a heteronucleotide, a bridged nucleotide (BNA for short) or an acyclic nucleotide. The "methoxy-modified nucleotide" refers to a nucleotide in which the 2' -hydroxyl group of the ribosyl group is substituted with a methoxy group.
In the present context, the expressions "complementary" or "reverse complementary" are used interchangeably and have the meaning well known to the person skilled in the art, i.e. in a double-stranded nucleic acid molecule the bases of one strand are each paired in a complementary manner with the bases on the other strand. In DNA, the purine base adenine (a) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair comprises a purine and a pyrimidine. Two strands are considered to be complementary to each other when adenine on one strand always pairs with thymine (or uracil) on the other strand and guanine always pairs with cytosine, and the sequence of that strand can be deduced from the sequence of its complementary strand. Accordingly, "mismatch" in the art means that in a double-stranded nucleic acid, the bases at the corresponding positions are not paired in a complementary fashion.
In the above and below, unless otherwise specified, "substantially reverse complementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; "substantially reverse complementary" means that no more than 1 base mismatch exists between two nucleotide sequences; "completely reverse complementary" means that there is no base mismatch between two nucleotide sequences.
In the above and below, the "nucleotide difference" between one nucleotide sequence and another nucleotide sequence means that the former has a change in the base type of the nucleotide at the same position as compared with the latter, for example, in the latter, when one nucleotide base is A, in the case where the corresponding nucleotide base at the same position of the former is U, C, G or T, it is considered that there is a nucleotide difference between the two nucleotide sequences at that position. In some embodiments, when a nucleotide in situ is replaced with a nucleotide without a base or its equivalent, it is also believed that a nucleotide difference is created at that position.
In the above and the following, particularly in describing the preparation method of the siRNA, the siRNA-containing pharmaceutical composition or the siRNA conjugate of the present disclosure, unless otherwise specified, the Nucleoside monomer (Nucleoside monomer) means modified or unmodified Nucleoside phosphoramidite monomers (sometimes referred to as Nucleoside phosphoramidites) used in solid phase synthesis of phosphoramidites, depending on the kind and order of nucleotides in the siRNA or siRNA conjugate to be prepared. Solid phase phosphoramidite synthesis is a method used in RNA synthesis well known to those skilled in the art. Nucleoside monomers for use in the present disclosure are all commercially available.
In the context of the present disclosure, "conjugated," means that two or more chemical moieties, each having a particular function, are linked to each other in a covalent linkage, unless otherwise indicated; accordingly, "conjugate" refers to a compound formed by covalent linkage between the various chemical moieties. Further, "siRNA conjugate" means a compound formed by covalently attaching one or more chemical moieties having a specific function to siRNA. Hereinafter, the siRNA conjugates of the present disclosure are also sometimes simply referred to as "conjugates". The siRNA conjugate is understood as a generic term of a plurality of siRNA conjugates or an siRNA conjugate represented by a certain chemical formula according to the context. In the context of the present disclosure, a "conjugate molecule" should be understood as a specific compound that can be conjugated to an siRNA by a reaction, ultimately forming an siRNA conjugate of the present disclosure.
As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally substituted" alkyl "includes" alkyl "and" substituted alkyl "as defined below. It will be understood by those skilled in the art that, for any group containing one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is sterically impractical, synthetically non-feasible, and/or inherently unstable.
As used herein, "alkyl" refers to straight and branched chains having the specified number of carbon atoms, typically from 1 to 20 carbon atoms, for example from 1 to 10 carbon atoms, such as from 1 to 8 or from 1 to 6 carbon atoms. E.g. C1-C6Alkyl groups include straight and branched chain alkyl groups of 1 to 6 carbon atoms. When referring to an alkyl residue having a particular number of carbons, it is intended to encompass all branched and straight chain forms having that number of carbons; thus, for example, "butyl" is meant to include n-butyl, sec-butyl, isobutyl, and tert-butyl; "propyl" includes n-propyl and isopropyl. Alkylene is a subset of alkyl and refers to the same as alkyl but with the proviso thatResidues with two points of attachment.
As used herein, "alkenyl" refers to an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon double bond obtained by removing a molecule of hydrogen from the adjacent carbon atom of the parent alkyl group. The group may be in the cis or trans configuration of the double bond. Typical alkenyl groups include, but are not limited to: a vinyl group; propenyl, such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-2-en-2-yl; butenyl, e.g., but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-en-1-yl, but-2-en-2-yl, but-1, 3-dien-1-yl, but-1, 3-dien-2-yl, and the like. In certain embodiments, alkenyl groups have 2 to 20 carbon atoms, and in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkenylene is a subset of alkenyl and refers to the same residue as alkenyl, but with two points of attachment.
As used herein, "alkynyl" refers to an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon triple bond obtained by removing two molecules of hydrogen from adjacent carbon atoms of the parent alkyl group. Typical alkynyl groups include, but are not limited to: an ethynyl group; propynyl groups, such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyl groups such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl and the like. In certain embodiments, alkynyl groups have 2 to 20 carbon atoms, and in other embodiments 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkynylene is a subset of alkynyl and refers to the same residue as alkynyl, but with two points of attachment.
As used herein, "alkoxy" refers to an alkyl group of the indicated number of carbon atoms attached through an oxygen bridge, e.g., methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentyloxy, 2-pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, 2-hexyloxy, 3-methylpentyloxy, and the like. Alkoxy groups typically have 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms connected by an oxygen bridge.
As used herein, "aryl" refers to a group derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by the removal of a hydrogen atom from a ring carbon atom. The aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbon of 6 to 18 carbon atoms, wherein at least one ring in the ring system is fully unsaturated, i.e. comprises a cyclic, delocalized (4n +2) pi-electron system according to Huckel theory. Aryl groups include, but are not limited to, phenyl, fluorenyl, naphthyl, and the like. Arylene is a subset of aryl and refers to the same residue as aryl, but with two points of attachment.
As used herein, "halogen substituent" or "halo" refers to fluoro, chloro, bromo, or iodo, and the term "halogen" includes fluoro, chloro, bromo, or iodo.
As used herein, "haloalkyl" refers to an alkyl group as defined above wherein the specified number of carbon atoms are substituted with one or more, up to the maximum allowable number of halogen atoms. Examples of haloalkyl include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl, or pentafluoroethyl.
"heterocyclyl" refers to a stable 3-to 18-membered non-aromatic ring radical containing 2-12 carbon atoms and 1-6 heteroatoms selected from nitrogen, oxygen, or sulfur. Unless otherwise indicated in the specification, heterocyclyl is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, and may include fused or bridged ring systems. The heteroatoms in the heterocyclic group may be optionally oxidized. One or more nitrogen atoms (if present) are optionally quaternized. Heterocyclyl groups are partially or fully saturated. The heterocyclyl group may be attached to the remainder of the molecule through any ring atom. Examples of such heterocyclic groups include, but are not limited to: dioxanyl, thienyl [1, 3] dithioyl (thienyl [1, 3] dithianyl), decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxapiperazinyl, 2-oxapiperidinyl, 2-oxapyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidinonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trithioyl (trithiofuranyl), tetrahydropyranyl, thiomorpholinyl (thiomorpholinyl), 1-oxothiomorpholinyl (1-oxo-thiomorpholinyl), and 1, 1-dioxothiomorpholinyl (1, 1-dioxothiomorpholinyl).
"heteroaryl" refers to a group derived from a 3-to 18-membered aromatic ring radical containing 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur. As used herein, a heteroaryl group can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, wherein at least one ring in the ring system is fully unsaturated, i.e., comprises a cyclic delocalized (4n +2) pi-electron system according to huckel theory. Heteroaryl includes fused or bridged ring systems. The heteroatoms in the heteroaryl group are optionally oxidized. One or more nitrogen atoms (if present) are optionally quaternized. The heteroaryl group is attached to the rest of the molecule through any ring atom. Examples of heteroaryl groups include, but are not limited to: azacyclotrienoyl, acridinyl, benzimidazolyl, benzindolyl, 1, 3-benzodioxazolyl, benzofuranyl, benzoxazolyl, benzo [ d ] thiazolyl, benzothiadiazolyl, benzo [ b ] [1, 4] dioxepinyl (benzo [ b ] [1, 4] dioxepinyl), benzo [ b ] [1, 4] oxazinyl (benzo [ b ] [1, 4] oxazinyl), 1, 4-benzodioxanyl (1, 4-benzodioxanyl), benzonaphthofuranyl, benzoxazolyl, benzodioxolyl (benzodioxolyl), benzodioxinyl (benzodioxanyl), benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothiophenyl, benzothieno [3, 2-d ] pyrimidinyl, benzotriazolyl, benzo [4, 6] imidazo [1, 2-a ] pyridinyl, Carbazolyl, cinnolinyl, cyclopenta [ d ] pyrimidinyl, 6, 7-dihydro-5H-cyclopenta [4, 5] thieno [2, 3-d ] pyrimidinyl, 5, 6-dihydrobenzo [ H ] quinazolinyl (5, 6-dihydrobenzo [ H ] quinazolinyl), 5, 6-dihydrobenzo [ H ] cinnolinyl (5, 6-dihydrobenzo [ H ] cinnolinyl), 6, 7-dihydro-5H-benzo [6, 7] cyclohepta [1, 2-c ] pyridazinyl, dibenzofuranyl, dibenzothienyl, furanyl, furanonyl, furo [3, 2-c ] pyridinyl, 5, 6, 7, 8, 9, 10-hexahydrocycloocta [ d ] pyrimidinyl, 5, 6, 7, 8, 9, 10-hexahydrocycloocta [ d ] pyridazinyl, 5, 6, 7, 8, 9, 10-hexahydrocycloocta [ d ] pyridazinyl, 7, 8, 9, 10-hexahydrocycloocta [ d ] pyridyl, isothiazolyl, imidazolyl, indazolyl (indazolyl), indolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, 5, 8-methanol-5, 6, 7, 8-tetrahydroquinazolinyl (5, 8-methano-5, 6, 7, 8-tetrahydroquinazolinyl), naphthyridinyl (naphthyridinyl), 1, 6-naphthyridinonyl (1, 6-naphthyridinonyl), oxadiazolyl, 2-oxazepinyl (2-oxoazepinyl), oxazolyl, oxacyclopropane (oxacinnanyl), 5, 6, 6a, 7, 8, 9, 10, 10 a-octahydrobenzo [ H ] quinazolinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, and oxazolyl, Phthalazinyl (phthalazinyl), pteridinyl (pteridinyl), purinyl, pyrrolyl, pyrazolyl, pyrazolo [3, 4-d ] pyrimidinyl, pyridyl, pyrido [3, 2-d ] pyrimidinyl, pyrido [3, 4-d ] pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl (quinoxalinyl), quinolinyl, tetrahydroquinolinyl, 5, 6, 7, 8-tetrahydroquinazolinyl, 5, 6, 7, 8-tetrahydrobenzo [4, 5] thieno [2, 3-d ] pyrimidinyl, 6, 7, 8, 9-tetrahydro-5H-cyclohepta [4, 5] thieno [2, 3-d ] pyrimidinyl, 5, 6, 7, 8-tetrahydropyrido [4, 5-c ] pyridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, Thieno [2, 3-d ] pyrimidinyl, thieno [3, 2-d ] pyrimidinyl, thieno [2, 3-c ] pyridinyl (thieno [2, 3-c ] pridinyl) and thienyl (thiophenyl/thiophenyl).
Various hydroxyl protecting groups may be used in the present disclosure. In general, protecting groups render a chemical functionality insensitive to particular reaction conditions, and can be added to and removed from the molecule at that functionality without substantially damaging the rest of the molecule. Representative hydroxyl protecting Groups are disclosed in Beaucage et al, Tetrahedron 1992, 48, 2223-2311, and Greenea and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed, John Wiley & Sons, New York, 1991, each of which is incorporated herein by reference in its entirety. In some embodiments, the protecting group is stable under basic conditions, but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxy protecting groups that may be used herein include Dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthen-9-yl (Pixyl), or 9- (p-methoxyphenyl) xanthen-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4, 4 '-dimethoxytrityl), or TMTr (4, 4', 4 "-trimethoxytrityl).
The term "subject", as used herein, refers to any animal, e.g., a mammal or a marsupial. Subjects of the present disclosure include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats, or any kind of poultry.
As used herein, "treatment" refers to a method of obtaining a beneficial or desired result, including but not limited to a therapeutic benefit. By "therapeutic benefit" is meant eradication or amelioration of the underlying disorder being treated. In addition, therapeutic benefit is achieved by eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, although the subject may still be afflicted with the underlying disorder.
As used herein, "prevention" refers to a method of obtaining a beneficial or desired result, including but not limited to a prophylactic benefit. To obtain a "prophylactic benefit," an siRNA conjugate or pharmaceutical composition can be administered to a subject at risk for a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though a diagnosis of the disease may not have been made.
In one aspect, the present disclosure provides first through sixth sirnas capable of inhibiting expression of TMPRSS6 gene. Which will be described in detail in turn below.
The sirnas of the present disclosure contain a nucleotide group as a basic structural unit, which is well known to those skilled in the art, and the nucleotide group contains a phosphate group, a ribose group and a base, which are not described in detail herein.
The siRNA of the present disclosure contains a sense strand and an antisense strand, the sense strand and the antisense strand being the same or different in length, the sense strand being 19-23 nucleotides in length and the antisense strand being 19-26 nucleotides in length. Thus, the length ratio of the sense strand and the antisense strand of the siRNA provided by the present disclosure may be 19/19, 19/20, 19/21, 19/22, 19/23, 19/24, 19/25, 19/26, 20/20, 20/21, 20/22, 20/23, 20/24, 20/25, 20/26, 21/20, 21/21, 21/22, 21/23, 21/24, 21/25, 21/26, 22/20, 22/21, 22/22, 22/23, 22/24, 22/25, 22/26, 23/20, 23/21, 23/22, 23/23, 23/24, 23/25 or 23/26. In some embodiments, the siRNA has a length ratio of sense strand to antisense strand of 19/21, 21/23, or 23/25.
First siRNA
According to the present disclosure, the siRNA may be a first siRNA.
The first siRNA comprises a sense strand and an antisense strand, each nucleotide in the first siRNA is a modified or unmodified nucleotide independently, wherein the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially complementary in a reverse direction to form a double-stranded region, wherein the nucleotide sequence I is complementary to the nucleotide sequence shown in SEQ ID NO: 1 is equal in length and differs by NO more than 3 nucleotides, and the nucleotide sequence II is identical to SEQ ID NO: 2 are equal in length and differ by no more than 3 nucleotides:
5′-CCGCCAAAGCCCAGAAGAZ1-3′(SEQ ID NO：1)；
5′-Z2UCUUCUGGGCUUUGGCGG-3′(SEQ ID NO：2)，
wherein Z is1Is U, Z2Is A, the nucleotide sequence I comprises a position corresponding to Z1Nucleotide Z of3The nucleotide sequence II comprises a position corresponding to Z2Nucleotide Z of4Z is the same as4Is the first nucleotide at the 5' end of the antisense strand.
In the above and below, "positional correspondence" means that they are at the same position in the nucleotide sequence from the same end of the nucleotide sequence. For example, the 1 st nucleotide at the 3' end of the nucleotide sequence I is a nucleotide sequence whose position corresponds to SEQ ID NO: 1, nucleotide of the 1 st nucleotide of the 3' end of 1.
In some embodiments, the sense strand comprises only nucleotide sequence I and the antisense strand comprises only nucleotide sequence II.
In some embodiments, the nucleotide sequence I is identical to SEQ ID NO: 1, and/or the nucleotide sequence II differs from the nucleotide sequence shown in SEQ ID NO: 2 by no more than 1 nucleotide difference.
In some embodiments, the nucleotide sequence II is identical to SEQ ID NO: 2 comprises Z4A difference at position, and Z4Selected from U, C or G. In some embodiments, the nucleotide difference is Z4Difference in position, Z4Selected from U, C or G. In some embodiments, Z3Is a reaction of with Z4A complementary nucleotide. The sirnas having the above nucleotide differences have higher target mRNA inhibitory ability of the sirnas, and these sirnas comprising the nucleotide differences are also within the scope of the present disclosure.
In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, or fully reverse complementary; by substantially reverse complementary is meant that no more than 3 base mismatches occur between two nucleotide sequences; the substantially reverse complement refers to the presence of no more than 1 base mismatch between two nucleotide sequences; by fully reverse complementary is meant that there is no base mismatch between the two nucleotide sequences.
In some embodiments, the nucleotide sequence I is SEQ ID NO: 3, and the nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 4:
5′-CCGCCAAAGCCCAGAAGAZ3-3′(SEQ ID NO：3)；
5′-Z4UCUUCUGGGCUUUGGCGG-3′(SEQ ID NO：4)，
wherein, Z is4Is the first nucleotide at the 5' end of the antisense strand, Z3Selected from A, U, G or C, and Z4Is a reaction of with Z3A complementary nucleotide; in some embodiments, Z3Is U, Z4Is A.
In some embodiments, the sense strand further comprises nucleotide sequence III and the antisense strand further comprises nucleotide sequence IV, each of nucleotide sequence III and nucleotide sequence IV being 1-4 nucleotides in length; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or fully reverse complementary; the nucleotide sequence III is connected at the 5 'end of the nucleotide sequence I, and the nucleotide sequence IV is connected at the 3' end of the nucleotide sequence II. In some embodiments, the nucleotide sequence IV is substantially reverse complementary or fully reverse complementary to a second nucleotide sequence that is complementary to a nucleotide sequence in the target mRNA consisting of SEQ ID NO: 1 and a nucleotide sequence which is adjacent to the 5' end of the nucleotide sequence shown in the expression 1 and has the same length as the nucleotide sequence IV.
In some embodiments, the length of each of nucleotide sequence III and nucleotide sequence IV is 1 nucleotide, the base of nucleotide sequence III is a, and the base of nucleotide sequence IV is U; at this time, the length ratio of the sense strand to the antisense strand is 20/20; or, the length of the nucleotide sequences III and IV is 2 nucleotides, the base composition of the nucleotide sequence III is AA, and the base composition of the nucleotide sequence IV is UU according to the direction from the 5 'end to the 3' end; in this case, the length ratio of the sense strand to the antisense strand is 21/21; or, the length of the nucleotide sequences III and IV is 3 nucleotides, the base composition of the nucleotide sequence III is AAA, and the base composition of the nucleotide sequence IV is UUUU according to the direction from the 5 'end to the 3' end; at this time, the length ratio of the sense strand to the antisense strand was 22/22; or, the length of the nucleotide sequences III and IV is 4 nucleotides, the base composition of the nucleotide sequence III is GAAA, and the base composition of the nucleotide sequence IV is UUUUC according to the direction from the 5 'end to the 3' end; in this case, the length ratio of the sense strand to the antisense strand is 23/23. In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are 2 nucleotides in length, and the base composition of the nucleotide sequence III is AA and the base composition of the nucleotide sequence IV is UU in the direction from the 5 'end to the 3' end; in this case, the length ratio of the sense strand to the antisense strand was 21/21.
In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are fully complementary in reverse orientation, such that, given the bases of the nucleotide sequence III, the bases of the nucleotide sequence IV are defined.
Second siRNA
According to the present disclosure, the siRNA may be a second siRNA.
The second siRNA comprises a sense strand and an antisense strand, each nucleotide in the second siRNA is a modified or unmodified nucleotide independently, wherein the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially complementary in a reverse direction to form a double-stranded region, wherein the nucleotide sequence I is complementary to the nucleotide sequence shown in SEQ ID NO: 61 and NO more than 3 nucleotides different, and the nucleotide sequence II is identical to the nucleotide sequence shown in SEQ ID NO: 62 are equal in length and differ by no more than 3 nucleotides:
5′-AGGCACUGCUGGUGGAGGZ5-3′(SEQ ID NO：61)；
5′-Z6CCUCCACCAGCAGUGCCU-3′(SEQ ID NO：62)，
wherein Z is5Is A, Z6Is U, the nucleotide sequence I comprises a position corresponding to Z5Nucleotide Z of7The nucleotide sequence II comprises a position corresponding to Z6Nucleotide Z of8Z is the same as8Is the first nucleotide at the 5' end of the antisense strand.
In the above and below, "positional correspondence" means that they are at the same position in the nucleotide sequence from the same end of the nucleotide sequence. For example, the 1 st nucleotide at the 3' end of the nucleotide sequence I is a nucleotide sequence whose position corresponds to SEQ ID NO: 61, 3' of the nucleotide of the 1 st nucleotide.
In some embodiments, the sense strand comprises only nucleotide sequence I and the antisense strand comprises only nucleotide sequence II.
In some embodiments, the nucleotide sequence I is identical to SEQ ID NO: 61, and/or the nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO: 62 is no more than 1 nucleotide different.
In some embodiments, the nucleotide sequence II is identical to SEQ ID NO: 62 comprises Z8A difference at position, and Z8Selected from A, C or G. In some embodiments, the nucleotide difference is Z8Difference in position, Z8Selected from A, C or G. In some embodiments, Z7Is a reaction of with Z8A complementary nucleotide. The sirnas having the above nucleotide differences have higher target mRNA inhibitory ability of the sirnas, and these sirnas comprising the nucleotide differences are also within the scope of the present disclosure.
In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, or fully reverse complementary.
In some embodiments, the nucleotide sequence I is SEQ ID NO: 63 and nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 64, and (b) the nucleotide sequence shown in 64:
5′-AGGCACUGCUGGUGGAGGZ7-3′(SEQ ID NO：63)；
5′-Z8CCUCCACCAGCAGUGCCU-3′(SEQ ID NO：64)，
wherein, Z is8Is the first nucleotide at the 5' end of the antisense strand, Z7Selected from A, U, G or C, and Z8Is a reaction of with Z7A complementary nucleotide; in some embodiments, Z 7Is A, Z8Is U.
In some embodiments, the sense strand further comprises nucleotide sequence III and the antisense strand further comprises nucleotide sequence IV, each of nucleotide sequence III and nucleotide sequence IV being 1-4 nucleotides in length; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or fully reverse complementary; the nucleotide sequence III is connected to the 5 'end of the nucleotide sequence I, the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II, the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary with a second nucleotide sequence, and the second nucleotide sequence refers to a nucleotide sequence which is complementary with the nucleotide sequence shown in SEQ ID NO: 61 and a nucleotide sequence having the same length as the nucleotide sequence IV and being adjacent to the 5' -end of the nucleotide sequence.
In some embodiments, the length of each of nucleotide sequence III and nucleotide sequence IV is 1 nucleotide, the base of nucleotide sequence III is C, and the base of nucleotide sequence IV is G; at this time, the length ratio of the sense strand to the antisense strand is 20/20; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and the base composition of the nucleotide sequence III is GC and the base composition of the nucleotide sequence IV is GC according to the direction from the 5 'end to the 3' end; in this case, the length ratio of the sense strand to the antisense strand is 21/21; or, the length of the nucleotide sequences III and IV is 3 nucleotides, the base composition of the nucleotide sequence III is UGC, and the base composition of the nucleotide sequence IV is GCA according to the direction from the 5 'end to the 3' end; at this time, the length ratio of the sense strand to the antisense strand was 22/22; or, the length of the nucleotide sequences III and IV is 4 nucleotides, the base composition of the nucleotide sequence III is GUGC and the base composition of the nucleotide sequence IV is GCAC according to the direction from the 5 'end to the 3' end; in this case, the length ratio of the sense strand to the antisense strand is 23/23. In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are 2 nucleotides in length, and the base composition of the nucleotide sequence III is GC and the base composition of the nucleotide sequence IV is GC in the direction from the 5 'end to the 3' end; in this case, the length ratio of the sense strand to the antisense strand was 21/21.
In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are fully complementary in reverse orientation, such that, given the bases of the nucleotide sequence III, the bases of the nucleotide sequence IV are defined.
Third type of siRNA
According to the present disclosure, the siRNA may be a third siRNA.
The third siRNA comprises a sense strand and an antisense strand, each nucleotide in the third siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially complementary in reverse direction to form a double-stranded region, wherein the nucleotide sequence I is complementary to the nucleotide sequence of SEQ ID NO: 121 and NO more than 3 nucleotides different, and the nucleotide sequence II is identical to SEQ ID NO: 122 are equal in length and differ by no more than 3 nucleotides:
5′-GGAGGCAGAAGUAUGAUUZ9-3′(SEQ ID NO：121)；
5′-Z10AAUCAUACUUCUGCCUCC-3′(SEQ ID NO：122)，
wherein Z is9Is U, Z10Is A, the nucleotide sequence I comprises a position corresponding to Z9Nucleotide Z of11The nucleotide sequence II comprises a position corresponding to Z10Nucleotide Z of12Z is the same as12Is the first nucleotide at the 5' end of the antisense strand.
In the above and below, "positional correspondence" means that they are at the same position in the nucleotide sequence from the same end of the nucleotide sequence. For example, the 1 st nucleotide at the 3' end of the nucleotide sequence I is a nucleotide sequence whose position corresponds to SEQ ID NO: 121 at the 1 st nucleotide of the 3' end.
In some embodiments, the sense strand comprises only nucleotide sequence I and the antisense strand comprises only nucleotide sequence II.
In some embodiments, the nucleotide sequence I is identical to SEQ ID NO: 121, and/or the nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO: 122 by no more than 1 nucleotide difference.
In some embodiments, the nucleotide sequence II is identical to SEQ ID NO: 122 comprises Z12A difference at position, and Z12Selected from U, C or G. In some casesIn embodiments, the nucleotide difference is Z12Difference in position, Z12Selected from U, C or G. In some embodiments, Z11Is a reaction of with Z12A complementary nucleotide. The sirnas having the above nucleotide differences have higher target mRNA inhibitory ability of the sirnas, and these sirnas comprising the nucleotide differences are also within the scope of the present disclosure.
In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, or fully reverse complementary.
In some embodiments, the nucleotide sequence I is SEQ ID NO: 123, and nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 124, and the nucleotide sequence shown in the sequence No:
5′-GGAGGCAGAAGUAUGAUUZ11-3′(SEQ ID NO：123)；
5′-Z12AAUCAUACUUCUGCCUCC-3′(SEQ ID NO：124)，
wherein, Z is12Is the first nucleotide at the 5' end of the antisense strand, Z11Selected from A, U, G or C, and Z12Is a reaction of with Z11A complementary nucleotide; in some embodiments, Z11Is U, Z12Is A.
In some embodiments, the sense strand further comprises nucleotide sequence III and the antisense strand further comprises nucleotide sequence IV, each of nucleotide sequence III and nucleotide sequence IV being 1-4 nucleotides in length; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or fully reverse complementary; the nucleotide sequence III is connected to the 5 'end of the nucleotide sequence I, the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II, the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to a second nucleotide sequence which is complementary to a nucleotide sequence in the target mRNA represented by SEQ ID NO: 121 and a nucleotide sequence having the same length as the nucleotide sequence IV and having adjacent 5' ends.
In some embodiments, the length of each of nucleotide sequence III and nucleotide sequence IV is 1 nucleotide, the base of nucleotide sequence III is a, and the base of nucleotide sequence IV is U; at this time, the length ratio of the sense strand to the antisense strand is 20/20; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and according to the direction from the 5 'end to the 3' end, the base composition of the nucleotide sequence III is GA, and the base composition of the nucleotide sequence IV is UC; in this case, the length ratio of the sense strand to the antisense strand is 21/21; or, the length of the nucleotide sequences III and IV is 3 nucleotides, the base composition of the nucleotide sequence III is UGA and the base composition of the nucleotide sequence IV is UCA according to the direction from the 5 'end to the 3' end; at this time, the length ratio of the sense strand to the antisense strand was 22/22; or, the length of the nucleotide sequences III and IV is 4 nucleotides, and according to the direction from the 5 'end to the 3' end, the base composition of the nucleotide sequence III is CUGA, and the base composition of the nucleotide sequence IV is UCAG; in this case, the length ratio of the sense strand to the antisense strand is 23/23. In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are 2 nucleotides in length, the base composition of the nucleotide sequence III is GA, the base composition of the nucleotide sequence IV is UC, in the direction from the 5 'end to the 3' end; in this case, the length ratio of the sense strand to the antisense strand was 21/21.
In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are fully complementary in reverse orientation, such that, given the bases of the nucleotide sequence III, the bases of the nucleotide sequence IV are defined.
Fourth siRNA
In accordance with the present disclosure, the siRNA can be a fourth siRNA.
The fourth siRNA comprises a sense strand and an antisense strand, each nucleotide in the fourth siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially complementary in reverse direction to form a double-stranded region, wherein the nucleotide sequence I is complementary to the nucleotide sequence of SEQ ID NO: 181 and NO more than 3 nucleotides different, and the nucleotide sequence II is identical to SEQ ID NO: 182 are equal in length and differ by no more than 3 nucleotides:
5′-GAGUUCCUCUGUUCUGUGZ13-3′(SEQ ID NO：181)；
5′-Z14CACAGAACAGAGGAACUC-3′(SEQ ID NO：182)，
wherein Z is13Is A, Z14Is U, the nucleotide sequence I comprises a position corresponding to Z13Nucleotide Z of15The nucleotide sequence II comprises a position corresponding to Z14Nucleotide Z of16Z is the same as16Is the first nucleotide at the 5' end of the antisense strand.
In the above and below, "positional correspondence" means that they are at the same position in the nucleotide sequence from the same end of the nucleotide sequence. For example, the 1 st nucleotide at the 3' end of the nucleotide sequence I is a nucleotide sequence whose position corresponds to SEQ ID NO: 181 at the 3' end of the sequence of nucleotide 1.
In some embodiments, the sense strand comprises only nucleotide sequence I and the antisense strand comprises only nucleotide sequence II.
In some embodiments, the nucleotide sequence I is identical to SEQ ID NO: 181, and/or the nucleotide sequence II differs from the nucleotide sequence shown in SEQ ID NO: 182 by no more than 1 nucleotide difference.
In some embodiments, the nucleotide sequence II is identical to SEQ ID NO: 182 comprises Z16A difference at position, and Z16Selected from A, C or G. In some embodiments, the nucleotide difference is Z16Difference in position, Z16Selected from A, C or G. In some embodiments, Z15Is a reaction of with Z16A complementary nucleotide. The sirnas having the above nucleotide differences have higher target mRNA inhibitory ability of the sirnas, and these sirnas comprising the nucleotide differences are also within the scope of the present disclosure.
In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, or fully reverse complementary.
In some embodiments, the nucleotide sequence I is SEQ ID NO: 183 and nucleotide sequence II is SEQ ID NO: 184, and (b):
5′-GAGUUCCUCUGUUCUGUGZ15-3′(SEQ ID NO：183)；
5′-Z16CACAGAACAGAGGAACUC-3′(SEQ ID NO：184)，
wherein, Z is16Is the first nucleotide at the 5' end of the antisense strand, Z15Selected from A, U, G or C, and Z16Is a reaction of with Z15A complementary nucleotide; in some embodiments, Z15Is A, Z16Is U.
In some embodiments, the sense strand further comprises nucleotide sequence III and the antisense strand further comprises nucleotide sequence IV, each of nucleotide sequence III and nucleotide sequence IV being 1-4 nucleotides in length; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or fully reverse complementary; the nucleotide sequence III is connected to the 5 'end of the nucleotide sequence I, the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II, the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to a second nucleotide sequence which is complementary to a nucleotide sequence in the target mRNA represented by SEQ ID NO: a nucleotide sequence which is adjacent to the 5' end of the nucleotide sequence shown in 181 and has the same length as the nucleotide sequence IV.
In some embodiments, the length of each of nucleotide sequence III and nucleotide sequence IV is 1 nucleotide, the base of nucleotide sequence III is a, and the base of nucleotide sequence IV is U; at this time, the length ratio of the sense strand to the antisense strand is 20/20; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and according to the direction from the 5 'end to the 3' end, the base composition of the nucleotide sequence III is GA, and the base composition of the nucleotide sequence IV is UC; in this case, the length ratio of the sense strand to the antisense strand is 21/21; or, the length of the nucleotide sequences III and IV is 3 nucleotides, the base composition of the nucleotide sequence III is GGA and the base composition of the nucleotide sequence IV is UCC according to the direction from the 5 'end to the 3' end; at this time, the length ratio of the sense strand to the antisense strand was 22/22; or, the length of the nucleotide sequences III and IV is 4 nucleotides, and according to the direction from the 5 'end to the 3' end, the base composition of the nucleotide sequence III is UGGA, and the base composition of the nucleotide sequence IV is UCCA; in this case, the length ratio of the sense strand to the antisense strand is 23/23. In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are 2 nucleotides in length, the base composition of the nucleotide sequence III is GA, the base composition of the nucleotide sequence IV is UC, in the direction from the 5 'end to the 3' end; in this case, the length ratio of the sense strand to the antisense strand was 21/21.
In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are fully complementary in reverse orientation, such that, given the bases of the nucleotide sequence III, the bases of the nucleotide sequence IV are defined.
Fifth siRNA
According to the present disclosure, the siRNA may be a fifth siRNA.
