WO2023284763A1 - 双链寡核苷酸、含双链寡核苷酸的组合物与缀合物及制备方法和用途 - Google Patents

双链寡核苷酸、含双链寡核苷酸的组合物与缀合物及制备方法和用途 Download PDF

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WO2023284763A1
WO2023284763A1 PCT/CN2022/105340 CN2022105340W WO2023284763A1 WO 2023284763 A1 WO2023284763 A1 WO 2023284763A1 CN 2022105340 W CN2022105340 W CN 2022105340W WO 2023284763 A1 WO2023284763 A1 WO 2023284763A1
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nucleotide sequence
nucleotide
double
stranded oligonucleotide
nucleotides
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French (fr)
Chinese (zh)
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梁子才
张鸿雁
高山
李海涛
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Suzhou Ribo Life Science Co Ltd
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Suzhou Ribo Life Science Co Ltd
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Priority to US18/576,589 priority Critical patent/US20240309378A1/en
Priority to JP2024502054A priority patent/JP2024528634A/ja
Priority to CN202280046071.8A priority patent/CN117580952A/zh
Priority to EP22841396.9A priority patent/EP4372086A1/en
Publication of WO2023284763A1 publication Critical patent/WO2023284763A1/zh
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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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    • 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
    • C12N15/1131—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 against viruses
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Definitions

  • the present disclosure relates to a double-stranded oligonucleotide with reduced off-target effects and pharmaceutical compositions and oligonucleotide conjugates containing the double-stranded oligonucleotide.
  • the present disclosure also relates to methods of preparation and use of these double-stranded oligonucleotides, pharmaceutical compositions and oligonucleotide conjugates.
  • the present disclosure provides a double-stranded oligonucleotide comprising a sense strand and an antisense strand, the sense strand comprising nucleotide sequence I, and the antisense strand comprising Nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are both composed of 19 nucleotides, each core of the nucleotide sequence I and the nucleotide sequence II Nucleotides are modified or unmodified nucleotides, the nucleotide sequence I and the nucleotide sequence II are at least partly reverse complementary to form a double-stranded region, and the nucleotide sequence II is at least partly with the second nucleotide sequence A nucleotide sequence is reverse complementary, and the first nucleotide sequence is a 19-nucleotide nucleotide sequence in the mRNA expressed by the target gene; according to the sequence from the 5' end to the 3' end direction, at
  • the present disclosure also provides a pharmaceutical composition, which contains the double-stranded oligonucleotide provided in the present disclosure and a pharmaceutically acceptable carrier.
  • the present disclosure also provides a method for treating and/or preventing diseases or symptoms related to the mRNA level of target gene expression, the method comprising administering the double-stranded oligosaccharide of the present disclosure to a subject in need thereof.
  • Nucleotides, pharmaceutical compositions and/or oligonucleotide conjugates are provided.
  • the present disclosure also provides a kit comprising the double-stranded oligonucleotide, the pharmaceutical composition and/or the oligonucleotide conjugate of the present disclosure.
  • the double-stranded oligonucleotides, pharmaceutical compositions and/or oligonucleotide conjugates of the present disclosure have good stability, high target gene expression regulation activity, and low off-target effects.
  • the details are as follows.
  • the liver weight hardly increased, and showed significantly lower hepatic steatosis and inflammation than the reference Toxic responses than siRNA conjugates.
  • the siRNA conjugates provided by the present disclosure have an inhibition rate of off-target target sequences of no higher than 25% in the in vitro siCHECK system, showing significant Lower off-target effects.
  • mice at a dose of 30 mg/kg, in mice administered with the siRNA conjugate of the present disclosure, hepatic steatosis showed a toxic response close to that of the blank control group, and significantly lower than that of the blank control group.
  • mice administered a reference conjugate that did not contain a stabilizing modified nucleotide For another example, in mice given the siRNA conjugate of the present disclosure at a dose of 100 mg/kg, the blood biochemical indicators were significantly reduced, and there was no obvious abnormality compared with the blank control group; and compared with the reference siRNA conjugate, given Mice given the siRNA conjugates of the present disclosure did not show more than moderate inflammatory cell infiltration and necrosis, and showed significantly lower toxicity in histopathology. For another example, even at a high dose of 300 mg/kg, there was no significant difference in serum ALT compared with the blank control group, and 6 of the mice given the reference conjugate showed inflammation in histopathological sections.
  • the expression inhibition rate of the target sequence is at least 38.92nM, up to 67.54%; at a concentration of 0.1nM, the expression inhibition rate of the target sequence is at least 84.73%, up to 89.35%; meanwhile, The level of target sequence inhibitory activity was comparable to that of the reference siRNA conjugate that did not contain the stabilizing modified nucleotide.
  • the siRNA conjugate of the present disclosure has high target sequence inhibitory activity in the in vitro siCHECK system, with an IC 50 between 6.89-8.55pM; at the same time, it is identical to the rest of the sequence but does not contain stabilizing modified nucleotides
  • the reference conjugate has close target inhibitory activity.
  • Figure 1 is a histogram of the relative expression level of HBV mRNA in primary liver cells of 44Bri mice after transfection of siRNA of the present disclosure and reference siRNA respectively.
  • Fig. 2 is a histogram of relative expression levels of HBV mRNA in primary liver cells of 44Bri mice after free intake of siRNA conjugates of the present disclosure or reference siRNA conjugates and reference siRNA NC.
  • 3A and 3B are scatter plots of the relative expression levels of HBV mRNA in the liver of 44Bri mice after administration of different concentrations of siRNA conjugates of the present disclosure or reference siRNA conjugates and PBS, respectively.
  • 8A and 8B are line graphs showing the changes in serum TG levels or serum CHO levels over time after administration of siRNA conjugates of the present disclosure, reference siRNA conjugates or PBS, respectively.
  • Figures 9A and 9B are line graphs showing changes in serum TG levels or serum CHO levels over time after administration of siRNA conjugates of the present disclosure or PBS, respectively.
  • the expressions "complementary” or “reverse complementary” can be used interchangeably and have the meaning known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, the bases of one strand are each linked to the bases of the other strand. The bases on the base pair up in a complementary fashion.
  • the purine base adenine (A) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA);
  • the purine base guanine (C) is always paired with the pyrimidine base Cytosine (G) is paired.
  • Each base pair consists of a purine and a pyrimidine.
  • the double-stranded oligonucleotide is siRNA
  • the mRNA expressed by the target gene is selected from the mRNA expressed by the hepatitis B virus gene (HBV) and the mRNA expressed by the angiopoietin-like protein 3 (ANGPTL3) gene.
  • the present disclosure also provides a second double-stranded oligonucleotide, the double-stranded oligonucleotide comprises a sense strand and an antisense strand, and each nucleotide of the sense strand and the antisense strand All are modified nucleotides, wherein, the sense strand comprises nucleotide sequence I, the antisense strand comprises nucleotide sequence II, and both the nucleotide sequence I and the nucleotide sequence II are composed of Composed of 19 nucleotides, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse-complementary to form a double-stranded region, and the nucleotide sequence II is at least partially connected to the first nucleotide sequence The sequence is reverse complementary, and the first nucleotide sequence is a 19-nucleotide nucleotide sequence in the mRNA expressed by the target gene; according to the direction from the 5
  • P1 is VP, Ps or P representing a specific modification
  • the letter combination VP indicates that the adjacent nucleotide on the right side of the letter combination VP is vinyl phosphate (5'-(E)- Vinylphosphonate, E-VP) modified nucleotides
  • the letter combination Ps indicates that the adjacent nucleotide on the right side of the letter combination Ps is a phosphorothioate modified nucleotide
  • the capital letter P indicates that the right side of the letter P is the same
  • the adjacent nucleotide is a 5'-phosphate nucleotide.
  • each U in the above sequence can be replaced by T, and this replacement will not significantly reduce the gene expression regulation activity and/or off-target effect inhibition ability of the double-stranded oligonucleotide.
  • the cation is selected from one or more of alkali metal ions, ammonium cations formed from tertiary amines, and quaternary ammonium cations.
  • the alkali metal ions may be K + and/or Na +
  • the cations formed by tertiary amines may be ammonium ions formed by triethylamine and/or ammonium ions formed by N,N-diisopropylethylamine.
  • the double-stranded oligonucleotides or oligonucleotide conjugates described in the present disclosure may exist at least in part in the form of a salt.
  • the blood stability of the double-stranded oligonucleotides of the present disclosure can be further improved, their targeting, solving In vivo delivery problems of double-stranded oligonucleotides of the present disclosure, etc.
  • carriers or conjugated molecules that can impart or improve targeting will be very beneficial, which will greatly improve the efficiency of double-stranded oligonucleotides in regulating the expression of target genes and reduce potential side effects.
  • the double-stranded oligonucleotides also need to be able to function at the target site, that is, the encapsulation/conjugation of the carrier or conjugated molecules cannot affect the double-stranded oligonucleotides.
  • the activity of the acid itself (for example, in the case where the double-stranded oligonucleotide is siRNA, cannot affect the RNAi machinery that siRNA is loaded into the cell, ie the RISC complex).
