Double-stranded oligonucleotide agents and their uses

Abstract

This invention provides double-stranded oligonucleotide agents and their applications. The double-stranded oligonucleotide agents contain specific cleavage sites of endonucleases, and the cleaved double-stranded oligonucleotide agents can inhibit the expression of target genes via RNA interference (RNAi).

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to China Patent Application No. 202310900474.0, filed on 21 July 2023, China Patent Application No. 202310904516.8, filed on 21 July 2023, China Patent Application No. 202311532296.7, filed on 16 November 2023, and China Patent Application No. 202410396475.0, filed on 2 April 2024, all of which are incorporated herein by reference.

[0002] This specification discloses double-stranded oligonucleotide agents that can inhibit the expression of target genes via RNA interference (RNAi) and their applications. [Background technology]

[0003] RNA interference (RNAi) technology is a highly conserved, efficient, and specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA) during evolution. dsRNA inhibits the expression of target genes by disrupting target mRNA. Because RNAi technology can specifically eliminate or turn off the expression of target genes, it is rapidly becoming one of the most useful research tools in the discovery of gene function and therapeutic drugs for treating many diseases, including metabolic disorders, infectious diseases, and malignancies. [Overview of the project]

[0004] This specification provides double-stranded oligonucleotide agents comprising a sense strand and an antisense strand. The sense strand and antisense strand form a double-stranded portion of 15-27 base pairs in length and a 5' extension within the antisense strand. The 5' extension is at least 3 nucleotides long and can be cleaved from the 3' nucleotide of the 5' extension, and the cleaved double-stranded oligonucleotide agent can silence the target RNA or inhibit the expression of the target gene via RNA interference.

[0005] This specification uses formula (C): [ka] We provide a double-stranded oligonucleotide agent containing the structure shown in formula (C), The first fragment in the antisense strand and the second fragment in the sense strand form a double-stranded portion by base pairing, and the first and second fragments are of equal length. The length of the 5' extension is at least 3 nucleotides. The antisense strand includes a cleavage region containing the last 5' nucleotide (X2) of the first fragment and two last 3' nucleotides (YZ) of the 5' extension, the cleavage region containing a nucleotide sequence that can be cleaved between X2 and Y, as shown in formula A: (3'-5')X2-YZ, where formula A is defined as follows: The sense strand length is 15-35, 15-23, 15-22, or 15-21 nucleotides. The length of the antisense strand is 25-35, 26-35, 26-30, 25-27, or 26-27 nucleotides.

[0006] The double-stranded oligonucleotide agent shown in formula (C) does not require the formation of blunt ends in the complementary and target regions. The target region may include a 3' overhang, or the complementary region may include a 5' extension, or both the complementary and target regions may form blunt ends.

[0007] This specification uses formula (D): [ka] We provide a double-stranded oligonucleotide agent containing the structure shown in formula (D), The first fragment in the antisense strand and the second fragment in the sense strand form a double-stranded portion by base pairing, and the first and second fragments are of equal length. The length of the 5' extension is at least 3 nucleotides. The antisense strand includes a cleavage region that includes the most 5'-nucleotide (X2) of the first fragment and the two most 3'-nucleotides (Y-Z) of the 5'-extension. The cleavage region is cleavable between X2 and Y as shown in formula A: (3'-5')X2-Y-Z. When cleaved, it includes a nucleotide sequence in which the 5'-extension is removed from the most 3'-nucleotide (Y). Formula A is defined as follows: The length of the sense strand is 15-35, 15-23, 15-22, 15-21, 16-25, 17-23, 18-23, 19-23, 19-21, 20-23, 20-21, 21-23, for example, 17, 18, 19, 20, 21, 22 or 23 nucleotides, The length of the antisense strand is 25-35, 25-30, 26-35, 26-30, 26-27, for example 25, 26, 27, 28, 29 or 30 nucleotides.

[0008] This specification provides a double-stranded oligonucleotide agent comprising a double-stranded oligonucleotide operably linked to a blocking group, the blocking group comprising M03 or M06.

[0009] This specification provides a double-stranded oligonucleotide agent comprising a double-stranded oligonucleotide operably linked to a ligand, wherein the ligand comprises a chemical structure selected from the group consisting of VSDL-01, VSDL-01A, VSDL-02, VSDL-02A, VSDL-03, VSDL-03A, VSDL-04, VSDL-04A, VSDL-05, VSDL-05A, VSDL-06, VSDL-06A, VSDL-07, VSDL-07A, VSDL-08, VSDL-08A, VSDL-09, VSDL-10, VSDL-11, VSDL-12, VSDL-13, and VSDL-14.

[0010] This specification provides a pharmaceutical composition comprising the double-stranded oligonucleotide agent disclosed herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0011] This specification provides a method for inhibiting the expression of a target gene in a subject in need thereof, which comprises administering to the subject a pharmaceutically effective amount of the double-stranded oligonucleotide agent disclosed herein or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition disclosed herein.

[0012] This specification provides a method for treating a disease or disorder of a subject in need thereof, which comprises administering to the subject a pharmaceutically effective amount of the double-stranded oligonucleotide agent disclosed herein or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition disclosed herein.

[0013] Both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present invention. Also, the accompanying drawings incorporated herein and constituting a part thereof illustrate embodiments of the present invention and are for the purpose of explaining the principles of the present invention.

[0014] As will be apparent to those skilled in the art, each of the embodiments described and illustrated herein has individual components and features that can be readily separated from or combined with any of the features of some other embodiments without departing from the scope or spirit of the present disclosure.

Brief Description of the Drawings

[0015] The accompanying drawings referred to herein constitute a part of this specification. The features shown in the accompanying drawings show only some embodiments of the present application, not all embodiments, unless specifically described in detail or implied to the contrary by the reader of this specification.

[0016] [Figure 1] Shows the relative AGT mRNA expression in the liver of test compounds (ds101, ds100). [[ID=https: / / www.drugbank.ca / drugs / DB0000?utm_source=patents&utm_medium=link&utm_campaign=api&utm_content=text&utm_term=DB0000114]]

[0017] [Figure 2]This shows the relative SOD-1 expression of the test compounds (ds110, ds111, ds112, ds113) in different tissues (frontal cortex, hippocampus, striatum, and heart).

[0018] [Figure 3] This shows the relative SOD-1 expression of the test compounds (ds114, ds115) in different tissues (prefrontal cortex and lumbar spinal cord).

[0019] [Figure 4] The relative MTTR expression of the test compounds (ds118, ds119) is shown.

[0020] [Figure 5] The results of the liver homogenate treatment reaction of the test compound are shown.

[0021] [Figure 6] This shows the plasma relative AGT levels of the test compounds (ds101, ds86) in the NHP model. [Modes for carrying out the invention]

[0022] The following description of the Disclosure is intended solely to illustrate various embodiments of the Disclosure. Therefore, any specific modifications described herein should not be construed as limiting the scope of the Disclosure. It will be obvious to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the Disclosure, and such equivalent embodiments should be included within the Disclosure. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0023] In this application, unless otherwise specified, the use of the singular form includes the plural form. In this application, unless otherwise specified, “or” means “and / or.” The use of other forms of the terms such as “include,” “contain,” and “included” is not limited. The terms “element” or “component” mean both an element or component containing one unit and an element or component containing multiple subunits, unless otherwise specified. Furthermore, the section headings used herein are for organizational purposes only and should not be construed as limiting the issues described. definition

[0024] In this specification, unless otherwise specified, and unless otherwise clearly contradicted, the definite and indefinite articles such as “a,” “an,” and “the” used in the context of the present invention (particularly in the context of the claims) shall be interpreted as including both singular and plural forms.

[0025] Values ​​or parameters preceded by "approximately" include (describe) embodiments relating to the value or parameter itself. For example, the description of "approximately X" includes the description of "X". Numerical ranges include the numerical values ​​that define the range. In general, the term "approximately" refers to the given value of a variable and all values ​​of the variable that are within the experimental error of the given value (e.g., within the 95% confidence interval of the mean) or within 10% of the given value, whichever is greater.

[0026] In this application, the terms “optional” or “at will” mean both cases in which the described event or condition occurs and cases in which it does not occur, and include both. For example, “optionally modified” includes both unmodified and modified nucleotides, and further, “optionally modified nucleotide” includes both unmodified and modified nucleotides.

[0027] In this disclosure, terms such as “include,” “contain,” “contain,” “possess,” “have,” and “possess” are intended to be comprehensive or non-exclusive and do not exclude additional, undescribed elements or steps of the process.

[0028] In this specification, the term "disorder" means a disease, disorder, or condition that impairs the normal function of a subject (e.g., a human).

[0029] The term "effective dose" refers to the amount of a drug that achieves a desirable local or systemic therapeutic effect in a reasonable benefit / risk ratio applicable to any treatment, either alone or in combination with additional doses. When treating a specific disorder, the desired local or systemic therapeutic effect preferably refers to inhibition of the progression of the disorder. This includes slowing the progression of the disorder, in particular interrupting or reversing its progression. When administered for the prevention of a disorder, the dose is sufficient to avoid or delay the onset of the disorder. The effective dose does not need to cure the disorder or always prevent the onset of the disease. The effective dose of a drug described herein depends on the individual parameters of the patient, including the symptoms being treated, the severity of the disorder, age, physiological state, physique, and weight, the duration of treatment, the type of combination therapy (if any), the specific route of administration, and similar factors. Therefore, the dosage of a drug described herein depends on these various parameters. Also, if the patient's response to the initial dose is insufficient, a higher dose (or a substantially higher dose achieved by another local route of administration) may be used. In some embodiments, the effective dose of a drug depends on its therapeutic indicators, solubility, etc.

[0030] In this specification, the term "inhibition" is used synonymously with "reduction," "silencing," "decrease," "suppression," and other similar terms, and includes all levels of inhibition, in particular statistically significant or clinically significant inhibition.

[0031] In this specification, the term "inhibition of expression" for a target gene refers to a significant reduction in the level of expression of the target gene in cells, cell populations, or tissues treated with the drug of interest, compared to untreated cells, cell populations, or tissues. Gene expression levels can be measured, for example, by the level of mRNA transcript of the target gene or the level of protein expressed from the target gene. In some embodiments, inhibition of target gene expression results in a clinically relevant inhibition of the target gene's expression level, e.g., sufficient inhibition to allow for an effective therapeutic response.

[0032] In this specification, the term “pharmaceutically acceptable” means that a substance or composition is chemically and / or toxicologically compatible with other components, including the formulation, and / or the subject being treated with them.

[0033] In this specification, the term “pharmaceutically acceptable salt” includes, unless otherwise specified, salts that retain the biological efficacy of the free acid and free base of a particular compound and are not biologically or otherwise undesirable. Possible forms of pharmaceutically acceptable salts include, but are not limited to, mono, bis, tris, and tetrakis. Pharmaceutically acceptable salts are nontoxic with respect to dosage and concentration. By preparing such salts, the physical properties of a compound can be altered without interfering with the exertion of its physiological effects, thereby facilitating its pharmacological use. Useful alterations to physical properties include lowering the melting point to facilitate transmucosal administration and increasing solubility to facilitate high-concentration administration of the drug. Pharmaceutically acceptable salts may include acid addition salts containing sulfates, chlorides, fumarates, maleates, phosphates, sulfamates, acetates, citrates, lactates, tartrates, malonates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, cyclohexylsulfamates, and quinates. Pharmaceutically acceptable salts can be obtained from acids such as sulfuric acid, hydrochloric acid, fumaric acid, maleic acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, and quinic acid.