The fifth siRNA comprises a sense strand and an antisense strand, each nucleotide in the fifth siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially complementary in reverse direction to form a double-stranded region, wherein the nucleotide sequence I is complementary to the nucleotide sequence of SEQ ID NO: 241 are equal in length and differ by NO more than 3 nucleotides, and said nucleotide sequence II is identical to SEQ ID NO: 242 are equal in length and differ by no more than 3 nucleotides:
5′-GGAUGUGCAGUUGAUCCCZ17-3′(SEQ ID NO：241)；
5′-Z18GGGAUCAACUGCACAUCC-3′(SEQ ID NO：242)，
wherein Z is17Is A, Z18Is U, the nucleotide sequence I comprises a position corresponding to Z17Nucleotide Z of19The nucleosideThe inclusion positions in sequence II correspond to Z18Nucleotide Z of20Z is the same as20Is the first nucleotide at the 5' end of the antisense strand.
In the above and below, "positional correspondence" means that they are at the same position in the nucleotide sequence from the same end of the nucleotide sequence. For example, the 1 st nucleotide at the 3' end of the nucleotide sequence I is a nucleotide sequence whose position corresponds to SEQ ID NO: 241 to the 3' -end of the nucleotide sequence 1.
In some embodiments, the sense strand comprises only nucleotide sequence I and the antisense strand comprises only nucleotide sequence II.
In some embodiments, the nucleotide sequence I is identical to SEQ ID NO: 241 and/or said nucleotide sequence II differs from the nucleotide sequence shown in SEQ ID NO: 242 by no more than 1 nucleotide.
In some embodiments, the nucleotide sequence II is identical to SEQ ID NO: 242 comprises Z20A difference at position, and Z20Selected from A, C or G. In some embodiments, the nucleotide difference is Z20Difference in position, Z20Selected from A, C or G. In some embodiments, Z19Is a reaction of with Z20A complementary nucleotide. The sirnas having the above nucleotide differences have higher target mRNA inhibitory ability of the sirnas, and these sirnas comprising the nucleotide differences are also within the scope of the present disclosure.
In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, or fully reverse complementary.
In some embodiments, the nucleotide sequence I is SEQ ID NO: 243, and nucleotide sequence II is SEQ ID NO: 244, and the nucleotide sequence shown in SEQ ID NO:
5′-GGAUGUGCAGUUGAUCCCZ19-3′(SEQ ID NO：243)；
5′-Z20GGGAUCAACUGCACAUCC-3′(SEQ ID NO：244)，
whereinZ is the same as20Is the first nucleotide at the 5' end of the antisense strand, Z19Selected from A, U, G or C, and Z20Is a reaction of with Z19A complementary nucleotide; in some embodiments, Z19Is A, Z20Is U.
In some embodiments, the sense strand further comprises nucleotide sequence III and the antisense strand further comprises nucleotide sequence IV, each of nucleotide sequence III and nucleotide sequence IV being 1-4 nucleotides in length; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or fully reverse complementary; the nucleotide sequence III is connected to the 5 'end of the nucleotide sequence I, the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II, the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to a second nucleotide sequence which is complementary to a nucleotide sequence in the target mRNA represented by SEQ ID NO: 241 and a nucleotide sequence having the same length as the nucleotide sequence IV and adjacent to the 5' -end of the nucleotide sequence.
In some embodiments, the length of each of nucleotide sequence III and nucleotide sequence IV is 1 nucleotide, the base of nucleotide sequence III is U, the base of nucleotide sequence IV is a; at this time, the length ratio of the sense strand to the antisense strand is 20/20; or, the length of the nucleotide sequences III and IV is 2 nucleotides, the base composition of the nucleotide sequence III is GU and the base composition of the nucleotide sequence IV is AC according to the direction from the 5 'end to the 3' end; in this case, the length ratio of the sense strand to the antisense strand is 21/21; or, the length of the nucleotide sequences III and IV is 3 nucleotides, the base composition of the nucleotide sequence III is AGU and the base composition of the nucleotide sequence IV is ACU according to the direction from the 5 'end to the 3' end; at this time, the length ratio of the sense strand to the antisense strand was 22/22; or, the length of the nucleotide sequences III and IV is 4 nucleotides, the base composition of the nucleotide sequence III is AAGU and the base composition of the nucleotide sequence IV is ACUU according to the direction from the 5 'end to the 3' end; in this case, the length ratio of the sense strand to the antisense strand is 23/23. In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are 2 nucleotides in length, and in the direction from the 5 'end to the 3' end, the base composition of the nucleotide sequence III is GU, and the base composition of the nucleotide sequence IV is AC; in this case, the length ratio of the sense strand to the antisense strand was 21/21.
In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are fully complementary in reverse orientation, such that, given the bases of the nucleotide sequence III, the bases of the nucleotide sequence IV are defined.
Sixth siRNA
According to the present disclosure, the siRNA may be a sixth siRNA.
The sixth siRNA comprises a sense strand and an antisense strand, each nucleotide in the sixth siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially complementary in reverse directions to form a double-stranded region, wherein the nucleotide sequence I is complementary to the nucleotide sequence of SEQ ID NO: 301 is of equal length and differs by NO more than 3 nucleotides, and said nucleotide sequence II is identical to SEQ ID NO: 302 are equal in length and differ by no more than 3 nucleotides:
5′-UAACUUGGGAUCUGGGAAZ21-3′(SEQ ID NO：301)；
5′-Z22UUCCCAGAUCCCAAGUUA-3′(SEQ ID NO：302)，
wherein Z is21Is U, Z22Is A, the nucleotide sequence I comprises a position corresponding to Z21Nucleotide Z of23The nucleotide sequence II comprises a position corresponding to Z22Nucleotide Z of24Z is the same as24Is the first nucleotide at the 5' end of the antisense strand.
In some embodiments, the sense strand comprises only nucleotide sequence I and the antisense strand comprises only nucleotide sequence II.
In some embodiments, the nucleotide sequence I is identical to SEQ ID NO: 301, and/or said nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO: 302 does not differ by more than 1 nucleotide.
In some embodiments, the nucleotide sequence II is identical to SEQ ID NO: the nucleotide difference between the nucleotide sequences set forth in 302 comprises Z24A difference at position, and Z24Selected from U, C or G. In some embodiments, the nucleotide difference is Z24Difference in position, Z24Selected from U, C or G. In some embodiments, Z23Is a reaction of with Z24A complementary nucleotide. The sirnas having the above nucleotide differences have higher target mRNA inhibitory ability of the sirnas, and these sirnas comprising the nucleotide differences are also within the scope of the present disclosure.
In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, or fully reverse complementary.
In some embodiments, the nucleotide sequence I is SEQ ID NO: 303, and nucleotide sequence II is SEQ ID NO: 304:
5′-UAACUUGGGAUCUGGGAAZ23-3′(SEQ ID NO：303)；
5′-Z24UUCCCAGAUCCCAAGUUA-3′(SEQ ID NO：304)，
wherein, Z is24Is the first nucleotide at the 5' end of the antisense strand, Z23Selected from A, U, G or C, and Z24Is a reaction of with Z23A complementary nucleotide; in some embodiments, Z23Is U, Z24Is A.
In some embodiments, the sense strand further comprises nucleotide sequence III and the antisense strand further comprises nucleotide sequence IV, each of nucleotide sequence III and nucleotide sequence IV being 1-4 nucleotides in length; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or fully reverse complementary; the nucleotide sequence III is connected to the 5 'end of the nucleotide sequence I, the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II, the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to a second nucleotide sequence which is complementary to a nucleotide sequence in the target mRNA represented by SEQ ID NO: a nucleotide sequence represented by 301, the 5' end of which is adjacent to the nucleotide sequence and the length of which is the same as the nucleotide sequence IV.
In some embodiments, the length of each of nucleotide sequence III and nucleotide sequence IV is 1 nucleotide, the base of nucleotide sequence III is C, and the base of nucleotide sequence IV is G; at this time, the length ratio of the sense strand to the antisense strand is 20/20; or, the length of the nucleotide sequences III and IV is 2 nucleotides, the base composition of the nucleotide sequence III is UC and the base composition of the nucleotide sequence IV is GA according to the direction from 5 'end to 3' end; in this case, the length ratio of the sense strand to the antisense strand is 21/21; or, the length of the nucleotide sequences III and IV is 3 nucleotides, the base composition of the nucleotide sequence III is GUC and the base composition of the nucleotide sequence IV is GAC according to the direction from the 5 'end to the 3' end; at this time, the length ratio of the sense strand to the antisense strand was 22/22; or, the length of the nucleotide sequences III and IV is 4 nucleotides, the base composition of the nucleotide sequence III is GGUC and the base composition of the nucleotide sequence IV is GACC according to the direction from the 5 'end to the 3' end; in this case, the length ratio of the sense strand to the antisense strand is 23/23. In some embodiments, the nucleotide sequence III and nucleotide sequence IV are 2 nucleotides in length, the base composition of nucleotide sequence III is UC and the base composition of nucleotide sequence IV is GA in the 5 'to 3' direction; in this case, the length ratio of the sense strand to the antisense strand was 21/21.
In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are fully complementary in reverse orientation, such that, given the bases of the nucleotide sequence III, the bases of the nucleotide sequence IV are defined.
Pendant end and modification of siRNA
Hereinafter, the description of the nucleotide sequence V, the nucleic acid sequence, the nucleotide modification in the siRNA, and the modified sequence is applicable to any one of the first to sixth sirnas described above. That is, if not specified, the following description of siRNA shall be regarded as that the first siRNA, the second siRNA, the third siRNA, the fourth siRNA, the fifth siRNA and the sixth siRNA are described one by one. For example, unless a specific siRNA is specifically indicated, "the siRNA further contains a nucleotide sequence V" means "the first siRNA, the second siRNA, the third siRNA, the fourth siRNA, the fifth siRNA or the sixth siRNA further contains a nucleotide sequence V".
In some embodiments, the sense strand and the antisense strand are different in length, and the antisense strand further comprises a nucleotide sequence V, 1 to 3 nucleotides in length, attached at the 3 'end of the antisense strand to form a 3' overhang of the antisense strand. Thus, the length ratio of the sense strand and the antisense strand of the siRNA provided by the present disclosure may be 19/20, 19/21, 19/22, 20/21, 20/22, 20/23, 21/22, 21/23, 21/24, 22/23, 22/24, 22/25, 23/24, 23/25, or 23/26. In some embodiments, the nucleotide sequence V is 2 nucleotides in length, and thus, the ratio of the lengths of the sense and antisense strands of the sirnas provided by the present disclosure can be 19/21, 21/23, or 23/25.
Each nucleotide in the nucleotide sequence V can be any nucleotide, and for the convenience of synthesis and the saving of synthesis cost, the nucleotide sequence V is continuous 2 thymidylate ribonucleotides (dTdT) or continuous 2 uracil ribonucleotides (UU); alternatively, to increase the affinity of the siRNA antisense strand to the target mRNA, the nucleotide sequence V is complementary to the nucleotide at the corresponding position of the target mRNA. Thus, in some embodiments, the siRNA of the present disclosure has a ratio of the length of the sense strand to the length of the antisense strand of 19/21 or 21/23, when the siRNA of the present disclosure has better mRNA silencing activity.
The nucleotide at the corresponding position of the target mRNA means a nucleotide or a nucleotide sequence adjacent to the 5' -end of the third nucleotide sequence of the target mRNA, which is substantially reverse-complementary or fully reverse-complementary to the nucleotide sequence II, or a nucleotide sequence consisting of the nucleotide sequence II and the nucleotide sequence IV.
In some embodiments, for the first siRNA, the sense strand of the siRNA comprises a sequence as set forth in SEQ ID NO: 5, and the antisense strand comprises the nucleotide sequence shown as SEQ ID NO: 6:
5′-CCGCCAAAGCCCAGAAGAZ3-3′(SEQ ID NO：5)；
5′-Z4UCUUCUGGGCUUUGGCGGUU-3′(SEQ ID NO：6)；
alternatively, the sense strand of the siRNA comprises the sequence set forth in SEQ ID NO: 7, and the antisense strand comprises the nucleotide sequence shown as SEQ ID NO: 8, and the nucleotide sequence is as follows:
5′-AACCGCCAAAGCCCAGAAGAZ3-3′(SEQ ID NO：7)；
5′-Z4UCUUCUGGGCUUUGGCGGUUUC-3′(SEQ ID NO：8)；
wherein, Z is4Is the first nucleotide at the 5' end of the antisense strand, Z3Selected from A, U, G or C, and Z4Is a reaction of with Z3A complementary nucleotide.
In some embodiments, for the second siRNA, the sense strand of the siRNA comprises a sequence as set forth in SEQ ID NO: 65, and the antisense strand comprises the nucleotide sequence shown as SEQ ID NO: 66, and the nucleotide sequence shown in (b):
5′-AGGCACUGCUGGUGGAGGZ7-3′(SEQ ID NO：65)；
5′-Z8CCUCCACCAGCAGUGCCUGC-3′(SEQ ID NO：66)，
alternatively, the sense strand of the siRNA comprises the sequence set forth in SEQ ID NO: 67, and the antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 68:
5′-GCAGGCACUGCUGGUGGAGGZ7-3′(SEQ ID NO：67)；
5′-Z8CCUCCACCAGCAGUGCCUGCAC-3′(SEQ ID NO：68)，
wherein, Z is8Is the first nucleotide at the 5' end of the antisense strand, Z7Selected from A, U, G or C, and Z8Is a reaction of with Z7A complementary nucleotide.
In some embodiments, for the third siRNA, the sense strand of the siRNA comprises a sequence as set forth in seq id NO: 125, and the antisense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO: 126, and (b) the nucleotide sequence shown in (a):
5′-GGAGGCAGAAGUAUGAUUZ11-3′(SEQ ID NO：125)；
5′-Z12AAUCAUACUUCUGCCUCCUC-3′(SEQ ID NO：126)，
alternatively, the sense strand of the siRNA comprises the sequence set forth in SEQ ID NO: 127, wherein the antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 128:
5′-GAGGAGGCAGAAGUAUGAUUZ11-3′(SEQ ID NO：127)；
5′-Z12AAUCAUACUUCUGCCUCCUCAG-3′(SEQ ID NO：128)，
wherein, Z is12Is the first nucleotide at the 5' end of the antisense strand, Z11Selected from A, U, G or C, and Z12Is a reaction of with Z11A complementary nucleotide.
In some embodiments, for the fourth siRNA, the sense strand of the siRNA comprises a sequence as set forth in SEQ ID NO: 185, and the antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 186 by the nucleotide sequence shown in seq id no:
5′-GAGUUCCUCUGUUCUGUGZ15-3′(SEQ ID NO：185)；
5′-Z16CACAGAACAGAGGAACUCUC-3′(SEQ ID NO：186)，
alternatively, the sense strand of the siRNA comprises the sequence set forth in SEQ ID NO: 187, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 188 by sequence number:
5′-GAGAGUUCCUCUGUUCUGUGZ15-3′(SEQ ID NO：187)；
5′-Z16CACAGAACAGAGGAACUCUCCA-3′(SEQ ID NO：188)，
wherein, Z is16Is the first nucleotide at the 5' end of the antisense strand, Z15Selected from A, U, G or C, and Z16Is a reaction of with Z15A complementary nucleotide.
In some embodiments, for the fifth siRNA, the sense strand of the siRNA comprises a sequence as set forth in SEQ ID NO: 245, and the antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 246 by the sequence given in seq id no:
5′-GGAUGUGCAGUUGAUCCCZ19-3′(SEQ ID NO：245)；
5′-Z20GGGAUCAACUGCACAUCCAC-3′(SEQ ID NO：246)，
alternatively, the sense strand of the siRNA comprises the sequence set forth in SEQ ID NO: 247, and the antisense strand of said siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 248, and (b) the nucleotide sequence shown in (b):
5′-GUGGAUGUGCAGUUGAUCCCZ19-3′(SEQ ID NO：247)；
5′-Z20GGGAUCAACUGCACAUCCACUU-3′(SEQ ID NO：248)，
wherein, Z is20Is the first nucleotide at the 5' end of the antisense strand, Z19Selected from A, U, G or C, and Z20Is a reaction of with Z19A complementary nucleotide.
In some embodiments, for the sixth siRNA, the sense strand of the siRNA comprises the sequence set forth as SEQ ID NO: 305 and the antisense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO: 306 of the sequence set forth in seq id no:
5′-UAACUUGGGAUCUGGGAAZ23-3′(SEQ ID NO：305)；
5′-Z24UUCCCAGAUCCCAAGUUAGA-3′(SEQ ID NO：306)，
alternatively, the sense strand of the siRNA comprises the sequence set forth in SEQ ID NO: 307, and the antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 308, and (b) the nucleotide sequence shown in (b):
5′-UCUAACUUGGGAUCUGGGAAZ23-3′(SEQ ID NO：307)；
5′-Z24UUCCCAGAUCCCAAGUUAGACC-3′(SEQ ID NO：308)，
wherein, Z is24Is the first nucleotide at the 5' end of the antisense strand, Z23Selected from A, U, G or C, and Z24Is a reaction of with Z23A complementary nucleotide.
In some embodiments, the siRNA of the present disclosure is siTMPa1, siTMPa2, siTMPb1, siTMPb2, siTMPc1, siTMPc2, siTMPd1, siTMPd2, siTMPe1, siTMPe2, siTMPf1, or siTMPf2 listed in tables 1a-1 f.
As previously described, the nucleotides in the sirnas of the present disclosure are each independently modified or unmodified nucleotides. In some embodiments, the nucleotides in the sirnas of the present disclosure are unmodified nucleotides; in some embodiments, some or all of the nucleotides in the sirnas of the present disclosure are modified nucleotides, and such modifications on the nucleotide groups do not result in a significant impairment or loss of the function of the sirnas of the present disclosure to inhibit expression of the TMPRSS6 gene.
In some embodiments, the sirnas of the present disclosure contain at least 1 modified nucleotide. In the context of the present disclosure, the term "modified nucleotide" is used to refer to a nucleotide or nucleotide analog in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with another group, or a nucleotide having a modified base. The modified nucleotides do not result in significant impairment or loss of the function of the siRNA to inhibit gene expression. For example, j.k.watts, g.f.deleavey, and m.j.damha, chemical ly modified siRNA: drug Discov Today, 2008, 13 (19-20): 842-55.
In some embodiments, at least one nucleotide in the sense strand or the antisense strand of an siRNA provided by the present disclosure is a modified nucleotide, and/or at least one phosphate group is a phosphate group having a modifying group; in other words, at least a portion of the phosphate groups and/or ribosyl groups in the phosphate-sugar backbone of at least one single strand of the sense strand and the antisense strand are phosphate groups having a modifying group and/or ribosyl groups having a modifying group.
In some embodiments, all of the nucleotides in the sense strand and/or the antisense strand are modified nucleotides. In some embodiments, each nucleotide in the sense strand and the antisense strand of the sirnas provided by the present disclosure is independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide.
The inventors of the present disclosure surprisingly found that the sirnas described in the present disclosure achieved a high balance of stability in plasma and gene silencing efficiency in animal experiments.
In some embodiments, the fluoro-modified nucleotide is located in nucleotide sequence I and nucleotide sequence II, and at least the 7 th, 8 th, 9 th nucleotides of the nucleotide sequence I are fluoro-modified nucleotides in the direction from the 5 'end to the 3' end; at least the 2 nd, 6 th, 14 th and 16 th nucleotides of the nucleotide sequence II are fluoro-modified nucleotides according to the direction from the 5 'end to the 3' end.
In some embodiments, the fluoro-modified nucleotide is located in nucleotide sequence I and nucleotide sequence II, the fluoro-modified nucleotide is no more than 5 in nucleotide sequence I, and at least the 7 th, 8 th, 9 th nucleotides of nucleotide sequence I are fluoro-modified nucleotides in the direction from the 5 'end to the 3' end; the number of the fluorinated modified nucleotides in the nucleotide sequence II is not more than 7, and at least the 2 nd, 6 th, 14 th and 16 th nucleotides in the nucleotide sequence II are fluorinated modified nucleotides.
In some embodiments, in the direction from the 5 'end to the 3' end, in the sense strand, the 7 th, 8 th, 9 th or 5 th, 7 th, 8 th, 9 th nucleotide of the nucleotide sequence I is a fluorinated modified nucleotide, and the remaining nucleotides in the sense strand are non-fluorinated modified nucleotides; according to the direction from the 5 'end to the 3' end, in the antisense strand, the nucleotides at the 2 nd, 6 th, 14 th and 16 th positions or the nucleotides at the 2 nd, 6 th, 8 th, 9 th, 14 th and 16 th positions of the nucleotide sequence II are fluorine-modified nucleotides, and the nucleotides at the rest positions in the antisense strand are non-fluorine-modified nucleotides.
In the context of the present disclosure, "fluoro-modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with fluorine, which has a structure represented by the following formula (7). "non-fluorinated modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with a non-fluorinated group, or a nucleotide analog. In some embodiments, each non-fluorinated modified nucleotide is independently selected from one of a nucleotide or a nucleotide analog in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with a non-fluorinated group.
The nucleotide in which the hydroxyl group at the 2 '-position of the ribosyl group is substituted with a non-fluorine group is known to those skilled in the art, and the nucleotide may be one selected from the group consisting of a 2' -alkoxy-modified nucleotide, a2 '-substituted alkoxy-modified nucleotide, a 2' -alkyl-modified nucleotide, a2 '-substituted alkyl-modified nucleotide, a 2' -amino-modified nucleotide, a2 '-substituted amino-modified nucleotide, and a 2' -deoxynucleotide.
In some embodiments, the 2 ' -alkoxy modified nucleotide is a2 ' -methoxy (2 ' -OMe) modified nucleotide, as shown in formula (8). In some embodiments, the 2 ' -substituted alkoxy modified nucleotide, for example, can be a2 ' -O-methoxyethyl (2 ' -MOE) modified nucleotide, as shown in formula (9). In some embodiments, 2 '-amino (2' -NH)2) The modified nucleotide is shown as formula (10). In some embodiments, the 2' -Deoxynucleotide (DNA) is according to formula (11):
Figure BDA0002498737070000251
a nucleotide analog refers to a group that can replace a nucleotide in a nucleic acid, but that differs in structure from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. In some embodiments, the nucleotide analog can be a heteronucleotide, a bridged nucleotide, or an acyclic nucleotide.
Bridged Nucleic Acid (BNA) refers to a constrained or inaccessible nucleotide. BNAs may contain five-membered, six-membered, or seven-membered ring bridged structures with "fixed" C3' -endo-sugar pull-down. The bridge is typically incorporated at the 2 '-, 4' -position of the ribose to provide a2 ', 4' -BNA nucleotide. In some embodiments, BNA may be LNA, ENA, cET BNA, etc., where LNA is as shown in equation (12), ENA is as shown in equation (13), and cET BNA is as shown in equation (14):
Figure BDA0002498737070000261
acyclic nucleotides are a class of nucleotides in which the sugar ring of the nucleotide is opened. In some embodiments, the acyclic nucleotide can be an Unlocked Nucleic Acid (UNA) or a Glycerol Nucleic Acid (GNA), wherein UNA is represented by formula (15) and GNA is represented by formula (16):
Figure BDA0002498737070000262
in the above formulae (15) and (16), R is selected from H, OH or an alkoxy group (O-alkyl group).
An isonucleotide is a compound formed by changing the position of a base in a nucleotide on a ribose ring. In some embodiments, the isonucleotides can be compounds in which the base moves from the 1 ' -position to the 2 ' -position or the 3 ' -position of the ribose ring, as shown in formula (17) or (18).
Figure BDA0002498737070000263
In the compounds represented by the above-mentioned formulae (17) to (18), Base represents a nucleic acid Base such as A, U, G, C or T; r is selected from H, OH, F or a non-fluorine group as described above.
In some embodiments, the nucleotide analog is selected from one of a heteronucleotide, LNA, ENA, cET, UNA, and GNA. In some embodiments, each of the non-fluorinated modified nucleotides is a methoxy modified nucleotide, which refers to a nucleotide in which the 2' -hydroxyl group of the ribosyl group is substituted with a methoxy group, both supra and infra.
In the above and hereinafter, "fluoro-modified nucleotide", "2 '-fluoro-modified nucleotide", "nucleotide in which 2' -hydroxyl group of ribose group is substituted with fluorine" and "nucleotide having 2 '-fluoro-ribosyl group" have the same meaning, and all refer to a compound having a structure represented by formula (7) in which 2' -hydroxyl group of nucleotide is substituted with fluorine; the terms "methoxy-modified nucleotide", "2 '-methoxy-modified nucleotide", "nucleotide in which 2' -hydroxyl group of ribose group is substituted with methoxy group" and "nucleotide having 2 '-methoxy ribosyl group" are the same, and refer to a compound having a structure represented by formula (8) in which 2' -hydroxyl group of ribose group of nucleotide is substituted with methoxy group.
In some embodiments, the siRNA of the present disclosure is an siRNA with the following modifications: in the direction from the 5 'end to the 3' end, in the sense strand, the nucleotides at the 7 th, 8 th and 9 th positions or the 5 th, 7 th, 8 th and 9 th positions of the nucleotide sequence I are fluorine-modified nucleotides, and the nucleotides at the rest positions in the sense strand are methoxy-modified nucleotides; in the antisense strand, the 2 nd, 6 th, 14 th, 16 th or 2 nd, 6 th, 8 th, 9 th, 14 th, 16 th nucleotide of the nucleotide sequence II is a fluoro-modified nucleotide, and the rest nucleotides in the antisense strand are methoxy-modified nucleotides.
In some embodiments, the siRNA of the present disclosure is an siRNA with the following modifications: the nucleotides at the 5 th, 7 th, 8 th and 9 th positions of the nucleotide sequence I in the sense strand of the siRNA are fluorine-modified nucleotides, the nucleotides at the rest positions of the sense strand of the siRNA are methoxy-modified nucleotides, and the nucleotides at the 2 nd, 6 th, 8 th, 9 th, 14 th and 16 th positions of the nucleotide sequence II in the antisense strand of the siRNA are fluorine-modified nucleotides, and the nucleotides at the rest positions of the antisense strand of the siRNA are methoxy-modified nucleotides, in the direction from the 5 'end to the 3' end;
or, according to the direction from 5 'end to 3' end, the nucleotides at the 5 th, 7 th, 8 th and 9 th positions of the nucleotide sequence I in the sense strand of the siRNA are fluorine modified nucleotides, the nucleotides at the rest positions of the sense strand of the siRNA are methoxy modified nucleotides, and, according to the direction from 5 'end to 3' end, the nucleotides at the 2 nd, 6 th, 14 th and 16 th positions of the nucleotide sequence II in the antisense strand of the siRNA are fluorine modified nucleotides, and the nucleotides at the rest positions of the antisense strand of the siRNA are methoxy modified nucleotides;
or, according to the direction from 5 'end to 3' end, the nucleotides at the 7 th, 8 th and 9 th positions of the nucleotide sequence I in the sense strand of the siRNA are fluorine modified nucleotides, the nucleotides at the rest positions of the sense strand of the siRNA are methoxy modified nucleotides, and according to the direction from 5 'end to 3' end, the nucleotides at the 2 nd, 6 th, 14 th and 16 th positions of the nucleotide sequence II in the antisense strand of the siRNA are fluorine modified nucleotides, and the nucleotides at the rest positions of the antisense strand of the siRNA are methoxy modified nucleotides.
In some embodiments, the siRNA provided by the present disclosure is any one of siTMPa1-M1, siTMPa1-M2, siTMPa1-M3, siTMPa2-M1, siTMPa2-M2, siTMPa2-M3, siTMPb1-M1, siTMPb1-M2, siTMPb1-M3, siTMPb2-M1, siTMPb2-M2, siTMPb2-M3, siTMPcl-M1, siTMPcl-M2, siTMPc1-M3, siTMPcl 2-M1, siTMPc2-M2, siTMPc2-M2, siTMPf2-M2, PesM 2-Pf 2, PemP 36si 2-Pf 2, PemSi 36si 2-Pf 36si 2, PemP 36si 2-36si 2, PemSi 36si 2-36si 2, PemSi 36si 2, PemP 36si 2, PemSi 36si 2, Pem36si 36si 2, PemSi 2, PemP 36si 2-Pf 2, Pem36si 2-Pem36si 2, PemSi 36si 2, PemSi 36si 2, Pem.
The modified siRNA is low in cost, and can ensure that ribonuclease in blood does not easily cut nucleic acid, so that the stability of the nucleic acid is improved, and the nucleic acid has stronger resistance to nuclease hydrolysis.
In some embodiments, the present disclosure provides sirnas wherein at least a portion of the phosphate groups in the phosphate-sugar backbone of at least one single strand of the sense and antisense strands are phosphate groups having a modifying group. In some embodiments, the phosphate group having a modifying group is a phosphorothioate group formed by substituting at least one oxygen atom in a phosphodiester bond in the phosphate group with a sulfur atom; in some embodiments, the phosphate group having a modifying group is a phosphorothioate group having a structure as shown in formula (1):
Figure BDA0002498737070000281
the modification can stabilize the double-stranded structure of siRNA and maintain the high specificity and high affinity of base pairing.
In some embodiments, the present disclosure provides sirnas wherein the phosphorothioate-based linkage is present at least one of the group consisting of: between the first and second nucleotides at either end of the sense or antisense strand; between the second and third nucleotides at either end of the sense or antisense strand; or any combination of the above. In some embodiments, phosphorothioate-based linkages are present at all of the above positions except at the 5' end of the sense strand. In some embodiments, phosphorothioate-based linkages are present at all of the above positions except at the 3' end of the sense strand. In some embodiments, the phosphorothioate-based linkage is present in at least one of the following positions:
between the 1 st and 2 nd nucleotides of the 5' terminus of the sense strand;
between the 2 nd and 3 rd nucleotides at the 5' end of the sense strand;
between the 1 st and 2 nd nucleotides of the 3' terminus of the sense strand;
between the 2 nd and 3 rd nucleotides at the 3' terminus of the sense strand;
between the 1 st and 2 nd nucleotides of the 5' terminus of the antisense strand;
between the 2 nd and 3 rd nucleotides of the 5' terminus of the antisense strand;
between the 1 st and 2 nd nucleotides of the 3' terminus of the antisense strand; and
between the 2 nd and 3 rd nucleotides of the 3' terminus of the antisense strand.
In some embodiments, the siRNA provided by the present disclosure is any one of siTMPa-M1, siTMPa-M2, siTMPa-M3, siTMPb-M1, siTMPb-M2, siTMPb-M3, siTMPb-M1, siTMPb-M2, siTMPb-M3, siTMPc-M1, siTMPc-M2, siTMPc-M3, siTMPd-M1, siTMPd-M2, siTMPd-M3, siTMPe-M1, siTMPe-M2, siTMPe-M3, Pf-M1, Pf-M2, Pf TMPf-M3, Pf-M2, Pf-M3, and Pf-M3, or Pf-Pesi-M3 listed in tables 1a-1 f.
In some embodiments, the 5 ' terminal nucleotide of the siRNA antisense strand is a 5 ' -phosphate nucleotide or a 5 ' -phosphate analog modified nucleotide.
Commonly used nucleotides modified with said 5 ' -phosphate nucleotides or 5 ' -phosphate analogues are well known to the person skilled in the art, e.g. nucleotides 5 ' -phosphate may have the following structure:
Figure BDA0002498737070000291
for another example, Anastasia Khvorova and Jonathan K.Watts, The chemical evolution of oligonucleotide therapeutics of clinical utility, Nature Biotechnology, 2017, 35 (3): 238-48 discloses the following 4 5' -phosphate analogue modified nucleotides:
Figure BDA0002498737070000292
wherein R is selected from H, OH, methoxy and fluorine; base represents a nucleobase selected from A, U, C, G or T.
In some embodiments, the nucleotide 5 '-phosphate is a nucleotide containing a 5' -phosphate modification represented by formula (2), and the nucleotide 5 '-phosphate analog modification is a nucleotide containing a vinyl phosphate (5' - (E) -vinylphosphonate, E-VP) modification, represented by formula (3), or a phosphorothioate modification, represented by formula (5).
In some embodiments, the siRNAs provided by the present disclosure are siTMPa1-M1P1, siTMPa1-M2P1, siTMPa1-M3P1, siTMPa2-M1P1, siTMPa2-M2P1, siTMPa2-M3P1, siTMPa1-M1SP1, siTMPa1-M2SP1, siTMPa1-M3SP1, siTMPa2-M1SP1, siTMPa2-M2SP1, siTMPa2-M3SP1, siTMPb1-M1P1, TMSiP 1-P1, TMSiP 36siP 363-M1, sP 36sP 1-M363P 1, siP 36P 1, siP 36P 1-sP 1, sP 36sP 1-P36sP 1, sP 36sP 1-M36sP 1, sP1, TMSP 36sP 1-M36sP 1, sP 36sP 1, sP 36sP 1, TMP 36sP 1, sP 36sP 1-M1, TMP 36sP 1, TMP 36sP 1-M1, sP 36sP 1, TMP 36sP 1, sP 36sP 1, TMP 36sP 1, s, SiTMPd-M3P, siTMPd-M1P, siTMPd-M2P, siTMPd-M3P, siTMPd-M1 SP, siTMPd-M2 SP, siTMPd-M3 SP, siTMPe-M1P, siTMPe-M2P, siTMPe-M3P, siTMPe-M1 SP, siTMPe-M2 SP, siTMPe-M3 SP, siTMPe-M1 SP, siTMPe-M2 SP, siPe-M3 SP, siTMPf-M1P, siTMP-M2P, siTMP-M3 SP, siTMPf-M3P, siTMPf-M1P, siTMPf-M2 SP, siTMP-M3 SP, siTMPf-M3 SP, siPf-M2 SP, siM 3SP, siP, siTMP, siPf-M3 SP, siM 3SP, siP, siTMP-Pf-M2 SP, and siM 2 SP.