  • these targeting carriers or conjugated molecules are also required to have good biocompatibility and as low toxicity as possible.
  • the methods of the present disclosure can significantly reduce off-target effects of double-stranded oligonucleotides.
  • the method of the present disclosure significantly reduces the off-target effect of the double-stranded oligonucleotide while maintaining the target gene expression regulation ability of the double-stranded oligonucleotide.
  • the substitution is made to the 3rd or 5th nucleotide in the antisense strand in a 5' end to 3' end direction.
  • the substitutions are made to no more than 2 nucleotides of the 3rd to 9th nucleotides in the antisense strand in a 5' end to 3' end direction.
  • the double-stranded oligonucleotide obtained according to the method of the present disclosure can achieve an optimal balance between pharmaceutical activity and low off-target effects.
  • the substitution is made to the 3rd and/or 5th nucleotide in the antisense strand in a 5' end to 3' end direction.
  • the substitution is also performed on one of the 4th, 7th or 9th nucleotides of the antisense strand.
  • the definitions and selection ranges of the stabilizing modified nucleotides and stabilizing modifying groups are as described above.
  • the methods of the present disclosure comprise performing one of the following substitutions on the antisense strand in the direction from the 5' end to the 3' end:
  • the pharmaceutical composition there is no special requirement on the content of the double-stranded oligonucleotide and the pharmaceutically acceptable carrier.
  • the double-stranded oligonucleotide and the pharmaceutically acceptable carrier The weight ratio of the carrier can be 1:(1-500), and in some embodiments, the above weight ratio is 1:(1-50).
  • the pharmaceutical composition may also contain other pharmaceutically acceptable excipients, which may be one or more of various preparations or compounds routinely used in the art.
  • the other pharmaceutically acceptable excipients may include at least one of a pH buffer, a protective agent and an osmotic pressure regulator.
  • the protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose and glucose. Based on the total weight of the pharmaceutical composition, the content of the protective agent may be 0.01-30% by weight.
  • the osmotic pressure regulator may be sodium chloride and/or potassium chloride.
  • the content of the osmotic pressure regulator makes the osmotic pressure of the pharmaceutical composition 200-700 milliosmol/kg (mOsm/kg). According to the desired osmotic pressure, those skilled in the art can easily determine the content of the osmotic pressure regulator.
  • the dosage of the preparation made from the pharmaceutical composition will be adjusted due to different administration methods during administration.
  • the organic amine can be a compound represented by formula (201) or a pharmaceutically acceptable salt thereof described in Chinese patent application CN103380113A:
  • R 103 is a polyamine. In other embodiments, R 103 is a ketal. In some embodiments, each of R 101 and R 102 in formula (201) is independently any substituted or unsubstituted, branched or straight chain alkyl or alkenyl, the alkyl A radical or alkenyl group has 3 to about 20 carbon atoms, such as 8 to about 18 carbon atoms, and 0 to 4 double bonds, such as 0 to 2 double bonds.
  • R 103 can be any of the following formulas (204)-(213):
  • each "HCC” represents a hydrocarbon chain
  • each * shows that R 103 is the same as in formula (201) Possible points of attachment of the nitrogen atom in , where each H at any * position can be replaced to achieve attachment to the nitrogen atom in formula (201).
  • the organic amine is an organic amine shown in formula (214) and/or an organic amine shown in formula (215):
  • the amount of alcohol is such that the total mass concentration of the obtained lipid solution is 2-25mg/mL, For example, it can be 8-18 mg/mL.
  • the alcohol is selected from pharmaceutically acceptable alcohols, such as alcohols that are liquid around room temperature, for example, ethanol, propylene glycol, benzyl alcohol, glycerin, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400 One or more of, for example, can be ethanol.
  • the lipid solution and the double-stranded oligonucleotide aqueous solution are mixed, and the mixed product is incubated at 40-60° C. for at least 2 minutes, for example, 5-30 minutes, to obtain an incubated liposome preparation.
  • the volume ratio of lipid solution and double-stranded oligonucleotide aqueous solution is 1:(2-5), for example, it can be 1:4.
  • the present disclosure provides an oligonucleotide conjugate comprising the double-stranded oligonucleotide provided by the present disclosure or the double-stranded oligonucleotide obtained according to the method of the present disclosure. Nucleotides, and a conjugating group that is conjugated to the double-stranded oligonucleotide.
  • the conjugation group comprises a linker and a pharmaceutically acceptable targeting group and/or a delivery assisting group, and the double-stranded oligonucleotide, the linker and the target
  • the targeting group or the delivery auxiliary group is sequentially connected covalently or non-covalently, each targeting group is selected from ligands capable of binding to cell surface receptors, and each delivery auxiliary group is selected from ligands capable of increasing The biocompatible group of the oligonucleotide conjugate in the delivery target organ or tissue.
  • conjugate means that two or more chemical moieties each having a specific function are covalently linked to each other; accordingly, a “conjugate” is Refers to the compound formed by covalent linkage between the various chemical moieties.
  • oligonucleotide conjugate refers to a compound formed by covalently linking one or more chemical moieties with specific functions to an oligonucleotide. The oligonucleotide conjugate should be understood as a general term of multiple oligonucleotide conjugates or an oligonucleotide conjugate represented by a certain chemical formula according to the context.
  • the conjugation group comprises at least one targeting group and an optional linker that is pharmaceutically acceptable, and the double-stranded oligonucleotide, the linker and the targeting The groups are linked sequentially.
  • the double-stranded oligonucleotide molecule A1 may be non-covalently or covalently conjugated to the conjugating group, for example may be covalently conjugated to the conjugating group.
  • the conjugation site of the double-stranded oligonucleotide and the conjugate group can be at the 3' or 5' end of the sense strand of the double-stranded oligonucleotide, or at the 5' end of the antisense strand, or at the double-stranded oligonucleotide's sense strand. in the internal sequence of the chain oligonucleotide.
  • the conjugation site between the double-stranded oligonucleotide and the conjugating group is at the 3' end of the sense strand of the double-stranded oligonucleotide.
  • the conjugate group can be attached to the phosphate group, the 2'-position hydroxyl group or the base of the nucleotide. In some embodiments, the conjugate group can be connected to the hydroxyl group at the 3'-position, and at this time, the nucleotides are connected by 2'-5' phosphodiester bonds.
  • the conjugate group is usually connected to the phosphate group of the nucleotide; when the conjugate group is connected to the double-stranded oligonucleotide For internal sequences, the conjugating group is usually attached to the ribose sugar ring or the base.
  • the targeting group can be connected to the double-stranded oligonucleotide molecule through a suitable linker, and those skilled in the art can select a suitable linker according to the specific type of the targeting group.
  • suitable linker those skilled in the art can select a suitable linker according to the specific type of the targeting group.
  • the types of these linkers, targeting groups and connection methods with double-stranded oligonucleotides can be found in the disclosure of WO2015006740A2, the entire contents of which are incorporated herein by reference.
  • the targeting group can be a ligand routinely used in the field of double-stranded oligonucleotide administration, such as various ligands described in WO2009082607A2, the entire disclosure of which is incorporated by reference This article.
  • At least one or each of the targeting groups is selected from ligands capable of binding to cell surface receptors expressing the target gene.
  • At least one or each of the targeting groups is selected from ligands capable of binding to receptors on the surface of mammalian liver parenchymal cells.
  • each of said targeting groups is independently a ligand that has an affinity for an asialoglycoprotein receptor on the surface of mammalian liver cells.
  • each of said targeting groups is independently an asialoglycoprotein or a sugar.
  • 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, ⁇ -D-mannofuranose, ⁇ -D-mannose furanose, ⁇ -D-mannopyranose, ⁇ -D-mannopyranose , ⁇ -D-glucopyranose, ⁇ -D-glucopyranose, ⁇ -D-glucopyranose, ⁇ -D-glucofuranose, ⁇ -D-fructofuranose, ⁇ -D-fructopyranose, ⁇ -D- Galactopyranose, ⁇ -D-galactopyranose, ⁇ -D-galactofuranose, ⁇ -D-galactofuranose, glucosamine, sialic acid
  • the linker in the oligonucleotide conjugates of the present disclosure has a structure as shown in formula (301):
  • k is an integer of 1-3;
  • L A has a structure containing an amide bond as shown in formula (302)
  • L B has a structure containing N-acylpyrrolidine as shown in formula (303), containing carbonyl and oxygen atoms
  • LC is based on hydroxymethyl Linking groups for aminomethane, dimethylolaminomethane or trishydroxymethylaminomethane;
  • n 302 , q 302 and p 302 are each independently an integer of 2-6, optionally, n 302 , q 302 and p 302 are each independently 2 or 3; n 303 is an integer of 4-16, which can be Optionally, n 303 is an integer of 8-12, Indicates the site where the group is covalently attached.
  • the oligonucleotide conjugates provided by the present disclosure have a structure as shown in formula (305):
  • Nu represents the double-stranded oligonucleotide provided by the present disclosure, or the double-stranded oligonucleotide obtained according to the method of the present disclosure.