[0034] The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, such as non-human primates, mammals such as mice, rats, cats, rabbits, sheep, dogs, cattle, chickens, amphibians, and reptiles, and non-mammals.

[0035] In this specification, the terms “treatment,” “to treat,” or “to cure” a disorder mean managing, eliminating, reducing, or improving the disorder and / or its associated symptoms. Treatment of a disorder does not have to eliminate the disorder or its associated symptoms, although this is not excluded. In this specification, the term “treatment” may also include “preventive treatment” performed before the onset of symptoms or signs of a disorder in subjects who are not affected but at risk of the disorder, or who are prone to recurrence of the disorder, or who are at risk of or prone to recurrence of the disorder, in order to reduce the likelihood of the disorder occurring or recurring, or to reduce the likelihood of recurrence of a previously controlled disorder. Also, within the scope of the present invention, “treatment” includes the prevention or preventive phase of recurrence, as well as treatment for acute or chronic signs, symptoms, and / or functional impairments. Treatment may target symptoms, for example, to suppress them. Treatment may be short-term, medium-term, or long-term, such as maintenance therapy.

[0036] nucleotide

[0037] In this specification, the term “nucleotide” means a sugar-bonded base in which a phosphate group is covalently bonded to the sugar moiety, and is intended to encompass both natural (i.e., unmodified) nucleotides and modified nucleotides. In some embodiments, the nucleotide is an unmodified ribonucleotide. In some embodiments, the ribonucleotide is a 3'-ribonucleotide. In some embodiments, the ribonucleotide is a 5'-ribonucleotide. In some embodiments, the modified or unmodified nucleotide may optionally be further modified.

[0038] Natural nucleotides consist of a natural base, a natural sugar moiety, and a phosphate moiety. In this specification, natural nucleotides refer to adenine ribonucleotide, adenine deoxyribonucleotide, guanine ribonucleotide, guanine deoxyribonucleotide, cytosine ribonucleotide, cytosine deoxyribonucleotide, uracil ribonucleotide, thymine ribonucleotide, or thymine deoxyribonucleotide. "Ribonucleotide" means a nucleotide having a hydroxyl group at the 2' position of the sugar moiety. "Deoxyribonucleoside" means a nucleotide having a hydrogen atom at the 2' position of the sugar moiety.

[0039] The natural bases of RNA include A (adenine), G (guanine), C (cytosine), U (uracil), and T (thymine).

[0040] In this specification, unless otherwise specified, nucleotides "G", "C", "A", "T", and "U" refer to natural or modified nucleotides containing guanine, cytosine, adenine, thymidine, and uracil as bases, respectively.

[0041] Nucleotides may be substituted with analogues, including natural and unnatural analogues. Examples of guanosine analogues include 6-thioguanosine, 8-azaguanosine, 8-oxoguanosine, and 2-aminopurine riboside. Examples of adenosine analogues include cordycepin (3'-deoxyadenosine), N6-benzyladenosine, and 2-chloroadenosine. Examples of cytidine analogues include gemcitabine (2',2'-difluoro-2'-deoxycytidine), cytarabine (1-β-D-arabinofuranosylcytosine), and decitabine (5-aza-2'-deoxycytidine). Examples of uridine analogues include 5-fluorouridine, pseudouridine, 5-bromouridine, 4-thiouridine, and 5-aziduridine.

[0042] In this specification, "ribonucleic acid" (RNA) is a carrier of genetic information found in cells, some viruses, and viroids. RNA consists of ribonucleotides linked together via nucleotide-linking bonds that form a chain, and includes single-stranded RNA and double-stranded RNA. The natural nucleotide-linking bond is a phosphodiester bond.

[0043] In this specification, the term "modified nucleotide" refers to a nucleotide having at least one modified base, modified sugar, or modified phosphate group capable of forming a bond between modified nucleotides. In some embodiments, a modified nucleotide may have one, two, three or more modifications. In some embodiments, a nucleotide may have one modification. In some embodiments, a nucleotide may have two modifications. In some embodiments, a nucleotide may have three modifications.

[0044] Examples of modified bases include, but are not limited to, hypoxanthine (I), xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine (m5C), 5-hydroxymethylcytosine, N6-methyladenosine (m6A), 3-methyluridine (m3U), 5-methyluridine (m5U), pseudouridine, 2-thiouridine (s2U), and 5-propyluridine (5-pU).

[0045] Modified sugars include 2'-sugar modifications, 3'-sugar modifications, 5'-sugar modifications, for example, 2'-OMe (2'-O-methyl) modifications, 2'-F (2'-deoxy-2'-fluoro) modifications, 2'-O-MOE (2'-O-methoxyethyl) modifications, 2'-deoxy (2'-d) modifications, 5'-morpholine (5'-Mo) modifications, unlocked nucleic acid (UNA) modifications, glycol nucleic acid (GNA) modifications, locked nucleic acid (LNA) modifications, tricyclo-DNA (tcDNA) modifications, (S)-restricted ethyl cross-linked nucleic acid ((S)-cEt-BNA) modifications, 5'-(E)-vinyl phosphate (VP) modifications, 2'-O-C16 modifications, 2'-C16 modifications, and modifications at the 5' or 3' end. Examples of conjugation include, but are not limited to, conjugation with inverted debasic nucleotides (invAB), substitution with inverted debasic nucleotides (invAb), substitution with 2,4-difluorotolyl ribonucleotide (rF), substitution with (S)-glycerol nucleic acid, substitution with inosine (I), conjugation with M03 at the 5' or 3' end, conjugation with M06 at the 5' or 3' end, and conjugation with ligands such as GalNAc ligands, lipophilic ligands, or other receptor-targeting ligands that may promote endocytosis of siRNA conjugates (e.g., TfR-targeting ligands, LDL-R-targeting ligands, integrin-targeting ligands).

[0046] Modified nucleotide inter-bonding includes methylphosphonate (MP), methoxypropylmethylphosphonate (MOP), phosphorothioate (PS), phosphorodithioate (PS2), phosphoroselenoate, phosphorodiselenoate, phosphoranilothioate, phosphoraniladete, phosphoramidate, -OP(O)(OR)-O-, -OP(S)(OR)-O-, -OP(S)(SR)-O-, -SP(O)(OR)-O-, -OP(O)(OR)-S-, -SP(O)(OR)-S-, -OP(S)(OR)-S-, -SP(S)(OR)-O-, -OP(O)(R )-O-, -OP(S)(R)-O-, -SP(O)(R)-O-, -SP(S)(R)-O-, -SP(O)(R)-S-, -OP(S)(R)-S-, -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP( O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(0H)-O, -OP(O)(H)-O-, - OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, or -OP(S)(H)-S-, [ka] These are some examples, but are not limited to them. R is independently selected from H, any substituted alkyl, any substituted cycloalkyl, and any substituted aryl. See, for example, the following. LaPlanche et al., Nucleic Acid Research 14:9081 (1986), Stec et al., J.Am.Chem.Soc. 106:6077 (1984), Stein et al., Nucleic Acid Research 16:3209 (1988), Zon et al., Anticancer Drug Design 6:539 (1991), Zon et al., Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, ed., Oxford University Press, Oxford, UK (1991)), Stec et al., US No. 5151510, Uhlmann and Peyman, Chemical Reviews 90:543 (1990), RNA Biochemistry Handbook: 2nd Edition, Fully Revised and Expanded Edition (Roland K. Hartmann, Albrecht Bindereif, Astrid Schon, Eric Westhof, eds., Wiley-VCH Verlag) GmbH&Co.KGaA (2014), Nucleic Acids in Medicinal Chemistry and Chemical Biology: Drug Development and Clinical Application (Lihe Zhang, Xinjing Tang, Zhen Xi, Jyoti Chattopadhyaya, eds., John Willie & Sons (2023)). In some embodiments, the modified nucleotide bond is phosphorus-free and contains peptide bonds within peptide nucleic acids (PNA), or binding groups including carbamates, amides, linear and cyclic hydrocarbon groups. In some embodiments, the modified nucleotide bond is a phosphorothioate bond.

[0047] In this specification, the terms “small interfering RNA,” “siRNA,” and “iRNA agent” are used synonymously with agents that mediate the silencing of target RNA, such as mRNA and transcripts of protein-coding genes. For convenience, such mRNA is also called the silencing mRNA. Such genes are also called target genes. Generally, the silencing RNA is an endogenous gene or a pathogen gene. Other RNAs besides mRNA, such as tRNA and viral RNA, can also be targeted.

[0048] Typically, siRNA comprises a double-stranded region of 60, 50, 40, or fewer than 30 complementary base pairs, and preferably, siRNA comprises a double-stranded region of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 complementary base pairs.

[0049] In some embodiments of this application, the sense strand and / or antisense strand of the siRNA are independently 15 to 35 nucleotides long, forming complementary double-stranded regions of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 base pairs long. In some embodiments of this application, the sense strand of the siRNA is 15 to 35 nucleotides long, and the antisense strand of the siRNA is 25 to 35 nucleotides long, forming complementary double-stranded regions of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 base pairs long. In some embodiments of this application, the sense strand of the siRNA is 17 to 23 nucleotides long, and the antisense strand of the siRNA is 25 to 30 nucleotides long. In some embodiments of this application, the sense strand of the siRNA is 21 to 23 nucleotides long, and the antisense strand of the siRNA is 26 to 30 nucleotides long. In some embodiments of this application, the sense and antisense strands of the siRNA are fully complementary with a length of 15 to 30 base pairs. In some embodiments of this application, the sense and antisense strands of the siRNA are fully complementary with a length of 17, 18, 19, 20, 21, 22, or 23 base pairs.

[0050] In some embodiments, the siRNA (e.g., antisense strand) is sufficiently complementary to the target RNA to silence the target RNA and, for example, inhibit the production of the protein encoded by the target RNA.

[0051] The term "sufficiently complementary" is used to indicate a degree of complementarity sufficient to ensure stable and specific binding between siRNA, particularly its antisense strand, and the target RNA molecule. Such a degree of complementarity is sufficient to avoid nonspecific binding of the siRNA to non-target sequences under conditions where specific binding is desired, i.e., under physiological conditions in the case of assays or therapeutic procedures or in vitro assays, and under the conditions under which the assay is performed. Non-target sequences typically differ by at least 4 nucleotides. In some embodiments, non-target sequences differ by at least 8 nucleotides. Alternatively or additionally, such a degree of complementarity is sufficient for the siRNA to silence the target RNA, for example, reducing the production of the protein encoded by the target mRNA.

[0052] The term "complementary" in relation to two nucleotide sequences means that they are arranged antiparallel and specifically hybridize the two single-stranded nucleotide sequences. The degree to which one oligonucleotide complements another, i.e., complementarity, is measured by the proportion of bases in each strand that can form hydrogen bonds with each other, according to established base pairing rules. Oligonucleotide sequences do not need to be perfectly complementary to their corresponding nucleic acid sequences. For example, a first nucleotide sequence may be considered complementary to a second nucleotide sequence if it exhibits a certain level of sequence complementarity, such as at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. In an exemplary embodiment, 18 of the 20 nucleic acid bases in the first nucleotide sequence align with the corresponding region of the second nucleotide sequence, achieving 90% complementarity. Non-complementary nucleic acid bases, also known as inappropriate nucleic acid bases, are either grouped together or scattered among complementary bases and do not need to be adjacent to each other or to complementary nucleic acid bases.