The inventors of the present disclosure surprisingly found that the sirnas provided by the present disclosure not only have significantly enhanced plasma and lysosomal stability, but also show higher TMPRSS6 mRNA inhibitory activity.
The siRNA provided by the present disclosure can be obtained by methods conventional in the art for siRNA preparation, such as methods of solid phase synthesis and solution phase synthesis. Among them, solid phase synthesis has been commercially available as a custom service. Modified nucleotide groups can be introduced into the sirnas described in the present disclosure by using nucleotide monomers with corresponding modifications, and methods of preparing nucleotide monomers with corresponding modifications and methods of introducing modified nucleotide groups into sirnas are also well known to those skilled in the art.
Pharmaceutical composition
The present disclosure provides a pharmaceutical composition comprising the siRNA as described above as an active ingredient and a pharmaceutically acceptable carrier.
The pharmaceutically acceptable carrier may be a carrier conventionally used in the art of siRNA administration, such as, but not limited to, magnetic nanoparticlesParticles (magnetic nanoparticles, e.g. based on Fe)3O4Or Fe2O3Nanoparticles of (a), carbon nanotubes (carbon nanotubes), mesoporous silicon (mesopore silicon), calcium phosphate nanoparticles (calcium phosphate nanoparticles), Polyethyleneimine (PEI), Polyamidoamine (PAMAM) dendrimer), polylysine (L-lysine), PLL), chitosan (chitosan), 1, 2-dioleoyl-3-trimethyolpropane (1, 2-dioleoyl-3-trimethyoronium-propane, DOTAP), poly-D or L-type lactic acid/glycolic acid copolymer (D) glycolic acid copolymer (bmam), etc&L-lactic/glycolic acid) copolymer, PLGA, poly (2-aminoethylethylene phosphate), PPEEA, and poly (N, N-dimethylaminoethyl methacrylate), PDMAEMA, and derivatives thereof.
In some embodiments, the amount of siRNA and pharmaceutically acceptable carrier in the pharmaceutical composition is not particularly required, and in some embodiments, the weight ratio of siRNA to pharmaceutically acceptable carrier may be 1: 1 (1-500), and in some embodiments, the above weight ratio is 1: 1 (1-50).
In some embodiments, the pharmaceutical composition may further comprise other pharmaceutically acceptable excipients, which may be one or more of various formulations or compounds conventionally employed in the art. For example, the pharmaceutically acceptable additional excipients may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.
The pH buffer may be a tris hydrochloride buffer at a pH of 7.5 to 8.5 and/or a phosphate buffer at a pH of 5.5 to 8.5, for example a phosphate buffer at a pH of 5.5 to 8.5.
The protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. The content of the protective agent may be 0.01 to 30% by weight, based on the total weight of the pharmaceutical composition.
The osmotic pressure regulator may be sodium chloride and/or potassium chloride. The osmolality adjusting agent is present in an amount such that the osmolality of the pharmaceutical composition is 200-700 milliosmoles per kilogram (mOsm/kg). The content of the osmolality adjusting agent can be easily determined by the skilled person, depending on the desired osmolality.
In some embodiments, the pharmaceutical composition may be a liquid formulation, such as an injection solution; or can be lyophilized powder for injection, and can be mixed with liquid adjuvant to make into liquid preparation. The liquid preparation can be used for subcutaneous, intramuscular or intravenous injection, and can also be used for spraying administration to the lung or spraying administration to other organ tissues (such as liver). In some embodiments, the pharmaceutical composition is for intravenous administration.
In some embodiments, the pharmaceutical composition may be in the form of a liposomal formulation. In some embodiments, the pharmaceutically acceptable carrier used in the liposome formulation comprises an amine-containing transfection compound (which may also be referred to hereinafter as an organic amine), a helper lipid, and/or a pegylated lipid. Wherein the organic amine, helper lipid, and pegylated lipid can be individually selected from one or more of the amine-containing transfection compound described in CNl03380113A (incorporated herein by reference in its entirety), or a pharmaceutically acceptable salt or derivative thereof, a helper lipid, and a pegylated lipid.
In some embodiments, the organic amine may be a compound described in CN103380113A as shown in formula (201) or a pharmaceutically acceptable salt thereof:
Figure BDA0002498737070000311
wherein:
X101or X102Each independently O, S, N-A or C-A, wherein A is hydrogen or a C1-C20 hydrocarbon chain;
Y101or Z101Each independently is C O, C S, S O, CH OH or SO2；
R101、R102、R103、R104、R105、R106Or R107Each independently is hydrogen, a cyclic or acyclic, substituted or unsubstituted, branched or linear aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or linear heteroaliphatic group, a substituted or unsubstituted, branched or linear acyl group, a substituted or unsubstituted, branched or linear aryl group, a substituted or unsubstituted, branched or linear heteroaryl group;
x is an integer from 1 to 10;
n is an integer of 1 to 3, m is an integer of 0 to 20, p is 0 or 1; wherein if m ═ p ═ 0, then R102Is hydrogen;
and, if at least one of n or m is 2, then R103And the nitrogen in formula (201) forms a structure as shown in formula (202) or formula (203):
Figure BDA0002498737070000321
wherein g, e or f are each independently an integer of 1 to 6, "HCC" represents a hydrocarbon chain, and each N represents a nitrogen atom in formula (201).
In some embodiments, R103Is a polyamine. In other embodiments, R103Is a ketal. In some embodiments, R in formula (201)101And R102Each of which is independently any substituted or unsubstituted, branched or straight chain alkyl or alkenyl group having from 3 to about 20 carbon atoms, such as from 8 to about 18 carbon atoms, and from 0 to 4 double bonds, such as from 0 to 2 double bonds.
In some embodiments, if each of n and m independently has a value of 1 or 3, then R103May be any of the following formulae (204) to (213):
Figure BDA0002498737070000322
Figure BDA0002498737070000331
wherein, in formula (204) -formula (213), g, e and f are each independently an integer of 1 to 6, each "HCC" represents a hydrocarbon chain, and each indicates R103A possible point of attachment to the nitrogen atom in formula (201), wherein each H at any x position may be replaced to achieve attachment to the nitrogen atom in formula (201).
Among them, the compound represented by formula (201) can be prepared according to the description in CN 103380113A.
In some embodiments, the organic amine is an organic amine according to formula (214) and/or an organic amine according to formula (215):
Figure BDA0002498737070000332
the helper lipid is cholesterol, cholesterol analogue and/or cholesterol derivative;
the pegylated lipid is 1, 2-dipalmitoamide-sn-glycerol-3-phosphatidylethanolamine-N- [ methoxy (polyethylene glycol) ] -2000.
In some embodiments, the molar ratio of the organic amine, the helper lipid, and the pegylated lipid in the pharmaceutical composition is (19.7-80) to (0.3-50), and may be, for example, (50-70) to (20-40) to (3-20).
In some embodiments, the pharmaceutical composition particles formed from the sirnas of the present disclosure and the above-described amine-containing transfection reagents have an average diameter of about 30nm to about 200nm, typically about 40nm to about 135nm, more typically the liposome particles have an average diameter of about 50nm to about 120nm, about 50nm to about 100nm, about 60nm to about 90nm, or about 70nm to about 90nm, e.g., the liposome particles have an average diameter of about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, or 160 nm.
In some embodiments, the weight ratio (weight/weight ratio) of siRNA to total lipid (e.g., organic amine, helper lipid, and/or pegylated lipid) in a pharmaceutical composition formed from an siRNA of the present disclosure and an amine-containing transfection reagent described above is in the range of from about 1: 1 to about 1: 50, from about 1: 1 to about 1: 30, from about 1: 3 to about 1: 20, from about 1: 4 to about 1: 18, from about 1: 5 to about 1: 17, from about 1: 5 to about 1: 15, from about 1: 5 to about 1: 12, from about 1: 6 to about 1: 12, or from about 1: 6 to about 1: 10, for example, the weight ratio of siRNA of the present disclosure to total lipid is about 1: 5, 1: 6, 1: 7, 1: 8, 1: 9, 1: 10, 1: 11, 1: 12, 1: 13, 1: 14, 1: 15, 1: 16, 1: 17, or 1: 18.
In some embodiments, the pharmaceutical compositions may be sold with the components present separately and may be in the form of a liquid formulation for use. In some embodiments, the pharmaceutical composition of the siRNA provided by the present disclosure and the above pharmaceutically acceptable carrier can be prepared according to various known methods, except that the siRNA provided by the present disclosure is used to replace the existing siRNA; in some embodiments, the following methods may be used:
suspending organic amine, auxiliary lipid and pegylated lipid in alcohol according to the molar ratio and uniformly mixing to obtain a lipid solution; the amount of alcohol used is such that the total mass concentration of the resulting lipid solution is 2-25mg/mL, for example, 8-18 mg/mL. The alcohol is selected from pharmaceutically acceptable alcohols such as alcohols that are liquid at about room temperature, for example, one or more of ethanol, propylene glycol, benzyl alcohol, glycerol, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, which may be, for example, ethanol.
The siRNA provided by the present disclosure is dissolved in a buffered salt solution to obtain an siRNA aqueous solution. The concentration of the buffered salt solution is 0.05-0.5M, such as 0.1-0.2M, the pH of the buffered salt solution is adjusted to 4.0-5.5, such as 5.0-5.2, and the amount of buffered salt solution is such that the concentration of siRNA does not exceed 0.6mg/mL, such as 0.2-0.4 mg/mL. The buffer salt is selected from one or more of soluble acetate and soluble citrate, and can be sodium acetate and/or potassium acetate.
The lipid solution and the aqueous siRNA solution are mixed, and the resulting mixture is incubated at 40-60 ℃ for at least 2 minutes, which may be, for example, 5-30 minutes, to obtain a post-incubation liposome preparation. The volume ratio of the lipid solution to the siRNA aqueous solution is 1: 2-5.
Concentrating or diluting the incubated liposome preparation, removing impurities, and sterilizing to obtain the pharmaceutical composition provided by the disclosure, wherein the physicochemical parameters are that the pH value is 6.5-8, the entrapment rate is not less than 80%, the particle size is 40-200nm, the polydispersity index is not higher than 0.30, and the osmotic pressure is 250-400 mOsm/kg; for example, the physical and chemical parameters may be pH 7.2-7.6, encapsulation efficiency not less than 90%, particle size 60-100nm, polydispersity index not more than 0.20, and osmotic pressure 300-400 mOsm/kg.
Wherein the concentration or dilution may be performed before, after or simultaneously with the removal of the impurities. The impurities can be removed by various methods, such as ultrafiltration using a cut-phase flow system and a hollow fiber column under 100K Da conditions, and the ultrafiltration exchange solution is Phosphate Buffered Saline (PBS) with pH 7.4. The sterilization can be carried out by various methods, for example, by filtration sterilization on a 0.22 μm filter.
siRNA conjugates
The present disclosure provides an siRNA conjugate comprising the above siRNA and a conjugate group conjugated to the siRNA.
Generally, the conjugate group comprises at least one targeting group that is pharmaceutically acceptable and optionally a linker (linker), and the siRNA, the linker and the targeting group are linked in sequence. In some embodiments, the targeting group is 1-6. In some embodiments, the targeting group is 2-4. The siRNA molecule may be non-covalently or covalently conjugated to the conjugate group, e.g. may be covalently conjugated to the conjugate group. The conjugation site of the siRNA to the conjugate group may be at the 3 ' end or 5 ' end of the sense strand of the siRNA, or at the 5 ' end of the antisense strand, or within the internal sequence of the siRNA. In some embodiments, the site of conjugation of the siRNA to the conjugate group is at the 3' end of the sense strand of the siRNA.
In some embodiments, the conjugate group may be attached to the phosphate group, the hydroxyl group at the 2' -position, or the base of the nucleotide. In some embodiments, the conjugate group may also be attached to the hydroxyl group at the 3 ' -position, in which case 2 ' -5 ' phosphodiester linkages are used between nucleotides. When a conjugate group is attached to the end of the siRNA strand, the conjugate group is typically attached to the phosphate group of the nucleotide; when a conjugate group is attached to the internal sequence of the siRNA, the conjugate group is typically attached to a ribose sugar ring or base. Various ways of attachment can be found in the literature: sirna conjugates combining sequential hybridization approved tertiary N-acetyl amino linked tertiary amino acids ligating in vivo in contexts acs Chemical biology 2015, 10 (5): 1181-7.
In some embodiments, the siRNA may be attached to the conjugate group via acid labile, or reducible, chemical bonds that may degrade under the acidic environment of the cellular endosome, thereby leaving the siRNA in a free state. For non-degradable conjugation, a conjugation group can be attached to the sense strand of the siRNA, thereby minimizing the effect of conjugation on siRNA activity.
In some embodiments, the pharmaceutically acceptable targeting group can be a ligand conventionally used in the art of siRNA administration, such as the various ligands described in WO2009082607a2, the entire disclosure of which is incorporated herein by reference.
In some embodiments, the pharmaceutically acceptable targeting group may be selected from one or more of the following ligands formed by targeting molecules or derivatives thereof: lipophilic molecules such as cholesterol, bile acids, vitamins (e.g. vitamin E), lipid molecules of varying chain length; polymers, such as polyethylene glycol; polypeptides, such as membrane-penetrating peptides; an aptamer; an antibody; quantum dots; sugars such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folic acid (folate); ligands for receptors expressed by parenchymal hepatocytes, such as asialoglycoprotein, asialoglycoresidues, lipoproteins (e.g., high density lipoproteins, low density lipoproteins, etc.), glucagon, neurotransmitters (e.g., epinephrine), growth factors, transferrin, and the like.
In some embodiments, each ligand is independently selected from a ligand capable of binding to a cell surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a hepatocyte surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a mammalian cell surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a human hepatocyte surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to the liver surface asialoglycoprotein receptor (ASGPR). These ligand classes are known to those skilled in the art and generally function to bind to specific receptors on the surface of target cells and mediate the delivery of siRNA linked to the ligand to the target cell.
In some embodiments, the pharmaceutically acceptable targeting group can be any ligand that binds to asialoglycoprotein receptor (ASGPR) on the surface of a mammalian liver cell. In some embodiments, each ligand is independently a asialoglycoprotein, such as Asialoglycoprotein (ASOR) or Asialofetuin (ASF). In some embodiments, the ligand is a sugar or a derivative of a sugar.
In some embodiments, at least one ligand is a sugar. In some embodiments, each ligand is a sugar. In some embodiments, at least one ligand is a monosaccharide, a polysaccharide, a modified monosaccharide, a modified polysaccharide, or a sugar derivative. In some embodiments, at least one of the ligands may be a monosaccharide, disaccharide or trisaccharide. In some embodiments, at least one ligand is a modified sugar. In some embodiments, each ligand is a modified sugar. In some embodiments, each ligand is independently selected from a polysaccharide, a modified polysaccharide, a monosaccharide, a modified monosaccharide, a polysaccharide derivative, or a monosaccharide derivative. In some embodiments, each or at least one ligand is selected from the group consisting of: glucose and its derivatives, mannan and its derivatives, galactose and its derivatives, xylose and its derivatives, ribose and its derivatives, fucose and its derivatives, lactose and its derivatives, maltose and its derivatives, arabinose and its derivatives, fructose and its derivatives and sialic acid.
In some embodiments, each of the ligands can be independently selected from the group consisting of D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, alpha-D-mannofuranose, beta-D-mannofuranose, alpha-D-mannopyranose, beta-D-mannopyranose, alpha-D-glucopyranose, beta-D-glucopyranose, alpha-D-glucopyranose, beta-D-glucopyranose, alpha-D-fructopyranose, alpha-D-galactopyranose, beta-D-galacto, alpha-D-galactofuranose, beta-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-N-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O- [ (R) -1-carboxyethyl ] -2-deoxy-beta-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4, 6-dideoxy-4-carboxamido-2, 3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfonamido-D-glucopyranose, beta-galactofuranose, glucosamine, N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-N-butyrylgalactosamine, N-isobutyrylgalactosamine, N-glycolyl-alpha-neuraminic acid, 5-thio-beta-D-glucopyranose, 2, 3, 4-tri-O-acetyl-1-thio-6-O-trityl-alpha-D-glucopyranoside methyl ester, 4-thio-beta-D-galactopyranose, 3, 4, 6, 7-tetra-O-acetyl-2-deoxy-1, 5-dithio-alpha-D-glucopyranoside ethyl ester, 2, 5-anhydro-D-allositrile, ribose, D-4-thioribose, L-ribose or L-4-thioribose. Other options for such ligands can be found, for example, in the disclosure of CN105378082A, the entire disclosure of which is incorporated herein by reference.
In some embodiments, the pharmaceutically acceptable targeting group in the siRNA conjugate can be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule can be monovalent, divalent, trivalent, or tetravalent. It should be understood that the monovalent, divalent, trivalent, and tetravalent values as described herein mean that after the siRNA molecule and the conjugate group containing the galactose or N-acetylgalactosamine molecule as the targeting group form an siRNA conjugate, the siRNA conjugate has a molar ratio of the siRNA molecule to the galactose or N-acetylgalactosamine molecule of 1: 1, 1: 2, 1: 3, or 1: 4, respectively. In some embodiments, the pharmaceutically acceptable targeting group is N-acetylgalactosamine. In some embodiments, when the siRNA described in the present disclosure is conjugated to a conjugation group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the siRNA of the present disclosure is conjugated to a conjugation group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.
The targeting group can be attached to the siRNA molecule via a suitable linker, which one skilled in the art can select depending on the particular type of targeting group. The identity of these linkers, targeting groups, and the manner of attachment to the siRNA can be found in the disclosure of WO2015006740a2, which is incorporated by reference herein in its entirety.
In some embodiments, when the targeting group is N-acetylgalactosamine, a suitable linker may be of the structure shown in formula (301):
Figure BDA0002498737070000371
wherein the content of the first and second substances,
k is an integer of 1 to 3;
LAis a chain part containing amido bond with the structure as shown in formula (302), and each LAWith one of said targeting groups and said L at each end thereofCThe moieties are linked by an ether linkage:
Figure BDA0002498737070000372
LBis a chain part containing N-acyl pyrrolidine with a structure shown as a formula (303), wherein the chain part has carbonyl at one end and is connected with the LCPart is connected through amido bond, the other end has oxygen group and is connected with the siRNA through phosphate bond:
Figure BDA0002498737070000373
LCis a 2-4 valent linking group based on hydroxymethylaminomethane, dimethylolaminomethane or trimethylolpropane, said LCVia an oxygen atom with each of said LAThe moieties being linked by an ether bond and being linked to the L via a nitrogen atomBThe moieties are linked by amide bonds.
In some embodiments, when n is 3, LCIn the case of a 4-valent linking group based on tris, the linker is represented by the formula- (LA)3Tris-hydroxymethyl aminomethane-LB-linking the N-acetylgalactosamine molecule and the siRNA molecule to form an siRNA conjugate, which has the following structure (304):
Figure BDA0002498737070000381
in the formula, the double helix structure represents siRNA.
Similarly, the conjugation site of the siRNA to the conjugate group can be at the 3 ' end or 5 ' end of the sense strand of the siRNA, also at the 5 ' end of the antisense strand, and also in the internal sequence of the siRNA.
In some embodiments, the 3' end of the sense strand of the sirnas of the present disclosure is linked to the sense strand of the siRNA through a linker- (L)A)3Tris-hydroxymethyl aminomethane-LBCovalently conjugated with three molecules of N-acetylgalactosamine (GalNAc) to obtain a siRNA conjugate with a molar ratio of siRNA molecule to GalNAc molecule of 1: 3, hereinafter also referred to as (GalNAc)3-siRNA, having the structure represented by the following formula (305):
Figure BDA0002498737070000382
wherein the double helix structure represents the siRNA and the linker is attached to the 3' end of the sense strand of the siRNA.
In some embodiments, when the targeting group is N-acetylgalactosamine, a suitable linker may be of the structure shown in formula (306):
Figure BDA0002498737070000391
wherein the content of the first and second substances,
1 is an integer of 0 to 3;
*represents a site on the linker attached to the targeting group by an ether linkage;
#indicates the site on the linker to which the siRNA is attached via a phosphoester bond.
In some embodiments, when 1 ═ 2, the siRNA conjugate has a structure as shown in formula (307):
Figure BDA0002498737070000392
wherein the double helix structure represents the siRNA and the linker is attached to the 3' end of the sense strand of the siRNA.
The above-mentioned siRNA conjugates can be synthesized by methods that have been described in detail in the prior art. For example, methods for the preparation of various siRNA conjugates are described in detail in WO2015006740a 2. The siRNA conjugates of the present disclosure are obtained by means well known to those skilled in the art. As a method for preparing the structure of formula (305) described in WO2014025805A1, Rajeev et al describe a method for preparing the structure of formula (307) in ChemBiochem 2015, 16, 903-908.
In some embodiments, the siRNA conjugate has a structure as shown in formula (308):
Figure BDA0002498737070000401
wherein:
n1 is an integer selected from 1 to 3, n3 is an integer selected from 0 to 4;
m1, m2 or m3 are independently an integer selected from 2 to 10;
R10、R11、R12、R13、R14or R15Each independently is H, or is selected from the group consisting of: c1-C10Alkyl radical, C1-C10Haloalkyl and C1-C10An alkoxy group;
R3a group of the structure shown in formula a 59:
Figure BDA0002498737070000402
wherein E is1Is OH, SH or BH2Nu is a siRNA of the present disclosure;
R2is a straight chain alkylene group of 1 to 20 carbon atoms in length, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: c (O), NH, O, S, CH-N, S (O)2、C2-C10Alkenylene radical, C2-C10Alkynylene, C6-C10Arylene radical, C3-C18Heterocyclylene and C5-C10A heteroarylene group; and wherein R2May optionally have a substituent of any one or more of the group consisting of: c1-C10Alkyl radical, C6-C10Aryl radical, C5-C10Heteroaryl group, C1-C10Haloalkyl, -OC1-C10Alkyl, -OC1-C10Alkylphenyl, -C1-C10alkyl-OH, -OC1-C10Haloalkyl, -SC1-C10Alkyl, -SC1-C10Alkylphenyl, -C1-C10alkyl-SH, -SC1-C10Haloalkyl, halogen substituents, -OH, -SH, -NH2、-C1-C10alkyl-NH2、-N(C1-C10Alkyl) (C1-C10Alkyl), -NH (C)1-C10Alkyl group), N (C)1-C10Alkyl) (C1-C10Alkylphenyl), NH (C)1-C10Alkylphenyl), cyano, nitro, -CO2H、-C(O)O(C1-C10Alkyl), -CON (C)1-C10Alkyl) (C1-C10Alkyl), -CONH (C)1-C10Alkyl), -CONH2，-NHC(O)(C1-C10Alkyl), -NHC (O) (phenyl), -N (C)1-C10Alkyl) C (O) (C)1-C10Alkyl), -N (C)1-C10Alkyl) C (O) (phenyl), -C (O) C1-C10Alkyl, -C (O) C1-C10Alkylphenyl, -C (O) C1-C10Haloalkyl, -OC (O) C1-C10Alkyl, -SO2(C1-C10Alkyl), -SO2(phenyl), -8O2(C1-C10Haloalkyl), -SO2NH2、-SO2NH(C1-C10Alkyl), -SO2NH (phenyl), -NHSO2(C1-C10Alkyl), -NHSO2(phenyl) and-NHSO2(C1-C10Haloalkyl);
each L1Is a straight chain alkylene group of 1 to 70 carbon atoms in length, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: c (O), NH, O, S, CH-N, S (O)2、C2-C10Alkenylene radical, C2-C10Alkynylene, C6-C10Arylene radical, C3-C18Heterocyclylene and C5-C10A heteroarylene group; and wherein L1 may optionally have substituents of any one or more of the group consisting of: c1-C10Alkyl radical, C6-C10Aryl radical, C5-C10Heteroaryl group, C1-C10Haloalkyl, -OC1-C10Alkyl, -OC1-C10Alkylphenyl, -C1-C10alkyl-OH, -OC1-C10Haloalkyl, -SC1-C10Alkyl, -SC1-C10Alkylphenyl, -C1-C10alkyl-SH, -SC1-C10Haloalkyl, halogen substituents, -OH, -SH, -NH2、-C1-C10alkyl-NH2、-N(C1-C10Alkyl) (C1-C10Alkyl), -NH (C)1-C10Alkyl group), N (C)1-C10Alkyl) (C1-C10Alkylphenyl), NH (C)1-C10Alkylphenyl), cyano, nitro, -CO2H、-C(O)O(C1-C10Alkyl), -CON (C)1-C10Alkyl) (C1-C10Alkyl), -CONH (C)1-C10Alkyl), -CONH2，-NHC(O)(C1-C10Alkyl), -NHC (O) (phenyl), -N (C)1-C10Alkyl) C (O) (C)1-C10Alkyl), -N (C)1-C10Alkyl) C (O) (phenyl), -C (O) C1-C10Alkyl, -C (O) C1-C10Alkylphenyl, -C (O) C1-C10Haloalkyl, -OC (O) C1-C10Alkyl, -SO2(C1-C10Alkyl), -SO2(phenyl), -SO2(C1-C10Haloalkyl), -SO2NH2、-SO2NH(C1-C10Alkyl), -SO2NH (phenyl), -NHSO2(C1-C10Alkyl), -NHSO2(phenyl) and-NHSO2(C1-C10Haloalkyl).
In some embodiments, L1Can be selected from the group consisting of (A1) - (A26) or any combination thereof, wherein the structures and definitions of (A1) - (A26) are as follows:
Figure BDA0002498737070000411
Figure BDA0002498737070000421
wherein j1 is an integer from 1 to 20; j2 is an integer from 1 to 20;
r' is C1-C10An alkyl group;
ra is selected from the group consisting of groups represented by formulas (A27) - (A45) or any combination thereof:
Figure BDA0002498737070000422
Figure BDA0002498737070000431
rb is C1-C10An alkyl group;
Figure BDA0002498737070000432
indicates the site at which the group is covalently attached.
The skilled person will understand that although for convenience L is used1Is defined as a linear alkylene group, but it may not be a linear group or differ in name, for example, an amine or an alkenyl group resulting from the above substitutions and/or substitutions. For purposes of this disclosure, L1Is the number of atoms in the chain connecting the two points of attachment. For this purpose, a ring (e.g., a heterocyclylene or heteroarylene) obtained by substituting a carbon atom of the linear alkylene group is counted as one atom.
M1Refers to targeting groups, which are defined and alternative to the same scope as the targeting groups described above. In some embodiments, each M is1Independently selected from one of the ligands having affinity for asialoglycoprotein receptors on the surface of mammalian liver cells.
When M is1In the case of ligands having affinity for asialoglycoprotein receptors on the surface of mammalian liver cells, n1 can be an integer from 1 to 3 and n3 can be an integer from 0 to 4 in some embodiments, ensuring that M is present in the siRNA conjugate1The number of targeting groups is at least2; in some embodiments, n1+ n3 ≧ 2, which can result in M1The number of targeting groups is at least 3, such that M1The targeting group is more easily combined with the liver surface asialoglycoprotein receptor, thereby facilitating the siRNA conjugate to enter cells through endocytosis. Experiments show that when M is used 1When the number of targeting groups is more than 3, M1The increased ease of binding of the targeting group to the hepatic surface asialoglycoprotein receptor is not significant, and thus, in some embodiments, n1 is an integer from 1 to 2, n3 is an integer from 0 to 1, and n1+ n3 is 2 to 3, all taken together from the aspects of ease of synthesis, structure/process cost, and delivery efficiency.
In some embodiments, when M1, M2, or M3 is independently selected from an integer of 2 to 10, a plurality of M may be used1Spatial position between targeting groups is adapted to M1In order to make the siRNA conjugates provided by the present disclosure simpler, easier to synthesize, and/or less costly, the binding of the targeting group to the liver surface asialoglycoprotein receptor, in some embodiments, m1, m2, or m3 are each independently integers from 2 to 5, and in some embodiments, m1 ═ m2 ═ m 3.
It will be understood by those skilled in the art that when R is present10、R11、R12、R13、R14Or R15Each independently selected from H, C1-C10Alkyl radical, C1-C10Haloalkyl, and C1-C10One of the alkoxy groups, without changing the properties of the siRNA conjugates of the present disclosure, can achieve the objects of the present disclosure. In some embodiments, R10、R11、R12、R13、R14Or R15Each independently selected from H, methyl or ethyl. In some embodiments, Ri0、R11、R12、R13、R14And R15Are all H.
R3A group of the structure shown as formula A59, wherein E1Is OH, SH or BH2In some embodiments, E is based on considerations of ready availability of starting materials for preparation1Is OH or SH.
R2Is selected to effect attachment to the N atom of the nitrogen-containing backbone to a 59. In the context of the present disclosure, "nitrogen-containing backbone" means a linkage with R10、R11、R12、R13、R14And R15A chain structure in which carbon atoms and N atoms are linked to each other. Thus, R2May be any linking group capable of linking the a59 group to the N atom on the nitrogen-containing backbone in a suitable manner. In some embodiments, where the siRNA conjugate represented by formula (308) is prepared by a process of solid phase synthesis, R is2The group desirably contains both a linking site to the N atom of the nitrogen-containing skeleton and a linking site to R3The P atom in (a) to which the linking site is attached. In some embodiments, R2Wherein the site attached to the N atom of the nitrogen-containing backbone forms an amide bond with the N atom, and said site is attached to R3The site to which the P atom is attached forms a phosphoester bond with the P atom; in some embodiments, R2May be B5, B6, B5 'or B6':
Figure BDA0002498737070000441
wherein the content of the first and second substances,
Figure BDA0002498737070000451
indicates the site at which the group is covalently attached.
q2Can be an integer from 1 to 10, and in some embodiments, q is2Is an integer of 1 to 5.
L1Has the effect of mixing M1The targeting group is linked to N on the nitrogen-containing backbone to provide a liver targeting function for the siRNA conjugate shown in formula (308). In some embodiments, L1One or more connecting combinations selected from the group of the formulas A1-A26. In some embodiments, L1A combination of one or more linkages selected from a1, a4, a5, a6, A8, a10, a11, and a 13. In some embodiments, L1A linked combination of at least 2 selected from a1, a4, A8, a10, and a 11. In some embodimentsIn the formula, L1At least 2 connecting combinations selected from A1, A8 and A10.
In some embodiments, L1Can be 3-25 atoms, 3-20 atoms, 4-15 atoms, or 5-12 atoms in length. In some embodiments, L1The length of (a) is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60 atoms.
In some embodiments j1 is an integer from 2 to 10, and in some embodiments j1 is an integer from 3 to 5. In some embodiments j2 is an integer from 2 to 10, and in some embodiments j2 is an integer from 3 to 5. R' is C1-C4Alkyl, and in some embodiments, R' is one of methyl, ethyl, and isopropyl. Ra is one of a27, a28, a29, a30, and a31, and in some embodiments, Ra is a27 or a 28. Rb is C1-C5And in some embodiments, Rb is one of methyl, ethyl, isopropyl, and butyl. In some embodiments, j1, j2, R', Ra, Rb are each selected in formulas A1-A26 to achieve M1The targeting group being attached to the N atom of the nitrogen-containing skeleton and M being bonded1The spatial position between the targeting groups is more suitable for M1The targeting group binds to the hepatic surface asialoglycoprotein receptor.
In some embodiments, the siRNA conjugate has a structure represented by formula (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421), or (422):
Figure BDA0002498737070000452
Figure BDA0002498737070000461
Figure BDA0002498737070000471
Figure BDA0002498737070000481
Figure BDA0002498737070000491
Figure BDA0002498737070000501
Figure BDA0002498737070000511
Figure BDA0002498737070000521
in some embodiments, the P atom in formula a59 can be attached to any possible position in the siRNA sequence, for example, the P atom in formula a59 can be attached to any one nucleotide of the sense or antisense strand of the siRNA; in some embodiments, the P atom in formula a59 is attached to any one nucleotide of the sense strand of the siRNA. In some embodiments, the P atom in formula a59 is attached to the end of the sense or antisense strand of the siRNA; in some embodiments, the P atom in formula a59 is attached to the end of the sense strand of the siRNA. The end refers to the first 4 nucleotides of the sense strand or the antisense strand from one end thereof. In some embodiments, the P atom in formula a59 is attached to the end of the sense or antisense strand of the siRNA; in some embodiments, the P atom in formula a59 is attached to the 3' end of the sense strand of the siRNA. In the case of the above position of the sense strand linked to siRNA, after the siRNA conjugate shown in formula (308) enters the cell, upon unwinding, the separate siRNA antisense strand can be released to block the process of protein translation of TMPRSS6 mRNA, inhibiting TMPRSS6 gene expression.