  • the linker in the oligonucleotide conjugates of the present disclosure has the structure shown in formula (306):
  • n 306 is an integer of 0-3, and each p 306 is independently an integer of 1-6, Indicates the site where the group is covalently attached;
  • the linking group is connected by an ether bond with the targeting group through the oxygen atom marked by *;
  • the linking group is connected by at least one of the oxygen atoms marked by # One is connected to the double-stranded oligonucleotide by forming a phosphate bond or a phosphorothioate bond, and the rest is connected to a hydrogen atom with an oxygen atom marked by # to form a hydroxyl group, or to a C 1 -C 3 alkyl group to form a C 1 -C 3 alkoxy;
  • Nu represents the double-stranded oligonucleotide provided by the present disclosure, or the double-stranded oligonucleotide obtained according to the method of the present disclosure.
  • the oligonucleotide conjugates of the present disclosure have the structure shown in formula (308):
  • n1 is an integer selected from 1-3, and n3 is an integer selected from 0-4;
  • Each m1, m2 or m3 is independently an integer selected from 2-10;
  • R 10 , R 11 , R 12 , R 13 , R 14 or R 15 are each independently H, or are selected from the group consisting of the following groups: C 1 -C 10 alkyl, C 1 -C 10 haloalkane and C 1 -C 10 alkoxy;
  • R 3 has the structure shown in formula A59:
  • E 1 is OH, SH or BH 2
  • Nu represents the double-stranded oligonucleotide provided by the present disclosure, or the double-stranded oligonucleotide obtained according to the method of the present disclosure
  • n1 is an integer of 1-2
  • n3 is an integer of 0-1
  • n1+n3 2-3.
  • the spatial position between a plurality of M1 ligands can be suitable for the M1 ligand and the liver surface asialoglycoprotein receptor
  • R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are each independently selected from H, C 1 -C 10 alkyl, C 1 -C 10 haloalkyl, and C
  • R 10 , R 11 , R 12 , R 13 , R 14 , and R 15 are each independently selected from H, methyl, and ethyl.
  • R 10 , R 11 , R 12 , R 13 , R 14 , and R 15 are all H.
  • R 3 is a group with the structure shown in formula A59, wherein E 1 is OH, SH or BH 2 , based on the consideration of the availability of raw materials for preparation, in some embodiments In, E 1 is OH or SH.
  • R2 is selected to achieve linkage to N and A59 on the nitrogen-containing backbone.
  • nitrogen-containing skeleton refers to a chain structure in which carbon atoms connected with R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are connected to N.
  • R2 may be any linking group capable of linking the A59 group to the N on the nitrogen - containing backbone in an appropriate manner.
  • the R2 group needs to contain both the linking site connected to the N on the nitrogen-containing backbone and the Linkage site for P phase linkage in R3 .
  • R 2 is B5, B6, B5' or B6':
  • the value range of q 2 may be an integer of 1-10, and in some embodiments, q 2 is an integer of 1-5.
  • L1 is selected from a linked combination of one or more of the groups of formulas A1-A26.
  • L 1 is selected from the connection combination of one or more of A1, A4, A5, A6, A8, A10, A11 and A13; in some embodiments, L 1 is selected from A1, A4, A connection combination of at least 2 of A8, A10, and A11; in some embodiments, L1 is selected from a connection combination of at least 2 of A1, A8, and A10.
  • an oligonucleotide conjugate of the present disclosure has the formula (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421) or (422) structure:
  • P in Formula A59 is connected to the end of the double-stranded oligonucleotide sense strand or the antisense strand; in some embodiments, P in Formula A59 is connected to the double-stranded oligonucleotide sense strand the end of. The terminus refers to the first 4 nucleotides counted from one end of the sense strand or the antisense strand. In some embodiments, P in Formula A59 is connected to the end of the double-stranded oligonucleotide sense strand or the antisense strand; in some embodiments, P in Formula A59 is connected to the end of the double-stranded oligonucleotide sense strand. 3' end.
  • P in formula A59 can be connected to any possible position on the nucleotide in the double-stranded oligonucleotide, for example, the 5' position of the nucleotide, the 2' position of the nucleotide, the 3' position of the nucleotide or nucleotide bases.
  • P in formula A59 can be linked to the 2' position, the 3' position or the 5' position of the nucleotides in the double-stranded oligonucleotide by forming a phosphodiester bond.
  • the P in the formula A59 is connected to the oxygen atom formed after the dehydrogenation of the 3' hydroxyl of the 3' terminal nucleotide of the sense strand of the double-stranded oligonucleotide, or the P in the formula A59 is replaced by a double-stranded
  • the hydrogen in the 2'-hydroxyl group of one nucleotide in the sense strand of the oligonucleotide is attached to the nucleotide, or P in formula A59 is replaced by the 5'-terminal nucleotide of the sense strand of the double-stranded oligonucleotide. 'The hydrogen in the hydroxyl group is attached to the nucleotide.
  • the double-stranded oligonucleotide contained in the oligonucleotide conjugate of the present disclosure may be siRNA, and in this case, the oligonucleotide conjugate of the present disclosure is also referred to as siRNA conjugate.
  • the double-stranded oligonucleotides comprised by the oligonucleotide conjugates of the present disclosure can be, for example, siRNAs listed in Table 1. Oligonucleotide conjugates containing these siRNAs exhibited low off-target effects and high mRNA inhibitory activity of target gene expression.
  • oligonucleotide conjugates can be synthesized by methods that have been described in detail in the prior art.
  • WO2015006740A2 describes the preparation methods of various siRNA conjugates in detail.
  • the oligonucleotide conjugates of the present disclosure can also be obtained by means well known to those skilled in the art.
  • the preparation method of the structure represented by formula (305) is described in WO2014025805A1
  • the preparation method of the structure represented by formula (307) is described by Rajeev et al. in ChemBioChem 2015, 16, 903-908.
  • Chinese patent application CN110959011A also discloses in detail the method for preparing the oligonucleotide conjugate represented by formula (308).
  • the contents of the above documents are incorporated herein in their entirety by reference.
  • the oligonucleotide conjugates of the present disclosure can also be used in combination with other pharmaceutically acceptable adjuvants.
  • the adjuvants can be one or more of various preparations or compounds routinely used in the art. For details, please refer to the above-mentioned Description of the pharmaceutical compositions of the present disclosure.
  • the present disclosure provides double-stranded oligonucleotides provided by the present disclosure, double-stranded oligonucleotides obtained according to the methods of the present disclosure, pharmaceutical compositions and/or oligonucleotide conjugates for use in Use in medicines for treating and/or preventing diseases or symptoms associated with target gene expression mRNA levels.
  • the specific gene is a gene abnormally expressed in hepatocytes.
  • the specific gene is an endogenous gene expressed in the liver.
  • the specific gene is a gene of a pathogen that propagates in the liver.
  • the specific gene is a gene expressed in lung epithelial cells.
  • the specific gene is a gene expressed in the central nervous system. In some embodiments, the specific gene is a gene expressed in tumor cells. In some embodiments, the mRNA expressed by the target gene is selected from one of the mRNAs transcribed from the following genes: ACE2, AGT, ANGPTL3, ApoA, ApoB, ApoC, AR, ASK1, C3, C5, Col1A1, CTGF , Ebola, FOXO1, FTO, FVII, FXI, FXII, GCGR, HBV, HCV, HSD, p53, PCSK9, PNP, PLG, PKK, KNG, RAGE, RPTOR, SARS-CoV-2, SCD1, SCNN1A, SOD1, STAT3 , TIMP-1, TMPRSS6, XO.
  • the mRNA expressed by the target gene is selected from the mRNA expressed by the hepatitis B virus gene (HBV), the mRNA expressed by the angiopoietin-like protein 3 (ANGPTL3) gene, or the mRNA expressed by the apolipoprotein C3 (ApoC3) gene mRNA.
  • the disease or symptom associated with the mRNA level of target gene expression is chronic liver disease, hepatitis, liver fibrotic disease, liver proliferative disease and/or dyslipidemia.
  • the disease or symptom associated with the mRNA level of target gene expression is hepatitis B or dyslipidemia.
  • the dyslipidemia is hypercholesterolemia, hypertriglyceridemia, or atherosclerosis.
  • the present disclosure provides a method for treating and/or preventing diseases or symptoms related to the mRNA level of target gene expression, the method comprising administering to a subject in need thereof an effective amount of the present disclosure provides The double-stranded oligonucleotide, the double-stranded oligonucleotide obtained according to the method of the present disclosure, the pharmaceutical composition and/or the oligonucleotide conjugate.
  • the mRNA expressed by the target gene is selected from one of the mRNAs transcribed from the following genes: ACE2, AGT, ANGPTL3, ApoA, ApoB, ApoC, AR, ASK1, C3, C5, Col1A1, CTGF , Ebola, FOXO1, FTO, FVII, FXI, FXII, GCGR, HBV, HCV, HSD, p53, PCSK9, PNP, PLG, PKK, KNG, RAGE, RPTOR, SARS-CoV-2, SCD1, SCNN1A, SOD1, STAT3 , TIMP-1, TMPRSS6, XO.
  • the mRNA expressed by the target gene is selected from the mRNA expressed by the hepatitis B virus gene (HBV), the mRNA expressed by the angiopoietin-like protein 3 (ANGPTL3) gene, or the mRNA expressed by the apolipoprotein C3 (ApoC3) gene mRNA.