[0053] In this specification, “improper” means 1) Pairing of two opposite nucleotides other than AT, AU, or GC (independently, natural or unnatural), 2) No hydrogen bond is formed between two opposite nucleotides (independently, natural or unnatural), and 3) This includes, but is not limited to, a deficiency of a base between two opposing (independently natural or unnatural) nucleotides.

[0054] In some embodiments, the improper bases include wobble base pairs and Hoogsteen base pairs.

[0055] The term "fully complementary" means that the first and second nucleotide sequences form a hybrid consisting solely of Watson-Crick base pairs in their fully complementary region.

[0056] A “sufficiently complementary” oligonucleotide may contain an internal region (e.g., at least 7, 8, 9, or 10 nucleotides) that is fully complementary to the target RNA.

[0057] In this specification, the term "oligonucleotide" refers to nucleic acid molecules (RNA or DNA) having a length of, for example, less than 100, 200, 300, or 400 nucleotides.

[0058] In this application, “monomer” refers to a class of compounds that can be incorporated into a ribonucleic acid chain and perform a specific function. In this application, “monomer” includes, but is not limited to, natural nucleotides, non-natural nucleotides (e.g., modified nucleotides, nucleotide analogs, debased deoxyribonucleotides, GNAs, LNAs, etc.), blocking groups, M03 as disclosed herein, and M06 as disclosed herein.

[0059] In this application, "bonding" refers to the linking of residues between two monomers (such as nucleotides), such as nucleotide residues, via a single bond or a bonding group (such as a phosphodiester bond, phosphorothioate bond, or phosphorodithioate bond). In some embodiments, "bonding" refers to the linking of residues between two monomers (such as nucleotides) via internucleotide bonds such as phosphodiester bonds, phosphorothioate bonds, or phosphorodithioate bonds.

[0060] In this specification, the term “nucleotide interbonding” means a bond (e.g., a bond or binding group) between two parts of an oligonucleotide disclosed herein, such as two monomers, and includes bonds between two nucleotides, bonds between one nucleotide and one ligand, bonds between one nucleotide and one blocking group, and bonds between one nucleotide and a debased nucleotide of an oligonucleotide disclosed herein.

[0061] In this specification, the terms "alternating motif" or "alternating pattern" refer to a motif having one or more modifications on a single-stranded alternating nucleotide. Alternating nucleotides refer to those with alternating nucleotides every other nucleotide, every three nucleotides, or similar patterns. For example, if A, B, and C each show one type of modification to a nucleotide, the alternating motif may be "ABABABABABAB…", "AABBAABBAABB…", "AABAABAABAAB…", "AAABAAABAAAB…", "AAABBBAAABBB…", or "ABCABCABCABC…".

[0062] The types of modifications included in the alternating motif may be the same or different. For example, if A, B, C, and D each represent one type of modification to a nucleotide, the alternating pattern, i.e., the modifications to every other nucleotide, may be the same, but each of the sense strand or antisense strand may be selected from several possible modifications within the alternating motif, such as "ABABA...", "ACACAC...", "BDBDBD...", "CDCDCD...".

[0063] In this specification, the verb term “conjugate” or “to conjugate” refers to the joining of residues of two molecules (e.g., two nucleotides), such as two nucleotide residues, via a bond (e.g., a single bond) or a binding group (e.g., a phosphodiester, phosphorothioate, or phosphorodithioate). Correspondingly, the noun term “conjugate” refers to a compound or complex formed by covalent linkage between various chemical moieties. For example, “double-stranded RNA conjugate” refers to a compound or complex formed by covalent linkage of one or more chemical moieties (e.g., a conjugating group, a ligand group, or a delivery system) to double-stranded RNA. In this specification, the double-stranded RNA conjugate disclosed herein is also referred to as “conjugate” for brevity, and this should be understood as the whole term “double-stranded RNA conjugate” in context. In some embodiments, the delivery system, ligand group, or conjugated group may be attached to any available position on any nucleotide of any oligonucleotide agent, comprising a phosphate group, a sugar ring (including covalent linkage of the delivery system, ligand group, or conjugated group to the atom at the 3' or 5'' position of the sugar ring of the nucleotide via a phosphodiester bond), a 2'-hydroxyl group, a 5'-hydroxyl group, and a base. In some embodiments, the ligand group or delivery system may be attached to the 3'-position of the nucleotide, and unless otherwise specified, the nucleotide is linked via a 3'-5' phosphodiester bond. In some embodiments, the delivery system, ligand group, or conjugated group may be attached to the 2'-position of the nucleotide, and unless otherwise specified, the nucleotide is linked via a 2'-5' phosphodiester bond.

[0064] Ligand

[0065] The double-stranded oligonucleotide agents disclosed herein may optionally be conjugated with one or more ligands. The ligands may be attached to the sense strand, the antisense strand, or to the 3'-terminus, 5'-terminus, or both ends of both strands. In some embodiments, the ligand may be conjugated to the sense strand, in particular to the 3'-terminus of the sense strand. In some embodiments, the ligand is conjugated to the antisense strand, in particular to the 5'-terminus of the antisense strand.

[0066] A wide variety of entities can be bound to the oligonucleotides disclosed herein. Preferably, the ligand is covalently bound directly or indirectly via an intervening tether.

[0067] In a preferred embodiment, a ligand alters the distribution, targeting, or lifespan of the molecule it incorporates. In a preferred embodiment, a ligand increases the affinity of a selected target, such as a molecule, cell, cell type, compartment, or receptor (e.g., a cell or organ compartment, tissue, organ, or body region), compared to species in which such a ligand is absent. A ligand that has increased affinity for a selected target is also called a target ligand.

[0068] Some ligands possess endomoric properties. Endomolitic ligands facilitate the lysis of endosomes and / or the transport of the disclosed compositions or components thereof from endosomes to the cytoplasm of cells.

[0069] Ligands can improve transport, hybridization, and specific properties, and may improve the nuclease resistance of polymer molecules containing the resulting natural or modified oligonucleotides, or any combination of monomers and / or natural or modified ribonucleotides described herein.

[0070] Generally, ligands can include, for example, therapeutic modifiers to promote uptake, diagnostic compounds or reporter groups to monitor distribution, crosslinking agents, and nuclease resistance-constituting moieties. Common examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptide mimetic compounds. Ligands can also include, for example, target groups such as cell or tissue targeting agents such as lectins, glycoproteins, lipids, or proteins, and antibodies that bind to specific cell types such as kidney cells.

[0071] Other ligands include dyes, inserts (e.g., acridine), crosslinking agents (e.g., psoralen, mitomycin C), porphyrins (e.g., TPPC4, texaphylline, saffrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases or chelating agents (e.g., EDTA), lipophilic molecules (e.g., cholesterol, cholic acid, adamantane acetate, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O-(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl or phenoxazine), peptide conjugates (e.g., Antennapedia peptide, Tat peptide), alkylating agents, phosphates, amino groups, mercapto groups, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino groups, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole cluster, acridine-imidazole conjugate, Eu tetraaza macrocyclic compounds) 3+ Examples include complexes, dinitrophenyl, HRP, or AP.

[0072] Ligands can be proteins such as glycoproteins, peptides such as molecules having a specific affinity for co-ligands, or antibodies that bind to specific cell types such as cancer cells, endothelial cells, or osteocytes. Ligands can be substances such as drugs that can increase the uptake of iRNA agents into cells by disrupting the cytoskeleton of cells, for example, by disrupting microtubules, microfilaments, and / or intermediate filaments. Ligands can increase the uptake of oligonucleotides into cells, for example, by activating an inflammatory response. Exemplary ligands having such effects include tumor necrosis factor α (TNFα), interleukin-1β, or γ-interferon. In one embodiment, the ligand is a lipid or lipid molecule. Lipid ligands can be used, for example, to modulate, for example, control the binding of a conjugate to a target tissue. In another embodiment, the ligand is a portion, such as a vitamin, taken up by a target cell, such as a proliferating cell. In another embodiment, the ligand is a cell permeabilizer, preferably a helical cell permeabilizer. Ligands can be peptides or peptide mimes. In one embodiment, the target peptide may be an amphiphilic α-helical peptide.

[0073] A target ligand can be a ligand capable of targeting a specific receptor. Examples include sugar clusters such as folic acid, GalNAc, galactose, mannose, mannose-6P, GalNAC clusters, mannose clusters, and galactose clusters, or aptamers. A cluster is a combination of two or more sugar units. Target ligands also include integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands. Ligands can also be nucleic acid-based, such as aptamers. Aptamers may be unmodified or have any combination of the modifications disclosed herein.

[0074] Other ligand conjugates applicable to the present invention are described in U.S. Patent Application No. 10 / 916185, U.S. Patent Application No. 10 / 946873, U.S. Patent Application No. 10 / 833934, U.S. Patent Application No. 11 / 115989, and U.S. Patent Application No. 11 / 944227, the contents of which are incorporated herein by reference for all purposes.

[0075] When two or more ligands are present, the ligands may all have the same properties, all have different properties, or some may have the same properties and some may have different properties. For example, a ligand may have targeting properties, endomolitotic activity, or PK modulation properties. In some embodiments, all ligands have different properties.

[0076] The ligand can be bound to different sites on the oligonucleotide, e.g., the 3'-terminus, 5'-terminus, and / or an internal position. In a preferred embodiment, the ligand is attached to the oligonucleotide via an intervening tether. When the monomer is incorporated into the growth chain, the ligand or tethered ligand may be present on the monomer. In some embodiments, the ligand may be incorporated by binding to the “precursor” monomer after the “precursor” monomer has been incorporated into the growth chain. For example, TAP-(CH2) n Monomers having an amino-terminal tether such as NH2 (i.e., without an associated ligand) may be incorporated into the growing oligonucleotide chain. In the subsequent operation, i.e., after the incorporation of the precursor monomer into the chain, a ligand having an electrophilic group such as a pentafluorophenyl ester or an aldehyde group may subsequently be attached to the precursor monomer by binding the electrophilic group of the ligand to the terminal nucleophilic group of the precursor monomer's tether.

[0077] In other examples, monomers having chemical groups suitable for click reactions may be incorporated with azide or alkyne terminal tethers / linkers. Following the subsequent operation, i.e., the incorporation of the precursor monomer into the chain, ligands having complementary chemical groups such as alkynes or azides may be attached to the precursor monomer by binding to the alkyne and azide.

[0078] In the case of double-stranded oligonucleotides, the ligand can be attached to one strand or both strands. In some embodiments, the double-stranded iRNA agent contains a ligand conjugated to the sense strand. In other embodiments, the double-stranded iRNA agent contains a ligand conjugated to the antisense strand.

[0079] In some embodiments, ligands can be conjugated to bases, sugars, or internucleotide bonds of nucleic acid molecules. Conjugation with purine bases or their derivatives can occur at any position, including intra-ring and extra-ring atoms. In some embodiments, the 2-, 6-, 7-, or 8-position of the purine base is attached to the conjugated moiety. Conjugation with pyrimidine bases or their derivatives can also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of the pyrimidine base can be substituted with the conjugated moiety. Conjugation with nucleoside sugars can occur at any carbon atom. Carbon atoms of the sugar moiety that can be attached to the conjugated moiety include those with carbon 2', 3', and 5'. The 1' position can also be attached to a conjugated moiety, such as a debasic residue. Internucleotide bonds may have a conjugated moiety. In the case of phosphorus-containing bonds (e.g., phosphodiesters, phosphorothioates, phosphorodithioates, phosphoramidates), the conjugated moiety can be directly attached to the phosphorus atom or to an O, N, or S atom bonded to the phosphorus atom. In the case of amine or amide-containing nucleotide bonds (e.g., PNA), the conjugated moiety may be attached to the nitrogen atom or adjacent carbon atom of the amine or amide.