In some embodiments, P in formula a59 can be attached to any possible position on a nucleotide in the siRNA, e.g., the 5 ' position of the nucleotide, the 2 ' position of the nucleotide, the 3 ' position of the nucleotide, or the base of the nucleotide. In some embodiments, the P atom in formula a59 can be attached to the nucleotide in the siRNA at the 2 ' position, 3 ' position, or 5 ' position by forming a phosphodiester bond. In some embodiments, the P atom in formula a59 is attached to an oxygen atom formed after dehydrogenation of the 3 ' hydroxyl group of the 3 ' terminal nucleotide of the siRNA sense strand (in which case the P atom in a59 can also be considered to be the P atom in the phosphate group contained in the siRNA), or the P atom in formula a59 is attached to the nucleotide by substitution of a hydrogen in the 2 ' -hydroxyl group of one nucleotide in the siRNA sense strand, or the P atom in formula a59 is attached to the nucleotide by substitution of a hydrogen in the 5 ' hydroxyl group of the 5 ' terminal nucleotide in the siRNA sense strand.
The inventors of the present disclosure surprisingly found that the siRNA conjugates of the present disclosure also exhibit higher TMPRSS6 mRNA silencing activity while having significantly improved stability in plasma, low off-target effect. In some embodiments, the siRNA of the present disclosure may be one of the sirnas shown in tables 1a-1 f. siRNA conjugates containing these sirnas showed higher TMPRSS6 mRNA silencing activity.
TABLE 1A first siRNA sequence of the present disclosure
Figure BDA0002498737070000531
Figure BDA0002498737070000541
TABLE 1b second siRNA sequences of the present disclosure
Figure BDA0002498737070000551
Figure BDA0002498737070000561
TABLE 1c third siRNA sequences of the present disclosure
Figure BDA0002498737070000562
Figure BDA0002498737070000571
Figure BDA0002498737070000581
TABLE 1d fourth siRNA sequences of the present disclosure
Figure BDA0002498737070000582
Figure BDA0002498737070000591
Figure BDA0002498737070000601
TABLE 1e fifth siRNA sequence of the present disclosure
Figure BDA0002498737070000602
Figure BDA0002498737070000611
Figure BDA0002498737070000621
TABLE 1f sixth siRNA sequences of the present disclosure
Figure BDA0002498737070000622
Figure BDA0002498737070000631
Figure BDA0002498737070000641
In the siRNA or siRNA conjugate, each adjacent nucleotide is connected by phosphodiester bond or phosphorothioate diester bond, non-bridging oxygen atom or sulfur atom in the phosphodiester bond or phosphorothioate diester bond has negative charge, and the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or phosphorothioate diester bond can exist in the form of hydroxyl or sulfhydryl, and hydrogen ions in the hydroxyl or sulfhydryl can be partially or completely replaced by cations. The cation may be any cation, such as a metal cation, ammonium NH4 +One of organic ammonium cations. For the purpose of enhancing solubility, in one embodiment, the cation is selected from one or more of alkali metal ions, tertiary amine forming ammonium cations, and quaternary ammonium cations. The alkali metal ion may be K+And/or Na+The cation formed by the tertiary amine may be an ammonium ion formed by triethylamine and/or an ammonium ion formed by N, N-diisopropylethylamine. Thus, the siRNA or siRNA conjugate of the present disclosure may be at least partially present in the form of a salt. In one mode, the non-bridging oxygen or sulfur atoms in the phosphodiester or phosphorothioate linkages are at least partially bound to sodium ions and the sirnas or siRNA conjugates of the present disclosure are present as sodium salts or partial sodium salts.
It is clear to one skilled in the art that modified nucleotide groups can be introduced into the sirnas described in the present disclosure by using nucleoside monomers with corresponding modifications. Methods for preparing nucleoside monomers with corresponding modifications and methods for introducing modified nucleotide groups into siRNA are also well known to those skilled in the art. All modified nucleoside monomers are commercially available or can be prepared by known methods.
Preparation of siRNA conjugate represented by formula (308)
Any reasonable synthetic route can be used to prepare the siRNA conjugates represented by formula (308).
In some embodiments, the siRNA conjugate represented by formula (308) can be prepared by a method comprising sequentially linking nucleoside monomers in a3 'to 5' direction according to the nucleotide types and the order of the sense strand and the antisense strand of the siRNA, respectively, under the conditions of phosphoramidite solid phase synthesis, the linking of each nucleoside monomer comprising four steps of deprotection, coupling, capping, oxidation, or sulfurization; separating a sense strand and an antisense strand of the siRNA, and annealing, wherein the siRNA is the siRNA of the present disclosure;
and, the method further comprises contacting the compound represented by formula (321) with a nucleoside monomer or a nucleotide sequence attached to a solid support in the presence of a coupling reagent under coupling reaction conditions to allow the compound represented by formula (321) to be attached to the nucleotide sequence via a coupling reaction. Hereinafter, the compound represented by formula (321) is also referred to as a conjugate molecule.
Figure BDA0002498737070000651
Wherein:
R4is a group capable of binding to the siRNA represented by Nu in the compound represented by the formula (308). In some embodiments, R4Is a group capable of binding to the siRNA represented by Nu through a covalent bond. In some embodiments, R4A group which is capable of being conjugated to any functional group of the siRNA represented by Nu through a phosphodiester bond by a reaction;
each S1Independently is M1Wherein all active hydroxyl groups are substituted with YCOO-groups, wherein each Y is independently selected fromOne of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl and alkylphenyl; in some embodiments, Y is methyl.
n1、n3、m1、m2、m3、R10、R11、R12、R13、R14、R15、L1、M1The respective definitions and alternative ranges are as described above.
R4Is selected to achieve attachment to the N atom of the nitrogen-containing backbone and to provide a suitable reaction site for the synthesis of the siRNA conjugate shown in formula (308). In some embodiments, R4Including R2Linking groups or protected R2A linking group, and a functional group that can react with the siRNA to form the structure shown as A59.
In some embodiments, R4Comprises a1 st functional group which can form a phosphite ester with a group on the siRNA or nucleoside monomer represented by Nu and a2 nd functional group which can react with a hydroxyl group or an amino group to form a covalent bond or a solid phase carrier connected by the covalent bond. In some embodiments, the 1 st functional group is a phosphoramidite, a hydroxyl, or a protected hydroxyl. In some embodiments, the 2 nd functional group is a phosphoramidite, a carboxyl, or a carboxylate. In some embodiments, the 2 nd functional group is a solid support attached to the rest of the molecule via a covalent bond formed from a hydroxyl or amino group. In some embodiments, the solid support is linked via a phosphate ester linkage, a carboxylate ester linkage, or an amide linkage. In some embodiments, the solid support is a resin.
In some embodiments, the 1 st functional group contains a hydroxyl group, -ORkOr a group of formula (C3); the 2 nd functional group has a structure represented by formula (C1), (C2), (C3), (C1 ') or (C3'):
Figure BDA0002498737070000661
in the formula, q1Is an integer of 1 to 4, X is O or NH, M+Is a cation, RkIs a hydroxyl protecting group, SPS represents a solid phase carrier,
Figure BDA0002498737070000662
indicates the site at which the group is covalently attached.
In some embodiments, the 1 st functional group contains a phosphoramidite group, as shown in formula (C3), which can be coupled to a hydroxyl group at any position on a nucleotide, such as a hydroxyl group at the 2 'position or a hydroxyl group at the 3' position, to form a phosphite, and oxidized or sulfurized to form a phosphodiester or phosphorothioate linkage as shown in formula a59, to conjugate the conjugation molecule to the siRNA. At this time, even if the 2 nd functional group is not present, the compound represented by formula (321) can be conjugated to a nucleotide without affecting the obtainment of the siRNA conjugate represented by formula (308). In this case, after obtaining the sense strand or the antisense strand of the siRNA via a phosphoramidite solid phase synthesis or the like, the compound represented by formula (321) is reacted with a hydroxyl group on the terminal nucleotide in the nucleotide sequence and forms a phosphodiester linkage or a phosphorothioate linkage in a subsequent oxidation or sulfurization process, and the compound represented by formula (321) is conjugated to the siRNA.
In some embodiments, the 1 st functional group contains a protected hydroxyl group. In some embodiments, the 2 nd functional group comprises a group that can react with a solid support, the reaction providing a conjugate molecule comprising a solid support. In some embodiments, the 2 nd functional group contains a carboxyl group, a carboxylate salt, or a phosphoramidite, as shown in formula (C1), (C2), or (C3), and when the 2 nd functional group contains a carboxyl group or a carboxylate salt, the compound represented by formula (321) undergoes an esterification reaction or an amidation reaction with a hydroxyl group or an amino group on a solid support, e.g., a resin, to form a carboxylate-linked conjugate molecule comprising a solid support. When the 2 nd functional group comprises a phosphoramidite functional group, the compound represented by formula (321) undergoes a coupling reaction with a hydroxyl group on a common solid support, e.g., a resin, and is oxidized to form a phosphodiester linked conjugate molecule comprising a solid support. Subsequently, the nucleoside monomers are sequentially linked according to a phosphoramidite solid phase synthesis method by using the product after the solid phase carrier is linked as the starting material to obtain the sense strand or the antisense strand of the siRNA with the conjugated group. During solid phase phosphoramidite synthesis, deprotection of the 1 st functional group occurs, followed by coupling with a phosphoramidite group on a nucleoside monomer under coupling reaction conditions.
In some embodiments, the 1 st functional group contains a hydroxyl group or a protected hydroxyl group; the 2 nd functional group contains a solid phase carrier connected by a carboxylate bond or an amide bond or a solid phase carrier connected by a phosphate bond, and is shown as a formula (C1 ') or (C3'). In this case, the compound represented by the formula (321) is used in place of the solid carrier
Initially, nucleoside monomers are sequentially linked according to a phosphoramidite solid phase synthesis method to obtain a sense strand or an antisense strand of siRNA having a conjugate group attached thereto.
In some embodiments, the carboxylate may be represented by-COO-M+Wherein M is+Is a cation, e.g. selected from the group consisting of metal cations, ammonium cations NH4 +One of organic ammonium cations. In one embodiment, the metal ion is selected from one of the alkali metal ions, such as K+Or Na+. In view of the solubility enhancement and the ease of reaction, in some embodiments, the organic ammonium ion is an ammonium cation formed from a tertiary amine or a quaternary ammonium cation, such as an ammonium ion formed from triethylamine or an ammonium ion formed from N, N-diisopropylethylamine. In some embodiments, the carboxylate is triethylamine carboxylate or N, N-diisopropylethylamine carboxylate.
In some embodiments, R4Contains a structure represented by formula (B9), (B10), (B9 '), (B10'), (B11), (B12), (B11 ') or (B12'):
Figure BDA0002498737070000671
Figure BDA0002498737070000681
wherein q is1Is an integer of 1 to 4, q2Is an integer of 1 to 10, X is O or NH, M+Is a cation, RkIs a hydroxyl protecting group, SPS represents a solid phase carrier,
Figure BDA0002498737070000682
indicates the site at which the group is covalently attached. In some embodiments, q is1Is 1 or 2. In some embodiments, q is2Is an integer of 1 to 5. In some embodiments, R4Contains a structure represented by the formula (B9) or (B10). In some embodiments, R4Contains a structure represented by the formula (B11) or (B12).
In some embodiments, RkIs one or more of Tr (trityl), MMTr (4-methoxytrityl), DMTr (4, 4 '-bismethoxytrityl), TMTr (4, 4' -trimethoxytrityl). In some embodiments, RkMay be DMTr, i.e. 4, 4 '-dimethoxytrityl (4, 4' -dimethoxytrityl).
L1As defined above.
In some embodiments, L1Is used for M1The targeting group is attached to the N atom on the nitrogen-containing backbone, thereby providing a liver targeting function to the siRNA conjugate shown in formula (308). In some embodiments, L1Comprising any one of formula (a1) -formula (a26), or a combination thereof.
From the above description, it is easily understood by those skilled in the art that the siRNA conjugate represented by formula (308) that links a conjugate molecule to any possible position of a nucleotide sequence, for example, the conjugate molecule is linked to the end of the nucleotide sequence and the conjugate molecule is linked to the end of the nucleotide sequence, can be obtained through the above-described 1 st functional group and optionally the 2 nd functional group, compared to the solid phase synthesis method of phosphoramidite known in the art. Accordingly, unless otherwise indicated, in the following description relating to the preparation of siRNA conjugates and/or conjugate molecules, when referring to "deprotection", "coupling", "capping", "oxidation", "sulfurization", etc. reactions, it is to be understood that reaction conditions and reagents involved in solid phase methods of phosphoramidite nucleic acid synthesis known in the art are equally applicable to these reactions. Exemplary reaction conditions and reagents will be described in detail hereinafter.
In some embodiments, each S is1Independently is M1. In some embodiments, each S is1Independently is M1Wherein at least one active hydroxyl group is protected by a hydroxyl protecting group. In some embodiments, each S is1Independently is M1Any active hydroxyl groups present in (a) are all protected by a hydroxyl protecting group. In some embodiments, any hydroxy protecting group known to those skilled in the art may be used to protect M1Active hydroxyl group in (1). In some embodiments, the protected hydroxy group may be represented by the formula YCOO-, wherein each Y is independently selected from the group consisting of C1-C10Alkyl and C6-C10Aryl group, said C1-C10Alkyl and C6-C10Aryl is optionally substituted with one or more substituents selected from the group consisting of halogen and C1-C6 alkyl. In some embodiments, each Y is independently selected from the group consisting of: methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl, and C1-C6An alkyl phenyl group.
In some embodiments, each S is1Each independently selected from the group consisting of formula A46-A54:
Figure BDA0002498737070000691
Figure BDA0002498737070000701
in some embodiments, S1Is of the formula A49Or a 50.
In some embodiments, each Y is independently selected from one of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, chloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl, and alkylphenyl; in some embodiments, Y is methyl.
As described above, the preparation method of the siRNA conjugate represented by formula (308) further comprises the steps of: synthesizing the other strand of the siRNA (for example, when the sense strand of the siRNA to which the conjugate molecule is linked is synthesized in the above-mentioned step, synthesizing the antisense strand of the siRNA according to a solid phase synthesis method and vice versa is also included), separating the sense strand and the antisense strand, and annealing. Specifically, in the separation step, the solid support attached to the nucleotide sequence and/or conjugate molecule is cleaved off while the necessary protecting group is removed (at this time, each S in the compound represented by formula (321)1Conversion of the group to the corresponding M1Targeting group) to obtain a sense strand (or antisense strand) and a corresponding antisense strand (or sense strand) of the siRNA linked with the conjugate molecule, the sense strand and the antisense strand annealing to form a double-stranded RNA structure, obtaining the siRNA conjugate shown in formula (308).
In some embodiments, the method of preparing the siRNA conjugate represented by formula (308) comprises the steps of: contacting a compound shown in a formula (321) with a first nucleoside monomer at the 3 ' end of a sense strand or an antisense strand under a coupling reaction condition and in the presence of a coupling reagent, connecting the first nucleotide in a connecting sequence to the compound shown in the formula (321), and sequentially connecting the nucleoside monomers in the 3 ' to 5 ' direction according to the type and the sequence of the nucleotide of the desired sense strand or antisense strand under the condition of phosphoramidite solid phase synthesis to synthesize the sense strand or antisense strand of the siRNA; wherein the compound represented by the formula (321) is R4The compound contains a1 st functional group and a2 nd functional group, wherein the 1 st functional group contains protected hydroxyl, the 2 nd functional group has a structure shown as a formula (C1 ') or (C3'), and the compound shown as a formula (321) is subjected to deprotection before being connected with a first nucleoside monomer; the connection of each nucleoside monomer comprises four steps of deprotection, coupling, capping, oxidation or sulfuration; to obtain a conjugated groupThe sense strand or the antisense strand of the nucleic acid of (a); under the condition of solid phase synthesis of phosphoramidite, nucleoside monomers are connected in sequence according to the nucleotide types and the sequence of an antisense strand or a sense strand and in the 3 'to 5' direction to synthesize the antisense strand or the sense strand of nucleic acid; the connection of each nucleoside monomer comprises four steps of deprotection, coupling, capping, oxidation or sulfuration; removing protecting group, cutting with solid phase carrier, separating and purifying to obtain sense strand and antisense strand, and annealing.
In some embodiments, the method of preparing the siRNA conjugate represented by formula (308) comprises the steps of: according to the nucleotide types and the sequence of a sense strand or an antisense strand in the double-stranded siRNA, nucleoside monomers are sequentially connected in a 3 'to 5' direction to synthesize the sense strand and the antisense strand, wherein the connection of each nucleoside monomer comprises four steps of deprotection, coupling, capping, oxidation or sulfuration, and the sense strand connected to a solid phase carrier and the antisense strand connected to the solid phase carrier are obtained; contacting the compound represented by the formula (321) with a sense strand attached to a solid support or an antisense strand attached to a solid support in the presence of a coupling reagent under coupling reaction conditions, and attaching the compound represented by the formula (321) to the sense strand or the antisense strand, wherein the compound represented by the formula (321) is R4A compound represented by the formula (321) wherein the 1 st functional group is a phosphoramidite group; removing protecting groups, cutting with a solid phase carrier, respectively separating and purifying to obtain a sense strand or an antisense strand of the siRNA, and annealing, wherein the sense strand or the antisense strand of the siRNA is connected with a conjugate group.
In some embodiments, the P atom in formula a59 is attached to the 3' end of the sense strand in the siRNA, and the method of preparing the siRNA conjugate represented by formula (308) comprises:
(1) removing the compound represented by the formula (321) (wherein the compound represented by the formula (321) is R4Contains a1 st functional group and a2 nd functional group, the 1 st functional group contains a protected hydroxyl group ORkThe 2 nd functional group is a compound having a structure represented by the formula (C1 ') or (C3')k(ii) a Contacting the deprotected product with a nucleoside monomer under coupling reaction conditions and in the presence of a coupling reagent to obtain a product obtained by conjugationA nucleoside monomer with a synthon attached to a solid support;
(2) synthesizing a sense strand of the siRNA by a phosphoramidite solid phase synthesis method in a 3 '-5' direction starting with the nucleoside monomer linked to the solid phase support by the conjugate molecule;
(3) Synthesizing an antisense strand of the siRNA by a phosphoramidite solid phase synthesis method;
(4) the sense strand and the antisense strand of the siRNA are isolated and annealed to obtain an siRNA conjugate represented by formula (308).
Wherein, in the step (1), the protecting group R in the compound represented by the above formula (321) is removedkThe method of (1) includes contacting the compound represented by the formula (321) with a deprotection reagent under deprotection conditions. Deprotection conditions include temperatures of 0 to 50 deg.C, in some embodiments 15 to 35 deg.C, reaction times of 30 to 300 seconds, in some embodiments 50 to 150 seconds, and the deprotection reagent may be selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, monochloroacetic acid, and in some embodiments dichloroacetic acid. The molar ratio of deprotecting reagent to compound represented by formula (321) is from 10: 1 to 1000: 1, and in some embodiments from 50: 1 to 500: 1.
The coupling reaction conditions and coupling reagents may use any conditions and reagents suitable for the above-described coupling reaction. In some embodiments, the same conditions and reagents can be used as for the coupling reaction in the solid phase synthesis method employed.
In some embodiments, the conditions of the coupling reaction include a reaction temperature of from 0 to 50 ℃, in some embodiments from 15 to 35 ℃. The molar ratio of the compound represented by formula (321) to the nucleoside monomer is 1: 1 to 1: 50, and in some embodiments 1: 2 to 1: 5; the molar ratio of the compound represented by formula (321) and the coupling reagent may be in the range of 1: 1 to 1: 50, and in some embodiments 1: 3 to 1: 10, with a reaction time in the range of 200 to 3000 seconds, and in some embodiments, 500 to 1500 seconds. The coupling reagent is selected from one or more of 1H-tetrazole, 5-ethylthio 1H-tetrazole, and 5-benzylthio 1H-tetrazole, and in some embodiments is 5-ethylthio 1H-tetrazole. The coupling reaction may be carried out in an organic solvent selected from one or more of anhydrous acetonitrile, anhydrous DMF, anhydrous dichloromethane, and in some embodiments, anhydrous acetonitrile. The organic solvent is used in an amount of 3 to 50L/mol, and in some embodiments, 5 to 20L/mol, relative to the compound represented by formula (321).
In step (2), the sense strand of the siRNA conjugate is synthesized in the 3 '-5' direction by a method of solid phase synthesis of phosphoramidite nucleic acid, starting with the nucleoside monomer attached to the solid support by the conjugate molecule prepared in the above step. At this point, the conjugate group is attached to the 3' end of the resulting sense strand.
Other conditions of the solid phase synthesis in the steps (2) and (3) include deprotection conditions of nucleoside monomers, types and amounts of deprotection reagents, coupling reaction conditions, types and amounts of coupling reagents, capping reaction conditions, types and amounts of capping reagents, oxidation reaction conditions, types and amounts of oxidation reagents, vulcanization reaction conditions, and types and amounts of vulcanization reagents, which are various reagents, amounts and conditions conventionally used in the art.
For example, in some embodiments, the solid phase synthesis in steps (2) and (3) may use the following conditions:
the nucleoside monomer deprotection conditions include a temperature of 0 to 50 deg.C, in some embodiments 15 to 35 deg.C, a reaction time of 30 to 300 seconds, in some embodiments 50 to 150 seconds, and the deprotection reagent may be selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, monochloroacetic acid, and in some embodiments dichloroacetic acid. The molar ratio of deprotecting reagent to 4, 4' -dimethoxytrityl protecting group on the solid support can be from 2: 1 to 100: 1, and in some embodiments from 3: 1 to 50: 1.
The coupling reaction conditions include a temperature of 0 to 50 deg.C, and in some embodiments 15 to 35 deg.C, and the molar ratio of nucleic acid sequence attached to the solid support to nucleoside monomer can be 1: 1 to 1: 50, and in some embodiments 1: 5 to 1: 15; the molar ratio of nucleic acid sequence attached to the solid support to coupling reagent is from 1: 1 to 1: 100, and in some embodiments from 1: 50 to 1: 80, and the reaction time and choice of coupling reagent are the same as described above.
Capping reaction conditions include a temperature of 0-50 deg.C, in some embodiments 15-35 deg.C, and a reaction time of 5-500 seconds, in some embodiments 10-100 seconds, with the same selection of capping reagents as previously described. The molar ratio of the total amount of capping reagent to nucleic acid sequence attached to the solid support is 1: 100 to 100: 1, and in some embodiments 1: 10 to 10: 1. In the case where equimolar amounts of acetic anhydride and N-methylimidazole are used as the capping reagent, the molar ratio of acetic anhydride, N-methylimidazole and nucleic acid sequence attached to the solid support may be 1: 10 to 10: 1, and in some embodiments 1: 2 to 2: 1.
The oxidation reaction conditions include a temperature of from 0 to 50 deg.C, in some embodiments from 15 to 35 deg.C, a reaction time of from 1 to 100 seconds, in some embodiments from 5 to 50 seconds, and the oxidizing agent, in some embodiments, iodine (in some embodiments, provided in the form of iodine water). The molar ratio of oxidizing reagent to nucleic acid sequence attached to the solid support in the coupling step can be from 1: 1 to 100: 1, and in some embodiments from 5: 1 to 50: 1. In some embodiments, the oxidation reaction is carried out in a mixed solvent of tetrahydrofuran, water, pyridine, 3: 1 to 1: 3. The sulfurization reaction conditions include a temperature of from 0 to 50 deg.C, in some embodiments from 15 to 35 deg.C, a reaction time of from 50 to 2000 seconds, in some embodiments from 100 to 1000 seconds, and a sulfurizing agent, in some embodiments hydrogenated flavonones. The molar ratio of the sulfurizing reagent to the nucleic acid sequence attached to the solid support in the coupling step is from 10: 1 to 1000: 1, and in some embodiments from 10: 1 to 500: 1. In some embodiments, the sulfurization reaction is carried out in a mixed solvent of acetonitrile to pyridine of 1: 3 to 3: 1.
After ligating all nucleoside monomers, the method further comprises isolating the sense and antisense strands of the siRNA prior to annealing. Isolation procedures are well known to those skilled in the art and generally involve cleaving the synthesized nucleotide sequence from the solid support, removing protecting groups on the base, phosphate and ligand, purification and desalting.
Cleaving the synthesized nucleotide sequence from the solid supportNext, the removal of the protecting groups on the base, phosphate and ligand can be performed according to the conventional cleavage and deprotection methods in siRNA synthesis. For example, the obtained nucleotide sequence with the solid support attached thereto is contacted with concentrated ammonia water; during deprotection, the protecting group YCOO-of the A46-A54 group is converted into a hydroxyl group, S1Conversion of the group to the corresponding M1And (3) generating the siRNA conjugate shown in the formula (308). Wherein, the concentrated ammonia water can be 25-30 wt% ammonia water, and the dosage of the concentrated ammonia water can be 0.2 ml/mu mol-0.8 ml/mu mol compared with the target siRNA sequence.
When there is at least one 2 '-TBDMS protection on the synthesized nucleotide sequence, the method further comprises contacting the nucleotide sequence with the solid support removed with triethylamine trihydrofluoride to remove the 2' -TBDMS protection. At this time, the corresponding nucleotide in the obtained target siRNA sequence has a free 2' -hydroxyl group. The amount of the triethylamine trihydrofluoride pure product can be 0.4 ml/mu mol-1.0 ml/mu mol compared with the target siRNA sequence. This gave an siRNA conjugate represented by the formula (308).
Methods of purification and desalination are well known to those skilled in the art. For example, purification of nucleic acids can be accomplished by gradient elution with NaBr or NaCl using a preparative ion chromatography purification column; the products can be desalted by adopting a reverse phase chromatographic purification column after being collected and combined.
In the siRNA conjugate represented by the formula (308) thus obtained, the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or phosphorothioate diester bond between nucleotides is substantially bound to sodium ions, and the siRNA conjugate represented by the formula (308) exists substantially in the form of a sodium salt. Other forms of siRNA conjugates represented by formula (308) can be obtained by replacing the sodium ions with hydrogen ions and/or other cations using well known ion exchange methods. The cations are as described above.
The purity and molecular weight of the nucleic acid sequence can be readily determined during synthesis to better control the quality of the synthesis, and such methods are well known to those skilled in the art. For example, nucleic acid purity can be detected by ion exchange chromatography and molecular weight determined by liquid chromatography-mass spectrometry (LC-MS).
Methods of annealing are also well known to those skilled in the art. For example, the synthesized sense strand (S strand) and antisense strand (AS strand) can be simply mixed in equimolar ratio in water for injection and heated to 70-95 ℃ followed by cooling at room temperature to allow formation of a double-stranded structure by hydrogen bonding. This gave an siRNA conjugate represented by the formula (308).
After obtaining the siRNA conjugate, in some embodiments, the synthesized siRNA conjugate shown in formula (308) can be further characterized by molecular weight detection and the like using a method such as liquid chromatography-mass spectrometry, and the synthesized siRNA conjugate is determined to be the siRNA conjugate shown in formula (308) designed for the target, and the sequence of the synthesized siRNA is the sequence of the desired siRNA, for example, one of the sequences listed in tables 1a-1 f.
The compound represented by the formula (321) can be obtained by the following production method: the method comprises the following steps of contacting a compound shown as a formula (313) with a cyclic acid anhydride in an organic solvent under esterification reaction conditions in the presence of a base and an esterification catalyst, carrying out ion exchange, and separating to obtain a compound shown as a formula (321):
Figure BDA0002498737070000731
wherein, n1, n3, m1, m2, m3 and R10、R11、R12、R13、R14、R15、L1、S1The respective definitions and alternative ranges are as described above;
R6to provide R in formula (321)4A group of (a); in some embodiments, R6Has a structure represented by formula (A61):
Figure BDA0002498737070000741
wherein R isiTo enable connection to N atoms of nitrogen-containing skeleton, to RkO is linked to and is linked to an optional radical of a free hydroxyl group, RkIs a hydroxyl protecting group. In this case, R is obtained4The 2 nd functional group contains a 1 st functional group and a 2 nd functional group as hydroxyl protecting groups, and the 2 nd functional group contains a compound represented by a formula (321) having a structure represented by a formula (C1) or (C2).
The esterification reaction conditions include a reaction temperature of 0-100 ℃ and a reaction time of 8-48 hours, and in some embodiments, the esterification reaction conditions are a reaction temperature of 10-40 ℃ and a reaction time of 20-30 hours.
In some embodiments, the organic solvent comprises one or more of an epoxy-based solvent, an ether-based solvent, a haloalkane-based solvent, dimethyl sulfoxide, N-dimethylformamide, and N, N-diisopropylethylamine. In some embodiments, the epoxy-based solvent is dioxane and/or tetrahydrofuran, the ether-based solvent is diethyl ether and/or methyl tert-butyl ether, and the haloalkane-based solvent is one or more of dichloromethane, chloroform, and 1, 2-dichloroethane. In some embodiments, the organic solvent is dichloromethane. The organic solvent is used in an amount of 3 to 50L/mol, and in some embodiments, 5 to 20L/mol, relative to the compound represented by formula (313).
In some embodiments, the cyclic anhydride is one of succinic anhydride, glutaric anhydride, adipic anhydride, or pimelic anhydride, and in some embodiments succinic anhydride. The molar ratio of the cyclic anhydride to the compound of formula (313) is from 1: 1 to 10: 1, and in some embodiments from 2: 1 to 5: 1.
The esterification catalyst may be any catalyst that catalyzes the esterification reaction, for example, the catalyst may be 4-dimethylaminopyridine. The molar ratio of the catalyst to the compound of formula (313) is from 1: 1 to 10: 1, and in some embodiments from 2: 1 to 5: 1.
In some embodiments, the base can be any inorganic base, organic base, or combination thereof. The base may be, for example, a tertiary amine in view of solubility and product stability. In some embodiments, the tertiary amine is triethylamine or N, N-diisopropylethylamine. The molar ratio of the tertiary amine to the compound of formula (313) is from 1: 1 to 20: 1, and in some embodiments from 3: 1 to 10: 1.
The ion exchange is a process for converting the compound represented by formula (321) into the desired carboxylic acid or carboxylate salt form, and the ion exchange is well known to those skilled in the art, and suitable ion exchange solutions and exchange conditions can be used to obtain a compound having M+The cationic conjugate molecule will not be described in detail. In some embodiments, the ion exchange reaction is performed using a triethylamine phosphate solution having a concentration of 0.2 to 0.8M, in some embodiments, 0.4 to 0.6M, in an amount of 3 to 6L/mol, and in further embodiments, 4 to 5L/mol, relative to the compound represented by formula (313).
The compound represented by formula (321) can be isolated from the reaction mixture using any suitable isolation method. In some embodiments, the compound represented by formula (321) may be isolated by evaporation of the solvent followed by chromatographic methods, e.g., using two chromatographic conditions: (1) normal phase purification of silica gel: silica gel filler of 200-300 meshes, and the gradient elution is carried out by using dichloromethane containing 1 wt% of triethylamine and methanol, wherein the ratio of the dichloromethane to the methanol is 100: 18-100: 20; or (2) reversed-phase purification: c18, C8 reversed phase packing, eluting with a gradient of methanol to acetonitrile 0.1: 1 to 1: 0.1. In some embodiments, the solvent may be removed directly to provide a crude compound of formula (321) which may be used directly in a subsequent reaction.
In some embodiments, the method for preparing a compound represented by formula (321) further comprises contacting the product obtained by the above ion exchange reaction with a solid support containing an amino group or a hydroxyl group in an organic solvent in the presence of a condensing agent, a condensation catalyst and a tertiary amine under condensation reaction conditions. In this case, R is obtained4The compound contains a 1 st functional group and a 2 nd functional group, wherein the 1 st functional group contains a hydroxyl protecting group, and the 2 nd functional group contains a compound represented by a formula (321) having a structure represented by a formula (C1').
The solid phase carrier is one of carriers used in solid phase synthesis of siRNA, some of which are well known to those skilled in the art. For example, the solid support may be selected from solid supports containing reactive hydroxyl or amino functional groups, and in some embodiments, the solid support is an amino resin or a hydroxyl resin. In some embodiments, the amino or hydroxyl resin has the following parameters: the grain diameter is 100-400 meshes (mesh), and the surface amino or hydroxyl loading is 0.2-0.5 mmol/g. The dosage ratio of the compound shown in the formula (321) to the solid phase carrier is 10-400 mu mol of the compound per gram of the solid phase carrier (mu mol/g). In some embodiments, the compound of formula (321) is present in an amount of 50 to 200. mu. mol/g relative to the solid support.
The organic solvent may be any suitable solvent or mixture of solvents known to those skilled in the art. In some embodiments, the organic solvent is one or more of acetonitrile, an epoxy-based solvent, an ether-based solvent, a haloalkane-based solvent, dimethyl sulfoxide, N-dimethylformamide, and N, N-diisopropylethylamine. In some embodiments, the epoxy-based solvent is dioxane and/or tetrahydrofuran, the ether-based solvent is diethyl ether and/or methyl tert-butyl ether, and the haloalkane-based solvent is one or more of dichloromethane, chloroform, and 1, 2-dichloroethane. In some embodiments, the organic solvent is acetonitrile. The organic solvent is used in an amount of 20 to 200L/mol, and in some embodiments, 50 to 100L/mol, relative to the compound represented by formula (321).