  • the disease or symptom associated with the mRNA level of target gene expression is chronic liver disease, hepatitis, liver fibrotic disease, liver proliferative disease and/or dyslipidemia.
  • the disease or symptom associated with the mRNA level of target gene expression is hepatitis B or dyslipidemia.
  • the dyslipidemia is hypercholesterolemia, hypertriglyceridemia, or atherosclerosis.
  • the conjugates provided by the present disclosure can also be used to treat other liver diseases, including diseases characterized by unwanted cell proliferation, blood diseases, metabolic diseases and diseases characterized by inflammation.
  • a proliferative disease of the liver may be a benign or malignant disease, such as cancer, hepatocellular carcinoma (HCC), liver metastases, or hepatoblastoma.
  • a hematological or inflammatory disease of the liver may be a disease involving coagulation factors, complement mediated inflammation or fibrosis. Metabolic disorders of the liver include dyslipidemia and irregularities in glucose regulation.
  • the disease is treated by administering one or more double-stranded oligonucleotides with high homology to gene sequences involved in the disease.
  • the mRNA expressed by the target gene is selected from one of the mRNAs transcribed from the following genes: ACE2, AGT, ANGPTL3, ApoA, ApoB, ApoC, AR, ASK1, C3, C5, Col1A1, CTGF, Ebola , FOXO1, FTO, FVII, FXI, FXII, GCGR, HBV, HCV, HSD, p53, PCSK9, PNP, PLG, PKK, KNG, RAGE, RPTOR, SARS-CoV-2, SCD1, SCNN1A, SOD1, STAT3, TIMP -1, TMPRSS6, XO.
  • the regulation refers to inhibiting the expression of the target gene in the cell
  • the mRNA expressed by the target gene is selected from the mRNA expressed by the hepatitis B virus gene (HBV), the expression of the angiopoietin-like protein 3 (ANGPTL3) gene mRNA or mRNA expressed by apolipoprotein C3 (ApoC3) gene.
  • HBV hepatitis B virus gene
  • ANGPTL3 angiopoietin-like protein 3
  • ApoC3 apolipoprotein C3
  • the double-stranded oligonucleotide provided by the present disclosure, the double-stranded oligonucleotide obtained according to the method of the present disclosure, the pharmaceutical composition and/or the oligonucleotide conjugate can be used to prevent and/or treat the pathology Condition or disease, or for the manufacture of a medicament for the prevention and/or treatment of a pathological condition or disease as described herein.
  • administration/administration refers to an action by causing at least partly positioning a double-stranded oligonucleotide, a pharmaceutical composition and/or an oligonucleotide conjugate at a desired site to produce a desired effect.
  • Routes of administration suitable for the methods of the present disclosure include topical and systemic administration.
  • topical administration results in delivery of more double-stranded oligonucleotides, pharmaceutical compositions and/or oligonucleotide conjugates to a specific site compared to the subject's entire body; whereas systemic administration results in The double-stranded oligonucleotide, pharmaceutical composition and/or oligonucleotide conjugate is delivered to substantially the entire body of the subject.
  • the doses of double-stranded oligonucleotides, pharmaceutical compositions and/or oligonucleotide conjugates described in the present disclosure can be conventional doses in the art, and the doses can be based on various parameters, especially the subjects. determined by age, weight and sex. Toxicity and efficacy can be determined by standard pharmaceutical procedures in cell culture or experimental animals, such as determining the LD50 (the dose that causes 50% of the population to die) and the ED50 (the dose that can cause 50% of the maximum response intensity in quantitative response, and in quantitative response). Middle refers to the dose that causes 50% of the test subjects to have a positive reaction).
  • a range of dosage for use in humans can be derived based on the data obtained from cell culture assays and animal studies.
  • the cells are hepatocytes.
  • the hepatocytes may be cells selected from hepatoma cell lines such as Hep3B, HepG2, Huh7, etc. or isolated primary hepatocytes, in some embodiments, primary hepatocytes.
  • the present disclosure provides a kit comprising the double-stranded oligonucleotide provided by the present disclosure, the double-stranded oligonucleotide obtained according to the method of the present disclosure, a pharmaceutical composition and/or an oligonucleotide conjugate compound.
  • kits described herein may provide a double-stranded oligonucleotide, a pharmaceutical composition, and/or a conjugate in one container.
  • a kit described herein may comprise a container providing a pharmaceutically acceptable excipient.
  • the kit may also contain other components, such as stabilizers or preservatives.
  • the kits described herein may comprise at least one additional therapeutic agent in a container other than the container in which the double-stranded oligonucleotides, pharmaceutical compositions, and/or conjugates described herein are provided .
  • the double-stranded oligonucleotide and the pharmaceutically acceptable carrier and/or adjuvant and the pharmaceutical composition and/or conjugate, and/or the pharmaceutically acceptable adjuvant can be Provided in any form, eg liquid form, dry form or lyophilized form.
  • the double-stranded oligonucleotide and pharmaceutically acceptable carrier and/or adjuvant and the pharmaceutical composition and/or conjugate and optional pharmaceutically acceptable adjuvant are substantially pure and/or sterile.
  • sterile water can be provided in kits of the present disclosure.
  • the siRNA, the pharmaceutical composition comprising siRNA, and the siRNA conjugate in these embodiments are also referred to as the siRNA of the present disclosure, the pharmaceutical composition of the present disclosure, and the siRNA conjugate of the present disclosure. compound.
  • the double-stranded oligonucleotide of the present disclosure can only be siRNA, on the contrary, the double-stranded oligonucleotide can be other variants disclosed herein or known to those skilled in the art, such as small activating RNA ( saRNA) and so on.
  • compositions comprising siRNA, and siRNA conjugates
  • other double-stranded oligonucleotides will be used alone, or to form pharmaceutical compositions and/or oligonucleotides described in the present disclosure.
  • Acid conjugates function similarly.
  • reagents and medium used in the following examples are commercially available, and the operations such as nucleic acid electrophoresis and real-time PCR used are all referred to in Molecular Cloning (Cold Spring Harbor LBboratory Press (1989)). method to proceed.
  • the uppercase letters C, G, U, A, and T represent the base composition of nucleotides;
  • the lowercase letter m indicates that the adjacent nucleotide on the left side of the letter m is a methoxy-modified nucleotide;
  • the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a fluorinated nucleotide;
  • the underlined lowercase letter combination moe indicates that the nucleotide adjacent to the left of the letter combination is ribose 2'-O -Methoxyethyl-modified nucleotides;
  • a lowercase letter s indicates that there is a phosphorothioate linkage between the two nucleotides to the left and right of the letter s.
  • the sense and antisense strands corresponding to the siRNAs numbered as reference siRNA1, reference siRNA2, and NC in Table 2 were synthesized by solid-phase synthesis method. Use DEPC water to dissolve the obtained equimolar sense strand and antisense strand respectively, and then anneal to obtain the reference siRNA, coded as NC.
  • the uppercase letters C, G, U, A, and T represent the base composition of nucleotides;
  • the lowercase letter m indicates that the adjacent nucleotide on the left side of the letter m is a methoxy-modified nucleotide;
  • the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a fluorinated nucleotide;
  • the underlined letter combination moe indicates that the nucleotide adjacent to the left of the letter combination moe is ribose 2'-O -Methoxyethyl-modified nucleotides;
  • the lowercase letter s indicates that the two nucleotides on the left and right of the letter s are connected by a phosphorothioate group, and
  • VP indicates that the nucleotide on the right side of the letter VP is 5' - Vinyl phosphate modified nucleotides;
  • P indicates that the one nucleotide to the right
  • Each of the above-prepared conjugate 5, conjugate 19, reference conjugate 10 and reference conjugate 11 was prepared as a 0.02 mg/mL solution with 1 ⁇ PBS buffer as the test product solution.
  • the double-strand thermal dissociation temperature Tm was calculated from the first derivative of the temperature-absorbance curve according to the specification of the spectrophotometer. Tm value and ⁇ Tm value result are shown in following table 4:
  • ⁇ Tm value (conjugate to be tested) Tm (conjugate to be tested) - Tm (reference conjugate 10);
  • ⁇ Tm value (conjugate to be tested) Tm (conjugate to be tested) - Tm (reference conjugate 11).
  • the double-stranded oligonucleotide comprising a stabilized modified nucleotide of the present disclosure and its conjugate have a higher double-stranded thermal dissociation temperature.
  • modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect.
  • Nucleic Acids Research, 6(708) In the method described in 2136-2151, a detection plasmid is constructed, and the detection plasmid and the siRNA to be tested are co-transfected into HEK293A cells, and the target sequence inhibitory activity of the siRNA is reflected by the expression level of the dual luciferase reporter gene. Specific steps are as follows:
  • a detection plasmid was constructed using psiCHECK TM -2 (Promega TM ) plasmid, which contained a target sequence 1, ie, the siRNA target sequence.
  • the target sequence 1 is as follows:
  • the target sequence 1 is the complete complementary sequence of the detected siRNA antisense strand, so the inhibitory effect of each siRNA on the target sequence 1 can reflect the inhibitory ability of the detected siRNA to target gene expression.