[0080] Any suitable ligand in the field of RNA interference is typically a carbohydrate such as monosaccharides (e.g., GalNAc), disaccharides, trisaccharides, tetrasaccharides, or polysaccharides, and may be used.

[0081] Linkers that conjugate ligands to nucleic acids include those described above. For example, the ligand may be one or more GalNAc (N-acetylglucosamine) derivatives attached via a divalent or trivalent branched linker.

[0082] In some embodiments, the ligand is conjugated to an oligonucleotide (e.g., the 5' end of an antisense strand) via an internucleotide bond, and the internucleotide bond is optionally modified as described above.

[0083] In some embodiments, the ligand is a GalNAc ligand, a lipophilic ligand, or another ligand that targets a receptor that may promote endocytosis of an siRNA-conjugate (such as a TfR-targeted ligand, an LDL-R-targeted ligand, or an integrin-targeted ligand).

[0084] Blocking group

[0085] The double-stranded oligonucleotide agents disclosed herein may optionally be conjugated with one or more blocking groups. The blocking groups may be attached to the sense strand, the antisense strand, or to the 3'-terminus, 5'-terminus, or both ends of both strands. In some embodiments, the blocking group is conjugated to the antisense strand, particularly to the 5'-terminus of the antisense strand.

[0086] In some embodiments, the blocking group is conjugated to an oligonucleotide (e.g., the 5' end of an antisense strand) via an internucleotide bond, and the internucleotide bond is optionally modified as described above. In some embodiments, the blocking group is conjugated to a double-stranded oligonucleotide agent via a phosphorothioate. In some embodiments, the blocking group is conjugated to a double-stranded oligonucleotide agent via a phosphodiester.

[0087] In this specification, “blocking group” refers to a group, for example, at the 5' end of an antisense strand, that is conjugated to the oligonucleotide disclosed herein and reduces or inhibits exonuclease degradation. In some embodiments, the blocking group may reduce or inhibit the RNA interference effect of the oligonucleotide. In some embodiments, the blocking group is cleaved from the oligonucleotide before it can impart an RNA interference effect.

[0088] Blocking groups include, but are not limited to, debasic residues, inverse debasic residues, M03, and M06.

[0089] I. Double-stranded oligonucleotide preparations

[0090] This disclosure provides a double-stranded oligonucleotide agent comprising a sense strand and an antisense strand. The sense strand and antisense strand form a double-stranded portion and a 5' extension within the antisense strand. In one embodiment, the length of the 5' extension is at least 3 nucleotides.

[0091] The inventors have surprisingly discovered that the 5' extension in the antisense strand can be designed to be cleaved at a specific cleavage site. In particular, the double-stranded oligonucleotide agent provided herein is especially advantageous in that the 5' extension can be cleaved from the 3' nucleotide of the 5' extension. In other words, once the double-stranded oligonucleotide agent reaches the target tissue, it can be converted into a cleavage product. Such cleaved double-stranded oligonucleotide agents have been found to be able to inhibit the expression of target genes via RNA interference.

[0092] In this specification, “RNA interference” refers to the ability to sequence-specifically silence a target RNA via double-stranded short interfering RNA (siRNA). The first step of RNA interference (RNAI) is the activation of the RNA-induced silencing complex (RISC), which requires the degradation of the sense strand of a double-stranded RNA (dsRNA) duplex. This sense strand acts as the initial substrate for RISC and is cleaved by Argonaut 2 (Ago2) in the middle of the duplex region. Once the cleaved 5'- and 3'-terminal fragments of the sense strand are removed by Ago2, RISC is activated by the antisense strand (Rand et al., (2005) Cell 123, 621).

[0093] In some embodiments, the double-stranded oligonucleotide agents provided herein exhibit improved target gene expression inhibitory activity compared to reference siRNAs having similar antisense sequences other than the 5' extension or cleavage region provided herein.

[0094] In some embodiments, the double-stranded oligonucleotide agent provided herein consists essentially of ribonucleic acid (RNA). In some embodiments, the sense strand of the double-stranded oligonucleotide agent provided herein consists essentially of RNA. In some embodiments, the antisense strand of the double-stranded oligonucleotide agent provided herein consists essentially of RNA.

[0095] In some embodiments, the double-stranded oligonucleotide preparations provided herein may optionally be converted to siRNA after being delivered to a target tissue or target cell. In some embodiments, the double-stranded oligonucleotide preparations provided herein may be converted to siRNA by an enzyme, or optionally by RNAase III, or optionally by a dicer. In some embodiments, the cleaved double-stranded oligonucleotide preparations provided herein are siRNA.

[0096] a. Double-stranded portion and 5' extension

[0097] In one embodiment, the double-stranded portion is formed by base pairing of a first fragment in the antisense strand and a second fragment in the sense strand, wherein the first and second fragments are of equal length.

[0098] In some embodiments, the base pairing between the first and second fragments may include Watson-Crick base pairings and / or other types of base pairings capable of forming a stable double helix. In the case of Watson-Crick-Watt base pairings, adenine (A) pairs with thymine (T) in DNA and uracil (U) in RNA, and guanine (G) pairs with cytosine (C). Base pairings may be formed with non-Watson-Crick base pairings, including but not limited to GU wobble base pairings and Hoogsteen base pairings. In some embodiments, nucleotides containing hypoxanthine as a base may pair with nucleotide bases containing adenine, cytosine, or uracil. In some embodiments, nucleotides containing uracil, guanine, or adenine may be substituted with, for example, nucleotides containing inosine (wherein "I" indicates a hypoxanthine base, inosine, or inosine-containing nucleotide, depending on the context). This substitution is also called "I modification." In some embodiments, adenine and cytosine at arbitrary positions in the oligonucleotide can be substituted with guanine and uracil, respectively, to form GU wobble base pairs with the target mRNA.

[0099] In some embodiments, the base pairs formed in the double-stranded portion may include or essentially consist of Watson-Crick base pairings and / or non-Watson-Crick base pairs such as GU wobble base pairs and Hoogsteen base pairs, or combinations thereof.

[0100] In some embodiments, the first fragment in the sense strand and the second fragment in the antisense strand are of equal length and complementary to form a double-stranded portion. In some embodiments, the first and second fragments are complementary by at least 80%, 85%, 90%, 95%, or 100%.

[0101] In one embodiment, the length of the double-stranded portion is 15 to 27 nucleotide pairs. In another embodiment, the length of the double-stranded portion is 16 to 27 nucleotide pairs, 16 to 26 nucleotide pairs, 16 to 25 nucleotide pairs, 16 to 24 nucleotide pairs, 16 to 23 nucleotide pairs, 16 to 22 nucleotide pairs, 16 to 21 nucleotide pairs, 16 to 20 nucleotide pairs, 17 to 23 nucleotide pairs, 17 to 22 nucleotide pairs, 17 to 21 nucleotide pairs, 17 to 20 nucleotide pairs, 18 to 23 nucleotide pairs, 18 to 22 nucleotide pairs, 18 to 21 nucleotide pairs, 18 to 20 nucleotide pairs, 19 to 23 nucleotide pairs, 19 to 22 nucleotide pairs, or 19 to 21 nucleotide pairs. In yet another embodiment, the length of the double-stranded portion is 16 nucleotide pairs, 17 nucleotide pairs, 18 nucleotide pairs, 19 nucleotide pairs, 20 nucleotide pairs, 21 nucleotide pairs, or 22 nucleotide pairs or 23 nucleotide pairs.

[0102] In one embodiment, the sense strand length of the double-stranded oligonucleotide agent is 15-35 nucleotides (nt), 16-35, 16-30, 16-27, 16-26, 16-25, 16-24, 16-23, 16-22, 16-21, 17-35, 17-30, 17-25, 17-23, 17-22, 17-21, 18-35, 18-30, 18-25, 18-23, 18-22, 18-21, 19-35, 19-30, 19-25, 19-23, 19- 22, 19-21, 20-35, 20-30, 20-25, 20-23, 21-35, 21-30, 21-25, 21-23, for example, 35, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16 or 15nt.

[0103] In one embodiment, the sense strand length of the double-stranded oligonucleotide agent is 15-23, 15-22, or 15-21 nt.

[0104] In one embodiment, the length of the 5' extension is at least 3, 4, 5, 6, or 7 nucleotides.

[0105] In one embodiment, the double-stranded oligonucleotide agent further comprises a 3' extension in the antisense strand. In one embodiment, the length of the 3' extension is at least 1, 2 nucleotides or more. In one embodiment, the 3' extension may be an overhang with a length of at least 1, 2, 3, 4, or 5 nucleotides.

[0106] The length of the extension (e.g., the 3' extension) may be 1 to 6 nucleotides, for example, 2 to 6 nucleotides, 1 to 5 nucleotides, 2 to 5 nucleotides, 1 to 4 nucleotides, 2 to 4 nucleotides, 1 to 3 nucleotides, 2 to 3 nucleotides, or 1 to 2 nucleotides. The extension (e.g., the 5' extension or the 3' extension) may be the result of one chain being longer than the other, or of two chains of equal length being formed in a staggered pattern.

[0107] In one embodiment, each nucleotide within the extended region independently contains a 2'-sugar modification such as 2'-F or 2'-OMe, but may also be modified or unmodified nucleotides, and are not limited to these modifications.

[0108] The sense strand, the antisense strand, or the 5'- or 3'-extensions of both strands may be phosphorylated. In some embodiments, the extension region contains two nucleotides having a phosphorothioate bond between them, and the two nucleotides may be the same or different. In one embodiment, the 3'-extension is located in the antisense strand.

[0109] In one embodiment, the antisense chain lengths of the double-stranded oligonucleotide agent are at least 25-35, 25-34, 25-33, 25-32, 25-31, 25-30, 25-29, 25-28, 25-27, 25-26, 26-35, 26-34, 26-33, 26-32, 26-31, 26-30, 26-29, 26-28, and 26-27 nt. In another embodiment, the antisense chain lengths of the double-stranded oligonucleotide agent are 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, and 30 nt.

[0110] In one embodiment, the first fragment includes a target region that is sufficiently complementary to a portion of the target RNA, and optionally, mRNA encoding a target gene.

[0111] In one embodiment, the target region disclosed herein is at least 90%, at least 95%, or 100% complementary to a portion of the target RNA. In some embodiments, the target region binds to the target RNA and forms a double structure consisting only of Watson-Crick base pairs within the complementary region. In one embodiment, the target region may include an internal region (e.g., at least 10 nucleotides) that is 100% complementary to the target RNA. In one embodiment, the internal region that is 100% complementary to the target RNA may be a seed region from the 5' end of the target region in the antisense strand, from position 2 to position 8. In other embodiments, the target region is 100% complementary to the target RNA, and for example, the target RNA anneals to the dsRNA biform to form a hybrid consisting only of Watson-Crick base pairs in the fully complementary region. Also in some embodiments, the dsRNA agent of the present invention specifically recognizes single nucleotide differences. In this case, the dsRNA agent mediates RNAi only if full complementarity is observed in the region of the single nucleotide difference (e.g., within 7 nucleotides).

[0112] b. Cutting area

[0113] In one embodiment, the antisense strand of the double-stranded oligonucleotide agent includes a cleavage region that allows for specific cleavage from the most 3' nucleotide in the 5' extension.