In some embodiments, the condensing agent may be benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), 3-diethoxyphosphoryl-1, 2, 3-benzoxazole 4(3H) -one (3- (Diethoxyphosphoryloxy) -1, 2, 3-benzotriazine-4 (3H) -one, dept), and/or O-benzotriazol-tetramethyluronium hexafluorophosphate (O-benzotriazol-1-yl-tetramethyluronium hexafluorophosphate), which in some embodiments is O-benzotriazol-tetramethyluronium hexafluorophosphate. The molar ratio of the condensing agent to the compound of formula (321) is 1: 1 to 20: 1, and in other embodiments 1: 1 to 5: 1.
In some embodiments, the tertiary amine is triethylamine and/or N, N-diisopropylethylamine, in some embodiments N, N-diisopropylethylamine; the molar ratio of the tertiary amine to the compound of formula (321) is from 1: 1 to 20: 1, and in some embodiments from 1: 1 to 5: 1.
In some embodiments, the method for preparing the compound represented by formula (321) may further comprise contacting the resulting condensation product with a capping reagent and an acylation catalyst in an organic solvent under capping reaction conditions to isolate the compound represented by formula (321). The capping reaction serves to remove any reactive functional groups that have not reacted to completion to avoid the production of unwanted by-products in subsequent reactions. The capping reaction conditions include a reaction temperature of 0 to 50 deg.C, in some embodiments 15 to 35 deg.C, and a reaction time of 1 to 10 hours, in some embodiments 3 to 6 hours. The capping reagent may be one used in solid phase synthesis of siRNA, and the capping reagent used in solid phase synthesis of siRNA is well known to those skilled in the art.
In some embodiments, the capping reagent consists of capping reagent 1(cap1) and capping reagent 2(cap2), wherein capping reagent 1 is N-methylimidazole, and in some embodiments is provided as a pyridine/acetonitrile mixed solution of N-methylimidazole, wherein the volume ratio of pyridine to acetonitrile is 1: 10 to 1: 1, and in some embodiments 1: 3 to 1: 1, and the volume ratio of the total volume of pyridine to acetonitrile to N-methylimidazole is 1: 1 to 10: 1, and in some embodiments 3: 1 to 7: 1. The capping reagent 2 is acetic anhydride. In some embodiments, the capping reagent 2 is provided as a solution of acetic anhydride in acetonitrile, wherein the volume ratio of acetic anhydride to acetonitrile is from 1: 1 to 1: 10, and in further embodiments from 1: 2 to 1: 6.
In some embodiments, the ratio of the volume of the pyridine/acetonitrile mixed solution of N-methylimidazole to the mass of the compound represented by formula (321) is 5ml/g to 50ml/g, and in some embodiments 15ml/g to 30 ml/g. The ratio of the volume of the acetonitrile solution of acetic anhydride to the mass of the compound represented by formula (321) is 0.5ml/g to 10ml/g, and in some embodiments 1ml/g to 5 ml/g.
In some embodiments, the capping reagent uses equimolar amounts of acetic anhydride and N-methylimidazole. In some embodiments, the organic solvent is one or more of acetonitrile, an epoxy-based solvent, an ether-based solvent, a haloalkane-based solvent, dimethyl sulfoxide, N-dimethylformamide, and N, N-diisopropylethylamine. In some embodiments, the organic solvent is acetonitrile. The organic solvent is used in an amount of 10 to 50L/mol, and in some embodiments, 5 to 30L/mol, relative to the compound represented by formula (321).
In some embodiments, the acylation catalyst may be selected from any catalyst useful for esterification condensation or amidation condensation, such as a basic heterocyclic compound. In some embodiments, the acylation catalyst is 4-dimethylaminopyridine. The mass ratio of the catalyst to the compound of formula (321) is from 0.001: 1 to 1: 1, and in some embodiments from 0.01: 1 to 0.1: 1.
In some embodiments, the compound represented by formula (321) may be isolated from the reaction mixture using any suitable separation method. In some embodiments, the compound represented by formula (321) may be obtained by washing well with an organic solvent selected from acetonitrile, dichloromethane, methanol, and in some embodiments acetonitrile, and filtering to remove unreacted reactants, excess capping reagent, and other impurities.
In some embodiments, a method of preparing a conjugate molecule of formula (321) comprises contacting a compound of formula (313) with a phosphoramidite in an organic solvent under coupling reaction conditions and in the presence of a coupling reagent, and isolating the compound of formula (321). In this case, R is obtained4The compound contains a 1 st functional group and a 2 nd functional group, wherein the 1 st functional group contains a hydroxyl protecting group, and the 2 nd functional group contains a compound represented by a formula (321) with a structure shown as a formula (C3).
In some embodiments, the coupling reaction conditions include a temperature that may range from 0 to 50 deg.C, such as from 15 to 35 deg.C, and a molar ratio of the compound represented by formula (313) to the phosphorodiamidite that may range from 1: 1 to 1: 50, such as from 1: 5 to 1: 15; the molar ratio of the compound represented by formula (313) to the coupling reagent may be from 1: 1 to 1: 100, for example from 1: 50 to 1: 80; the reaction time may be 200 to 3000 seconds, for example 500 to 1500 seconds. The phosphorodiamidite may be, for example, bis (diisopropylamino) (2-cyanoethoxy) phosphine, which is commercially available or synthesized according to a method well known in the art. The coupling reagent is one or more selected from 1H-tetrazole, 5-ethylthio 1H-tetrazole, and 5-benzylthio 1H-tetrazole, such as 5-ethylthio 1H-tetrazole. The coupling reaction can be carried out in an organic solvent selected from one or more of anhydrous acetonitrile, anhydrous DMF, and anhydrous dichloromethane, for example, anhydrous acetonitrile. In some embodiments, the organic solvent is used in an amount of 3 to 50L/mol, for example, 5 to 20L/mol, relative to the compound represented by formula (313). By carrying out this coupling reaction, the hydroxyl group in the compound represented by formula (313) reacts with the phosphoramidite to form a phosphoramidite group. In some embodiments, the solvent may be removed directly to provide a crude compound of formula (321) which may be used directly in a subsequent reaction.
In some embodiments, the method of preparing a compound represented by formula (321) further comprises the steps of: the isolated product is further contacted with a solid support comprising hydroxyl groups under coupling reaction conditions in an organic solvent and in the presence of a coupling reagent. Subsequently, the compound represented by the formula (321) is isolated by capping reaction and oxidation reaction. In this case, R is obtained4The compound contains a 1 st functional group and a 2 nd functional group, wherein the 1 st functional group contains a hydroxyl protecting group, and the 2 nd functional group has a structure shown as a formula (C3'), and is represented by a formula (321).
In some embodiments, the solid phase support is a solid phase support known in the art and useful for solid phase synthesis of nucleic acids, e.g., a commercially available general-purpose solid phase support after deprotection reaction (c)
Figure BDA0002498737070000772
HL UnyLinkerTM300 oligonucleotid Synthesis Support, Kinovate Life Sciences, having the structure shown in formula B80):
Figure BDA0002498737070000771
deprotection reactions are well known to those skilled in the art. In some embodiments, the deprotection conditions include a temperature of 0 to 50 ℃, e.g., 15 to 35 ℃; the reaction time is from 30 to 300 seconds, for example from 50 to 150 seconds. The deprotection agent may be selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, monochloroacetic acid, and in some embodiments, the deprotection agent is dichloroacetic acid. The molar ratio of deprotecting reagent to-DMTr (4, 4' -dimethoxytrityl) protecting group on the stationary phase is from 2: 1 to 100: 1, for example from 3: 1 to 50: 1. By carrying out the deprotection, free hydroxyl groups with reactivity are obtained on the surface of the solid phase carrier, so that subsequent coupling reaction is facilitated.
The coupling reaction conditions and the choice of coupling reagents may be as described above. By carrying out this coupling reaction, the free hydroxyl group formed in the deprotection reaction reacts with the phosphoramidite group to form a phosphite linkage.
In some embodiments, capping reaction conditions include a temperature of 0 to 50 ℃, e.g., 15 to 35 ℃, and a reaction time of 5 to 500 seconds, e.g., 10 to 100 seconds, the capping reaction being carried out in the presence of a capping reagent. The selection and amount of capping reagent may be as described above.
The oxidation reaction conditions include a temperature of from 0 to 50 deg.C, for example, from 15 to 35 deg.C, a reaction time of from 1 to 100 seconds, for example, from 5 to 50 seconds, and an oxidizing agent, for example, iodine (in some embodiments, provided in the form of iodine water). In some embodiments, the molar ratio of oxidizing agent to nucleic acid sequence attached to the solid support is from 1: 1 to 100: 1, and can be, for example, from 5: 1 to 50: 1. In some embodiments, the oxidation reaction is carried out in a mixed solvent of tetrahydrofuran, water, pyridine, 3: 1 to 1: 3.
In some embodiments, R6Is one of the groups of formula B7 or B8,
Figure BDA0002498737070000781
wherein q is2The definition of (a) is as described above,
in this case, the compound represented by formula (313) can be obtained by the following production method: contacting a compound represented by the formula (314) with a compound represented by the formula (A-1) or a compound represented by the formula (A-2) in an organic solvent under amidation reaction conditions in the presence of an amidation reaction condensing agent and a tertiary amine, followed by separation:
Figure BDA0002498737070000782
wherein, n1, n3, m1, m2, m3 and R10、R11、R12、R13、R14、R15、L1、S1、q2And RkThe respective definitions and alternative ranges are as described above.
The amidation reaction conditions may include a reaction temperature of 0 to 100 ℃ and a reaction time of 1 to 48 hours, and in some embodiments, the amidation reaction conditions are a reaction temperature of 10 to 40 ℃ and a reaction time of 2 to 16 hours.
In some embodiments, the organic solvent is one or more of an alcohol solvent, an epoxy solvent, an ether solvent, a halogenated alkane solvent, dimethyl sulfoxide, N-dimethylformamide, and N, N-diisopropylethylamine. The alcoholic solvent is in some embodiments one or more of methanol, ethanol, propanol, and in some embodiments ethanol. The epoxy-based solvent is dioxane and/or tetrahydrofuran in some embodiments. The ethereal solvent is, in some embodiments, diethyl ether and/or methyl tert-butyl ether. The haloalkane-based solvent is, in some embodiments, one or more of dichloromethane, trichloromethane and 1, 2-dichloroethane. In some embodiments, the organic solvent is dichloromethane. The amount of the organic solvent used is 3 to 50L/mol, and in a further embodiment 3 to 20L/mol, relative to the compound represented by formula (314).
In some embodiments, the amidation reaction condensing agent is benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, 3-diethoxyphosphoryl-1, 2, 3-benzazole-4 (3H) -one, 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride, 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline (EEDQ), or O-benzotriazol-tetramethyluronium hexafluorophosphate, and in further embodiments 3-diethoxyphosphoryl-1, 2, 3-benzazole-4 (3H) -one. The molar ratio of the amidation condensation agent to the compound of formula (314) may be from 1: 1 to 10: 1, and in some embodiments from 2.5: 1 to 5: 1.
In some embodiments, the tertiary amine is triethylamine or N, N-diisopropylethylamine, and in further embodiments is N, N-diisopropylethylamine. The molar ratio of the tertiary amine to the compound of formula (314) is from 3: 1 to 20: 1, and in some embodiments from 5: 1 to 10: 1.
In some embodiments, the compounds represented by formula (A-1) and formula (A-2) may be prepared by any suitable means. For example, when R iskIn the case of DMTr group, the compound represented by the formula (A-1) can be prepared by reacting calcium glycerate with DMTrCl; similarly, the compound represented by formula (A-2) can be prepared by first contacting 3-amino-1, 2-propanediol with a cyclic anhydride, which may be a cyclic anhydride having from 4 to 13 carbon atoms, and in some embodiments, from 4 to 8 carbon atoms, and then reacting with DMTrCl. As is readily understood by those skilled in the art, the cyclic anhydride is selected so as to correspond to q in the compound represented by (A-2)2Different values of (A), e.g. when the cyclic anhydride is succinic anhydride, q2When the cyclic anhydride is glutaric anhydride, q is 12And so on for 2.
In some variations, the compound represented by formula (313) may also be prepared by reacting the compound represented by formula (314) with the cyclic anhydride, 3-amino-1, 2-propanediol, and DMTrCl, in that order. It is easily understood by those skilled in the art that these modifications do not affect the structure and function of the compound represented by formula (313), and that these modifications are easily accomplished by those skilled in the art based on the above-described method.
Similarly to the above, the compound represented by formula (313) can be isolated from the reaction mixture using any suitable separation method. In some embodiments, the compound represented by formula (313) may be isolated by removing the solvent by evaporation followed by chromatographic methods, e.g., separation may be performed using two chromatographic conditions: (1) normal phase purification of silica gel: silica gel filler of 200-300 meshes is subjected to gradient elution by using petroleum ether, ethyl acetate, dichloromethane and N, and N-dimethylformamide is 1: 0.5-1: 0.6; and (2) reversed-phase purification: c18, C8 reversed phase packing, eluting with a gradient of methanol to acetonitrile 0.1: 1 to 1: 0.1. In some embodiments, the solvent may be removed directly to provide a crude compound of formula (313), which may be used directly in a subsequent reaction.
In some embodiments, the compound of formula (314) may be prepared by the following method: the method comprises the steps of contacting a compound shown as a formula (320) with a compound shown as a formula (316) in an organic solvent in the presence of an amidation reaction condensing agent and tertiary amine under a condensation reaction condition, and then separating:
S1-L1-OH
formula (316)
Figure BDA0002498737070000801
Wherein, n1, n3, m1, m2, m3 and R10、R11、R12、R13、R14、R15The respective definitions and alternative ranges are as described above.
The compounds represented by formula (316) may be prepared using, for example, the compounds disclosed in j.am. chem.soc.2014, 136, 169581-16961, or the compounds represented by formula (316) may be prepared by various methods by those skilled in the art, for example, certain compounds represented by formula (316) may be prepared by reference to the methods disclosed in US patent 8,106,022B 2, example 1, the entire contents of which are incorporated herein by reference in their entirety.
In some embodiments, the condensation reaction conditions include a reaction temperature of 0 to 100 ℃ and a reaction time of 0.1 to 24 hours, in some embodiments a reaction temperature of 10 to 40 ℃ and a reaction time of 0.5 to 16 hours.
In view of the structure of the desired product compound represented by formula (314), the molar ratio of the compound represented by formula (316) to the compound represented by formula (320) should be determined based on the sum of n1 and n3 in formula (320). In some embodiments, for example, when n1+ n3 is 3, the molar ratio of the compound of formula (316) to the compound of formula (320) may be 3: 1 to 3.5: 1, and in some embodiments 3.01: 1 to 3.15: 1, in order to ensure that the reaction is complete and not excessive.
In some embodiments, the organic solvent is one or more of acetonitrile, an epoxy-based solvent, in some embodiments dioxane and/or tetrahydrofuran, an ether-based solvent, in some embodiments diethyl ether and/or methyl tert-butyl ether, an ether-based solvent, in some embodiments one or more of dichloromethane, chloroform and 1, 2-dichloroethane, an alkyl halide-based solvent, in some embodiments dichloromethane, an ethyl halide-based solvent, in some embodiments dioxane, and N, N-diisopropylethylamine. The organic solvent is used in an amount of 3 to 50L/mol, and in some embodiments, 5 to 20L/mol, relative to the compound represented by formula (320).
In some embodiments, the amidation reaction condensing agent is one or more of benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate, 3-diethoxyphosphoryl-1, 2, 3-benzoxazole 4(3H) -one (DEPBT), O-benzotriazol-tetramethyluronium hexafluorophosphate, 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride, or 1-hydroxybenzotriazole, in a further embodiment a mixture of benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate and 1-hydroxybenzotriazole, wherein benzotriazole-1-yl-oxytriazolidinyl phosphonium hexafluorophosphate and 1-hydroxybenzotriazole are used in equimolar amounts. The molar ratio of the total amidation condensation agent to the compound of formula (316) may be from 1: 1 to 3: 1, and in some embodiments from 1.05: 1 to 1.5: 1.
The tertiary amine may be N-methylmorpholine, triethylamine or N, N-diisopropylethylamine, in some embodiments N-methylmorpholine; the molar ratio of the tertiary amine to the compound of formula (316) may be from 2: 1 to 10: 1, and in some embodiments from 2: 1 to 5: 1.
Similarly to the above, the compound represented by formula (314) may be isolated from the reaction mixture using any suitable separation method. In some embodiments, the compound represented by formula (314) may be isolated by removing the solvent by evaporation followed by chromatographic methods, e.g., the isolation may be performed using two chromatographic conditions: (1) normal phase purification of silica gel: silica gel filler of 200-300 meshes, and gradient elution is carried out by using dichloromethane and methanol at the ratio of 100: 5-100: 7; and (2) reversed-phase purification: c18, C8 reversed phase packing, eluting with a gradient of methanol to acetonitrile 0.1: 1 to 1: 0.1. In some embodiments, the solvent may be removed directly to provide a crude compound of formula (314), which may be used directly in a subsequent reaction.
The compound represented by formula (320) is commercially available or obtained by a person skilled in the art using known methods. For example, when m1 ═ m2 ═ m3 ═ 3, n1 ═ 1, n3 ═ 2, and each R is10、R11、R12、R13、R14、R15In the case of both H, the compound represented by formula (320) is commercially available from Afahesar.
The siRNA conjugates of the present disclosure may also be combined with other pharmaceutically acceptable excipients, which may be one or more of a variety of formulations or compounds conventionally employed in the art, for details see the description above for the pharmaceutical compositions of the present disclosure.
siRNA, pharmaceutical composition and application of siRNA conjugate of the disclosure
In some embodiments, there is provided a use of an siRNA and/or a pharmaceutical composition and/or an siRNA conjugate of the present disclosure in the manufacture of a medicament for the treatment and/or prevention of an iron overload complication.
In some embodiments, the present disclosure provides a method of preventing and/or treating iron overload complications comprising administering to a subject in need thereof an effective amount of an siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure.
By administering the siRNA active ingredients of the present disclosure to a subject in need thereof, the prevention and/or treatment of iron overload complications can be achieved through the mechanism of RNA interference. Accordingly, the siRNA and/or the pharmaceutical composition and/or the siRNA conjugate of the present disclosure may be used for preventing and/or treating iron overload complications, or for the preparation of a medicament for preventing and/or treating iron overload complications. In some embodiments, complications of iron overload include, but are not limited to, tissue cell damage and impaired organ function caused by excessive iron deposition in tissue organs. In some embodiments, the manifestations of iron overload complications include, but are not limited to, symptoms of heart failure, liver fibrosis, diabetes, infertility, growth and development disorders due to excessive iron deposition in the body.
The term "administering" as used herein refers to placing an siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure into a subject by a method or route that results in at least partially positioning the siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure at a desired site to produce a desired effect. Routes of administration suitable for the methods of the present disclosure include local administration and systemic administration. In general, topical administration results in delivery of more siRNA conjugate to a particular site as compared to the systemic circulation of the subject; whereas systemic administration results in delivery of the siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure to the subject's basal systemic circulation. In view of the present disclosure directed to providing a means for preventing and/or treating iron overload complications, in some embodiments a mode of administration capable of delivering the drug to the liver is employed.
Administration to a subject can be by any suitable route known in the art, including but not limited to: oral or parenteral routes, such as intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal and topical (including buccal and sublingual) administration. The frequency of administration may be 1 or more times per day, week, month, half or year.
The dosage of the siRNA, pharmaceutical composition or siRNA conjugate described in the present disclosure may be a dosage that is conventional in the art, and the dosage may be determined according to various parameters, particularly age, weight and sex of the subject. Toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining LD50(lethal dose to death of 50% of the population), ED50(in quantitative response, the dose which gives rise to 50% of the maximal response intensity, in qualitative response, the dose which gives rise to 50% of the positive responses in the test subjects) or IC50(concentration of inhibitor/drug at which the quantitative response is half inhibited). The range of human doses can be derived based on data obtained from cell culture analysis and animal studies.
In administering the sirnas, pharmaceutical compositions, and/or siRNA conjugates described in the present disclosure, for example, for male or female, C57BL/6J mice at 6-12 weeks of age, weighing 18-25g (sometimes referred to herein simply as "C57 mice"), the following are calculated as sirnas: (i) for siRNA conjugates, the amount of siRNA may range from 0.001 to 100mg/kg body weight, in some embodiments from 0.01 to 50mg/kg body weight, in some embodiments from 0.05 to 20mg/kg body weight, in other embodiments from 0.1 to 15mg/kg body weight, and in other embodiments from 0.1 to 10mg/kg body weight; (ii) for pharmaceutical compositions of siRNA and a pharmaceutically acceptable carrier, the amount of siRNA may be from 0.001 to 50mg/kg body weight, in some embodiments from 0.01 to 10mg/kg body weight, in some embodiments from 0.05 to 5mg/kg body weight, and in some embodiments, from 0.1 to 3mg/kg body weight.
In some embodiments, the present disclosure provides a method of inhibiting TMPRSS6 gene expression in a hepatocyte, the method comprising contacting the hepatocyte with an effective amount of an siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure, introducing the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure into the hepatocyte, for the purpose of inhibiting TMPRSS6 gene expression in the hepatocyte by a mechanism of RNA interference. The liver cell can be selected from liver cancer cell lines such as SMMC-7721, HepG2, Huh7 and the like or isolated primary liver cells. In some embodiments, the cell is a HEK293A cell or a human primary hepatocyte.
The modified siRNA, pharmaceutical composition and/or siRNA conjugate provided generally have an amount of siRNA that is such that, using the methods provided by the present disclosure to inhibit expression of the TMPRSS6 gene in a cell: it is sufficient to reduce the expression of the target gene and result in an extracellular concentration at the surface of the target cell of 1pM to 1 μ M, or 0.01nM to 100nM, or 0.05nM to 50nM or 0.05nM to about 5 nM. The amount required to achieve this local concentration will vary depending on a variety of factors including the method of delivery, the site of delivery, the number of cell layers between the site of delivery and the target cell or tissue, the route of delivery (local versus systemic), and the like. The concentration at the delivery site may be significantly higher than the concentration at the surface of the target cell or tissue.
Reagent kit
The present disclosure provides a kit comprising an effective amount of at least one of a modified siRNA of the present disclosure, a pharmaceutical composition, and an siRNA conjugate.
In some embodiments, the kits described herein can provide modified siRNA in one container. In some embodiments, a kit described herein may comprise one container providing a pharmaceutically acceptable excipient. In some embodiments, the kit may further comprise other ingredients, such as stabilizers or preservatives and the like. In some embodiments, the kits described herein can comprise at least one additional therapeutic agent in a container other than the container providing the modified siRNA described herein. In some embodiments, the kit may comprise instructions for mixing the modified siRNA with a pharmaceutically acceptable carrier and/or adjuvant or other ingredients (if any).
In the kits of the present disclosure, the modified siRNA and the pharmaceutically acceptable carrier and/or adjuvant and the modified siRNA, the pharmaceutical composition and/or the siRNA conjugate, and/or the pharmaceutically acceptable adjuvant may be provided in any form, such as a liquid form, a dried form or a lyophilized form. In some embodiments, the modified siRNA and pharmaceutically acceptable carrier and/or adjuvant and the pharmaceutical composition and/or siRNA conjugate and optionally pharmaceutically acceptable adjuvant are substantially pure and/or sterile. In some embodiments, sterile water may be provided in the kits of the present disclosure.
The present disclosure is further illustrated by the following examples, but is not to be construed as being limited thereby.
Examples
Unless otherwise specified, reagents and media used in the following examples are commercially available, and the procedures for nucleic acid electrophoresis, real-time PCR and the like used therein are performed by the methods described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)).
Unless otherwise stated, the reagent ratios provided below are calculated as volume ratios (v/v).
The experimental data are as follows
Figure BDA0002498737070000831
Data analysis was performed using Graphpad prism5.0 statistical analysis software.
Preparation example 1 preparation of Cy 5-conjugate 1
This preparation example synthesized Cy 5-conjugate 1, in which siRNA conjugates conjugated with sense and antisense strand sequences corresponding to Cy 5-conjugate 1 in table 3.
(1-1) Synthesis of L-10 Compound
The L-10 compound was synthesized according to the following method:
Figure BDA0002498737070000841
synthesis of conjugated end segment GAL-5
Figure BDA0002498737070000851
Synthesis of (1-1-1a) GAL-2
100.0g GAL-1 (N-acetyl-D-galactosamine hydrochloride, CAS number: 1772-03-8, available from Ningbo Honghong Biochemical company, 463.8mmol) was dissolved in 1000ml of anhydrous pyridine, 540ml of acetic anhydride (available from Enox company, 5565.6mmol) was added under ice-water bath, and the reaction was stirred at room temperature for 1.5 hours. Pouring the reaction solution into 10L of ice water, carrying out suction filtration under reduced pressure, washing a filter cake with 2L of ice water, adding an acetonitrile/toluene mixed solvent (the volume ratio of acetonitrile to toluene is 1: 1) until the acetonitrile/toluene is completely dissolved, and evaporating the solvent to dryness to obtain a white solid product GAL-2130.0 g.
Synthesis of (1-1-1b) GAL-3
GAL-2(35.1g, 90.0mmol) obtained in step (1-1-1a) was dissolved in 213ml of anhydrous 1, 2-dichloroethane, and 24.0g of TMSOTf (CAS No.: 27607-77-8, available from Michael corporation, 108.0mmol) was added under ice water bath and nitrogen protection, and reacted at room temperature overnight.
The reaction solution was diluted with 400ml of dichloromethane, filtered through celite, and then 1L of saturated aqueous sodium bicarbonate was added, stirred well, the organic phase was separated, the aqueous phase was extracted twice with 300ml of dichloroethane, the organic phases were combined, washed with 300ml of saturated aqueous sodium bicarbonate and 300ml of saturated brine, respectively, the organic phase was separated, dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness under reduced pressure to obtain light yellow viscous syrup product GAL-326.9 g.
(1-1-1c) Synthesis of GAL-4
GAL-3(26.9g, 81.7mmol) obtained in step (1-1-1b) was dissolved in 136ml of anhydrous 1, 2-dichloroethane, and dried
Figure BDA0002498737070000852
30g of molecular sieve powder was added, 9.0g of 5-hexen-1-ol (CAS number: 821-41-0, available from Adamas-beta, 89.9mmol) was added, and the mixture was stirred at room temperature for 30 minutes, and 9.08g of TMSOTf (40.9mmol) was added under ice bath and nitrogen protection, and the reaction was stirred at room temperature overnight. Filtering to remove
Figure BDA0002498737070000853
Molecular sieve powder, diluting the filtrate with 300ml dichloromethane, filtering with diatomaceous earth, and adding 500ml saturated sodium bicarbonate waterThe solution was stirred for 10 minutes and washed, the organic phase was separated, the aqueous phase was extracted once with 300ml of dichloroethane, the organic phases were combined and washed with 300ml of saturated aqueous sodium bicarbonate and 300ml of saturated brine, respectively, the organic phase was separated, dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness under reduced pressure to give GAL-441.3 g as a yellow syrup product, which was directly subjected to the next oxidation reaction without purification.
Synthesis of (1-1-1d) GAL-5
GAL-4(14.9g, 34.7 mmol) obtained by the method described in step (1-1-1c) was dissolved in a mixed solvent of 77ml of methylene chloride and 77ml of acetonitrile, 103ml of deionized water and 29.7g of sodium periodate (CAS No.: 7790-28-5, available from Alantin, 138.8mmol) were added, respectively, stirred for 10 minutes in an ice water bath, ruthenium trichloride (CAS No.: 14898-67-0, available from Annona, 238mg, 1.145mmol) was added, and the reaction was allowed to proceed overnight at room temperature. The reaction mixture was diluted with 300ml of water and stirred, saturated sodium bicarbonate was added to adjust the pH to about 7.5, the organic phase was separated and discarded, and the aqueous phase was extracted three times with 200ml portions of dichloromethane and the organic phase was discarded. The aqueous phase was adjusted to pH about 3 with citric acid solid, extracted three times with 200ml each time with dichloromethane, the organic phases combined, dried over anhydrous sodium sulfate and the solvent evaporated under reduced pressure to dryness to give GAL-56.85 g as a white foamy solid product.1H NMR(400MHz，DMSO)12.01(br，1H)，7.83(d，J＝9.2Hz，1H)，5.21(d，J＝3.2Hz，1H)，4.96(dd，J＝11.2，3.2Hz，1H)，4.49(d，J＝8.4Hz，1H)，4.07-3.95(m，3H)，3.92-3.85(m，1H)，3.74-3.67(m，1H)，3.48-3.39(m，1H)，2.20(t，J＝6.8Hz，2H)，2.11(s，3H)，2.00(s，3H)，1.90(s，3H)，1.77(s，3H)，1.55-1.45(m，4H).
(1-1-2) Synthesis of L-8:
Figure BDA0002498737070000861
j-0(9.886g, 52.5mmol, commercially available from Afahisat) and GAL-5(72.819g, 162.75mmol, from a combination of batches) obtained in step (1-1-1) were dissolved in 525ml of dichloromethane and diisopropylethylamine (DIEA, 44.782g, 346.50mmol), benzotriazol-1-yl-amine (BCG-BC-oxytriazolidinylphosphonium hexafluorophosphate (PyBOP, 90.158g, 173.25mmol) and hydroxybenzotriazole (HOBt, 23.410g, 173.25mmol) were reacted at room temperature for 4h, 20ml of saturated sodium bicarbonate and 200ml of saturated brine were added for washing, the aqueous phase was extracted 2 times with dichloromethane, 100ml each time, the organic phases were combined, dried over anhydrous sodium sulfate, filtered and the solvent was evaporated under reduced pressure to give the crude product. Purifying with 200-300 mesh normal phase silica gel, neutralizing silica gel acidity with 10 wt% triethylamine, balancing column with 1 wt% triethylamine, gradient eluting with dichloromethane and methanol at ratio of 100: 25-100: 40, collecting product eluate, and evaporating solvent under reduced pressure to obtain pure product L-838.8 g.1H NMR(400MHz，DMSO)7.84(d，J＝9.0Hz，3H)，7.27-7.23(m，1H)，7.13-7.18(m，1H)，5.22(d，J＝3.1Hz，3H)，4.97(dd，J＝11.3，3.1Hz，3H)，4.48(d，J＝8.4Hz，3H)，4.09-3.98(m，9H)，3.88(dd，J＝19.3，9.3Hz，3H)，3.75-3.66(m，3H)，3.44-3.38(m，3H)，3.17-3.30(m，4H)，3.10-2.97(m，4H)，2.35-2.20(m，6H)，2.15-2.08(m，9H)，2.07-1.98(m，13H)，1.94-1.87(m，9H)，1.81-1.74(m，9H)，1.65-1.42(m，18H).MS m/z：C85H119N7O30，[M+H]+Theory: 1477.59, actually measuring: 1477.23.
(1-1-3a) Synthesis of A-1
Figure BDA0002498737070000871
Dissolving DMTrCl (4, 4' -bis (methoxy) trityl chloride, 101.65g and 300mmol) in 1000ml of anhydrous pyridine, adding DL-calcium glycerate hydrate (28.63g and 100mmol), reacting at 45 ℃ for 20h, filtering the reaction solution, leaching the filter cake with 200ml of DCM, concentrating the filtrate under reduced pressure to dryness, re-dissolving the residue with 500ml of dichloromethane, washing with 0.5M triethylamine phosphate (pH 7-8) for 2 times and 200ml each time, extracting the aqueous phase with dichloromethane for 2 times and 200ml each time, combining the organic phases, drying with anhydrous sodium sulfate, filtering, evaporating the solvent under reduced pressure, purifying with 200-300 mesh normal phase silica gel column, eluting with a gradient of petroleum ether, ethyl acetate, dichloromethane and methanol of 1: 0.35-1: 0.55, collecting the product, washing and purifying with silica gel column of 200-300 meshRemoving liquid, evaporating the solvent under reduced pressure, dissolving 600ml dichloromethane again, washing with 200ml 0.5M triethylamine phosphate for 1 time, extracting the water phase with 200ml dichloromethane for 1 time, combining the organic phases, drying with anhydrous sodium sulfate, filtering, evaporating the solvent under reduced pressure, and reducing the pressure of a vacuum oil pump overnight to obtain a white solid product A-150.7 g.1H NMR(400MHz，DMSO-d6)7.46(ddd，J＝6.5，2.3，1.1Hz，1H)，7.40-7.28(m，7H)，6.89-6.81(m，4H)，4.84(d，J＝5.0Hz，1H)，4.36-4.24(m，1H)，4.29(s，6H)，3.92(dd，J＝12.4，7.0Hz，1H)，3.67(dd，J＝12.3，7.0Hz，1H)，2.52(q，J＝6.3Hz，6H)，1.03(t，J＝6.3Hz，9H).MS m/z：C24H23O6，[M-H]-Theory: 407.15, actually measuring: 406.92.