  • the target sequence 1 and its complementary sequence were cloned into the Xho I/Not I site of the psiCHECK TM -2 plasmid.
  • fetal bovine serum FBS, Hyclone Company
  • penicillin-streptomycin Penicillin-Streptomycin, Gibco, Invitrogen Company
  • DMEM complete medium Hyclone Company
  • siRNAs Use DEPC water to dilute the above detection plasmid into 200ng/ ⁇ L detection plasmid working solution; use DEPC water to prepare each siRNA in the following siRNAs to 4000nM, 1000nM, 250nM, 62.5nM, 15.625nM, 3.91nM, 0.977nM, 0.244nM, 0.061nM, 0.0153nM and 0.0038nM siRNA working solution with 11 different concentrations, the siRNA used are siRNA1, siRNA2, siRNA3, siRNA4 prepared above and reference siRNA1, reference siRNA2 respectively.
  • each 2A1-2A11 solution contains 1 ⁇ L of siRNA working solution of the above 11 concentrations, 0.05 ⁇ L of detection plasmid working solution (including 10 ng of detection plasmid) and 10 ⁇ L of Opti-MEM culture solution. base.
  • each 2B solution contains 0.2 ⁇ L LipofectamineTM 2000 and 10 ⁇ L Opti-MEM medium.
  • each 2C solution contains 0.05 ⁇ L of detection plasmid working solution (containing 10 ng of detection plasmid) and 10 ⁇ L of Opti-MEM medium.
  • each siRNA transfection complex 2X1-2X11 were transfected into 3 culture wells to obtain co-transfection containing siRNA The mixture is recorded as the test group.
  • siRNA For each siRNA, 2 ⁇ 12 transfection complexes were added to the other 3 culture wells in an amount of 20 ⁇ L/well to obtain a transfection mixture without siRNA, which was recorded as a blank control group.
  • the co-transfection mixture containing siRNA and the co-transfection mixture without siRNA were respectively transfected in culture wells for 4 hours, and 100 ⁇ L of H-DMEM complete medium containing 20% FBS was added to each well.
  • the 96-well plate was placed in a CO 2 incubator to continue culturing for 24 hours.
  • the function is as follows,
  • Y is the ratio R, the relative residual activity of Renilla
  • Primary mouse liver cells were extracted from fresh liver tissue of 44Bri mice, and the density of primary mouse liver cells was adjusted to 2 ⁇ 105 cells in Opti-MEM (1X) medium (GIBCO, Cat. No. 31985-070) /mL to obtain mouse liver primary cell suspension. Then, the obtained mouse liver primary cell suspensions were respectively added to different culture wells of the 12-well plate, and the mouse liver primary cells were inoculated into the culture wells. The volume of primary mouse liver cell suspension added was 0.5 mL/well, and the number of primary mouse liver cells was 1 ⁇ 10 5 cells/well.
  • Each 4A solution contains 1.5 ⁇ L of the above siRNA working solution and 50 ⁇ L of Opti-MEM medium in turn.
  • the siRNA of the present disclosure shows excellent HBV mRNA inhibitory activity in 44Bri mouse primary liver cells, and at the siRNA concentration of 50nM, the HBV mRNA inhibitory rate is at least 73.92%, the highest can be reached 77.58%, and showed HBV mRNA inhibitory activity comparable to that of the corresponding reference siRNA1 that did not include stabilizing modified nucleotides.
  • the target sequence inhibitory activity of the reference siRNA2 with stabilizing modified nucleotides at the 2-position of the antisense strand was greatly reduced, and the HBV mRNA inhibition rate was only 36.31%.
  • Primary mouse liver cells were extracted from fresh liver tissue of 44Bri mice, and the density of primary mouse liver cells was adjusted to 1 ⁇ 105 cells in Opti-MEM (1X) medium (GIBCO, Cat. No. 31985-070) /mL to obtain mouse liver primary cell suspension. Then, the obtained mouse liver primary cell suspensions were respectively added to different culture wells of the 12-well plate, and the mouse liver primary cells were inoculated into the culture wells. The volume of the primary mouse liver cell suspension added was 1 mL/well, and the number of primary mouse liver cells was 1 ⁇ 10 5 cells/well.
  • Opti-MEM (1X) medium GIBCO, Cat. No. 31985-070
  • siRNA conjugate working solutions 4 ⁇ M (calculated as siRNA) siRNA conjugate working solutions, and the siRNA conjugates used are conjugate 5, conjugate 6 or reference than conjugate 4.
  • the reference siRNA NC was formulated into a 4 ⁇ M reference siRNA NC working solution.
  • siRNA conjugate working solution or the reference siRNA NC working solution of each conjugate to the different culture wells of mouse liver primary cell suspension mentioned above, mix evenly, and add 2.5 ⁇ L/well, each A siRNA conjugate or reference siRNA NC were transfected into 3 culture wells respectively to obtain a transfection mixture containing siRNA (calculated as siRNA, with a final concentration of 10 nM), which was recorded as the test group.
  • the mouse liver primary cell suspension in the other 3 culture wells was recorded as the blank control group.
  • Each transfection mixture containing siRNA and the blank control group were placed in an incubator containing 5% CO 2 and incubated at 37° C. for 24 h.
  • TRIZOL purchased from SIGMA, product number T9424 was used to extract the total RNA in the cells in each well according to the method described in the manual to obtain an aqueous solution of total RNA.
  • the reverse transcription conditions are: for each reverse transcription reaction system, incubate the reverse transcription reaction system at 50°C for 50 minutes, then incubate at 85°C for 5 minutes, and finally incubate at 4°C for 5 minutes. Add 80 ⁇ L of DEPC water to the system to obtain a solution containing cDNA.
  • each reverse transcription reaction system takes 5 ⁇ L of the above cDNA-containing solution as a template, use The reagents provided by the SYBR qPCR SuperMix Plus kit (purchased from Nearshore Protein Technology Co., Ltd., Cat. No. E096-01B) were used to prepare 15 ⁇ L of qPCR reaction system. 6, the final concentration of each primer was 0.25 ⁇ M.
  • SYBR qPCR SuperMix Plus kit purchased from Nearshore Protein Technology Co., Ltd., Cat. No. E096-01B
  • the final concentration of each primer was 0.25 ⁇ M.
  • Each qPCR reaction system was placed on an ABI StepOnePlus Real-Time PCR instrument and amplified using a three-step method. The amplification program was pre-denaturation at 95°C for 10 minutes, followed by denaturation at 95°C for 30s, annealing at 60°C for 25s, and extension at 72°C for 25s.
  • Figure 2 is a histogram of the relative expression levels of HBV mRNA in primary hepatocytes of 44Bri mice after free intake of conjugate 5, conjugate 6 or reference conjugate 4 and reference siRNA NC. Further, the inhibition rate of each siRNA conjugate or reference siRNA NC to HBV mRNA is summarized in Table 8.
  • Hepatitis B virus surface antigen diagnostic kit (enzyme-linked immunoassay) (Shanghai Kehua Biology) was used to detect the serum HbsAg content of 44Bri mice according to the method recorded in the instructions, and the mice with S/COV>10 were selected and randomly grouped (both Male), 5 mice in each group, numbered respectively, administered the conjugate 5, Conjugate 6 or reference conjugate 4, the siRNA conjugates were provided in the form of 1 ⁇ PBS solution containing 0.2mg/ml or 0.02mg/ml (calculated as siRNA) of siRNA conjugates respectively, and the administration volumes were 5ml/kg; each of the other two groups of mice was given 1 ⁇ PBS, and the administration volume was 5ml/kg, serving as a blank control group.
  • RNA later (Sigma Aldrich); 1mL Trizol (Sigma Company) was added to each liver tissue. ), crushed 3 times in a Tissuelyset II automatic tissue homogenizer, each time for 30s, to obtain liver tissue homogenate, add 0.2mL chloroform to it, and let it stand for 3min. Centrifuge at 12,000 rpm for 10 min at 4°C, and take 0.4 mL of the supernatant. Add 0.5mL isopropanol to the supernatant and let it stand at room temperature for 10min.
  • RNA in the liver tissue of each mouse take 10.5 ⁇ L of total RNA aqueous solution containing 1 ⁇ g of total RNA, use the reverse transcription kit Reverse Transcription System (purchased from Promega, Cat. No. A3500), and reverse The recording operation steps were prepared as 20 ⁇ L of reverse transcription reaction system, and the total RNA was reverse transcribed.
  • the reverse transcription conditions are: for each reverse transcription reaction system, incubate the reverse transcription reaction system at 42°C for 30 minutes, then incubate at 95°C for 5 minutes, and finally incubate at 4°C for 5 minutes. Add 80 ⁇ L of DEPC water to the recording reaction system to obtain a solution containing cDNA.
  • each reverse transcription reaction system takes 5 ⁇ L of the above cDNA-containing solution as a template, and use the reagents provided by the SYBR select Master Mix kit (Applied biosystem company) to prepare 20 ⁇ L of a qPCR reaction system, which is used to amplify the target gene HBV
  • the PCR primer sequences of X and the internal reference gene GAPDH are shown in Table 6, and the final concentration of each primer is 0.25 ⁇ M.