[0114] In some embodiments, the double-stranded oligonucleotide agent disclosed herein (e.g., 5' extension) undergoes enzyme-mediated cleavage (e.g., endonuclease) before mediating RNAi. In some embodiments, the cleavage is mediated by a ribonuclease (RNase). In some embodiments, the cleavage is mediated by an endonuclease. In some embodiments, the cleavage is mediated by RNAase III. RNAase III represents a class of endoribonucleases that prepare small RNAs for the RNA silencing pathway. In some embodiments, RNAase III is a dicer. In some embodiments, the cleavage is specific cleavage.

[0115] In this specification, the terms “specific” or “specifically” in relation to the cleavage of oligonucleotide agents (e.g., siRNA) refer to controlled or selective cleavage at a specific site, i.e., between two desired nucleotides of the oligonucleotide chain that has undergone specific cleavage. The products of specific cleavage may include multiple cleavage products, with the desired cleavage product (i.e., the product obtained by cleavage at a specific or desired site) being the most abundant cleavage product. In some embodiments, the desired cleavage product accounts for at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of all cleavage products (based on molar concentration or weight). In some embodiments, the amount of the desired cleavage product (based on molar concentration or weight) is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times that of any other cleavage product. While we do not wish to be bound by theory, specific cleavage is based on recognition by the enzyme mediating the cleavage of a particular sequence, feature, motif, or combination thereof of the oligonucleotide agent that has undergone cleavage.

[0116] In one embodiment, the cleavage region comprises a nucleotide sequence cleavable between X2 and Y, as shown in formula A:(3'-5')X2-YZ, where X2 is the furthest 5' nucleotide of the first fragment and YZ are the two furthest 3' nucleotides of the 5' extension.

[0117] In one embodiment, the cleavage region further comprises a fourth nucleotide (N1), which is a third nucleotide from the 3' end of the 5' extension, and the cleavage region comprises a nucleotide sequence that can be cleaved between X2 and Y, as shown in formula B:(3'-5')X2-YZ-N1.

[0118] In one embodiment, the cleavage region further comprises a third fragment (N), the third fragment comprising at least one nucleotide, where N1 is the furthest 3' nucleotide of the third fragment, and the cleavage region comprises a nucleotide sequence cleavable between X2 and Y, as shown in formula B':(3'-5')X2-YZn, where the length of N is 1 to 10 nucleotides, preferably 1 to 5 nucleotides, and more preferably 1 nucleotide.

[0119] While we do not wish to be bound by theory, it is conceivable that enzymatic cleavage can be determined using homogenate reaction assays of target tissues (e.g., liver, eyes, lungs, kidneys, brain, spinal cord, muscle, fat, etc.) (see, for example, the method described in Example 4.6) and dicer endonuclease therapeutic assays.

[0120] While I don't want to be constrained by theory, it seems that a certain nucleotide at a specific location within the cleavage region exhibits superior effects.

[0121] In one embodiment, Z is selected from G or A or their natural or unnatural analogues.

[0122] In one embodiment, X2 is selected from A or U or their natural or unnatural analogues.

[0123] While we do not wish to be constrained by theory, the double-stranded oligonucleotide agent disclosed herein can be specifically cleaved between X2 and Y to remove the 5' extension, thereby generating a cleaved double-stranded oligonucleotide agent. The cleaved double-stranded oligonucleotide is thought to be capable of mediating RNAi.

[0124] The cleaved double-stranded oligonucleotide agent has a blunt end at the 5' end of the antisense strand, and the first base pair at the 5' end of the antisense strand consists of X2 and its complementary nucleotide from the sense strand. In some embodiments, the first base pair at the 5' end of such an antisense strand is an AU base pair.

[0125] In one embodiment, formula A has a nucleotide sequence from 3'-5' selected from the group consisting of UUG, UAG, AUG, AAG, UUA, UAA, AUA, AAA, UCG, UGG, ACG, AGG, UCA, UGA, ACA, AGA, or natural or unnatural analogs thereof.

[0126] In one embodiment, nucleotide Y is selected from A or U or their natural or unnatural analogs.

[0127] In one embodiment, formula A has a nucleotide sequence from 3'-5' selected from the group consisting of UUG, UAG, AUG, AAG, UUA, UAA, AUA, and AAA.

[0128] In one embodiment, at least one nucleotide in the cleavage region is a modified nucleotide, and preferably, all nucleotides in the cleavage region are modified nucleotides.

[0129] In some embodiments, the modified nucleotide has a modified base, a modified sugar, or an inter-modified nucleotide bond. In some embodiments, the modified nucleotide has a modified base, optionally comprising hypoxanthine (I), xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine (m5C), 5-hydroxymethylcytosine, N6-methyladenosine (m6A), 3-methyluridine (m3U), 5-methyluridine (m5U), pseudouridine, 2-thiouridine (s2U), or 5-propyluridine (5-pU).

[0130] In one embodiment, the modified nucleotide has a modified sugar, optionally the modified sugar is 2'-sugar modified, 3'-sugar modified, 5'-sugar modified, for example, 2'-OMe (2'-O-methyl) modification, 2'-F (2'-deoxy-2'-fluoro) modification, 2'-O-MOE (2'-O-methoxyethyl) modification, 2'-deoxy (2'-d) modification, 5'-morpholine (5'-Mo) modification, unlocked nucleic acid (UNA) modification, glycol nucleic acid (GNA) modification, locked nucleic acid (LNA) modification, tricyclo-DNA (tcDNA) modification, (S)-restricted ethyl crosslinked nucleic acid ((S)-cEt-BNA) modification, 5'-(E)-vinyl phosphate (VP) modification, 2'-O -Includes C16 modification, conjugation with inverted debasic nucleotide (invAB) at the 5' or 3' end, substitution with inverted debasic nucleotide (invAb), substitution with 2,4-difluorotolyl ribonucleotide (rF), substitution with (S)-glycerol nucleic acid, substitution with inosine (I), conjugation with M03 at the 5' or 3' end, conjugation with M06 at the 5' or 3' end, and conjugation with ligands such as GalNAc ligands, lipophilic ligands, or other receptor-targeting ligands that may promote endocytosis of siRNA conjugates (e.g., TfR-targeting ligands, LDL-R-targeting ligands, integrin-targeting ligands).

[0131] In one embodiment, the modified nucleotide has a modified nucleotide interbond, and optionally, the modified nucleotide interbond includes methylphosphonate (MP), methoxypropylmethylphosphonate (MOP), phosphorothioate (PS), phosphorodithioate (PS2), phosphoroselenoate, phosphorodiselenoate, phosphoranilothioate, phosphoraniladete, phosphoramidate, and PNA.

[0132] In one embodiment, at least one nucleotide in the cleavage region is 2'-OMe modified or 2'-F modified. In one embodiment, all nucleotides in the cleavage region are modified nucleotides selected from 2'-OMe modification and 2'-F modification. In one embodiment, at least one nucleotide in the cleavage region is 2'-F modified. In one embodiment, two or fewer nucleotides in the cleavage region are 2'-F modified.

[0133] In one embodiment, Z in formula A or formula B is 2'-F modified, and optionally X2 in formula A or formula B is 2'-F modified. In one embodiment, N1 in formula A or formula B is 2'-F modified. In one embodiment, both X2 and Y in formula A or formula B are 2'-OMe modified, and both Z and N1 in formula A or formula B are 2'-F modified. In one embodiment, all of X2, Y and Z in formula A or formula B are 2'-OMe modified, and N1 in formula A or formula B is 2'-F modified.

[0134] While I don't want to be constrained by theory, it seems that certain nucleotide-nucleotide bonds at specific locations within the cleavage region exhibit superior effects.

[0135] In one embodiment, at least one of the internucleotide bonds between nucleotides within the cleavage region is not a phosphorothioate bond. In one embodiment, the internucleotide bond between X2 and Y is not a phosphorothioate bond. In one embodiment, the internucleotide bond between Y and Z is not a phosphorothioate bond. In one embodiment, none of the internucleotide bonds between nucleotides within the cleavage region are phosphorothioate bonds. In one embodiment, at least one of the internucleotide bonds between nucleotides within the cleavage region is a phosphodiester bond. In one embodiment, the internucleotide bond between X2 and Y is a phosphodiester bond. In one embodiment, the internucleotide bond between Y and Z is a phosphodiester bond. In one embodiment, each of the internucleotide bonds between nucleotides within the cleavage region is a phosphodiester bond.

[0136] c. Modification of double-stranded oligonucleotide agents

[0137] In one embodiment, the double-stranded oligonucleotide agent further comprises at least one phosphorothioate or methylphosphonate internucleotide bond.

[0138] In one embodiment, the double-stranded oligonucleotide agent provided herein comprises at least one phosphorothioate or methylphosphonate internucleotide bond at position 1 and / or position 2 (counting from its 5'-end) of the first fragment. In one embodiment, the double-stranded oligonucleotide agent provided herein comprises at least one phosphorothioate or methylphosphonate internucleotide bond at position 1 and / or position 2 (counting from its 3'-end) of the first fragment. In one embodiment, the double-stranded oligonucleotide agent provided herein comprises a phosphorothioate or methylphosphonate internucleotide bond between the first fragment (e.g., a nucleotide at position 1, counting from its 5'-end) and X2 in formula A or formula B.

[0139] In one embodiment, the double-stranded oligonucleotide agent provided herein comprises at least one phosphorothioate or methylphosphonate internucleotide bond at positions 1-8, 1-6, or 1 and / or 2 (counting from its 5'-terminus) of the second fragment. In one embodiment, the double-stranded oligonucleotide agent provided herein comprises at least one phosphorothioate or methylphosphonate internucleotide bond at positions 1-8, 1-6, or 1 and / or 2 (counting from its 3'-terminus) of the second fragment.

[0140] In one embodiment, each nucleotide of the first fragment of the antisense strand is modified. In another embodiment, each nucleotide of the antisense strand is modified. In another embodiment, each nucleotide of the second fragment of the sense strand is modified. In yet another embodiment, each nucleotide of the sense strand is modified.

[0141] In one embodiment, each nucleotide in the antisense strand is 2'-F modified or 2'-OMe modified, and / or each nucleotide in the sense strand is 2'-F modified or 2'-OMe modified.

[0142] In one embodiment, the sense strand further comprises a) optionally a motif of three consecutive 2'-F modified nucleotides located at positions 9, 10, and 11 counting from the 5' end of the sense strand, and / or b) a 2'-F modified nucleotide at positions 7 and / or 18 counting from the 5' end of the sense strand. In one embodiment, the sense strand further comprises an alternating motif at positions 9 to 13 counting from the 5' end, wherein the 2'-F modification and 2'-OMe modification occur on alternating nucleotides within the alternating motif.

[0143] In one embodiment, the first fragment of the antisense strand and / or the second fragment of the sense strand further comprises an alternating motif in which modifications occur on alternating nucleotides within the alternating motif, and optionally, 2'-F modifications occur on alternating nucleotides within the alternating motif, and / or 2'-OMe modifications occur on alternating nucleotides within the motif.