(1-1-3b) Synthesis of L-7:
Figure BDA0002498737070000872
mixing L-8(40g, 27.09mmol, obtained by combining several batches of product) obtained in step (1-1-2) and A-1(41.418g, 81.27mmol) obtained in step (1-1-3a), dissolving in 271ml of dichloromethane, adding 3-diethoxyphosphoryl-1, 2, 3-benzoxazole 4(3H) -one (DEPBT) (24.318g, 81.37mmol), adding diisopropylethylamine (21.007g, 162.54mmol), stirring at 25 ℃ for 1.5H, washing the organic phase with 800ml of saturated sodium bicarbonate, extracting the aqueous phase 3 times with dichloromethane, 50ml each time, the organic phase was washed with 150ml of saturated brine, the aqueous phase was extracted 1 time with 50ml of dichloromethane, the organic phases were combined and dried over anhydrous sodium sulfate, filtered and the solvent was evaporated under reduced pressure, foamed and dried overnight with a vacuum oil pump to give the crude product. The column purification uses 2kg of 200-300 mesh normal phase silica gel, the silica gel acidity is neutralized with 200ml of triethylamine, the column is equilibrated with petroleum ether containing 1 wt% of triethylamine, the gradient elution is carried out with petroleum ether, ethyl acetate, dichloromethane and N, N-dimethylformamide being 1: 0.5-1: 0.6, the product eluent is collected, and the solvent is evaporated to dryness under reduced pressure to obtain the pure product L-740.4 g. 1H NMR(400MHz，DMSO)7.90-7.78(m，4H)，7.75-7.64(m，1H)，7.38-7.18(m，9H)，6.91-6.83(m，4H)，5.25-5.10(m，4H)，4.97(dd，J＝11.2，3.2Hz，3H)，4.48-4.30(m，4H)，4.02(s，9H)，3.93-3.84(m，3H)，3.76-3.66(m，9H)，3.45-3.35(m，3H)，3.24-2.98(m，10H)，2.30-2.20(m，2H)，2.11-1.88(m，31H)，1.80-1.40(m，28H).MS m/z：C90H128N7O35，[M-DMTr]+Theory: 1564.65, actually measuring: 1564.88.
(1-1-4) Synthesis of L-9:
Figure BDA0002498737070000881
mixing L-7(40g, 21.4247mmol) obtained in step (1-1-3b), succinic anhydride (4.288g, 42.8494mmol) and 4-dimethylaminopyridine (DMAP, 5.235g, 42.8494mmol) and dissolving in 215ml of dichloromethane, adding diisopropylethylamine (DIEA, 13.845g, 107.1235mmol), stirring at 25 ℃ for 24h, washing the reaction solution with 800ml of 0.5M triethylamine phosphate, extracting the aqueous phase with dichloromethane 3 times, 5ml each time, combining the organic phases and evaporating to dryness under reduced pressure to obtain a crude product. The column purification uses 1kg of 200-300 mesh normal phase silica gel, 1 wt% of triethylamine is used for neutralizing the acidity of the silica gel, the column is balanced by dichloromethane, the product eluent is collected by gradient elution of dichloromethane containing 1 wt% of triethylamine and methanol which are 100: 18-100: 20, and the pure product L-9 conjugate molecule 31.0g is obtained by decompressing and evaporating the solvent.1H NMR(400MHz，DMSO)8.58(d，J＝4.2Hz，1H)，7.94-7.82(m，3H)，7.41-7.29(m，5H)，7.22(d，J＝8.1Hz，5H)，6.89(d，J＝8.3Hz，4H)，5.49-5.37(m，1H)，5.21(d，J＝3.0Hz，3H)，4.97(d，J＝11.1Hz，3H)，4.49(d，J＝8.2Hz，3H)，4.02(s，9H)，3.88(dd，J＝19.4，9.4Hz，3H)，3.77-3.65(m，9H)，3.50-3.39(m，6H)，3.11-2.90(m，5H)，2.61-2.54(m，4H)，2.47-2.41(m，2H)，2.26-2.17(m，2H)，2.15-1.95(m，22H)，1.92-1.84(m，9H)，1.80-1.70(m，10H)，1.65-1.35(m，17H)，1.31-1.19(m，4H)，0.96(t，J＝7.1Hz，9H).MS m/z：C94H132N7O38，[M-DMTr]+Theory: 1664.72, actually measuring: 1665.03.
(1-1-5) Synthesis of L-10 Compound:
Figure BDA0002498737070000891
in this step, the L-10 compound is prepared by attaching the L-9 conjugate molecule to a solid support.
Mixing the L-9 conjugated molecule (22.751g, 11mmol) obtained in step (1-1-4), O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU, 6.257g, 16.5mmol) and diisopropylethylamine (DIEA, 2.843g, 22mmol), dissolving in 900ml acetonitrile, stirring for 5 minutes at room temperature, adding aminomethyl resin (88g, 100-200 meshes, with an amino loading of 400 mu mol/g, purchased from Nankai Kagaku company), carrying out shaking table reaction at 25 ℃ at a rotation speed of 150 rpm, reacting for 18 hours, filtering, leaching a filter cake with DCM for 2 times (300 ml each time), leaching acetonitrile for 3 times (300 ml each time), drying for 18 hours by a vacuum oil pump, and then adding raw materials (CapA, CapB, 4-Dimethylaminopyridine (DMAP) and acetonitrile) according to the feeding ratio shown in Table 2 to carry out capping reaction. Placing the mixture on a shaking bed at 25 ℃, rotating at 150 revolutions per minute, reacting for 5 hours, filtering reaction liquid, leaching a filter cake for 3 times by using acetonitrile, wherein each time is 300ml, evaporating the solvent to dryness under reduced pressure, and drying overnight under reduced pressure by using a vacuum oil pump to obtain 102g of an L-10 compound (namely L-9 conjugated molecule connected with a solid phase carrier) with the loading capacity of 90.8 mu mol/g.
TABLE 2 charging ratio for cap reaction
Raw materials Dosage of Specification of Batch number Manufacturer of the product
CapA 1980ml —— —— ——
CapB 220ml —— —— ——
DMAP 1.100g Analytical purity 11422139 Aladdin
Acetonitrile 220ml Pure spectrum O15161001 Shanghai xing can
Wherein, the CapA and the CapB are capping reagent solutions, the CapA is a pyridine/acetonitrile mixed solution of 20 volume percent of N-methylimidazole, and the volume ratio of the pyridine to the acetonitrile is 3: 5; CapB is 20% acetic anhydride in acetonitrile.
(1-2) Synthesis of sense Strand of Cy 5-conjugate 1
Nucleoside monomers were linked one by one from the 3 '-5' direction according to the nucleotide arrangement order of the sense strand of siRNA corresponding to Cy 5-conjugate 1 in Table 3, using the initial cycle of the L-10 compound prepared in the above procedure by the solid phase phosphoramidite method. Each attachment of a nucleoside monomer involves a four-step reaction of deprotection, coupling, capping, oxidation or sulfurization. When two nucleotides are connected by adopting phosphate ester, and the next nucleoside monomer is connected, four-step reactions including deprotection, coupling, capping and oxidation are carried out. When two nucleotides are connected by phosphorothioate, and the next nucleoside monomer is connected, four-step reactions including deprotection, coupling, capping and sulfuration are carried out. The synthesis conditions are given as follows:
the nucleoside monomer was supplied as a 0.1M acetonitrile solution, the deprotection conditions were the same for each step, i.e., temperature was 25 deg.C, reaction time was 70 seconds, the deprotection reagent was dichloroacetic acid in dichloromethane (3% v/v), and the molar ratio of dichloroacetic acid to 4, 4' -dimethoxytrityl protecting group on the solid support was 5: 1.
The coupling reaction conditions in each step are the same, and the coupling reaction conditions comprise that the temperature is 25 ℃, the molar ratio of the nucleic acid sequence connected on the solid phase carrier to the nucleoside monomer is 1: 10, the molar ratio of the nucleic acid sequence connected on the solid phase carrier to the coupling reagent is 1: 65, the reaction time is 600 seconds, and the coupling reagent is 0.5M acetonitrile solution of 5-Ethylthio-1H-tetrazole (5- (ethyhio) -1H-tetrazole, ETT).
The capping conditions were the same for each step, including a temperature of 25 ℃ and a reaction time of 15 seconds. The capping reagent solution is a mixed solution of CapA and CapB with the molar ratio of 1: 1, and the molar ratio of the capping reagent to the nucleic acid sequence connected on the solid phase carrier is acetic anhydride, N-methylimidazole and the nucleic acid sequence connected on the solid phase carrier is 1: 1.
The oxidation reaction conditions in each step are the same, including the temperature of 25 ℃, the reaction time of 15 seconds, and the oxidizing agent of 0.05M iodine water. The molar ratio of iodine to nucleic acid sequence attached to the solid support in the coupling step is 30: 1. The reaction was carried out in a mixed solvent of tetrahydrofuran, water and pyridine at 3: 1.
The conditions of each step of sulfuration reaction are the same, including the temperature of 25 ℃, the reaction time of 300 seconds, and the sulfuration reagent of hydrogenated flavonol. The molar ratio of the sulfurizing reagent to the nucleic acid sequence attached to the solid support in the coupling step is 120: 1. The reaction was carried out in a mixed solvent of acetonitrile and pyridine at 1: 1.
After the last nucleoside monomer is connected, connecting a Cy5 phosphoramidite monomer (purchased from McMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcMcM.
Figure BDA0002498737070000901
The deprotection, coupling, capping and oxidation reaction conditions used for the attachment of the Cy5 phosphoramidite monomer to the 5' end of the sense strand are the same as described above, except that: 1) the deprotection reaction time is prolonged to 300 seconds; 2) the Cy5 coupling reaction time was extended to 900 seconds.
Cleavage and deprotection conditions were as follows: adding the synthesized nucleotide sequence connected with the carrier into an AMA solution (a mixed solution of 40 wt% methylamine water solution and 25 wt% ammonia water in a volume ratio of 1: 1), wherein the using amount of the AMA solution is 0.5 ml/mu mol, reacting for 2h under the condition of water bath at 25 ℃, filtering to remove the residual carrier, and concentrating the supernatant to be dry in vacuum.
The purification and desalting conditions were as follows: purification of nucleic acids was achieved by gradient elution with NaCl using a preparative ion chromatography purification column (Source 15Q). Specifically, the method comprises the following steps: eluent A: 20mM sodium phosphate (pH 8.1) in water/acetonitrile 9: 1 (volume ratio); eluent B: 1.5M sodium chloride, 20mM sodium phosphate (pH 8.1) in water/acetonitrile 9: 1 (volume ratio); elution gradient: eluting with eluent A and eluent B at ratio of 100: 0-50: 50. Collecting product eluates, mixing, desalting with reverse phase chromatography purification column, specifically desalting with Sephadex column, and eluting with deionized water, wherein the filler is Sephadex G25(Sephadex G25).
The detection method comprises the following steps: the purity of the sense strand was checked by ion exchange chromatography (IEX-HPLC) and the molecular weight was analyzed by liquid chromatography-mass spectrometry (LC-MS). Observed values are consistent with theoretical values, indicating that sense strand SS, 3' end conjugated to L-9 conjugate molecule, was synthesized.
(1-3) Synthesis of antisense strand of Cy 5-conjugate 1
By the solid phase phosphoramidite method, using a universal solid phase carrier (UnyLinker)TM loaded
Figure BDA0002498737070000911
HL Solid Supports, Kinovate Life Sciences) the antisense strand of Cy 5-conjugate 1 was synthesized in the nucleotide sequence of the antisense strand corresponding to the siRNA corresponding to Cy 5-conjugate 1 in table 3. Deprotection, coupling, capping, oxidation or sulfuration reaction conditions, cutting, deprotection, purification and desalting conditions in the solid phase phosphoramidite method are the same as those in the synthesis of a nucleoside monomer connected in a sense chain. Purity of the antisense strand was checked by ion exchange chromatography (IEX-HPLC) and molecular weight was analyzed by liquid chromatography-mass spectrometry (LC-MS). As a result, the observed value was matched with the theoretical value, indicating that the antisense strand AS having the target sequence was synthesized.
(1-4) Synthesis of Cy 5-conjugate 1
Respectively dissolving the sense strand and the antisense strand obtained in the steps (1-2) and (1-3) in water for injection to obtain a solution of 40mg/mL, mixing the solution in an equimolar ratio, heating the solution at 50 ℃ for 15min, cooling the solution at room temperature to obtain an annealed product, and freeze-drying the annealed product to obtain freeze-dried powder. After diluting the siRNA conjugate to a concentration of 0.2mg/mL using ultrapure water (Milli-Q ultrapure water meter, resistivity 18.2 M.OMEGA.. multidot.cm (25 ℃)), molecular weight measurement was performed using a Liquid chromatograph-Mass spectrometer (LC-MS, Liquid Chromatography-Mass Spectrometry, available from Waters, Inc., model: LCT Premier). The measured value is consistent with the theoretical value. The structure is shown as formula (403). The siRNA in Cy 5-conjugate 1 had the sense and antisense strand sequences corresponding to Cy 5-conjugate 1 shown in table 3.
Preparation example 2 preparation of Cy 5-conjugate 2 and conjugates 1-6
In the same manner as in preparation example 1, Cy 5-conjugate 2 was synthesized and subjected to molecular weight measurement, and the measured value was consistent with the theoretical value. Except that the siRNA according to the siRNA conjugate at the time of synthesis was a sequence having the sense strand and antisense strand corresponding to Cy 5-conjugate 2 shown in table 3.
Conjugates 1 to 6 were synthesized, respectively, in the same manner as in preparation example 1, except that (1) the sequences of siRNA based on which these siRNA conjugates were synthesized were the sequences of the sense strand and antisense strand of siRNA shown in table 3, which correspond to conjugates 1 to 6, in this order; (2) these conjugates do not contain the fluorophore Cy5 and therefore were prepared without the need for the step of attaching the Cy5 phosphoramidite monomer; (3) the cutting and deprotection conditions of the sense chain are different, and specifically comprise the following steps: the synthesized nucleotide sequence with the attached carrier was added to 25 wt% ammonia water in an amount of 0.5ml/μmol, reacted at 55 ℃ for 16 hours, the liquid was removed, and concentrated to dryness in vacuo. Molecular weight detection was performed on each of the synthesized conjugates 1 to 6, and found values were in agreement with theoretical values, indicating that siRNA conjugates having sequences of the sense strand and the antisense strand corresponding to the conjugates 1 to 6 in table 3, respectively, were obtained.
TABLE 3 siRNA conjugates
Figure BDA0002498737070000921
Figure BDA0002498737070000931
Wherein, the capital letters C, G, U, A represent the base composition of nucleotides; the lower case letter m indicates that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide; the lower case letter f indicates that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide; the lower case letter s indicates a phosphorothioate-based linkage between the two nucleotides to the left and right of the letter s.
Preparation example 3 Synthesis of siRNA sequences
The siRNA sequences listed in table 4 were synthesized separately by solid phase synthesis method, equimolar of the mutually complementary sense and antisense strands in table 4 were dissolved separately using DEPC water, followed by annealing to give siRNA1, siRNA2, siRNA3, siRNA4, siRNA5, siRNA6, and reference siRNA NC as provided by the present disclosure.
TABLE 4 siRNA sequences
Figure BDA0002498737070000932
Figure BDA0002498737070000941
Wherein, the capital letters C, G, U, A represent the base composition of nucleotides; the lower case letter m indicates that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide; the lower case letter f indicates that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide; the lower case letter s indicates that the phosphorothioate linkage is between the two nucleotides to the left and right of the letter s; dT represents thymidine.
Cy5-siRNA1 was prepared in the same manner as in the preparation of Cy 5-conjugate 1, except that a general solid carrier (UnyLinker) was usedTM loaded
Figure BDA0002498737070000942
HL Solid Supports, Kinovate Life Sciences company), synthesizes the sense strand of Cy5-siRNA 1. Deprotection, coupling, capping, oxidation or sulfurization reaction conditions, cleavage and deprotection, purification and desalting conditions in the solid phase synthesis method are the same as those for synthesizing the sense chain of Cy 5-conjugate 1.
Cy5-siRNA2 was prepared in the same manner as Cy5-siRNA1 except that the siRNA sequences according to the synthesis were the siRNA sequences corresponding to Cy5-siRNA2 as shown in Table 4.
The molecular weights of the synthesized Cy5-siRNA1 and Cy5-siRNA2 were measured, and the measured values were consistent with theoretical values, indicating that the synthesized siRNA was the expected siRNA with Cy5 fluorophore linked. The siRNA sequences in Cy5-siRNA1 and Cy5-siRNA2 are shown in Table 4.
The molecular weights of the above siRNAs were measured according to the method of preparation example 1, and the measured values were matched with theoretical values, confirming that the obtained siRNAs had sequences corresponding to the respective siRNAs shown in Table 4, respectively, and that the synthesized Cy5-siRNA1 and Cy5-siRNA2 were the expected Cy5 fluorophore-linked siRNAs.
Experimental example 1 target sequence inhibitory Activity of siRNA of the present disclosure in psiCHECK System
Complete medium (Hyclone) was prepared in H-DMEM supplemented with 20% fetal bovine serum (FBS, Hyclone) and 0.2% by volume of Streptomycin-Streptomycin (Hyclone) at 37 ℃ in 5% CO2HEK293A cells (purchased from Beijing Korea Biotech, Inc.) were cultured in a 95% air incubator.
Using psiCHECKTM-2(PromegaTM) The plasmids construct test plasmids, each of which contains a target sequence, i.e., an siRNA target sequence. For the siRNA to be evaluated, the target sequences are shown below:
the target sequence of siRNA1 is:
CCGCCAAAGCCCAGAAGAT(SEQ ID NO：383)
the target sequence of siRNA2 is:
AGGCACTGCTGGTGGAGGA(SEQ ID NO：384)
the target sequence of siRNA3 is:
GGAGGCAGAAGTATGATTT(SEQ ID NO：385)
the target sequence of siRNA4 is:
GAGTTCCTCTGTTCTGTGA(SEQ ID NO：386)
the target sequence of siRNA5 is:
GGATGTGCAGTTGATCCCA(SEQ ID NO：387)
the target sequence of siRNA6 is:
TAACTTGGGATCTGGGAAT(SEQ ID NO：388)
these target sequences are all sequence fragments of TMPRSS6 mRNA.
The target sequence of the reference siRNA NC is SEQ ID NO: 383 (b) under stringent conditions.
Cloning of target sequence to psiCHECKTM-Xho I/Not I site of plasmid 2.
HEK293A cells at 8X 103The cells/well were plated in 96-well plates, and when the cell growth density reached 70-80% after 16 hours, H-DMEM complete medium was aspirated from the wells, and 80. mu.l of Opti-MEM medium (GIBCO Co.) was added to each well to continue the culture for 1.5 hours.
For each siRNA, the corresponding test plasmid was diluted with DEPC water to 200 ng/. mu.l of test plasmid working solution. For each siRNA, siRNA working solutions were formulated with siRNA and DEPC water at concentrations (by siRNA) of 10nM, 3nM and 1nM, respectively.
For each siRNA, 1A1 solution was prepared, each 1A1 solution containing 1. mu.l of siRNA working solution at a concentration of 10nM, 0.05. mu.l of test plasmid working solution (containing 10ng of test plasmid), and 10. mu.l of Opti-MEM medium.
For each siRNA, 1A2 solution was prepared, each 1A2 solution containing 1. mu.l of siRNA working solution at a concentration of 3nM, 0.05. mu.l of test plasmid working solution (containing 10ng of test plasmid), and 10. mu.l of Opti-MEM medium.
For each siRNA, 1A3 solution was prepared, each 1A3 solution containing 1. mu.l of siRNA working solution at a concentration of 1nM, 0.05. mu.l of test plasmid working solution (containing 10ng of test plasmid), and 10. mu.l of Opti-MEM medium.
1B solution was prepared containing 0.2. mu.l of Lipofectamine per 1B solutionTM2000 and 10. mu.l of Opti-MEM medium.
1C solutions were prepared, each 1C solution containing 0.05. mu.l of working solution for the test plasmid (containing 10ng of test plasmid) and 10. mu.l of Opti-MEM medium.
For each siRNA, one portion of the 1B solution was mixed with one portion of the 1a1 solution, one portion of the 1a2 solution, and one portion of the 1A3 solution, respectively, and incubated at room temperature for 20min to obtain transfection complexes 1X1, 1X2, and 1X3, respectively, one portion of the 1B solution was mixed with one portion of the 1C solution, and incubated at room temperature for 20min to obtain transfection complex 1X 4.
For each siRNA, transfection complex 1X1 was added separately to each of the three culture wells and mixed well at 20 ul/well to give a final siRNA concentration of about 0.1nM of the co-transfection mixture containing siRNA, which was designated test group 1.
For each siRNA, in three additional culture wells, transfection complex 1X2 was added separately and mixed well at 20. mu.l/well to give a final siRNA concentration of about 0.03nM of the co-transfection mixture containing siRNA, designated test group 2.
For each siRNA, in three additional culture wells, transfection complex 1X3 was added separately and mixed well at 20. mu.l/well to give a final siRNA concentration of about 0.01nM of the siRNA-containing co-transfection mixture, designated test group 3.
In another three culture wells, the transfection complex 1X4 was added to obtain a transfection mixture without siRNA in an amount of 20. mu.l/well, which was designated as a control group.
After transfection of the siRNA-containing co-transfection mixture and siRNA-free transfection mixture for 4H in culture wells, each well was supplemented with 100. mu.l of H-DMEM complete medium containing 20% FBS. Place 96-well plates at 37 ℃ in 5% CO2The culture was continued for 24h in an incubator with 95% air.
The culture medium was aspirated from the culture wells and 150. mu.l of the medium was added to each well
Figure BDA0002498737070000961
Mixing Luciferase reagent and H-DMEM mixed solution (volume ratio is 1: 1), fully and uniformly mixing, after incubating for 10min at room temperature, transferring 120 mu l of mixed solution to each hole of a 96-hole enzyme label plate, and reading the chemiluminescence value (Fir) of Firefol in each culture hole on the 96-hole enzyme label plate by using a Synergy II multifunctional enzyme label instrument (BioTek company); then 60. mu.l of each well was added
Figure BDA0002498737070000962
Stop&
Figure BDA0002498737070000963
And (3) fully and uniformly mixing the reagents, incubating at room temperature for 10min, and reading chemiluminescence values (Ren) of Renilla in each culture hole on a 96-hole enzyme label plate by using an enzyme label instrument according to the arrangement mode of reading Fin.
Calculating the light-emitting Ratio Ren/Fin of each hole, wherein the light-emitting Ratio (test) or Ratio (control) of each test group or control group is the average value of the ratios of the three culture holes; the luminescence Ratio of each test group is normalized by taking the luminescence Ratio of the control group as a reference to obtain Ratio R of Ratio (test)/Ratio (control), so that the expression level of the Renilla reporter gene, namely the residual activity, is represented. The inhibition rate of the target sequence was (1-R) × 100%.
FIG. 1 shows the residual activity of Renilla reporter in HEK293A cells after transfection with siRNA1, siRNA2, siRNA3, siRNA4, siRNA5, siRNA6 or NC, respectively.
The results show that the siRNA1-6 of the present disclosure has better TMPRSS6 mRNA inhibitory activity, especially under the siRNA concentration of 0.1nM, the siRNA6 has the target sequence inhibition rate as high as 70.98%, which shows that the siRNA of the present disclosure shows good effect of inhibiting TMPRSS6 mRNA.
Experimental example 2siRNA conjugates were tested for their inhibitory efficiency against TMPRSS6 mRNA in human primary hepatocytes.
In 24-well plates, each 500. mu.L of OptiPlate Heatote Media (XENOTECH, K8200) was incubated at 37 ℃ in a medium containing 5% CO2Culture of human Primary hepatocytes H1000.H15C + (purchased from Shanghai Haoyang Biotech Co., Ltd.) in an incubator with 95% air at a cell count of 4X 10 per well5Cells/well. After 6h the medium was discarded and 500. mu.L OptiC. mu.Lture Hepatocyte Media (XENOTECH, K8300) culture medium was added to each well at 37 ℃ in 5% CO2Incubate overnight in a 95% air incubator.
The medium was discarded and 500. mu.L OptiC. mu.Lture Hepatocyte Media was added to each well.
The siRNA conjugates 1 to 6 in Table 3 and NC in Table 4 were prepared into siRNA conjugate working solutions and NC working solutions of 20. mu.M (in terms of siRNA) respectively with DEPC water.
A1A solution was prepared, and for each siRNA conjugate, a 1A solution was prepared, each 1A solution containing 1.5. mu.l of the above siRNA conjugate working solution and 50. mu.l of Opti-MEM medium (GIBCO Co., Ltd.) in this order.
1B solution was prepared, each 1B solution containing 1. mu.l LipofectamineTM2000 and 50. mu.l of Opti-MEM medium.
A1C solution was prepared, which contained 1.5. mu.l of NC working solution and 50. mu.l of Opti-MEM medium.
One portion of the 1B solution was mixed with the obtained 1A solution of each siRNA conjugate, respectively, and incubated at room temperature for 20min, respectively, to obtain 1X transfection complex for each siRNA conjugate.
One part of the 1B solution was mixed with one part of the 1C solution and incubated at room temperature for 20min to obtain a transfection complex 1X'.
In the culture well, 1X transfection complex of each siRNA conjugate was added separately and mixed uniformly in an amount of 100. mu.l/well to obtain a transfection complex with a final concentration (in terms of siRNA) of about 50nM for each siRNA conjugate, and the transfection mixture containing siRNA conjugates was obtained and designated as test group.
In another 1 culture well, add the transfection complex 1X', add 100. mu.l/well, get the transfection mixture containing the reference siRNA NC, which is marked as negative control group.
After transfection mixtures containing siRNA conjugates and transfection mixtures containing reference siRNA NC were transfected for 4h in culture wells, each well was supplemented with 1ml OptiC. mu. Ltube Hepatocyte Media medium. The 24-well plates were placed in an incubator containing 5% CO 2/95% air and incubated at 37 ℃ for an additional 24 h.
Subsequently, total RNA was extracted from each well cell using RNAVzol (available from wiggle biotechnology (beijing) ltd, product number N002) according to the method described in the specification to obtain solutions containing total RNA, respectively.
For each well of cells, a solution containing 1. mu.g of total RNA was taken separately, and a reverse transcription kit, golden star, was usedTMRT6 cDNA Synthesis Kit (available from New Biotechnology Ltd of Beijing Ongjingkong, cat # TSK301M) provided reagent, wherein Goldnstar was selectedTM Oligo(dT)17As a primer, 20. mu.l of a reverse transcription reaction system is prepared according to the reverse transcription operation steps in the kit specification, and the total RNA of each well cell is subjected to reverse transcription. The reverse transcription conditions were: for each reverse transcription reaction system, the reverse transcription reaction system is incubated at 50 ℃ for 50min, then at 85 ℃ for 5min, and finally at 4 ℃ for 30s, and after the reaction is finished, 80 μ l of DEPC water is added into the reverse transcription reaction system to obtain a solution containing cDNA.
For each reverse transcription reaction system, 5. mu.l of the above cDNA-containing solution was used as a template
Figure BDA0002498737070000981
Reagent configuration qPCR reactant provided by SYBR qPCR Supermix Plus kit (purchased from near shore protein science and technology Co., Ltd., product No. E096-01B)20 μ l of the strain, wherein the PCR primer sequences for amplifying the target gene TMPRSS6 and the internal reference gene GAPDH are shown in Table 5, and the final concentration of each primer is 0.25 μ M. And (3) placing each qPCR reaction system on an ABI StepOnePlus Real-Time PCR instrument, amplifying by using a three-step method, wherein the amplification procedure is pre-denaturation at 95 ℃ for 10min, then denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s, and repeating the denaturation, annealing and extension processes for 40 times to obtain a product W containing the amplified target gene TMPRSS6 and the internal reference gene GAPDH. And (3) incubating the product W at 95 ℃ for 15s, 60 ℃ for 1min and 95 ℃ for 15s in sequence, and respectively collecting the dissolution curves of the target gene TMPRSS6 and the internal reference gene GAPDH in the product W by using a real-time fluorescent quantitative PCR instrument to obtain the Ct values of the target gene TMPRSS6 and the internal reference gene GAPDH.
The above quantitative PCR assay was performed three times for each of the test group and the control group.
TABLE 5 primer information
Figure BDA0002498737070000982
The comparative Ct (delta. Ct) method is adopted to carry out relative quantitative calculation on the target gene TMPRSS6 in each test group, and the calculation method is as follows:
Δ Ct (test group) ═ Ct (test group target gene) -Ct (test group reference gene)
Δ Ct (control group) ═ Ct (control group target gene) -Ct (control group reference gene)
Δ Δ Ct (test group) ═ Δ Ct (test group) — Δ Ct (control group average)
Δ Δ Ct (control group) ═ Δ Ct (control group) - Δ Ct (control group average)
Wherein, the delta Ct (average of the control group) is the arithmetic mean of the delta Ct (average of the control group) measured by three times of detection of the control group. Thus, each test result corresponds to a Δ Δ Ct value for each test group and control group.
Normalizing the expression level of TMPRSS6 mRNA in the test group based on the control group to define the expression level of TMPRSS6 mRNA in the control group as 100%,
test group TMPRSS6 mRNA relativityExpression level 2Δ Δ Ct (test group)×100％
Test group TMPRSS6 mRNA inhibition rate (1-test group TMPRSS6 mRNA relative expression level) × 100%
Figure 2 is a bar graph of the relative expression levels of TMPRSS6 mRNA in human primary hepatocytes after sequential transfection with siRNA conjugates 1-6 of the present disclosure and reference siRNA NCs. Further, the inhibition rate of TMPRSS6 mRNA by each siRNA conjugate is summarized in table 6.
TABLE 6 inhibition of TMPRSS6 mRNA in human primary hepatocytes
Figure BDA0002498737070000983
Figure BDA0002498737070000991
As can be seen from the results of table 6, the siRNA conjugates provided by the present disclosure showed higher TMPRSS6 mRNA inhibitory activity in human primary hepatocytes, and at 50nM siRNA concentration, the TMPRSS6 mRNA inhibitory rate was at least 53.63%, and even up to 67.07%.
Experimental example 3 distribution of siRNA conjugates in organs of C57 mice
Cy5-siRNA1, Cy 5-conjugate 1, Cy5-siRNA2 or Cy 5-conjugate 2 were dissolved in a1 XPBS solution to a concentration of 0.6mg/ml (in terms of siRNA), respectively.
6C 57BL/6J female mice (purchased from Beijing Wintolite laboratory animal technology Co., Ltd.) aged 6-7 weeks were selected and randomly grouped, 2 mice per group were subcutaneously injected with 1 XPBS and the above-prepared solutions of Cy5-siRNA1, Cy 5-conjugate 1, Cy5-siRNA2 or Cy 5-conjugate 2, respectively, and all animals were dosed with 5ml/kg of mouse body weight in a volume of 3mg/kg of mouse body weight in terms of siRNA.
After 48h of administration, all mice were sacrificed and 5 organs of heart, lung, liver, spleen and kidney were removed from each mouse and subjected to fluorescence imaging in IVIS lumine Series III. One animal was selected from each group of mice, and the above 5 organs were arranged in the longitudinal direction in this order, and the organs of the mice to which 1 XPBS, Cy5-siRNA1 or Cy 5-conjugate 1 was administered were photographed in the same visual field, and as a result, as shown in FIG. 3A, the organs of the mice to which 1 XPBS, Cy5-siRNA2 or Cy 5-conjugate 2 was administered were photographed in the same visual field, and as a result, as shown in FIG. 3B.
As can be seen from fig. 3A and 3B, only Cy 5-conjugate 1 and Cy 5-conjugate 2 were able to aggregate in large amounts in the liver, Cy5-siRNA1 and Cy5-siRNA2 were able to aggregate in small amounts in the kidney, and were not aggregated in other organs, indicating that the siRNA conjugates of the present disclosure were able to efficiently deliver siRNA specifically to the liver; further, in combination with the results of experimental example 2, it was shown that the siRNA conjugates provided by the present disclosure can specifically inhibit the expression of TMPRSS6 gene in liver.
Some embodiments of the present disclosure are described in detail above, however, the present disclosure is not limited to the specific details in the above embodiments, and many simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all belong to the protection scope of the present disclosure.
It should be noted that, in some embodiments, the various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not further described.
In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.