  • Each qPCR reaction system was placed on the ABI StepOnePlus Real-Time PCR instrument and amplified using a three-step method.
  • the amplification program was pre-denaturation at 95°C for 10 minutes, followed by denaturation at 95°C for 30s, annealing at 60°C for 30s, and extension at 72°C for 30s. After repeating the above-mentioned processes of denaturation, annealing and extension a total of 40 times, a product W containing amplified target gene HBV X and internal reference gene GAPDH was obtained. Product W was then incubated at 95°C for 1min, 55°C for 30s, and 95°C for 30s. The real-time fluorescent quantitative PCR instrument collected the melting curves of the target gene HBV X and the internal reference gene GAPDH in the product W respectively, and obtained the target gene HBV X and the internal reference gene GAPDH. Ct value.
  • Figure 3A and Figure 3B are respectively given 1mg/kg or 0.1mg/kg (calculated as siRNA) conjugate 5, conjugate 6 or reference conjugate 4 and PBS, the relative HBV mRNA in the liver of 44Bri mice Scatterplot of expression levels.
  • PBS represents the blank control group.
  • the inhibition rate of each siRNA conjugate to HBV mRNA is summarized in Table 9.
  • the siRNA conjugates of the present disclosure exhibit excellent HBV mRNA inhibitory effects in mice, and at a dose of 0.1 mg/kg, the HBV mRNA inhibitory rate is at least 63.18%. At a dose of 1 mg/kg, the HBV mRNA inhibition rate could even be as high as 96.31%, and showed an HBV mRNA inhibitory activity comparable to that of the corresponding reference conjugate 4 that did not include stabilizing modified nucleotides.
  • % and the preceding numbers represent the percentage difference between the corresponding index and the reference blank control group.
  • ⁇ stands for increase.
  • ⁇ 5.43% means that the liver weight increased by 5.43% compared with the blank control group.
  • the siRNA conjugates of the present disclosure can effectively reduce the liver toxicity caused by off-target effects, so in the preparation of drugs for the treatment and/or prevention of HBV diseases or symptoms Shows significantly higher safety and has excellent development prospects.
  • Figure 4 shows that the free intake of conjugate 5, conjugate 6, conjugate 22, conjugate 23, reference conjugate 4, reference conjugate 10, reference conjugate 12, reference Histogram of relative expression levels of HBV mRNA in primary hepatocytes of 44Bri mice after conjugate 13 or reference siRNA NC. Further, the inhibition rate of each siRNA conjugate or reference siRNA NC to HBV mRNA is summarized in Table 11.
  • siRNA conjugates 5, 6, 22 and 23 of the present disclosure showed excellent HBV mRNA inhibitory activity in 44Bri mouse primary liver cells, and at a siRNA concentration of 10 nM, HBV The mRNA inhibition rate was at least 78.47%, up to 85.97%, and the HBV mRNA inhibition activity was comparable to that of the reference conjugate 4, and significantly higher than that of the reference conjugates 10, 12 and 13.
  • the corresponding position in reference conjugate 4 is a non-stabilizing modified nucleotide
  • the corresponding position in reference conjugate 10 is an unmodified nucleotide
  • reference conjugates 12 and 13 are on the antisense strand In addition to the 3-9 position in the direction of the 5'-3' end, it also contains stabilizing modified nucleotides.
  • each mouse in the test group and the blank control group was subjected to orbital blood collection.
  • the blood collection volume was 0.6 mL. Centrifuge at 3000rpm for 15min to obtain serum.
  • concentration of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum was further detected by PM1P000/3 automatic serum biochemical analyzer (SABA, Italy). See Figure 6A and Figure 6B for the results.
  • PBS represents a blank control group.
  • Figure 6A and Figure 6B are the scatter points of ALT and AST concentrations in mouse serum after administration of 30 mg/kg of Conjugate 9, Conjugate 10, Reference Conjugate 5, Reference Conjugate 15 or PBS, respectively picture. It can be seen from Figure 6A and Figure 6B that, compared with the blank control group, after administration of the reference conjugate 5 that does not contain stabilized modified nucleotides, serum ALT and AST concentrations increased; while administration of the siRNA conjugate of the present disclosure After the siRNA conjugate, the serum ALT and AST concentrations were comparable to those of the blank control group, indicating that the siRNA conjugate of the present disclosure has low liver toxicity.
  • the concentrations of ALT and AST in mouse serum were significantly increased, indicating that the The reference conjugate may produce higher hepatotoxicity.
  • mice were sacrificed and dissected, preserved in 10% neutral buffered formalin fixative, and pathological sections were made. The severity of hepatic steatosis in the pathological sections was evaluated and graded, and a relative comparison was made.
  • mice given the reference conjugate 5 that did not contain stabilized modified nucleotides 4 mice exhibited severe hepatocyte degeneration, which was manifested by extensive liver tissue.
  • the cytoplasm of the cells was loose, and many hepatocytes were balloon-like degeneration, the cells were swollen, and the cytoplasm was vacuolated.
  • One mouse showed moderate hepatocyte degeneration. There was vacuolar degeneration, tiny round vacuoles were seen in the cytoplasm, a small number of local hepatocytes were necrotic, and the nuclei were condensed or fragmented.
  • One mouse showed mild hepatocyte degeneration, specifically manifested as more hepatocyte Hepatic steatosis was more severe than that in the blank control group.
  • mice given the reference conjugate 15 containing a stabilizing modified nucleotide only at the 7th position of the 5'-3' end of the antisense strand showed severe hepatic steatosis,
  • mice administered with siRNA conjugate 9 of the present disclosure 2 mice showed moderate hepatic steatosis, 3 mice showed mild hepatic steatosis, and no severe or severe hepatocyte degeneration was observed.
  • mice administered with siRNA conjugate 10 of the present disclosure 3 mice showed moderate hepatic steatosis, 2 mice showed mild hepatic steatosis, and no severe or severe hepatocyte degeneration was seen.
  • Mice administered the conjugates of the present disclosure exhibited a lower degree of hepatic steatosis compared to the reference conjugate.
  • the siRNA conjugates of the present disclosure can effectively reduce the liver toxicity caused by off-target effects, so in the preparation of drugs for the treatment and/or prevention of HBV diseases or symptoms Shows significantly higher safety and has excellent development prospects.
  • the in vitro siCHECK system was used to detect conjugate 11, conjugate 12, conjugate 13, conjugate 14, conjugate 15, conjugate 16, reference conjugate 6, reference Compared the inhibitory activity of conjugate 7, reference conjugate 8 or reference siRNA NC on the target sequence in the in vitro siCHECK system.
  • fetal bovine serum FBS, Hyclone Company
  • penicillin-streptomycin Penicillin-Streptomycin, Gibco, Invitrogen Company
  • DMEM complete medium Hyclone Company
  • each 12A1-12A3 solution contains 1 ⁇ L of the siRNA conjugate working solution or reference siRNA NC working solution of the above three concentrations respectively, and detect 0.05 ⁇ L of plasmid working solution (containing 10 ng of detection plasmid) and 10 ⁇ L of Opti-MEM medium.
  • each 12B solution contains 0.2 ⁇ L Lipofectamine TM 2000 and 10 ⁇ L Opti-MEM medium.
  • each 12C solution contains 0.05 ⁇ L of detection plasmid working solution (containing 10 ng of detection plasmid) and 10 ⁇ L of Opti-MEM medium.
  • each siRNA conjugate or reference siRNA NC in the culture well, add the transfection complex 12X1-12X3 of each siRNA conjugate or reference siRNA NC respectively, mix evenly, and the addition amount is 20 ⁇ L/well, and obtain the final transfection complex of each siRNA conjugate or reference siRNA NC Transfection complexes with concentrations of about 0.1nM, 0.03nM and 0.01nM (calculated as siRNA), each siRNA conjugate or reference siRNA NC transfection complex 12X1-12X3 respectively transfected 3 culture wells, Obtain the co-transfection mixture containing siRNA conjugate or reference siRNA NC, which is recorded as the test group.
  • transfection complex 12X4 For each siRNA conjugate or reference siRNA NC, in the other 3 culture wells, add transfection complex 12X4 respectively, the addition amount is 20 ⁇ L/well, to obtain the transfection mixture without siRNA, which is recorded as the blank control group .
  • the co-transfection mixture containing siRNA and the co-transfection mixture without siRNA were respectively transfected in culture wells for 4 hours, and 100 ⁇ L of H-DMEM complete medium containing 20% FBS was added to each well.
  • the 96-well plate was placed in a CO 2 incubator to continue culturing for 24 hours.
  • the luminescence ratio Ratio Ratio (test) or Ratio (control) of each test group or control group is the average value of three culture wells Ratio; The ratio was used as the benchmark, and the luminescence ratio of each test group was normalized to obtain the ratio R of Ratio (test)/Rati (control), which represented the relative expression level of the Renilla reporter gene, that is, the residual activity.
  • the inhibition rate of each siRNA conjugate or reference siRNA NC to the target sequence 4 (1-R) ⁇ 100%.