[0144] In one embodiment, the first fragment of the antisense strand and / or the second fragment of the sense strand may be modified with 2'-OMe (2'-O-methyl), 2'-F (2'-deoxy-2'-fluoro), 2'-O-MOE (2'-O-methoxyethyl), 2'-deoxy (2'-d), 5'-morpholine (5'-Mo), unlocked nucleic acid (UNA), glycol nucleic acid (GNA), locked nucleic acid (LNA), or tricyclo-DNA (tcD This includes modifications selected from the group consisting of (NA) modification, (S)-restricted ethyl cross-linked nucleic acid ((S)-cEt-BNA) modification, 5'-(E)-vinyl phosphate (VP) modification, 2'-O-C16 modification, conjugation with inverse debasic nucleotide (invAB) at the 5' or 3' end, substitution with inverse debasic nucleotide (invAb), substitution with 2,4-difluorotolyl ribonucleotide (rF), substitution with (S)-glycerol nucleic acid, and substitution with inosine (I).

[0145] d. Blocking groups

[0146] The double-stranded oligonucleotide agents disclosed herein may optionally be bound to one or more blocking groups. The blocking groups may be attached to the sense strand, the antisense strand, or to the 3'-terminus, 5'-terminus, or both ends of both strands. In some embodiments, the blocking group is bound to the antisense strand, particularly to the 5'-terminus of the antisense strand.

[0147] In some embodiments, the blocking group is bound to the oligonucleotide (e.g., the 5' end of the antisense strand) via an internucleotide bond, and the internucleotide bond is optionally modified as described above. In some embodiments, the blocking group is bound to the double-stranded oligonucleotide agent via a phosphorothioate. In some embodiments, the blocking group is bound to the double-stranded oligonucleotide agent via a phosphodiester.

[0148] For example, a nucleotide bound to an inverted debasic deoxyribonucleotide (e.g., bound to the 5' or 3' end) [ka] It has the structure of [the object].

[0149] For example, a nucleotide bound to M03 (e.g., bound to the 5' or 3' end) [ka] It has the structure. In some embodiments, conjugation with M03 is nucleotide and [ka] This is due to the reaction.

[0150] For example, a nucleotide bound to M06 (e.g., bound to the 5' or 3' end) [ka] It has the structure. In some embodiments, the binding with M06 is a nucleotide and [ka] This is due to the reaction.

[0151] Furthermore, this specification provides a method for reducing or inhibiting the exonuclease degradation of an oligonucleotide (e.g., a double-stranded oligonucleotide such as siRNA) by conjugating or binding a blocking group to the oligonucleotide. In some embodiments, the blocking group is a debasic residue, an inverted debasic residue, M03, or M06. In some embodiments, the blocking group (e.g., M03 or M06) is bound to the antisense strand of the oligonucleotide, particularly to the 5' or 3' end of the antisense strand. In some embodiments, the blocking group (e.g., M03 or M06) is bound to the 5' end of the antisense strand. In some embodiments, the blocking group may reduce or inhibit the RNA interference effect of the oligonucleotide.

[0152] Furthermore, this specification provides applications of blocking groups in reducing or inhibiting the exonuclease degradation of oligonucleotides (e.g., double-stranded oligonucleotides such as siRNA), wherein the blocking group is conjugated to or bound to the 5' or 3' end of the oligonucleotide, particularly the antisense strand. In some embodiments, the blocking group is a debasic residue, an inverted debasic residue, M03, or M06. In some embodiments, the blocking group is M03 or M06. In some embodiments, the blocking group is M03 or M06. In some embodiments, the blocking group (e.g., M03 or M06) is bound to the 5' end of the antisense strand. In some embodiments, the blocking group may reduce or inhibit the RNA interference effect of the oligonucleotide.

[0153] e. Specific examples of double-stranded oligonucleotide agents

[0154] In one embodiment, the double-stranded oligonucleotide agent provided herein is of formula (C): [ka] The structure includes the structure shown in formula (C), The first fragment in the antisense strand and the second fragment in the sense strand form a double-stranded portion by base pairing, and the first and second fragments are of equal length. The length of the 5' extension is at least 3 nucleotides. The antisense strand includes a cleavage region comprising the last 5' nucleotide (X2) of the first fragment and two last 3' nucleotides (YZ) of the 5' extension, the cleavage region comprising a nucleotide sequence cleavable between X2 and Y, as shown in formula A: (3'-5')X2-YZ, where formula A is as defined herein. The sense strand length is 15-35, 15-23, 15-22, or 15-21 nucleotides. The length of the antisense strand is 25-35, 26-35, 26-30, 25-27, or 26-27 nucleotides.

[0155] In some embodiments, the sense strand length of the double-stranded oligonucleotide agent shown in formula (C) is 17-23, 17-22, or 17-21 nucleotides, and the antisense strand length of the double-stranded oligonucleotide agent shown in formula (C) is 20-26, 20-25, and 20-24 nucleotides.

[0156] In some embodiments, the lengths of the sense strand and the antisense strand of the double-stranded oligonucleotide agent shown in formula (C) are a) 17 nt and 20 nt, respectively; b) 18 nt and 21 nt, respectively; c) 19 nt and 22 nt, respectively; d) 20 nt and 23 nt, respectively; or e) 21 nt and 24 nt, respectively.

[0157] In some embodiments, the double-stranded oligonucleotide agents disclosed herein may optionally be bound to one or more blocking groups. Any suitable blocking groups provided herein may be used. The blocking groups may be attached to the sense strand, the antisense strand, or to the 3'-terminus, 5'-terminus, or both ends of both strands. In some embodiments, the blocking group is attached to the antisense strand, particularly to the 5'-terminus of the antisense strand. In some embodiments, the blocking group is attached to the 5'-terminus of the 5' extension.

[0158] Furthermore, this specification includes formula (D): [ka] We provide a double-stranded oligonucleotide agent containing the structure shown in formula (D), The first fragment in the antisense strand and the second fragment in the sense strand form a double-stranded portion by base pairing, and the first and second fragments are of equal length. The length of the 5' extension is at least 3 nucleotides. The antisense strand includes a cleavage region containing the furthest 5' nucleotide (X2) of the first fragment and two furthest 3' nucleotides (YZ) of the 5' extension, the cleavage region containing a nucleotide sequence that is cleavable between X2 and Y, as shown in formula A: (3'-5')X2-YZ, and when cleaved, the 5' extension is removed from the furthest 3' nucleotide (Y), where formula A is as defined herein. The sense strand length is 15-35, 15-23, 15-22, 15-21, 16-25, 17-23, 18-23, 19-23, 19-21, 20-23, 20-21, 21-23, for example, 17, 18, 19, 20, 21, 22, or 23 nucleotides. The length of the antisense strand is 25-35, 25-30, 26-35, 26-30, 26-27, for example, 25, 26, 27, 28, 29, or 30 nucleotides.

[0159] In some embodiments, the sense strand length of the double-stranded oligonucleotide agent shown in formula (D) is 17-23, 17-22, or 17-21 nucleotides, and the antisense strand length of the double-stranded oligonucleotide agent shown in formula (D) is 22-28, 22-27, or 22-26 nucleotides.

[0160] In some embodiments, the lengths of the sense strand and the antisense strand of the double-stranded oligonucleotide agent shown in formula (D) are a) 17 nt and 22 nt, respectively; b) 18 nt and 23 nt, respectively; c) 19 nt and 24 nt, respectively; d) 20 nt and 25 nt, respectively; or e) 21 nt and 26 nt, respectively.

[0161] In some embodiments, the double-stranded oligonucleotide agents disclosed herein may optionally be bound to one or more blocking groups. Any suitable blocking groups provided herein may be used. The blocking groups may be attached to the sense strand, the antisense strand, or to the 3'-terminus, 5'-terminus, or both ends of both strands. In some embodiments, the blocking group is attached to the antisense strand, particularly to the 5'-terminus of the antisense strand. In some embodiments, the blocking group is attached to the 5'-terminus of the 5' extension.

[0162] In some embodiments, the sense chain of the oligonucleotide agent disclosed herein comprises at least 19, 20, 21, or 22 consecutive nucleotides, which is one of SEQ ID NOs: 1 to 97. In some embodiments, the sense chain of the oligonucleotide agent disclosed herein is one of SEQ ID NOs: 1 to 97.

[0163] In some embodiments, the antisense chain of the oligonucleotide agent disclosed herein comprises at least 19, 20, 21, or 22 consecutive nucleotides, which is one of SEQ ID NOs: 98 to 194. In some embodiments, the antisense chain of the oligonucleotide agent disclosed herein is one of SEQ ID NOs: 98 to 194.

[0164] In some embodiments, the antisense chain of the oligonucleotide agent disclosed herein comprises a pair of sense or antisense chains, as shown in Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0165] In some embodiments, the sense chain of the oligonucleotide agent disclosed herein is one of SEQ ID NOs: 1 to 97, and the antisense chain of the oligonucleotide agent disclosed herein is one of SEQ ID NOs: 98 to 194. a) At least one of the internucleotide bonds between nucleotides within the cleavage region is not a phosphorothioate bond, b) The internucleotide bond between X2 and Y is not a phosphorothioate bond, c) The internucleotide bond between Y and Z is not a phosphorothioate bond, d) The internucleotide bonds between nucleotides within the cleavage region are not phosphorothioate bonds, e) At least one of the internucleotide bonds between nucleotides within the cleavage region is a phosphodiester bond, f) The internucleotide bond between X2 and Y is a phosphodiester bond. g) The internucleotide bond between Y and Z is a phosphodiester bond, and / or h) Each of the internucleotide bonds between nucleotides within the cleavage region is a phosphodiester bond.

[0166] In some embodiments, the sense chain of the oligonucleotide agent disclosed herein is one of SEQ ID NOs: 1 to 97, and the antisense chain of the oligonucleotide agent disclosed herein is one of SEQ ID NOs: 98 to 194. a) At least one nucleotide within the cleavage region is 2'-OMe modified or 2'-F modified, b) Each nucleotide within the cleavage region is either 2'-OMe modified or 2'-F modified. c) At least one nucleotide within the cleavage region is 2'-F modified, d) Two or fewer nucleotides within the cleavage region are 2'-F modified, e) Z in formula A or formula B is modified by 2'-F, and optionally X2 in formula A or formula B is modified by 2'-F, f) N1 in formula A or formula B is modified by 2'-F, g) In formula A or formula B, X2 and Y are both modified by 2'-OMe, Z and N1 in formula A or formula B are both modified by 2'-F, and / or h) All of X2, Y, and Z in formula A or formula B are modified by 2'-OMe, and N1 in formula A or formula B is modified by 2'-F.

[0167] In some embodiments, the oligonucleotide agent disclosed herein is one of ds1 to ds121.

[0168] In some embodiments, the double-stranded oligonucleotide agents disclosed herein may optionally be bound to one or more blocking groups. Any suitable blocking groups provided herein may be used. The blocking groups may be attached to the sense strand, the antisense strand, or to the 3'-terminus, 5'-terminus, or both ends of both strands. In some embodiments, the blocking group is attached to the antisense strand, particularly to the 5'-terminus of the antisense strand.

[0169] f. Cleaved double-stranded oligonucleotide agent

[0170] In some embodiments, the cleaved double-stranded oligonucleotide agent includes a 3' extension on the antisense strand.

[0171] Each nucleotide of the cleaved double-stranded oligonucleotide agent may be independently modified or unmodified, as described above. In some embodiments, all nucleotides of the cleaved double-stranded oligonucleotide agent are independently modified. In some embodiments, all nucleotides of the cleaved double-stranded oligonucleotide agent are independently modified with 2'-F or 2'-OMe.

[0172] Each nucleotide bond in the cleaved double-stranded oligonucleotide agent may be independently modified or unmodified, as described above. In some embodiments, the cleaved double-stranded oligonucleotide agent includes at least one modified nucleotide bond. In some embodiments, the modified nucleotide bond is a phosphorothioate (PS) or phosphorodithioate (PS2).