Sequence listing
<110> Sa Ribo Biotechnology Ltd
<120> nucleic acid, pharmaceutical composition containing the same and siRNA conjugate, preparation method and application
<130> 17113RIBO-DJ
<150> 201910437175.1
<151> 2019-05-23
<160> 392
<170> SIPOSequenceListing 1.0
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<210> 127
<211> 21
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (21)..(21)
<223> n is a, u, g or c
<400> 127
gaggaggcag aaguaugauu n 21
<210> 128
<211> 23
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(1)
<223> n is a, u, g or c
<400> 128
naaucauacu ucugccuccu cag 23
<210> 129
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 129
ggaggcagaa guaugauuu 19
<210> 130
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 130
aaaucauacu ucugccuccu c 21
<210> 131
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 131
gaggaggcag aaguaugauu u 21
<210> 132
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 132
aaaucauacu ucugccuccu cag 23
<210> 133
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 133
ggaggcagaa guaugauuu 19
<210> 134
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 134
aaaucauacu ucugccuccu c 21
<210> 135
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 135
ggaggcagaa guaugauuu 19
<210> 136
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 136
aaaucauacu ucugccuccu c 21
<210> 137
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 137
ggaggcagaa guaugauuu 19
<210> 138
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 138
aaaucauacu ucugccuccu c 21
<210> 139
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 139
gaggaggcag aaguaugauu u 21
<210> 140
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 140
aaaucauacu ucugccuccu cag 23
<210> 141
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 141
gaggaggcag aaguaugauu u 21
<210> 142
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 142
aaaucauacu ucugccuccu cag 23
<210> 143
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 143
gaggaggcag aaguaugauu u 21
<210> 144
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 144
aaaucauacu ucugccuccu cag 23
<210> 145
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 145
ggaggcagaa guaugauuu 19
<210> 146
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 146
aaaucauacu ucugccuccu c 21
<210> 147
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 147
ggaggcagaa guaugauuu 19
<210> 148
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 148
aaaucauacu ucugccuccu c 21
<210> 149
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 149
ggaggcagaa guaugauuu 19
<210> 150
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 150
aaaucauacu ucugccuccu c 21
<210> 151
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 151
gaggaggcag aaguaugauu u 21
<210> 152
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 152
aaaucauacu ucugccuccu cag 23
<210> 153
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 153
gaggaggcag aaguaugauu u 21
<210> 154
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 154
aaaucauacu ucugccuccu cag 23
<210> 155
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 155
gaggaggcag aaguaugauu u 21
<210> 156
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 156
aaaucauacu ucugccuccu cag 23
<210> 157
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 157
ggaggcagaa guaugauuu 19
<210> 158
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 158
aaaucauacu ucugccuccu c 21
<210> 159
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 159
ggaggcagaa guaugauuu 19
<210> 160
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 160
aaaucauacu ucugccuccu c 21
<210> 161
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 161
ggaggcagaa guaugauuu 19
<210> 162
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 162
aaaucauacu ucugccuccu c 21
<210> 163
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 163
gaggaggcag aaguaugauu u 21
<210> 164
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 164
aaaucauacu ucugccuccu cag 23
<210> 165
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 165
gaggaggcag aaguaugauu u 21
<210> 166
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 166
aaaucauacu ucugccuccu cag 23
<210> 167
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 167
gaggaggcag aaguaugauu u 21
<210> 168
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 168
aaaucauacu ucugccuccu cag 23
<210> 169
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 169
ggaggcagaa guaugauuu 19
<210> 170
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 170
aaaucauacu ucugccuccu c 21
<210> 171
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 171
ggaggcagaa guaugauuu 19
<210> 172
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 172
aaaucauacu ucugccuccu c 21
<210> 173
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 173
ggaggcagaa guaugauuu 19
<210> 174
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 174
aaaucauacu ucugccuccu c 21
<210> 175
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 175
gaggaggcag aaguaugauu u 21
<210> 176
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 176
aaaucauacu ucugccuccu cag 23
<210> 177
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 177
gaggaggcag aaguaugauu u 21
<210> 178
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 178
aaaucauacu ucugccuccu cag 23
<210> 179
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 179
gaggaggcag aaguaugauu u 21
<210> 180
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 180
aaaucauacu ucugccuccu cag 23
<210> 181
<211> 19
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (19)..(19)
<223> n is a
<400> 181
gaguuccucu guucugugn 19
<210> 182
<211> 19
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(1)
<223> n is u
<400> 182
ncacagaaca gaggaacuc 19
<210> 183
<211> 19
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (19)..(19)
<223> n is a, u, g or c
<400> 183
gaguuccucu guucugugn 19
<210> 184
<211> 19
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(1)
<223> n is a, u, g or c
<400> 184
ncacagaaca gaggaacuc 19
<210> 185
<211> 19
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (19)..(19)
<223> n is a, u, g or c
<400> 185
gaguuccucu guucugugn 19
<210> 186
<211> 21
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(1)
<223> n is a, u, g or c
<400> 186
ncacagaaca gaggaacucu c 21
<210> 187
<211> 21
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (21)..(21)
<223> n is a, u, g or c
<400> 187
gagaguuccu cuguucugug n 21
<210> 188
<211> 23
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(1)
<223> n is a, u, g or c
<400> 188
ncacagaaca gaggaacucu cca 23
<210> 189
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 189
gaguuccucu guucuguga 19
<210> 190
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 190
ucacagaaca gaggaacucu c 21
<210> 191
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 191
gagaguuccu cuguucugug a 21
<210> 192
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 192
ucacagaaca gaggaacucu cca 23
<210> 193
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 193
gaguuccucu guucuguga 19
<210> 194
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 194
ucacagaaca gaggaacucu c 21
<210> 195
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 195
gaguuccucu guucuguga 19
<210> 196
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 196
ucacagaaca gaggaacucu c 21
<210> 197
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 197
gaguuccucu guucuguga 19
<210> 198
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 198
ucacagaaca gaggaacucu c 21
<210> 199
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 199
gagaguuccu cuguucugug a 21
<210> 200
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 200
ucacagaaca gaggaacucu cca 23
<210> 201
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 201
gagaguuccu cuguucugug a 21
<210> 202
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 202
ucacagaaca gaggaacucu cca 23
<210> 203
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 203
gagaguuccu cuguucugug a 21
<210> 204
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 204
ucacagaaca gaggaacucu cca 23
<210> 205
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 205
gaguuccucu guucuguga 19
<210> 206
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 206
ucacagaaca gaggaacucu c 21
<210> 207
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 207
gaguuccucu guucuguga 19
<210> 208
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 208
ucacagaaca gaggaacucu c 21
<210> 209
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 209
gaguuccucu guucuguga 19
<210> 210
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 210
ucacagaaca gaggaacucu c 21
<210> 211
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 211
gagaguuccu cuguucugug a 21
<210> 212
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 212
ucacagaaca gaggaacucu cca 23
<210> 213
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 213
gagaguuccu cuguucugug a 21
<210> 214
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 214
ucacagaaca gaggaacucu cca 23
<210> 215
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 215
gagaguuccu cuguucugug a 21
<210> 216
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 216
ucacagaaca gaggaacucu cca 23
<210> 217
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 217
gaguuccucu guucuguga 19
<210> 218
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 218
ucacagaaca gaggaacucu c 21
<210> 219
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 219
gaguuccucu guucuguga 19
<210> 220
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 220
ucacagaaca gaggaacucu c 21
<210> 221
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 221
gaguuccucu guucuguga 19
<210> 222
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 222
ucacagaaca gaggaacucu c 21
<210> 223
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 223
gagaguuccu cuguucugug a 21
<210> 224
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 224
ucacagaaca gaggaacucu cca 23
<210> 225
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 225
gagaguuccu cuguucugug a 21
<210> 226
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 226
ucacagaaca gaggaacucu cca 23
<210> 227
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 227
gagaguuccu cuguucugug a 21
<210> 228
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 228
ucacagaaca gaggaacucu cca 23
<210> 229
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 229
gaguuccucu guucuguga 19
<210> 230
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 230
ucacagaaca gaggaacucu c 21
<210> 231
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 231
gaguuccucu guucuguga 19
<210> 232
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 232
ucacagaaca gaggaacucu c 21
<210> 233
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 233
gaguuccucu guucuguga 19
<210> 234
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 234
ucacagaaca gaggaacucu c 21
<210> 235
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 235
gagaguuccu cuguucugug a 21
<210> 236
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 236
ucacagaaca gaggaacucu cca 23
<210> 237
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 237
gagaguuccu cuguucugug a 21
<210> 238
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 238
ucacagaaca gaggaacucu cca 23
<210> 239
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 239
gagaguuccu cuguucugug a 21
<210> 240
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 240
ucacagaaca gaggaacucu cca 23
<210> 241
<211> 19
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (19)..(19)
<223> n is a
<400> 241
ggaugugcag uugaucccn 19
<210> 242
<211> 19
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(1)
<223> n is u
<400> 242
ngggaucaac ugcacaucc 19
<210> 243
<211> 19
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (19)..(19)
<223> n is a, u, g or c
<400> 243
ggaugugcag uugaucccn 19
<210> 244
<211> 19
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(1)
<223> n is a, u, g or c
<400> 244
ngggaucaac ugcacaucc 19
<210> 245
<211> 19
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (19)..(19)
<223> n is a, u, g or c
<400> 245
ggaugugcag uugaucccn 19
<210> 246
<211> 21
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(1)
<223> n is a, u, g or c
<400> 246
ngggaucaac ugcacaucca c 21
<210> 247
<211> 21
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (21)..(21)
<223> n is a, u, g or c
<400> 247
guggaugugc aguugauccc n 21
<210> 248
<211> 23
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(1)
<223> n is a, u, g or c
<400> 248
ngggaucaac ugcacaucca cuu 23
<210> 249
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 249
ggaugugcag uugauccca 19
<210> 250
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 250
ugggaucaac ugcacaucca c 21
<210> 251
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 251
guggaugugc aguugauccc a 21
<210> 252
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 252
ugggaucaac ugcacaucca cuu 23
<210> 253
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 253
ggaugugcag uugauccca 19
<210> 254
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 254
ugggaucaac ugcacaucca c 21
<210> 255
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 255
ggaugugcag uugauccca 19
<210> 256
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 256
ugggaucaac ugcacaucca c 21
<210> 257
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 257
ggaugugcag uugauccca 19
<210> 258
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 258
ugggaucaac ugcacaucca c 21
<210> 259
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 259
guggaugugc aguugauccc a 21
<210> 260
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 260
ugggaucaac ugcacaucca cuu 23
<210> 261
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 261
guggaugugc aguugauccc a 21
<210> 262
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 262
ugggaucaac ugcacaucca cuu 23
<210> 263
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 263
guggaugugc aguugauccc a 21
<210> 264
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 264
ugggaucaac ugcacaucca cuu 23
<210> 265
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 265
ggaugugcag uugauccca 19
<210> 266
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 266
ugggaucaac ugcacaucca c 21
<210> 267
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 267
ggaugugcag uugauccca 19
<210> 268
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 268
ugggaucaac ugcacaucca c 21
<210> 269
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 269
ggaugugcag uugauccca 19
<210> 270
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 270
ugggaucaac ugcacaucca c 21
<210> 271
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 271
guggaugugc aguugauccc a 21
<210> 272
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 272
ugggaucaac ugcacaucca cuu 23
<210> 273
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 273
guggaugugc aguugauccc a 21
<210> 274
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 274
ugggaucaac ugcacaucca cuu 23
<210> 275
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 275
guggaugugc aguugauccc a 21
<210> 276
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 276
ugggaucaac ugcacaucca cuu 23
<210> 277
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 277
ggaugugcag uugauccca 19
<210> 278
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 278
ugggaucaac ugcacaucca c 21
<210> 279
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 279
ggaugugcag uugauccca 19
<210> 280
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 280
ugggaucaac ugcacaucca c 21
<210> 281
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 281
ggaugugcag uugauccca 19
<210> 282
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 282
ugggaucaac ugcacaucca c 21
<210> 283
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 283
guggaugugc aguugauccc a 21
<210> 284
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 284
ugggaucaac ugcacaucca cuu 23
<210> 285
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 285
guggaugugc aguugauccc a 21
<210> 286
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 286
ugggaucaac ugcacaucca cuu 23
<210> 287
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 287
guggaugugc aguugauccc a 21
<210> 288
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 288
ugggaucaac ugcacaucca cuu 23
<210> 289
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 289
ggaugugcag uugauccca 19
<210> 290
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 290
ugggaucaac ugcacaucca c 21
<210> 291
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 291
ggaugugcag uugauccca 19
<210> 292
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 292
ugggaucaac ugcacaucca c 21
<210> 293
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 293
ggaugugcag uugauccca 19
<210> 294
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 294
ugggaucaac ugcacaucca c 21
<210> 295
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 295
guggaugugc aguugauccc a 21
<210> 296
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 296
ugggaucaac ugcacaucca cuu 23
<210> 297
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 297
guggaugugc aguugauccc a 21
<210> 298
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 298
ugggaucaac ugcacaucca cuu 23
<210> 299
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 299
guggaugugc aguugauccc a 21
<210> 300
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 300
ugggaucaac ugcacaucca cuu 23
<210> 301
<211> 19
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(1)
<223> z is u
<220>
<221> misc_feature
<222> (19)..(19)
<223> n is a, c, g, t or u
<400> 301
uaacuuggga ucugggaan 19
<210> 302
<211> 19
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(1)
<223> z is a
<400> 302
nuucccagau cccaaguua 19
<210> 303
<211> 19
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (19)..(19)
<223> n is a, u, g or c
<400> 303
uaacuuggga ucugggaan 19
<210> 304
<211> 19
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(1)
<223> n is a, u, g or c
<400> 304
nuucccagau cccaaguua 19
<210> 305
<211> 19
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (19)..(19)
<223> n is a, u, g or c
<400> 305
uaacuuggga ucugggaan 19
<210> 306
<211> 21
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(1)
<223> n is a, u, g or c
<400> 306
nuucccagau cccaaguuag a 21
<210> 307
<211> 21
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (21)..(21)
<223> n is a, u, g or c
<400> 307
ucuaacuugg gaucugggaa n 21
<210> 308
<211> 23
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(1)
<223> n is a, u, g or c
<400> 308
nuucccagau cccaaguuag acc 23
<210> 309
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 309
uaacuuggga ucugggaau 19
<210> 310
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 310
auucccagau cccaaguuag a 21
<210> 311
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 311
ucuaacuugg gaucugggaa u 21
<210> 312
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 312
auucccagau cccaaguuag acc 23
<210> 313
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 313
uaacuuggga ucugggaau 19
<210> 314
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 314
auucccagau cccaaguuag a 21
<210> 315
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 315
uaacuuggga ucugggaau 19
<210> 316
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 316
auucccagau cccaaguuag a 21
<210> 317
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 317
uaacuuggga ucugggaau 19
<210> 318
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 318
auucccagau cccaaguuag a 21
<210> 319
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 319
ucuaacuugg gaucugggaa u 21
<210> 320
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 320
auucccagau cccaaguuag acc 23
<210> 321
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 321
ucuaacuugg gaucugggaa u 21
<210> 322
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 322
auucccagau cccaaguuag acc 23
<210> 323
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 323
ucuaacuugg gaucugggaa u 21
<210> 324
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 324
auucccagau cccaaguuag acc 23
<210> 325
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 325
uaacuuggga ucugggaau 19
<210> 326
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 326
auucccagau cccaaguuag a 21
<210> 327
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 327
uaacuuggga ucugggaau 19
<210> 328
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 328
auucccagau cccaaguuag a 21
<210> 329
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 329
uaacuuggga ucugggaau 19
<210> 330
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 330
auucccagau cccaaguuag a 21
<210> 331
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 331
ucuaacuugg gaucugggaa u 21
<210> 332
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 332
auucccagau cccaaguuag acc 23
<210> 333
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 333
ucuaacuugg gaucugggaa u 21
<210> 334
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 334
auucccagau cccaaguuag acc 23
<210> 335
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 335
ucuaacuugg gaucugggaa u 21
<210> 336
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 336
auucccagau cccaaguuag acc 23
<210> 337
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 337
uaacuuggga ucugggaau 19
<210> 338
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 338
auucccagau cccaaguuag a 21
<210> 339
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 339
uaacuuggga ucugggaau 19
<210> 340
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 340
auucccagau cccaaguuag a 21
<210> 341
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 341
uaacuuggga ucugggaau 19
<210> 342
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 342
auucccagau cccaaguuag a 21
<210> 343
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 343
ucuaacuugg gaucugggaa u 21
<210> 344
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 344
auucccagau cccaaguuag acc 23
<210> 345
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 345
ucuaacuugg gaucugggaa u 21
<210> 346
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 346
auucccagau cccaaguuag acc 23
<210> 347
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 347
ucuaacuugg gaucugggaa u 21
<210> 348
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 348
auucccagau cccaaguuag acc 23
<210> 349
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 349
uaacuuggga ucugggaau 19
<210> 350
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 350
auucccagau cccaaguuag a 21
<210> 351
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 351
uaacuuggga ucugggaau 19
<210> 352
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 352
auucccagau cccaaguuag a 21
<210> 353
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 353
uaacuuggga ucugggaau 19
<210> 354
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 354
auucccagau cccaaguuag a 21
<210> 355
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 355
ucuaacuugg gaucugggaa u 21
<210> 356
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 356
auucccagau cccaaguuag acc 23
<210> 357
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 357
ucuaacuugg gaucugggaa u 21
<210> 358
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 358
auucccagau cccaaguuag acc 23
<210> 359
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 359
ucuaacuugg gaucugggaa u 21
<210> 360
<211> 23
<212> RNA
<213> Artificial Sequence
<400> 360
auucccagau cccaaguuag acc 23
<210> 361
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 361
ccgccaaagc ccagaagau 19
<210> 362
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 362
aucuucuggg cuuuggcggu u 21
<210> 363
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 363
uaacuuggga ucugggaau 19
<210> 364
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 364
auucccagau cccaaguuag a 21
<210> 365
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 365
ccgccaaagc ccagaagau 19
<210> 366
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 366
aucuucuggg cuuuggcggu u 21
<210> 367
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 367
aggcacugcu gguggagga 19
<210> 368
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 368
uccuccacca gcagugccug c 21
<210> 369
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 369
ggaggcagaa guaugauuu 19
<210> 370
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 370
aaaucauacu ucugccuccu c 21
<210> 371
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 371
gaguuccucu guucuguga 19
<210> 372
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 372
ucacagaaca gaggaacucu c 21
<210> 373
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 373
ggaugugcag uugauccca 19
<210> 374
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 374
ugggaucaac ugcacaucca c 21
<210> 375
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 375
uaacuuggga ucugggaau 19
<210> 376
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 376
auucccagau cccaaguuag a 21
<210> 377
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 377
ccgccaaagc ccagaagau 19
<210> 378
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 378
aucuucuggg cuuuggcggu u 21
<210> 379
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 379
uaacuuggga ucugggaau 19
<210> 380
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 380
auucccagau cccaaguuag a 21
<210> 381
<211> 19
<212> RNA
<213> Artificial Sequence
<400> 381
uucuccgaac gugucacgu 19
<210> 382
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 382
acgugacacg uucggagaac u 21
<210> 384
<211> 19
<212> DNA/RNA
<213> Artificial Sequence
<400> 384
ccgccaaagc ccagaagat 19
<210> 385
<211> 19
<212> DNA/RNA
<213> Artificial Sequence
<400> 385
aggcactgct ggtggagga 19
<210> 385
<211> 19
<212> DNA/RNA
<213> Artificial Sequence
<400> 385
ggaggcagaa gtatgattt 19
<210> 386
<211> 19
<212> DNA/RNA
<213> Artificial Sequence
<400> 386
gagttcctct gttctgtga 19
<210> 387
<211> 19
<212> DNA/RNA
<213> Artificial Sequence
<400> 387
ggatgtgcag ttgatccca 19
<210> 388
<211> 19
<212> DNA/RNA
<213> Artificial Sequence
<400> 388
taacttggga tctgggaat 19
<210> 389
<211> 20
<212> DNA/RNA
<213> Artificial Sequence
<400> 389
gacgctggac aacaggctcg 20
<210> 390
<211> 22
<212> DNA/RNA
<213> Artificial Sequence
<400> 390
tcctcagtgc ataggcatca aa 22
<210> 391
<211> 19
<212> DNA/RNA
<213> Artificial Sequence
<400> 391
ggtcggagtc aacggattt 19
<210> 392
<211> 19
<212> DNA/RNA
<213> Artificial Sequence
<400> 392
ccagcatcgc cccacttga 19

Claims (14)

1. An siRNA conjugate, wherein said siRNA conjugate has a structure represented by formula (308):
Figure FDA0002498737060000011
wherein the content of the first and second substances,
n1 is an integer selected from 1 to 3, n3 is an integer selected from 0 to 4; each m1, m2, or m3 is independently an integer selected from 2 to 10; r10、R11、R12、R13、R14Or R15Each independently is H, or is selected from the group consisting of: c1-C10Alkyl radical, C1-C10Haloalkyl and C1-C10An alkoxy group;
R3a group of the structure shown in formula a 59:
Figure FDA0002498737060000012
wherein E is1Is OH, SH or BH2，
Nu is siRNA, the siRNA comprises a sense strand and an antisense strand, each nucleotide in the siRNA is a modified or unmodified nucleotide independently, wherein the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially complementary reversely to form a double-stranded region, and the nucleotide sequence I and the nucleotide sequence II are selected from one group of sequences shown in the following I) to vi):
i) the nucleotide sequence I is similar to SEQ ID NO: 1 is equal in length and differs by NO more than 3 nucleotides, and the nucleotide sequence II is identical to SEQ ID NO: 2 are equal in length and differ by no more than 3 nucleotides:
5′-CCGCCAAAGCCCAGAAGAZ1-3′(SEQ ID NO：1)；
5′-Z2UCUUCUGGGCUUUGGCGG-3′(SEQ ID NO：2)，
wherein Z is1Is U, Z2Is A, the nucleotide sequence I comprises a position corresponding to Z1Nucleotide Z of3The nucleotide sequence II comprises a position corresponding to Z2Nucleotide Z of4Z is the same as4Is the first nucleotide at the 5' end of the antisense strand;
ii) the nucleotide sequence I is identical to SEQ ID NO: 61 and NO more than 3 nucleotides different, and the nucleotide sequence II is identical to the nucleotide sequence shown in SEQ ID NO: 62 are equal in length and differ by no more than 3 nucleotides:
5′-AGGCACUGCUGGUGGAGGZ5-3′(SEQ ID NO：61)；
5′-Z6CCUCCACCAGCAGUGCCU-3′(SEQ ID NO：62)，
wherein Z is5Is A, Z6Is U, the nucleotide sequence I comprises a position corresponding to Z5Nucleotide Z of7The nucleotide sequence II comprises a position corresponding to Z6Nucleotide Z of8Z is the same as8Is the first nucleotide at the 5' end of the antisense strand;
iii) the nucleotide sequence I has the sequence shown in SEQ ID NO: 121 and NO more than 3 nucleotides different, and the nucleotide sequence II is identical to SEQ ID NO: 122 are equal in length and differ by no more than 3 nucleotides:
5′-GGAGGCAGAAGUAUGAUUZ9-3′(SEQ ID NO：121)；
5′-Z10AAUCAUACUUCUGCCUCC-3′(SEQ ID NO：122)，
wherein Z is9Is U, Z10Is A, the nucleotide sequence I comprises a position corresponding to Z9Nucleotide Z of11The nucleotide sequence II comprises a position corresponding to Z10Nucleotide Z of12Z is the same as12Is the first nucleotide at the 5' end of the antisense strand;
iv) the nucleotide sequence I has the sequence shown in SEQ ID NO: 181 and NO more than 3 nucleotides different, and the nucleotide sequence II is identical to SEQ ID NO: 182 are equal in length and differ by no more than 3 nucleotides:
5′-GAGUUCCUCUGUUCUGUGZ13-3′(SEQ ID NO：181)；
5′-Z14CACAGAACAGAGGAACUC-3′(SEQ ID NO：182)，
wherein Z is13Is A, Z14Is U, the nucleotide sequence I comprises a position corresponding to Z13Nucleotide Z of15The nucleotide sequence II comprises a position corresponding to Z14Nucleotide Z of16Z is the same as16Is the first nucleotide at the 5' end of the antisense strand;
v) the nucleotide sequence I has the sequence shown in SEQ ID NO: 241 are equal in length and differ by NO more than 3 nucleotides, and said nucleotide sequence II is identical to SEQ ID NO: 242 are equal in length and differ by no more than 3 nucleotides:
5′-GGAUGUGCAGUUGAUCCCZ17-3′(SEQ ID NO：241)；
5′-Z18GGGAUCAACUGCACAUCC-3′(SEQ ID NO：242)，
wherein Z is17Is A, Z18Is U, the nucleotide sequence I comprises a position corresponding to Z17Nucleotide Z of19The nucleotide sequence II comprises a position corresponding to Z18Nucleotide Z of20Z is the same as20Is the first nucleotide at the 5' end of the antisense strand; and is
vi) the nucleotide sequence I is identical to SEQ ID NO: 301 is of equal length and differs by NO more than 3 nucleotides, and said nucleotide sequence II is identical to SEQ ID NO: 302 are equal in length and differ by no more than 3 nucleotides:
5′-UAACUUGGGAUCUGGGAAZ21-3′(SEQ ID NO：301)；
5′-Z22UUCCCAGAUCCCAAGUUA-3′(SEQ ID NO：302)，
wherein Z is21Is U, Z22Is A, the nucleotide sequence I comprises a position corresponding to Z21Nucleotide Z of23The nucleotide sequence II comprises a positionIs arranged to correspond to Z22Nucleotide Z of24Z is the same as24Is the first nucleotide at the 5' end of the antisense strand;
R2is a straight chain alkylene group of 1 to 20 carbon atoms in length, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: c (O), NH, O, S, CH-N, S (O)2、C2-C10Alkenylene radical, C2-C10Alkynylene, C6-C10Arylene radical, C3-C18Heterocyclylene and C5-C10A heteroarylene group; and wherein R2May optionally have a substituent of any one or more of the group consisting of: c1-C10Alkyl radical, C6-C10Aryl radical, C5-C10Heteroaryl group, C1-C10Haloalkyl, -OC1-C10Alkyl, -OC1-C10Alkylphenyl, -C1-C10alkyl-OH, -OC1-C10Haloalkyl, -SC1-C10Alkyl, -SC1-C10Alkylphenyl, -C1-C10alkyl-SH, -SC1-C10Haloalkyl, halogen substituents, -OH, -SH, -NH2、-C1-C10alkyl-NH2、-N(C1-C10Alkyl) (C1-C10Alkyl), -NH (C)1-C10Alkyl), -N (C)1-C10Alkyl) (C1-C10Alkylphenyl), -NH (C)1-C10Alkylphenyl), cyano, nitro, -CO2H、-C(O)O(C1-C10Alkyl), -CON (C)1-C10Alkyl) (C1-C10Alkyl), -CONH (C)1-C10Alkyl), -CONH2、-NHC(O)(C1-C10Alkyl), -NHC (O) (phenyl), -N (C)1-C10Alkyl) C (O) (C)1-C10Alkyl), -N (C)1-C10Alkyl) C (O) (phenyl), -C (O) C1-C10Alkyl, -C (O) C1-C10Alkylphenyl, -C (O) C1-C10Haloalkyl, -OC (O) C1-C10Alkyl, -SO2(C1-C10Alkyl), -SO2(phenyl), -SO2(C1-C10Haloalkyl), -SO2NH2、-SO2NH(C1-C10Alkyl), -SO2NH (phenyl), -NHSO2(C1-C10Alkyl), -NHSO2(phenyl) and-NHSO2(C1-C10Haloalkyl);
each L1Independently a linear alkylene group of 1 to 70 carbon atoms in length, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: c (O), NH, O, S, CH-N, S (O)2、C2-C10Alkenylene radical, C2-C10Alkynylene, C6-C10Arylene radical, C3-C18Heterocyclylene and C5-C10A heteroarylene group; and wherein L1May optionally have a substituent of any one or more of the group consisting of: c1-C10Alkyl radical, C6-C10Aryl radical, C5-C10Heteroaryl group, C1-C10Haloalkyl, -OC1-C10Alkyl, -OC1-C10Alkylphenyl, -C1-C10alkyl-OH, -OC1-C10Haloalkyl, -SC1-C10Alkyl, -SC1-C10Alkylphenyl, -C1-C10alkyl-SH, -SC1-C10Haloalkyl, halogen substituents, -OH, -SH, -NH2、-C1-C10alkyl-NH2、-N(C1-C10Alkyl) (C1-C10Alkyl), -NH (C)1-C10Alkyl), -N (C)1-C10Alkyl) (C1-C10Alkylphenyl), -NH (C)1-C10Alkylphenyl), cyano, nitro, -CO2H、-C(O)O(C1-C10Alkyl), -CON: (C1-C10Alkyl) (C1-C10Alkyl), -CONH (C)1-C10Alkyl), -CONH2，-NHC(O)(C1-C10Alkyl), -NHC (O) (phenyl), -N (C)1-C10Alkyl) C (O) (C)1-C10Alkyl), -N (C)1-C10Alkyl) C (O) (phenyl), -C (O) C1-C10Alkyl, -C (O) C1-C10Alkylphenyl, -C (O) C1-C10Haloalkyl, -OC (O) C1-C10Alkyl, -SO2(C1-C10Alkyl), -SO2(phenyl), -SO2(C1-C10Haloalkyl), -SO2NH2、-SO2NH(C1-C10Alkyl), -SO2NH (phenyl), -NHSO2(C1-C10Alkyl), -NHSO2(phenyl) and-NHSO2(C1-C10Haloalkyl);
Figure FDA0002498737060000032
represents the site of covalent attachment of a group; m1Represents a targeting group.
2. The siRNA conjugate of claim 1, wherein each L is1Independently selected from the group consisting of (A1) - (A26) groups and any combination thereof:
Figure FDA0002498737060000031
Figure FDA0002498737060000041
wherein each j1 is independently an integer from 1-20; each j2 is independently an integer from 1-20;
each R' is independently C1-C10An alkyl group;
each Ra is selected from the group consisting of groups represented by formulas (a27) - (a45) or any combination thereof:
Figure FDA0002498737060000042
Figure FDA0002498737060000051
each Rb is independently C1-C10An alkyl group;
or L1A combination of one or more selected from A1, A4, A5, A6, A8, A10, A11 and A13;
or L1A linked combination of at least 2 selected from a1, a4, A8, a10, and a 11;
or, L1A combination of at least 2 linkages selected from a1, A8, a 10;
or, L1Is 3-25 atoms in length;
or, L1Is 4-15 atoms in length;
Figure FDA0002498737060000052
represents the site of covalent attachment of a group; or j1 is an integer of 2-10, j2 is an integer of 2-10, R' is C1-C4Alkyl, Ra is one of A27, A28, A29, A30 and A31, and Rb is C1-C5An alkyl group;
or j1 is an integer of 3-5, j2 is an integer of 3-5, R' is one of methyl, ethyl and isopropyl, Ra is a group shown in formula (A27) or a group shown in formula (A28), and Rb is one of methyl, ethyl, isopropyl and butyl; alternatively, n1 is an integer from 1 to 2, n3 is an integer from 0 to 1, and n1+ n3 is 2 to 3; alternatively, each m1, m2 or m3 is independently an integer from 2 to 5, and/or m1 ═ m2 ═ m 3; alternatively, each of said targeting groups is independently a ligand that has affinity for asialoglycoprotein receptors on the surface of mammalian hepatocytes;
alternatively, each of the targeting groups is independently an asialoglycoprotein or a sugar;
alternatively, each of the targeting groups is independently selected from D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, alpha-D-mannofuranose, beta-D-mannofuranose, alpha-D-mannopyranose, beta-D-mannopyranose, alpha-D-glucopyranose, beta-D-glucopyranose, alpha-D-glucopyranose, beta-D-glucofuranose, alpha-D-fructopyranose, alpha-D-galactopyranose, beta-D-galactopyranose, alpha-D-galactofuranose, beta-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-N-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O- [ (R) -1-carboxyethyl ] -2-deoxy-beta-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4, 6-dideoxy-4-carboxamido-2, 3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfonamido-D-glucopyranose, beta-galactofuranose, glucosamine, N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-N-butyrylgalactosamine, N-isobutyrylgalactosamine, N-glycolyl-alpha-neuraminic acid, 5-thio-beta-D-glucopyranose, 2, 3, 4-tri-O-acetyl-1-thio-6-O-trityl-alpha-D-glucopyranoside methyl ester, 4-thio-beta-D-galactopyranose, 3, 4, 6, 7-tetra-O-acetyl-2-deoxy-1, 5-dithio-alpha-D-glucopyranoside ethyl ester, 2, 5-anhydro-D-allositrile, ribose, D-4-thioribose, L-ribose, L-4-thioribose;
Alternatively, at least one or each of the targeting groups is galactose or N-acetylgalactosamine; or, R10、R11、R12、R13、R14Or R15Independently H, methyl or ethyl;
or R10、R11、R12、R13、R14Or R15Are all H; or, R2Containing both a linking site to the N on the nitrogen-containing backbone and a linking site to the R3The attachment site to which the P atom in (a) is attached;
or R2N of the nitrogen-containing skeletonThe site of attachment forms an amide bond with N, said bond being with R3The site to which the P atom is attached forms a phosphoester bond with P;
or R2Selected from the group represented by formula (B5), (B6), (B5 ') or (B6'); alternatively, the siRNA conjugate has a structure represented by formula (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421) or (422); alternatively, the P atom in formula a59 is attached to the end of the sense or antisense strand of the siRNA, which end refers to the first 4 nucleotides from one end of the sense or antisense strand;
or the P atom in formula A59 is connected to the end of the sense strand or antisense strand of the siRNA; or the P atom in formula a59 is attached to the 3' end of the siRNA sense strand;
or the P atom in formula a59 is linked to the 2 ', 3 ', or 5 ' position of the nucleotide in the siRNA through a phosphodiester bond.