  • Figure 7 is a histogram of the relative expression level of the target sequence 4 in the in vitro siCHECK system after co-transfection of the plasmid containing the target sequence 4 and the siRNA conjugate to be tested or the reference siRNA NC. Further, the expression inhibition rate of target sequence 4 by each siRNA conjugate or reference siRNA NC is summarized in Table 13.
  • the siRNA conjugates provided by the present disclosure have high target sequence inhibitory activity in the siCHECK system in vitro.
  • the expression inhibition rate of target sequence 4 is at least 38.92nM, up to 67.54%; at a concentration of 0.1nM, the expression inhibition rate of target sequence 4 is at least 84.73%, up to 89.35%.
  • it has a level of target sequence inhibitory activity close to that of reference conjugate 6, reference conjugate 7 or reference conjugate 8 that does not contain stabilizing modified nucleotides.
  • the inhibitory activity of Conjugate 11, Conjugate 12 and Reference Conjugate 6 in the in vitro siCHECK system was tested, the only difference being that Conjugate 11, Conjugate 12 or Reference Conjugate Conjugate 6 was used instead of the tested siRNA for detection;
  • the target sequence used in the detection plasmid was the following target sequence 5; 0.0370 ⁇ M, 0.0123 ⁇ M, 0.00412 ⁇ M, 0.00137 ⁇ M, 0.000457 ⁇ M, 0.000152 ⁇ M, 0.0000508 ⁇ M and 0.0000169 ⁇ M siRNA conjugate working solution with 11 different concentrations (all calculated as siRNA).
  • This target sequence 5 is the exact complement of the antisense strand of the siRNA in each conjugate tested.
  • siRNA conjugates of the present disclosure have high target sequence inhibitory activity in the in vitro siCHECK system, with IC 50 between 6.89-8.55 pM.
  • reference conjugate 6 which is identical to the rest of the sequence but does not contain stabilizing modified nucleotides, has an inhibitory activity close to that of the target sequence.
  • mice Human APOC3 transgenic mice Tg(APOC3)3707Bres (purchased from Jackson Laboratory, USA) with serum TG content > 2 mmol/L were selected for random grouping, with 6 mice in each group, half male and half male. Conjugate 12, reference conjugate 6 and PBS blank control were administered to each group of mice respectively. All animals were dosed according to their body weight, and administered in a single subcutaneous injection. The dosage of each siRNA conjugate (based on the amount of siRNA) was 3 mg/kg and 1 mg/kg of mouse body weight, and the administration volume was 5 ml. /kg. Each siRNA conjugate was provided in PBS aqueous solution, and the concentration that the conjugate should be prepared was calculated according to the dosage and volume of administration. Each of the mice in the other group was given 1 ⁇ PBS with a volume of 5 ml/kg, serving as a blank control group.
  • mice given reference conjugate 6 that does not contain stabilized modified nucleotides showed significant changes in blood biochemical indicators.
  • a high dose of 300mg/Kg female The concentrations of ALT and AST in mice increased by 212% and 36%, respectively.
  • the concentrations of ALT and AST in female mice given the conjugate 12 of the present disclosure only increased by 130% and 32%, respectively, compared with the reference conjugate 6, the concentration of ALT decreased significantly; No increase in the concentration of ALT and AST was found in the mice of the conjugate 11 of the present disclosure, and compared with the reference conjugate 6, the degree of change in blood biochemical indicators was significantly reduced.
  • the inhibitory IC 50 of conjugate 11 to target sequence 5 was 11.3pM, and the inhibitory rates to target sequence 6, 7 or 8 were less than 50% in all tested siRNA concentration ranges, that is, neither Off-target occurred; the inhibitory IC50 of conjugate 13 to target sequence 5 was 4.50pM, and the inhibition rate to target sequence 9, 10 or 11 was less than 50% in all tested siRNA concentration ranges, that is, no off-target occurred.
  • the siRNA conjugates of the present disclosure can effectively reduce the hepatotoxicity caused by off-target effects, so it is used in the preparation of drugs for the treatment and/or prevention of dyslipidemia-related diseases or symptoms. Drugs show significantly higher safety and have excellent development prospects.
  • siRNA conjugates used were conjugate 13, and the dosage of each siRNA conjugates ( The amount of siRNA) is 9 mg/kg, 3 mg/kg, 1 mg/kg, 0.5 mg/kg, 0.25 mg/kg, 0.1 mg/kg or 0.05 mg/kg of mouse body weight, and the administration volume is 5 ml/kg.
  • the amount of siRNA is 9 mg/kg, 3 mg/kg, 1 mg/kg, 0.5 mg/kg, 0.25 mg/kg, 0.1 mg/kg or 0.05 mg/kg of mouse body weight, and the administration volume is 5 ml/kg.
  • Each siRNA conjugate was provided in PBS aqueous solution, and the concentration that the conjugate should be prepared was calculated according to the dosage and volume of administration.
  • the administration time point was recorded as day 1, and blood was collected from the orbital venous plexus of mice on days 1, 8, 15, 22, 29, 36, 43, 50, 57 and 64 to detect the level of TG in serum. The results are shown in Figure 10.
  • mice Tg(APOC3)3707Bres purchased from Jackson Laboratory, USA
  • serum TG content > 2 mmol/L were selected for random grouping, with 8 mice in each group, half male and half male.
  • Conjugate 13, Conjugate 17 and PBS blank control were administered to each group of mice respectively. All animals were dosed according to their body weight, and administered in a single subcutaneous injection.
  • the dosage of each siRNA conjugate (based on the amount of siRNA) was 3 mg/kg and 1 mg/kg of mouse body weight, and the administration volume was 5 ml. /kg.
  • Each siRNA conjugate was provided in PBS aqueous solution, and the concentration that the conjugate should be prepared was calculated according to the dosage and volume of administration.
  • Each of the mice in the other group was given 1 ⁇ PBS with a volume of 5 ml/kg, serving as a blank control group.
  • the administration time point was recorded as the first day, and blood was collected from the orbital venous plexus of the mice on the 1st, 8th, 15th, 22nd, 29th, 36th, and 43rd days, 100 ⁇ L each time. After blood collection was left at room temperature for 30 minutes, it was centrifuged at 3000 rpm for 15 minutes at 4°C to obtain serum. Further use PM1P000/3 automatic serum biochemical analyzer (SABA, Italy) to detect the content of total cholesterol (CHO) and triglyceride (TG) in serum.
  • SABA automatic serum biochemical analyzer
  • Inhibition rate of blood lipid level (1-blood lipid content of test group after administration/blood lipid content of test group before administration) ⁇ 100%.
  • blood lipid refers to total cholesterol (CHO) or triglyceride (TG).
  • Conjugate 13 and Conjugate 17 were dissolved in PBS to a 30 mg/ml solution (calculated as siRNA conjugate).
  • ICR mice half male and half female, weighing 18-22 g, 5-6 weeks old, purchased from Speiford Co., Ltd.
  • mice half male and half female
  • the above-mentioned siRNA conjugate solution was administered to each mouse, and the administration volume was 10 mL/kg, as a test group; in addition, PBS was administered to each of a group of mice, and The drug volume was 10mL/kg, which was used as a blank control group.
  • the administration time point was recorded as day 1, and blood was collected from the orbital venous plexus of mice on days 1, 8, 15, and 22, 100 ⁇ L each time. After blood collection was left at room temperature for 30 minutes, it was centrifuged at 3000 rpm for 15 minutes at 4°C to obtain serum. Further use PM1P000/3 automatic serum biochemical analyzer (SABA, Italy) to detect the content of total cholesterol (CHO) and triglyceride (TG) in serum.
  • SABA automatic serum biochemical analyzer
  • Standardized blood lipid level (blood lipid content of the test group after administration/blood lipid content of the test group before administration) ⁇ 100%.
  • Inhibition rate of blood lipid level (1-blood lipid content of test group after administration/blood lipid content of test group before administration) ⁇ 100%.
  • blood lipid refers to total cholesterol (CHO) or triglyceride (TG).
  • FIG. 13A and 13B are line graphs showing changes in serum TG levels or serum CHO levels over time after administration of siRNA conjugates of the present disclosure, reference siRNA conjugates or PBS, respectively. Further, after administration of the siRNA conjugate of the present disclosure, the serum TG inhibition rate and serum CHO inhibition rate of mice at each time point are summarized in the following Table 21A and Table 21B:
  • test group Reference conjugate 8 Conjugate 15
  • Conjugate 16 dose 100mg/kg 100mg/kg 100mg/kg ALT 420% 116% 118% AST 126% - 32%
  • % and the preceding figures represent the difference between the concentration of alanine aminotransferase (ALT) or aspartate aminotransferase (AST) in the mouse serum and the concentration in the serum of the blank control group, and the difference with the concentration of the blank control group Percentage of concentration in group serum.
  • 420% in Table 22 means that the concentration of alanine aminotransferase in mice administered with reference conjugate 8 at 100 mg/Kg was 420% higher than that in the blank control group.
  • mice given the reference conjugate 8 that does not contain stabilized modified nucleotides showed obvious changes in blood biochemical indicators, and the concentration of ALT increased by 420%.
  • the concentration of AST increased by 126%.