[0173] In some embodiments, the cleaved double-stranded oligonucleotide agent includes alternating pattern modifications as described above.

[0174] In some embodiments, the agent comprises a cleaved double-stranded oligonucleotide agent, a phosphorothioate, or a methylphosphonate internucleotide bond. The phosphorothioate or methylphosphonate internucleotide bond modification may occur at any nucleotide on the sense strand or antisense strand, or at any position on both strands. For example, the internucleotide bond modification may occur on each nucleotide on the sense strand and / or antisense strand, each nucleotide bond modification may occur in an alternating pattern on the sense strand or antisense strand, or the sense strand or antisense strand may contain both nucleotide bond modifications in an alternating pattern. The alternating pattern of nucleotide bond modifications on the sense strand may be the same as or different from the alternating pattern of nucleotide bond modifications on the antisense strand, and the alternating pattern of nucleotide bond modifications on the sense strand may be shifted relative to the alternating pattern of nucleotide bond modifications on the antisense strand.

[0175] In one embodiment, the cleaved double-stranded oligonucleotide agent includes phosphorothioate or methylphosphonate internucleotide bond modifications within an extension region (e.g., 3' extension). For example, the extension region (e.g., 3' extension) includes two nucleotides having a phosphorothioate or methylphosphonate internucleotide bond between the two nucleotides. The internucleotide bond modification may also be performed to bond the extension nucleotide and the terminal pair nucleotide within the double region. For example, at least two, three, four, or all of the extension (e.g., 3' extension) nucleotides may be bonded via phosphorothioate or methylphosphonate internucleotide bonds, and optionally, additional phosphorothioate or methylphosphonate internucleotide bonds may be present that bond the extension nucleotide to the pair nucleotide adjacent to the extension nucleotide. For example, there may be at least two phosphorothioate internucleotide bonds between the three terminal nucleotides, where two of the three nucleotides are extension nucleotides and the third nucleotide is the pair nucleotide adjacent to the extension nucleotide. Preferably, these three terminal nucleotides may be at the 3' end of the antisense strand.

[0176] In one embodiment, the sense strand of the cleaved double-stranded oligonucleotide agent comprises 1 to 10 blocks of 2 to 10 phosphorothioate or methylphosphonate internucleotide bonds separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide bonds, one of which is positioned at any position in the oligonucleotide sequence, and the sense strand pairs with an antisense strand containing any combination of phosphorothioate internucleotide bonds, methylphosphonate internucleotide bonds and phosphate internucleotide bonds, or an antisense strand containing a phosphorothioate bond, a methylphosphonate bond, or a phosphate bond.

[0177] In one embodiment, the antisense chain of a cleaved double-stranded oligonucleotide agent comprises two blocks of two phosphorothioate or methylphosphonate internucleotide bonds separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide bonds, one of which is positioned at any position in the oligonucleotide sequence, and the antisense chain pairs with a sense chain containing any combination of phosphorothioate internucleotide bonds, methylphosphonate internucleotide bonds and phosphate internucleotide bonds, or with an antisense chain containing a phosphorothioate bond or a methylphosphonate bond or a phosphate bond.

[0178] In one embodiment, the cleaved double-stranded oligonucleotide agent further comprises 1 to 5 phosphorothioate or methylphosphonate internucleotide modifications at positions 1 to 5 of the sense strand (counted from the 5' end) and 1 to 5 phosphorothioate or methylphosphonate internucleotide modifications at positions 18 to 23, and 1 to 5 phosphorothioate or methylphosphonate internucleotide modifications at positions 1 and 2 of the antisense strand (counted from the 5' end) and 1 to 5 phosphorothioate or methylphosphonate internucleotide modifications at positions 18 to 23.

[0179] In one embodiment, in a cleaved double-stranded oligonucleotide agent, the alternating motif pattern of 2'-OMe and 2'-F modifications on the first sense strand is shifted relative to the alternating motif pattern of 2'-OMe and 2'-F modifications on the first antisense strand, i.e., 2'-OMe modified nucleotides on the sense strand pair with 2'-F modified nucleotides on the antisense strand, and vice versa. For example, the 2'-F modification may begin at position 1 of the sense strand and the 2'-OMe modification may begin at position 1 of the antisense strand. The initial modification pattern present in the sense strand and / or antisense strand is interrupted by introducing one or more motifs consisting of three identical modifications on three consecutive nucleotides into the sense strand and / or antisense strand. Surprisingly, interrupting the modification pattern of the sense strand and / or antisense strand by introducing one or more motifs consisting of three identical modifications on three consecutive nucleotides into the sense strand and / or antisense strand enhances gene silencing activity against the target gene.

[0180] g.ligand

[0181] In some embodiments, the double-stranded oligonucleotide agents disclosed herein may optionally be conjugated with one or more ligands. The ligands may be attached to the sense strand, the antisense strand, or the 3'-terminus, 5'-terminus, or both ends of both strands, or to any suitable nucleotide (e.g., on a sugar ring). In some embodiments, the ligand may be conjugated to the sense strand, in particular to the 3'-terminus of the sense strand. In some embodiments, the ligand is conjugated to the antisense strand, in particular to the 5'-terminus of the antisense strand.

[0182] In some embodiments, nucleotides (e.g., in the sense stand or antisense strand) are replaced with ligand-conjugated nucleotides. In this case, ligand-conjugated nucleotides are also included in the scope of “ligand”.

[0183] In some embodiments, the ligand is a GalNAc ligand, a lipophilic ligand, or another ligand that targets a receptor that may promote endocytosis of an siRNA-conjugate (such as a TfR-targeted ligand, an LDL-R-targeted ligand, or an integrin-targeted ligand).

[0184] In some embodiments, the ligand is conjugated to an oligonucleotide (e.g., the 5' end of an antisense strand) via an internucleotide bond, and the internucleotide bond is optionally modified as described above. In some embodiments, the ligand is conjugated to a double-stranded oligonucleotide agent via a phosphorothioate.

[0185] In some embodiments, the ligand is L96, [ka] It has the structure of [the object].

[0186] In some embodiments, the ligand is hexadecyloxy. For example, here it is abbreviated as "C16U". [ka] A C16 ligand having the structure shown may be conjugated with a uridine nucleotide at the 2' position.

[0187] In some embodiments, the ligand is VSDL-01. In some embodiments, the ligand is VSDL-01A. In some embodiments, the ligand is VSDL-02. In some embodiments, the ligand is VSDL-02A. In some embodiments, the ligand is VSDL-03. In some embodiments, the ligand is VSDL-03A. In some embodiments, the ligand is VSDL-04. In some embodiments, the ligand is VSDL-04A. In some embodiments, the ligand is VSDL-05. In some embodiments, the ligand is VSDL-05A. In some embodiments, the ligand is VSDL-06. In some embodiments, the ligand is VSDL-06A. In some embodiments, the ligand is VSDL-07. In some embodiments, the ligand is VSDL-07A. In some embodiments, the ligand is VSDL-08. In some embodiments, the ligand is VSDL-08A. In some embodiments, the ligand is VSDL-09. In some embodiments, the ligand is VSDL-10. In some embodiments, the ligand is VSDL-11. In some embodiments, the ligand is VSDL-12. In some embodiments, the ligand is VSDL-13. In some embodiments, the ligand is VSDL-14. VSDL-01, VSDL-01A, VSDL-02, VSDL-02A, VSDL-03, VSDL-03A, VSDL-04, VSDL-04A, VSDL-05, VSDL-05A, VSDL-06, VSDL-06A, VSDL-07, VSDL-07A, VSDL-08, VSDL-08A, VSDL-09, VSDL-10, VSDL-11, VSDL-12, VSDL-13, or VSDL-14 have the following structure, where X is S or or. [ka] [ka] For example, "5'-(VSDL-03A)*A-3'" is [ka] It has the structure of [the object].

[0188] h. Target gene

[0189] In some embodiments, the double-stranded oligonucleotide agents provided herein can inhibit the expression of target genes. In some embodiments, the target gene is selected from the group consisting of AGT, complement factor B, DGAT2, DUX, ANGPTL8, APOC3, F12, INHBE, PNPLA3, Serpinc1, APP, SOD1, TMPRSS6, KHK, PCSK9, VEGFA, ANGPTL3, ANGPTL4, C3, C5, TTR, IGF-1R, VEGFR, ANG2, GIPR, GPR75, ActRII, NUDT21, PLN, HSD17b13, CNOT6L, PTP1B, CFHR, ATX, CIDEB, mARC1, TSHR, CB1, and LPA.

[0190] In some embodiments, the target region contained in the first fragment provided herein is sufficiently complementary to a portion of the mRNA encoding the target gene. In some embodiments, the target region is at least 80%, 85%, 90%, or 95% complementary to a portion of the mRNA encoding the target gene. In some embodiments, the target region is 100% complementary (fully complementary) to a portion of the mRNA encoding the target gene.

[0191] II. Novel blocking groups and novel ligands

[0192] Furthermore, this specification provides a double-stranded oligonucleotide agent comprising a double-stranded oligonucleotide operably bonded to a blocking group, wherein the blocking group comprises M03 or M06.

[0193] In one embodiment, the double-stranded oligonucleotide may be any suitable siRNA or RNAi agent.

[0194] Furthermore, this specification provides a double-stranded oligonucleotide agent comprising a double-stranded oligonucleotide operably bound to a ligand, wherein the ligand comprises a chemical structure selected from the group consisting of VSDL-01, VSDL-01A, VSDL-02, VSDL-02A, VSDL-03, VSDL-03A, VSDL-04, VSDL-04A, VSDL-05, VSDL-05A, VSDL-06, VSDL-06A, VSDL-07, VSDL-07A, VSDL-08, VSDL-08A, VSDL-09, VSDL-10, VSDL-11, VSDL-12, VSDL-13, and VSDL-14. In one embodiment, the double-stranded oligonucleotide may be any suitable siRNA or RNAi agent.

[0195] In some embodiments, the double-stranded oligonucleotide agent operably bound to a blocking group or ligand includes a sense strand and an antisense strand. In some embodiments, the double-stranded oligonucleotide agent operably bound to a blocking group or ligand includes a blocking group conjugated to the 5' end of the antisense strand. In some embodiments, the double-stranded oligonucleotide agent operably bound to a blocking group or ligand includes a ligand conjugated to the 5' end of the antisense strand, the 5' end of the sense strand, or the 3' end of the sense strand. In some embodiments, the double-stranded oligonucleotide agent operably bound to a blocking group or ligand has one end operably bound to a blocking group and the other end operably bound to a ligand. In some embodiments, the double-stranded oligonucleotide agent operably bound to a blocking group or ligand can inhibit the expression of a target gene via RNA interference.

[0196] In some embodiments, the double-stranded oligonucleotide agent operably bound to a blocking group or ligand includes a sense strand and an antisense strand, each having 14 to 30 nucleotides.

[0197] In some embodiments, the antisense strand of a double-stranded oligonucleotide agent operably bound to a blocking group or ligand is sufficiently complementary to a portion of the mRNA encoding the target RNA or target gene, and the sense strand of the double-stranded oligonucleotide agent operably bound to a blocking group or ligand is sufficiently complementary to the target region of the antisense strand.

[0198] In some embodiments, the blocking group is conjugated to the 5' end of the antisense chain.

[0199] In some embodiments, the ligand is conjugated to the 5' end of the antisense chain, the 5' end of the sense chain, or the 3' end of the sense chain.