3. The siRNA conjugate of claim 1, wherein I) said nucleotide sequence I is identical to SEQ ID NO: 1, and/or the nucleotide sequence II differs from the nucleotide sequence shown in SEQ ID NO: 2 by no more than 1 nucleotide difference; or ii) the nucleotide sequence I is identical to SEQ ID NO: 61, and/or the nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO: 62 is no more than 1 nucleotide different; or, iii) the nucleotide sequence I has the sequence shown in SEQ ID NO: 121, and/or the nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO: 122 by no more than 1 nucleotide difference; or, iv) the nucleotide sequence I has the sequence shown in SEQ ID NO: 181, and/or the nucleotide sequence II differs from the nucleotide sequence shown in SEQ ID NO: 182 by no more than 1 nucleotide difference; or, v) the nucleotide sequence I has the sequence shown in SEQ ID NO: 241 differ by no more than 1 nucleotide, andand/or the nucleotide sequence II has the nucleotide sequence shown in SEQ ID NO: 242 by no more than 1 nucleotide difference between the nucleotide sequences set forth in seq id no; or vi) the nucleotide sequence I has the sequence shown in SEQ ID NO: 301, and/or said nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO: 302 is no more than 1 nucleotide different from one another; preferably, i) said nucleotide sequence II is identical to SEQ ID NO: 2 comprises Z4A difference at position, and Z4Selected from U, C or G; or II) the nucleotide sequence II is identical to SEQ ID NO: 62 comprises Z8A difference at position, and Z8Selected from A, C or G; or iii) the nucleotide sequence II is identical to SEQ ID NO: 122 comprises Z12A difference at position, and Z12Selected from U, C or G; or iv) the nucleotide sequence II is identical to the nucleotide sequence of SEQ ID NO: 182 comprises Z16A difference at position, and Z16Selected from A, C or G; or v) the nucleotide sequence II is identical to the nucleotide sequence of SEQ ID NO: 242 comprises Z20A difference at position, and Z20Selected from A, C or G; or vi) the nucleotide sequence II is identical to SEQ ID NO: the nucleotide difference between the nucleotide sequences set forth in 302 comprises Z24A difference at position, and Z24Selected from U, C or G; preferably, Z3Is a reaction of with Z4A complementary nucleotide; or Z7Is a reaction of with Z8A complementary nucleotide; or Z11Is a reaction of with Z12A complementary nucleotide; or Z15Is a reaction of with Z16A complementary nucleotide; or Z19Is a reaction of with Z20A complementary nucleotide; or Z23Is a reaction of with Z24A complementary nucleotide; preferably, said nucleotide sequence I and said nucleotide sequence II are substantially reverse complementary, substantially reverse complementary or fully reverse complementary; by substantially reverse complementary is meant that no more than 3 base mismatches occur between two nucleotide sequences; by substantially reverse complementary is meant that no more than 1 base is present between two nucleotide sequencesMismatch; perfect reverse complementarity means that there is no mismatch between the two nucleotide sequences.
4. The siRNA conjugate of any of claims 1 to 3, wherein said sense strand further comprises a nucleotide sequence III, said antisense strand further comprises a nucleotide sequence IV, said nucleotide sequence III and said nucleotide sequence IV being each independently 1 to 4 nucleotides in length, said nucleotide sequence III being linked at the 5 'end of nucleotide sequence I, said nucleotide sequence IV being linked at the 3' end of nucleotide sequence II, said nucleotide sequence III and said nucleotide sequence IV being of equal length and being substantially reverse complementary or fully reverse complementary; the substantially reverse complement refers to the presence of no more than 1 base mismatch between two nucleotide sequences; perfect reverse complementarity means that there is no mismatch between the two nucleotide sequences.
5. The siRNA conjugate of claim 4, wherein I) said nucleotide sequence I is SEQ ID NO: 3, and the nucleotide sequence II is SEQ ID NO: 4; the length of each of the nucleotide sequences III and IV is 1 nucleotide, and the base of the nucleotide sequence III is A; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and the base composition of the nucleotide sequence III is AA according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 3 nucleotides, and the base composition of the nucleotide sequence III is AAA according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 4 nucleotides, and the base composition of the nucleotide sequence III is GAAA according to the direction from the 5 'end to the 3' end; or
ii) the nucleotide sequence I is SEQ ID NO: 63, and the nucleotide sequence II is a nucleotide sequence shown in SEQ ID NO: 64; and the length of the nucleotide sequences III and IV is 1 nucleotide, and the base of the nucleotide sequence III is C; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and the base composition of the nucleotide sequence III is GC according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 3 nucleotides, and the base composition of the nucleotide sequence III is UGC according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 4 nucleotides, and the base composition of the nucleotide sequence III is GUGC according to the direction from the 5 'end to the 3' end; or
iii) the nucleotide sequence I is SEQ ID NO: 123, and the nucleotide sequence II is a nucleotide sequence shown in SEQ ID NO: 124; and the length of the nucleotide sequences III and IV is 1 nucleotide, and the base of the nucleotide sequence III is A; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and the base composition of the nucleotide sequence III is GA according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 3 nucleotides, and the base composition of the nucleotide sequence III is UGA according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 4 nucleotides, and the base composition of the nucleotide sequence III is CUGA according to the direction from the 5 'end to the 3' end; or
iv) the nucleotide sequence I is SEQ ID NO: 183, and the nucleotide sequence II is SEQ ID NO: 184; and the length of the nucleotide sequences III and IV is 1 nucleotide, and the base of the nucleotide sequence III is A; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and the base composition of the nucleotide sequence III is GA according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 3 nucleotides, and the base composition of the nucleotide sequence III is GGA according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 4 nucleotides, and the base composition of the nucleotide sequence III is UGGA according to the direction from the 5 'end to the 3' end; or
v) the nucleotide sequence I is SEQ ID NO: 243, and the nucleotide sequence II is SEQ ID NO: 244; the length of each of the nucleotide sequences III and IV is 1 nucleotide, and the base of the nucleotide sequence III is U; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and the base composition of the nucleotide sequence III is GU according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 3 nucleotides, and the base composition of the nucleotide sequence III is AGU according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 4 nucleotides, and the base composition of the nucleotide sequence III is AAGU according to the direction from the 5 'end to the 3' end; or
vi) the nucleotide sequence I is SEQ ID NO: 303, and the nucleotide sequence II is SEQ ID NO: 304; and the length of the nucleotide sequences III and IV is 1 nucleotide, and the base of the nucleotide sequence III is C; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and the base composition of the nucleotide sequence III is UC according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 3 nucleotides, and the base composition of the nucleotide sequence III is GUC according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 4 nucleotides, and the base composition of the nucleotide sequence III is GGUC according to the direction from the 5 'end to the 3' end; preferably, the nucleotide sequences III and IV are fully reverse complementary.
6. The siRNA conjugate of any of claims 1-5, wherein said antisense strand further comprises a nucleotide sequence V, having a length of 1 to 3 nucleotides, attached to the 3 'terminus of said antisense strand to form the 3' overhang of the antisense strand; or the nucleotide sequence V is 2 nucleotides in length; or the nucleotide sequence V is two continuous thymine deoxyribonucleotides or two continuous uracil ribonucleotides; or the nucleotide sequence V is complementary to the nucleotide at the corresponding position of the target mRNA; preferably, the sense strand of the siRNA comprises the sequence as set forth in SEQ ID NO: 5, and the antisense strand comprises the nucleotide sequence shown as SEQ ID NO: 6; or the sense strand of the siRNA contains the sequence shown as SEQ ID NO: 7, and the antisense strand comprises the nucleotide sequence shown as SEQ ID NO: 8;
or the sense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO: 65, and the antisense strand comprises the nucleotide sequence shown as SEQ ID NO: 66; or the sense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO: 67, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 68;
or the sense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO: 125, and the antisense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO: 126; or the sense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO: 127, wherein the antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 128;
Or the sense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO: 185, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 186; or the sense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO: 187, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 188;
or the sense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO: 245, and the antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 246; or the sense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO: 247, and the antisense strand of said siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 248;
or the sense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO: 305 and the antisense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO: 306; or the sense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO: 307, and the antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 308; preferably, the siRNA has a nucleotide sequence set forth in siTMPa1, siTMPa2, siTMPb1, siTMPb2, siTMPc1, siTMPc2, siTMPd1, siTMPd2, siTMPe1, siTMPe2, siTMPf1, or siTMPf 2; preferably, at least one nucleotide in the sense strand or the antisense strand is a modified nucleotide and/or at least one phosphate group is a phosphate group with a modifying group; preferably, each nucleotide in the sense and antisense strands is independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide;
or the fluorinated modified nucleotide is positioned in the nucleotide sequence I and the nucleotide sequence II, and at least the 7 th, 8 th and 9 th nucleotides of the nucleotide sequence I are fluorinated modified nucleotides according to the direction from the 5 'end to the 3' end; at least the 2 nd, 6 th, 14 th and 16 th nucleotides of the nucleotide sequence II are fluoro-modified nucleotides according to the direction from the 5 'end to the 3' end;
or according to the direction from 5 'end to 3' end, in the sense strand, the nucleotides at positions 7, 8 and 9 or 5, 7, 8 and 9 of the nucleotide sequence I are fluorine-modified nucleotides, and the nucleotides at the rest positions in the sense strand are non-fluorine-modified nucleotides; in the direction from the 5 'end to the 3' end, in the antisense strand, the nucleotides at positions 2, 6, 14 and 16 or 2, 6, 8, 9, 14 and 16 of the nucleotide sequence II are fluorine-modified nucleotides, and the nucleotides at the rest positions in the antisense strand are non-fluorine-modified nucleotides; preferably, each non-fluorinated modified nucleotide is independently selected from one of nucleotides or nucleotide analogs in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with a non-fluorinated group;
or the nucleotide formed by substituting the hydroxyl at the 2 '-position of the ribosyl of the nucleotide by a non-fluorine group is selected from one of 2' -alkoxy modified nucleotide, 2 '-substituted alkoxy modified nucleotide, 2' -alkyl modified nucleotide, 2 '-substituted alkyl modified nucleotide, 2' -amino modified nucleotide, 2 '-substituted amino modified nucleotide, 2' -deoxynucleotide; the nucleotide analogue is selected from one of isonucleotides, LNA, ENA, cET, UNA and GNA;
or each of the non-fluorinated modified nucleotides is a methoxy-modified nucleotide, which means a nucleotide in which the 2' -hydroxyl group of the ribosyl group is substituted with a methoxy group; preferably, the nucleotides at positions 5, 7, 8 and 9 of the nucleotide sequence I in the sense strand of the siRNA are fluoro-modified nucleotides, the nucleotides at the rest positions of the sense strand of the siRNA are methoxy-modified nucleotides, and the nucleotides at positions 2, 6, 8, 9, 14 and 16 of the nucleotide sequence II in the antisense strand of the siRNA are fluoro-modified nucleotides, and the nucleotides at the rest positions of the antisense strand of the siRNA are methoxy-modified nucleotides, in the direction from 5 'end to 3' end;
or, according to the direction from 5 'end to 3' end, the nucleotides at the 5 th, 7 th, 8 th and 9 th positions of the nucleotide sequence I in the sense strand of the siRNA are fluorine modified nucleotides, the nucleotides at the rest positions of the sense strand of the siRNA are methoxy modified nucleotides, and, according to the direction from 5 'end to 3' end, the nucleotides at the 2 nd, 6 th, 14 th and 16 th positions of the nucleotide sequence II in the antisense strand of the siRNA are fluorine modified nucleotides, and the nucleotides at the rest positions of the antisense strand of the siRNA are methoxy modified nucleotides;
or, according to the direction from 5 'end to 3' end, the nucleotides at the 7 th, 8 th and 9 th positions of the nucleotide sequence I in the sense strand of the siRNA are fluorine modified nucleotides, the nucleotides at the rest positions of the sense strand of the siRNA are methoxy modified nucleotides, and, according to the direction from 5 'end to 3' end, the nucleotides at the 2 nd, 6 th, 14 th and 16 th positions of the nucleotide sequence II in the antisense strand of the siRNA are fluorine modified nucleotides, and the nucleotides at the rest positions of the antisense strand of the siRNA are methoxy modified nucleotides; preferably, the siRNA is any one of siTMPa-M, siTMPb-M, siTMPc-M, siTMPd-M, siTMPe-M, siTMPePf-M, siTMPf-M; preferably, the phosphate group having a modifying group is a phosphorothioate group in which at least one oxygen atom in a phosphodiester bond in the phosphate group is substituted with a sulfur atom; or the phosphate group with the modification group is a thiophosphate group with a structure shown in the formula (1); preferably, in the siRNA, the phosphorothioate-based linkage is present at least one position in the group consisting of:
between the 1 st and 2 nd nucleotides of the 5' terminus of the sense strand;
between the 2 nd and 3 rd nucleotides at the 5' end of the sense strand;
between the 1 st and 2 nd nucleotides of the 3' terminus of the sense strand;
between the 2 nd and 3 rd nucleotides at the 3' terminus of the sense strand;
between the 1 st and 2 nd nucleotides of the 5' terminus of the antisense strand;
between the 2 nd and 3 rd nucleotides of the 5' terminus of the antisense strand;
between the 1 st and 2 nd nucleotides of the 3' terminus of the antisense strand; and
between the 2 nd and 3 rd nucleotides of the 3' terminus of the antisense strand; preferably, the siRNA is any one of siTMPa-M1, siTMPa-M2, siTMPa-M3, siTMPb-M1, siTMPb-M2, siTMPb-M3, siTMPc-M1, siTMPc-M2, siTMPc-M3, siTMPd-M1, siTMPd-M2, siTMPd-M3, siTMPe-M1, siTMPe-M2, siTMPe-M3, siTMPf-M3, Pf-M1, Pf-M2, Pf-M3, Pf-Si-M2, or Pf 2; preferably, the 5 ' terminal nucleotide of the siRNA antisense strand is a 5 ' -phosphate nucleotide or a 5 ' -phosphate analogue modified nucleotide;
or the 5 '-phosphate nucleotide is a nucleotide with a structure shown in the formula (2), the 5' -phosphate analogue modified nucleotide is selected from nucleotides with a structure shown in any one of the formulas (3) to (6),
Figure FDA0002498737060000111
wherein R is selected from H, OH, methoxy or fluorine; base represents a Base selected from A, U, C, G or T; preferably, the siRNA is siTMPa1-M1P1, siTMPa1-M2P1, siTMPa1-M3P1, siTMPa2-M1P1, siTMPa2-M2P1, siTMPa2-M3P1, siTMPa1-M1SP1, siTMPa1-M2SP1, siTMPa1-M3SP1, siTMPa1-M1SP1, siTMPa1-M1, siTMP 1-P1, siP 36p 1-P1, siP 36p 1-1, siP 36p 1-P36p 1, siP 36p 1-36p 1, siP 36p 1-36p 1, siP 36p 1, sTMP 36p 1-36p 1, sP 36p 1-36p 1, sP 36p 1-36p 1, sP 36p 1-1, sP 36p 1-1, sP 36sP 36s3672, sP 36sP 1, sP 36sP 1-1, sP 36sP 1, sP 36sP 1, TMP 36sP 1, sP 36sP 1-1, sP 36sP 1, sP 36sP 1, sP 36sP 1-1, sP 36sP 36s36sP 1, sP, SiTMPd-M1 SP, siTMPd-M2 SP, siTMPd-M3 SP, siTMPd-M1 SP, siTMPd-M2 SP, siTMPd-M3 SP, siTMPe-M1P, siTMPe-M2P, siTMPe-M3P, siTMPe-M1 SP, siTMPe-M2 SP, siTMPe-M3 SP, siTMPe-M1 SP, siTMPe-M2 SP, siTMP-M3 SP, siTMPf-M1P, siTMPf-M2P, siTMP-M3P, siTMSP-Pf-M1P, siTMP-M2P, siTMP-M3P, siTMSP-Pf-M1P, siTMPf-M2P, siTMP-Pf-M3P, siTMSP-Pf-M1 SP, siTMSP-Pf-M2 SP, siTMSP-Pf-M3 SP, siM 3SP, siTMSP-Pf-M2 SP, siM 3SP, siTMSP, siM 2SP, siTMP, siTMSP-Pf.
7. An siRNA comprising a sense strand and an antisense strand, each nucleotide in said sense strand and said antisense strand being independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide; the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially complementary reversely to form a double-stranded region, the fluorinated modified nucleotide is positioned in the nucleotide sequence I and the nucleotide sequence II, and according to the direction from 5 'end to 3' end, in the sense strand, the nucleotides at the 7 th, 8 th and 9 th positions of the nucleotide sequence I are fluorinated modified nucleotides, and the nucleotides at the rest positions in the sense strand are non-fluorinated modified nucleotides; in the direction from the 5 'end to the 3' end, in the antisense strand, the nucleotides at positions 2, 6, 14, 16 of the nucleotide sequence II are fluorine-modified nucleotides, the nucleotides at the remaining positions in the antisense strand are non-fluorine-modified nucleotides, and,
i) the nucleotide sequence I is similar to SEQ ID NO: 1 is equal in length and differs by NO more than 3 nucleotides, and the nucleotide sequence II is identical to SEQ ID NO: 2 are equal in length and have no more than 3 nucleotide differences, and the nucleotide sequence I comprises a nucleotide sequence with a position corresponding to Z1Nucleotide Z of3The nucleotide sequence II comprises a position corresponding to Z2Nucleotide Z of4Z is the same as4Is the first nucleotide at the 5' end of the antisense strand; alternatively, the first and second electrodes may be,
ii) the nucleotide sequence I is identical to SEQ ID NO: 61 and NO more than 3 nucleotides different, and the nucleotide sequence II is identical to the nucleotide sequence shown in SEQ ID NO: 62 are equal in length and do not differ by more than 3 nucleotides, and the nucleotide sequence I comprises a nucleotide sequence I with a position corresponding to Z5Nucleotide Z of7The nucleotide sequence II comprises a position corresponding to Z6Nucleotide Z of8Z is the same as8Is the first nucleotide at the 5' end of the antisense strand; alternatively, the first and second electrodes may be,
iii) the nucleotide sequence I has the sequence shown in SEQ ID NO: 121 and NO more than 3 nucleotides different, and the nucleotide sequence II is identical to SEQ ID NO: 122 are equal in length and differ by no more than 3 nucleotides in the nucleotide sequence I comprising a position corresponding to Z9Nucleotide Z of11The nucleotide sequence II comprises a position corresponding to Z10Nucleotide Z of12Z is the same as12Is the first nucleotide at the 5' end of the antisense strand; alternatively, the first and second electrodes may be,
iv) the nucleotide sequence I has the sequence shown in SEQ ID NO: 181 shown inIs equal in length and differs by NO more than 3 nucleotides, and the nucleotide sequence II is identical to SEQ ID NO: 182 are equal in length and differ by no more than 3 nucleotides, and the nucleotide sequence I comprises a position corresponding to Z13Nucleotide Z of15The nucleotide sequence II comprises a position corresponding to Z14Nucleotide Z of16Z is the same as16Is the first nucleotide at the 5' end of the antisense strand; alternatively, the first and second electrodes may be,
v) the nucleotide sequence I has the sequence shown in SEQ ID NO: 241 are equal in length and differ by NO more than 3 nucleotides, and said nucleotide sequence II is identical to SEQ ID NO: 242 are equal in length and differ by no more than 3 nucleotides, and the nucleotide sequence I comprises a position corresponding to Z17Nucleotide Z of19The nucleotide sequence II comprises a position corresponding to Z18Nucleotide Z of20Z is the same as20Is the first nucleotide at the 5' end of the antisense strand; alternatively, the first and second electrodes may be,
vi) the nucleotide sequence I is identical to SEQ ID NO: 301 is of equal length and differs by NO more than 3 nucleotides, and said nucleotide sequence II is identical to SEQ ID NO: 302 are equal in length and differ by no more than 3 nucleotides, and the nucleotide sequence I comprises a position corresponding to Z21Nucleotide Z of23The nucleotide sequence II comprises a position corresponding to Z22Nucleotide Z of24Z is the same as24Is the first nucleotide at the 5' end of the antisense strand.
8. The siRNA of claim 7, wherein each non-fluorinated modified nucleotide is independently selected from one of a nucleotide or a nucleotide analog in which a hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with a non-fluorinated group; preferably, the nucleotide in which the hydroxyl group at the 2 '-position of the ribosyl group of the nucleotide is substituted with a non-fluorine group is selected from the group consisting of a 2' -alkoxy-modified nucleotide, a 2 '-substituted alkoxy-modified nucleotide, a 2' -alkyl-modified nucleotide, a 2 '-substituted alkyl-modified nucleotide, and a 2' -amino-modified nucleotideOne of the nucleotide, 2 '-substituted amino-modified nucleotide, 2' -deoxynucleotide of (a); the nucleotide analogue is selected from one of isonucleotides, LNA, ENA, cET, UNA and GNA; preferably, each of the non-fluorinated modified nucleotides is a methoxy-modified nucleotide, which refers to a nucleotide in which the 2' -hydroxyl group of the ribosyl group is substituted with a methoxy group; preferably, I) said nucleotide sequence I is identical to SEQ ID NO: 1, and/or the nucleotide sequence II differs from the nucleotide sequence shown in SEQ ID NO: 2 by no more than 1 nucleotide difference; or ii) the nucleotide sequence I is identical to SEQ ID NO: 61, and/or the nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO: 62 is no more than 1 nucleotide different; or, iii) the nucleotide sequence I has the sequence shown in SEQ ID NO: 121, and/or the nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO: 122 by no more than 1 nucleotide difference; or, iv) the nucleotide sequence I has the sequence shown in SEQ ID NO: 181, and/or the nucleotide sequence II differs from the nucleotide sequence shown in SEQ ID NO: 182 by no more than 1 nucleotide difference; or, v) the nucleotide sequence I has the sequence shown in SEQ ID NO: 241 and/or said nucleotide sequence II differs from the nucleotide sequence shown in SEQ ID NO: 242 by no more than 1 nucleotide difference between the nucleotide sequences set forth in seq id no; or vi) the nucleotide sequence I has the sequence shown in SEQ ID NO: 301, and/or said nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO: 302 is no more than 1 nucleotide different from one another; preferably, i) said nucleotide sequence II is identical to SEQ ID NO: 2 comprises Z4A difference at position, and Z4Selected from U, C or G; or II) the nucleotide sequence II is identical to SEQ ID NO: 62 comprises Z8A difference at position, and Z8Selected from A, C or G; or iii) the coreNucleotide sequence II and SEQ ID NO: 122 comprises Z12A difference at position, and Z12Selected from U, C or G; or iv) the nucleotide sequence II is identical to the nucleotide sequence of SEQ ID NO: 182 comprises Z16A difference at position, and Z16Selected from A, C or G; or v) the nucleotide sequence II is identical to the nucleotide sequence of SEQ ID NO: 242 comprises Z20A difference at position, and Z20Selected from A, C or G; or vi) the nucleotide sequence II is identical to SEQ ID NO: the nucleotide difference between the nucleotide sequences set forth in 302 comprises Z24A difference at position, and Z24Selected from U, C or G; preferably, Z3Is a reaction of with Z4A complementary nucleotide; or Z7Is a reaction of with Z8A complementary nucleotide; or Z11Is a reaction of with Z12A complementary nucleotide; or Z15Is a reaction of with Z16A complementary nucleotide; or Z19Is a reaction of with Z20A complementary nucleotide; or Z23Is a reaction of with Z24A complementary nucleotide; preferably, said nucleotide sequence I and said nucleotide sequence II are substantially reverse complementary, substantially reverse complementary or fully reverse complementary; by substantially reverse complementary is meant that no more than 3 base mismatches occur between two nucleotide sequences; the substantially reverse complement refers to the presence of no more than 1 base mismatch between two nucleotide sequences; perfect reverse complement means that there is no mismatch between the two nucleotide sequences; preferably, the sense strand further comprises a nucleotide sequence III, the antisense strand further comprises a nucleotide sequence IV, the length of each of the nucleotide sequence III and the nucleotide sequence IV is independently 1 to 4 nucleotides, the nucleotide sequence III is linked to the 5 'end of the nucleotide sequence I, the nucleotide sequence IV is linked to the 3' end of the nucleotide sequence II, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or fully reverse complementary; the substantially reverse complement refers to the presence of no more than 1 base mismatch between two nucleotide sequences; perfect reverse complementarity means that there is no mismatch between the two nucleotide sequences.
9. The siRNA of any one of claims 7-8,
i) the nucleotide sequence I is SEQ ID NO: 3, and the nucleotide sequence II is SEQ ID NO: 4; the length of each of the nucleotide sequences III and IV is 1 nucleotide, and the base of the nucleotide sequence III is A; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and the base composition of the nucleotide sequence III is AA according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 3 nucleotides, and the base composition of the nucleotide sequence III is AAA according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 4 nucleotides, and the base composition of the nucleotide sequence III is GAAA according to the direction from the 5 'end to the 3' end; or
ii) the nucleotide sequence I is SEQ ID NO: 63, and the nucleotide sequence II is a nucleotide sequence shown in SEQ ID NO: 64; and the length of the nucleotide sequences III and IV is 1 nucleotide, and the base of the nucleotide sequence III is C; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and the base composition of the nucleotide sequence III is GC according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 3 nucleotides, and the base composition of the nucleotide sequence III is UGC according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 4 nucleotides, and the base composition of the nucleotide sequence III is GUGC according to the direction from the 5 'end to the 3' end; or
iii) the nucleotide sequence I is SEQ ID NO: 123, and the nucleotide sequence II is a nucleotide sequence shown in SEQ ID NO: 124; and the length of the nucleotide sequences III and IV is 1 nucleotide, and the base of the nucleotide sequence III is A; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and the base composition of the nucleotide sequence III is GA according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 3 nucleotides, and the base composition of the nucleotide sequence III is UGA according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 4 nucleotides, and the base composition of the nucleotide sequence III is CUGA according to the direction from the 5 'end to the 3' end; or
iv) the nucleotide sequence I is SEQ ID NO: 183, and the nucleotide sequence II is SEQ ID NO: 184; and the length of the nucleotide sequences III and IV is 1 nucleotide, and the base of the nucleotide sequence III is A; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and the base composition of the nucleotide sequence III is GA according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 3 nucleotides, and the base composition of the nucleotide sequence III is GGA according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 4 nucleotides, and the base composition of the nucleotide sequence III is UGGA according to the direction from the 5 'end to the 3' end; or
v) the nucleotide sequence I is SEQ ID NO: 243, and the nucleotide sequence II is SEQ ID NO: 244; the length of each of the nucleotide sequences III and IV is 1 nucleotide, and the base of the nucleotide sequence III is U; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and the base composition of the nucleotide sequence III is GU according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 3 nucleotides, and the base composition of the nucleotide sequence III is AGU according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 4 nucleotides, and the base composition of the nucleotide sequence III is AAGU according to the direction from the 5 'end to the 3' end; or
vi) the nucleotide sequence I is SEQ ID NO: 303, and the nucleotide sequence II is SEQ ID NO: 304; and the length of the nucleotide sequences III and IV is 1 nucleotide, and the base of the nucleotide sequence III is C; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and the base composition of the nucleotide sequence III is UC according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 3 nucleotides, and the base composition of the nucleotide sequence III is GUC according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 4 nucleotides, and the base composition of the nucleotide sequence III is GGUC according to the direction from the 5 'end to the 3' end; preferably, the nucleotide sequences III and IV are fully reverse complementary; preferably, the antisense strand further comprises a nucleotide sequence V, 1 to 3 nucleotides in length, linked at the 3 'end of the antisense strand to form the 3' overhang of the antisense strand; or the nucleotide sequence V is 2 nucleotides in length; or the nucleotide sequence V is two continuous thymine deoxyribonucleotides or two continuous uracil ribonucleotides; or the nucleotide sequence V is complementary to the nucleotide at the corresponding position of the target mRNA; preferably, the first and second electrodes are formed of a metal,
The sense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO: 5, and the antisense strand comprises the nucleotide sequence shown as SEQ ID NO: 6; or the sense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO: 7, and the antisense strand comprises the nucleotide sequence shown as SEQ ID NO: 8;
or the sense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO: 65, and the antisense strand comprises the nucleotide sequence shown as SEQ ID NO: 66; or the sense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO: 67, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 68;
or the sense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO: 125, and the antisense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO: 126; or the sense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO: 127, wherein the antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 128;
or the sense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO: 185, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 186; or the sense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO: 187, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 188;
or the sense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO: 245, and the antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 246; or the sense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO: 247, and the antisense strand of said siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 248;
or the sense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO: 305 and the antisense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO: 306; or the sense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO: 307, and the antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 308; preferably, the siRNA has a nucleotide sequence set forth in siTMPa1, siTMPa2, siTMPb1, siTMPb2, siTMPc1, siTMPc2, siTMPd1, siTMPd2, siTMPe1, siTMPe2, siTMPf1, or siTMPf 2; preferably, the siRNA is any one of siTMPa1-M1, siTMPa2-M1, siTMPb1-M1, siTMPb2-M1, siTMPc1-M1, siTMPc2-M1, siTMPd1-M1, siTMPd2-M1, siTMPe1-M1, siTMPe2-M1, siTMPf1-M1 or siTMPf 2-M1; preferably, at least one phosphate group in the sense strand or the antisense strand is a phosphate group having a modifying group; preferably, the phosphate group having a modifying group is a phosphorothioate group in which at least one oxygen atom in a phosphodiester bond in the phosphate group is substituted with a sulfur atom; or the phosphate group with the modification group is a thiophosphate group with a structure shown in the formula (1); preferably, in the siRNA, the phosphorothioate-based linkage is present at least one position in the group consisting of:
between the 1 st and 2 nd nucleotides of the 5' terminus of the sense strand;
between the 2 nd and 3 rd nucleotides at the 5' end of the sense strand;
between the 1 st and 2 nd nucleotides of the 3' terminus of the sense strand;
between the 2 nd and 3 rd nucleotides at the 3' terminus of the sense strand;
between the 1 st and 2 nd nucleotides of the 5' terminus of the antisense strand;
between the 2 nd and 3 rd nucleotides of the 5' terminus of the antisense strand;
between the 1 st and 2 nd nucleotides of the 3' terminus of the antisense strand; and
between the 2 nd and 3 rd nucleotides of the 3' terminus of the antisense strand; preferably, the siRNA is any one of siTMPa1-M1S, siTMPa2-M1S, siTMPb1-M1S, siTMPb2-M1S, siTMPc1-M1S, siTMPc2-M1S, siTMPd1-M1S, siTMPd2-M1S, siTMPe1-M1S, siTMPe2-M1S, siTMPf1-M1S or siTMPf 2-M1S; preferably, the 5 ' terminal nucleotide of the siRNA antisense strand is a 5 ' -phosphate nucleotide or a 5 ' -phosphate analogue modified nucleotide;
or the 5 '-phosphate nucleotide is a nucleotide with a structure shown in the formula (2), the 5' -phosphate analogue modified nucleotide is selected from nucleotides with a structure shown in any one of the formulas (3) to (6),
Figure FDA0002498737060000161
wherein R is selected from H, OH, methoxy or fluorine; base represents a Base selected from A, U, C, G or T; preferably, the siRNA is any one of siTMPa1-M1P1, siTMPa2-M1P1, siTMPa1-M1SP1, siTMPb1-M1P1, siTMPb1-M1SP1, siTMPc1-M1P1, siTMPc1-M1SP1, siTMPd1-M1SP1, siTMP 1-M1SP1, siTMP 1-P1, siTMP 1-Pf 1SP1, and PePf TMP 1-P1, or PePf 1SP 1-P1, siP 1, or Pep 1, siP 1, siTMP 1-P1, and Pep 361 SP 1; preferably, the siRNA is any one of siTMPa1-M1SP, siTMPb1-M1SP, siTMPc1-M1SP, siTMPd1-M1SP, siTMPe1-M1SP or siTMPf1-M1 SP.
10. A pharmaceutical composition comprising the siRNA of any one of claims 7 to 9 and a pharmaceutically acceptable carrier.
11. An siRNA conjugate comprising an siRNA of any one of claims 7 to 9 and a conjugate group conjugated to the siRNA.
12. Use of an siRNA conjugate according to any of claims 1 to 6 and 11, an siRNA according to any of claims 7 to 9 and/or a pharmaceutical composition according to claim 10 for the manufacture of a medicament for the treatment and/or prevention of complications of iron overload.
13. A method of inhibiting TMPRSS6 gene expression in a hepatocyte, the method comprising contacting the hepatocyte with an effective amount of an siRNA conjugate of any one of claims 1 to 6 and 11, an siRNA of any one of claims 7 to 9 and/or a pharmaceutical composition of claim 10.
14. A kit comprising an siRNA conjugate according to any of claims 1 to 6 and 11, an siRNA according to any of claims 7 to 9 and/or a pharmaceutical composition according to claim 10.
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