  • the concentration of ALT was only increased by 116% and 118%, respectively, and the concentration of AST was only increased by 32%, showing significantly decreased blood biochemical indicators.
  • mice given the conjugate 15 of the present disclosure only 2 mice showed mild hepatocyte inflammatory response, showing a small amount of inflammatory cell infiltration, and no moderate or above inflammatory response and necrosis.
  • mice given the conjugate 16 of the present disclosure only one mouse showed a mild hepatocyte inflammatory response, and no more than moderate inflammatory response and necrosis.
  • conjugates 15 and 16 exhibited significantly lower toxic responses.
  • the siRNA conjugates of the present disclosure can effectively reduce the hepatotoxicity caused by off-target effects, so it is used in the preparation of drugs for the treatment and/or prevention of dyslipidemia-related diseases or symptoms. Drugs show significantly higher safety and have excellent development prospects.
  • the target sequence 12 is a nucleotide sequence in the mRNA expressed by the human AMGPTL3 gene targeted by the detected siRNA, so the inhibitory effect of each siRNA conjugate on the target sequence 12 can reflect the siRNA in the detected siRNA conjugate Inhibition of target gene expression.
  • the target sequence 12 and its complementary sequence were cloned into the Xho I/Not I site of the psiCHECK TM -2 plasmid.
  • Figure 14 is a histogram of the relative expression level of the target sequence 12 in the in vitro siCHECK system after co-transfection of the plasmid containing the target sequence 12 and the siRNA conjugate to be tested or the reference siRNA NC. Further, the expression inhibition rate of target sequence 12 by each siRNA conjugate or reference siRNA NC is summarized in Table 23.
  • Table 23 The expression inhibition rate of target sequence 12 in the in vitro siCHECK system
  • the siRNA conjugates provided by the present disclosure have high target sequence inhibitory activity in the siCHECK system in vitro.
  • the expression inhibition rate of the target sequence is at least 51.56nM, up to 58.76%; at a concentration of 0.1nM, the expression inhibition rate of the target sequence can reach 87.92-88.84%.
  • it has a similar level of target sequence inhibitory activity compared to the reference conjugate 9 which does not contain stabilizing modified nucleotides.
  • the off-target sequence inhibitory activity of siRNA conjugates in the in vitro siCHECK system was determined, the only difference being that conjugate 18, conjugate 19, conjugate 20, conjugate 21.
  • the reference conjugate 9 or the reference conjugate 11 is determined instead of the tested siRNA conjugate; and, the target sequence used is the target sequence 12 or the target sequence 13 as shown below:
  • the target sequence 13 contains a nucleotide sequence complementary to the antisense strand of the siRNA in the siRNA conjugate to be tested, so the inhibitory effect of each siRNA conjugate on the target sequence 13 can reflect the degree of off-target effect. That is, the higher the inhibitory effect, the more likely the siRNA conjugate is off-target. On the other hand, the ratio of the inhibitory effect of each siRNA conjugate on target sequence 12 to the inhibitory effect on target sequence 13 can reflect the relative off-target tendency of the siRNA conjugate. The less likely off-target occurs, the lower the possibility of off-target toxicity when the same level of drug efficacy is obtained.
  • each siRNA conjugate containing stabilized modified nucleotides of the present disclosure not only The inhibitory activity against the on-target target sequence 12 was comparable or significantly higher, and all showed significantly lower off-target effects.
  • the ratio of off-target IC 25 /on-target IC 25 was at least 499, even as high as 2100, indicating that the same or similar
  • the off-target effect of the siRNA conjugates of the present disclosure is lower than that of the reference conjugate when the desired drug effect is obtained, so that the preparation of a drug for inhibiting ANGPTL3 that is more effective and less toxic due to off-target effects applications have shown excellent potential.
  • mice C57BL/6 mice (6-8 weeks old, purchased from Speyford Company) with serum TG content > 2 mmol/L were selected for random grouping, with 5 mice in each group, all of which were female.
  • Conjugate 18, Conjugate 19, Reference Conjugate 9 and PBS blank control were administered to each group of mice respectively. All animals were dosed according to their body weight, and administered in a single subcutaneous injection. The dose of each siRNA conjugate (based on the amount of siRNA) was 3 mg/kg mouse body weight, and the administration volume was 5 ml/kg. Each siRNA conjugate is provided in PBS aqueous solution, and the concentration that the conjugate should be prepared is calculated according to the dosage and volume of administration. Each of the other group of mice was given 1 ⁇ PBS with a volume of 5 ml/kg, serving as a blank control group.
  • RNA later Sigma Aldrich
  • 1 mL Trizol Sigma Company
  • Trizol Sigma Company
  • liver tissue homogenate to which 0.2mL chloroform was added, mixed evenly and allowed to stand for 10min.
  • Centrifuge at 12,000 rpm for 10 min at 4°C, and take 0.4 mL of the supernatant. Add 0.5mL isopropanol to the supernatant and let it stand at room temperature for 10min.
  • RNA concentration was determined with NANO DROP 2000 (Thermo Company) according to the method described in the manual.
  • RNA of the liver tissue of each mouse take the total RNA aqueous solution containing 1 ⁇ g total RNA respectively, the solution volume is 1000 ⁇ L/RNA concentration (ng/ ⁇ L), use the reverse transcription kit Reverse Transcription System (purchased from TSINGKE company) According to the reverse transcription operation steps in the kit instruction manual, 20 ⁇ L of reverse transcription reaction system was prepared, and the total RNA was reverse transcribed.
  • the reverse transcription conditions are: for each reverse transcription reaction system, incubate the reverse transcription reaction system at 42°C for 30 minutes, then incubate at 95°C for 5 minutes, and finally incubate at 4°C for 5 minutes. Add 80 ⁇ L of DEPC water to the recording reaction system to obtain a solution containing cDNA.
  • each reverse transcription reaction system 5 ⁇ L of the above cDNA-containing solution was used as a template, and 20 ⁇ L of the qPCR reaction system was prepared using the reagents provided by the 2 ⁇ Ultra SYBR Mixture (with ROX) kit (purchased from Beijing Kangwei Century Company).
  • the PCR primer sequences used to amplify the target gene mANGPTL3 and the internal reference gene mGAPDH are shown in Table 25, and the final concentration of each primer is 0.25 ⁇ M.
  • Each qPCR reaction system was placed on the ABI StepOnePlus Real-Time PCR instrument and amplified using a three-step method.
  • the amplification program was pre-denaturation at 95°C for 10 minutes, followed by denaturation at 95°C for 30s, annealing at 60°C for 30s, and extension at 72°C for 30s. After repeating the above-mentioned denaturation, annealing, and extension processes for a total of 40 times, a product W containing amplified target gene mANGPTL3 and internal reference gene mGAPDH was obtained. The product W was then incubated at 95°C for 1 min, 55°C for 30 s, and 95°C for 30 s.
  • the real-time fluorescent quantitative PCR instrument collected the melting curves of the target gene mANGPTL3 and the internal reference gene mGAPDH in the product W respectively, and obtained the Ct of the target gene mANGPTL3 and the internal reference gene mGAPDH. value.
  • the siRNA conjugates of the present disclosure exhibit excellent mANGPTL3 mRNA inhibitory effects in mice, and at a dose of 3 mg/kg, the inhibitory rate of mANGPTL3 mRNA is at least 70%, and can even be as high as 95%, and showed comparable or even higher mANGPTL3 mRNA inhibitory activity than the corresponding reference conjugate 9 that did not include stabilizing modified nucleotides.
  • % and the preceding figures represent the difference between the concentration of alanine aminotransferase (ALT) or aspartate aminotransferase (AST) in the mouse serum and the concentration in the serum of the blank control group, and the difference with the concentration of the blank control group Percentage of concentration in group serum.
  • the concentration of ALT in the serum of male mice and female mice given reference conjugate 9 that does not contain stabilized modified nucleotides increased by 630% and 840%, respectively
  • the concentrations of AST increased by 114% and 145%, respectively.
  • the concentration of ALT in the serum of male mice and female mice given the conjugate 18 of the present disclosure was only increased by 25.6% and 101%, and the concentration of AST was only increased by 10.8% and 37.0%, respectively.
  • mice given the reference conjugate 9 4 mice showed severe hepatocyte degeneration, which was specifically manifested as a large number of balloon-like degeneration of hepatocytes in the tissue, with swelling of the cells and centered nuclei.
  • Cytoplasmic vacuolization 2 cases showed severe hepatocyte degeneration, specifically manifested as extensive hepatocyte ballooning degeneration in the tissue, cell swelling, centered nucleus, and cytoplasmic vacuolation.
  • mice given the conjugate 18 of the present disclosure 3 mice showed mild hepatocyte degeneration, which was manifested in the integrity of tissue lobules, tight arrangement of hepatocytes, loose cytoplasm of a small number of hepatocytes, and no severe hepatocyte degeneration. or very severe hepatocellular degeneration.
  • the siRNA conjugates of the present disclosure can effectively reduce the hepatotoxicity caused by off-target effects, so it is used in the preparation of drugs for the treatment and/or prevention of dyslipidemia-related diseases or symptoms. Drugs show significantly higher safety and have excellent development prospects.

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