[0200] In some embodiments, the antisense strand of a double-stranded oligonucleotide agent operably bound to a blocking group or ligand has one end operably bound to the blocking group, the other end operably bound to the ligand, and / or In some embodiments, the antisense strand of a double-stranded oligonucleotide agent operably bound to a blocking group or ligand can silence target RNA or inhibit the expression of target genes via RNA interference.

[0201] In one embodiment, this specification discloses a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand having the structure shown in formula (I), [ka] The sense chain consists of region A1, region A2, and X1 (5'~3'). The antisense chain consists of regions N, Z, Y, X2, B2, and B1 (5'~3'). Region A1 consists of 0 to 15 nucleotides. Region B1 consists of 0-15 nucleotides. Regions A2 and B2 are of the same length and consist of at least one nucleotide. Regions A2 and B2 include complementary double-stranded regions. Each of X1, X2, Y, and Z is independently a single nucleotide, and X1 and X2 can form a hydrogen bond.

[0202] Region N is at least one nucleotide, The total length of the sense chain is 17-23 nt. The total length of the antisense chain is at least 26 nt. dsRNA contains 23 or fewer complementary base pairs. Each nucleotide in dsRNA can be modified independently and arbitrarily.

[0203] In some embodiments, the sense strand consists of region A1, region A2, and X1 (5'~3'), the antisense strand consists of region N, Z, Y, X2, region B2, and region B1 (5'~3'), region A1 is 1 to 6 nucleotides, region B1 is 0 to 6 nucleotides, regions A2 and B2 are of the same length and consist of at least 1 nucleotide, regions A2 and B2 include complementary double-stranded regions, each of X1, X2, Y, and Z is independently 1 nucleotide, X1 and X2 are complementary, region N is at least 1 nucleotide, the total length of the sense strand is 17 to 23 nt, the total length of the antisense strand is at least 26 nt, the dsRNA contains 23 or fewer complementary base pairs, and each nucleotide of the dsRNA is independently and optionally modified.

[0204] In some embodiments, X2 and Y are independently A, dA, U, or dU, and Z is G or dG.

[0205] In some embodiments, X2 and Y are independently A, dA, U, or dU, Z is G or dG, and each nucleotide of the dsRNA is independently modified.

[0206] In some embodiments, X2 is A or U, and Z is G.

[0207] In some embodiments, X2 is A or U, Z is G, and each nucleotide of the dsRNA is modified independently.

[0208] In some embodiments, Y is A or U.

[0209] In some embodiments, Y is A or U, and each nucleotide of the dsRNA is modified independently.

[0210] In some embodiments, X2 is A or U, and Y is A or U.

[0211] In some embodiments, X2 is A or U, Y is A or U, and each nucleotide of the dsRNA is modified independently.

[0212] In some embodiments, Y is A.

[0213] In some embodiments, Y is A, and each nucleotide of the dsRNA is modified independently.

[0214] In some embodiments, X1 is A or U.

[0215] In some embodiments, X1 is A, U, or I.

[0216] In some embodiments, X1 is A, U, G, or I.

[0217] In some embodiments, X1 is A, U, or I, and each of the nucleotides of the dsRNA is independently modified.

[0218] In some embodiments, Y, Z, and at least one nucleotide of region N are modified.

[0219] In some embodiments, region N consists of one nucleotide and a group X, where group X is a nucleotide, a blocking group, or a combination of a nucleotide and a blocking group, the blocking group being a structure resistant to nuclease degradation, the blocking group being bound to region N at its 5' end, and the blocking group being independently and optionally modified.

[0220] In some embodiments, the blocking group is selected from inverse debasing deoxyribonucleotide or RX, where RX is [ka] That is the case.

[0221] In some embodiments, region N consists of one nucleotide and one blocking group.

[0222] In some embodiments, one nucleotide and one blocking group of region N are linked via a phosphorothioate bond.

[0223] In some embodiments, region N consists of two nucleotides and one blocking group.

[0224] In some embodiments, two nucleotides and one blocking group of region N are linked via a phosphorothioate bond.

[0225] In some embodiments, region N consists of one nucleotide and two blocking groups.

[0226] In some embodiments, one nucleotide and two blocking groups in region N are linked via a phosphorothioate bond.

[0227] In some embodiments, region N consists of one nucleotide and two blocking groups.

[0228] In some embodiments, one nucleotide and two blocking groups in region N are linked via a bond, and the bond is by a single bond.

[0229] In some embodiments, one nucleotide and two blocking groups in region N are linked via a bond, and the bond is by a phosphodiester, phosphorothioate or phosphorodithioate bond.

[0230] In some embodiments, all nucleotides of the dsRNA are independently modified.

[0231] In some embodiments, nucleotide modifications include one, two or more selected from the group consisting of base modifications, sugar ring modifications, phosphate backbone modifications and terminal modifications. Base modifications include substitution of a stabilizing base, destabilizing base or base paired with an expanded library of partners, base removal (abasic nucleotide), or conjugated bases. Sugar modifications include modifications or sugar substitutions at the 2', 3' or � positions. Phosphate backbone modifications include modification or substitution of phosphodiester bonds. Terminal modifications include 5' end modifications (phosphorylation, conjugation, inverted linkage, etc.) and 3' end modifications (conjugation, DNA nucleotides, inverted linkage, etc.).

[0232] In some embodiments, nucleotide modifications include one, two or more selected from the group consisting of 2'-OMe modification, 2'-F modification, 2'-deoxy modification, VP modification, 5'-MP modification, PS modification, PS2 modification, MP modification, MOP modification, invAB modification, LNA modification, UNA modification, and modifications that enhance the affinity of dsRNA for ARGO protein.

[0233] In some embodiments, modifications that increase the affinity between dsRNA and ARGO protein are performed using nucleotides and [ka] Includes substitution with.

[0234] In some embodiments, the nucleotide modifications include one or more selected from the group consisting of 2'-OMe, 2'-F, 2'-deoxy, VP, 5'-MP, PS, PS2, MP, MOP, invAB, and modifications that increase the affinity between dsRNA and the ARGO protein.

[0235] In some embodiments, modifications that increase the affinity between dsRNA and the ARGO protein are located on X2.

[0236] In some embodiments, regions A1 and B1 include complementary double-stranded regions.

[0237] In some embodiments, region A1 is 1 nucleotide and region B1 is 3 nucleotides.

[0238] In some embodiments, the length of region A2 or B2 is 20 nt.

[0239] In some embodiments, region A1 is 0 nucleotides and region B1 is 2 nucleotides.

[0240] In some embodiments, the total length of the sense chain is 17–21 nt. In some embodiments, the total length of the sense chain is 18–21 nt.

[0241] In some embodiments, the total length of the antisense chain is 26–35 nt. In some embodiments, the total length of the antisense chain is 26–32 nt. In some embodiments, the total length of the antisense chain is 26–28 nt. In some embodiments, the total length of the antisense chain is 26–27 nt. In some embodiments, the total length of the antisense chain is 26 nt, 27 nt, 28 nt, 29 nt, or 30 nt.

[0242] In some embodiments, region A1 is 1 to 4 nucleotides.

[0243] In some embodiments, the total length of the antisense strand is 26-27 nt, and region A1 is 1-4 nucleotides.

[0244] In some embodiments, region N includes an inverted debasic deoxyribonucleotide (invAB).

[0245] In some embodiments, the total length of the antisense strand is 26-27 nt, region A1 is 1-4 nucleotides, and region N contains an inverted debasal deoxyribonucleotide (invAB).

[0246] In some embodiments, Z and region N are bound via a phosphorothioate bond.

[0247] In some embodiments, X2 and region B2 are bound via a phosphorothioate bond.

[0248] In some embodiments, Z and region N are linked via phosphorothioate bonds, and X2 and region B2 are linked via phosphorothioate bonds.

[0249] In some embodiments, the total length of the antisense strand is 26-27 nt, region A1 is 1-4 nucleotides, and regions Z and N are linked via phosphorothioate bonds.

[0250] In some embodiments, the full length of the antisense strand is 26-27 nt, region A1 is 1-4 nucleotides, and X2 and region B2 are linked via phosphorothioate bonds.

[0251] In some embodiments, the full length of the antisense strand is 26-27 nt, region A1 is 1-4 nucleotides, Z and region N are linked via phosphorothioate bonds, and X2 and region B2 are linked via phosphorothioate bonds.

[0252] In some embodiments, the full length of the antisense strand is 26-27 nt, region A1 is 1-4 nucleotides, region N contains inverted abasic deoxyribonucleotides (invAB), and Z and region N are linked via phosphorothioate bonds.

[0253] In some embodiments, the full length of the antisense strand is 26-27 nt, region A1 is 1-4 nucleotides, region N contains inverted abasic deoxyribonucleotides (invAB), and X2 and region B2 are linked via phosphorothioate bonds.

[0254] In some embodiments, the full length of the antisense strand is 26-27 nt, region A1 is 1-4 nucleotides, region N contains inverted abasic deoxyribonucleotides (invAB), Z and region N are linked via phosphorothioate bonds, and X2 and region B2 are linked via phosphorothioate bonds.

[0255] In some embodiments, the full length of the sense strand is 17-21 nt, the full length of the antisense strand is 26-27 nt, and region A1 is 1-4 nucleotides.

[0256] In some embodiments, the full length of the sense strand is 17-21 nt, and region N contains inverted abasic deoxyribonucleotides (invAB).

[0257] In some embodiments, the total length of the sense strand is 17 to 21 nt, region A1 is 1 to 4 nucleotides, and region N contains an inverse debasal deoxyribonucleotide (invAB).

[0258] In some embodiments, the total length of the antisense strand is 26-27 nt, region A1 is 1-4 nucleotides, and the 1st position counted from the 5' end of region N is substituted with RX, where RX is [ka] Z and region N are bound via phosphorothioate bonds.

[0259] In some embodiments, the total length of the antisense strand is 26-27 nt, region A1 is 1-4 nucleotides, and the 1st position counted from the 5' end of region N is substituted with RX, where RX is [ka] X2 and region B2 are bound via phosphorothioate bonds.

[0260] In some embodiments, the total length of the antisense strand is 26-27 nt, region A1 is 1-4 nucleotides, and the 1st position counted from the 5' end of region N is substituted with RX, where RX is [ka] Z and region N are bound via phosphorothioate bonds, and X2 and region B2 are bound via phosphorothioate bonds.

[0261] In some embodiments, the total length of the sense strand is 17–21 nt, region A1 is 1–4 nucleotides, and the 1st position counted from the 5' end of region N is substituted with RX, where RX is [ka] That is the case.

[0262] In some embodiments, the total length of the sense strand is 17-21 nt, the total length of the antisense strand is 26-27 nt, region A1 is 1-4 nucleotides, and the 1st position counted from the 5' end of region N is substituted with RX, where RX is [ka] That is the case.

[0263] In some embodiments, the total length of the sense chain is 17–21 nt, and regions Z and N are linked via phosphorothioate bonds.

[0264] In some embodiments, the total length of the sense chain is 17–21 nt, and X2 and region B2 are linked via phosphorothioate bonds.

[0265] In some embodiments, the total length of the sense chain is 17–21 nt, with Z and region N linked via phosphorothioate bonds, and X2 and region B2 linked via phosphorothioate bonds.

[0266] In some embodiments, the total length of the sense strand is 17–21 nt, the total length of the antisense strand is 26–27 nt, region A1 is 1–4 nucleotides, and regions Z and N are linked via phosphorothioate bonds.

[0267] In some embodiments, the total length of the sense strand is 17–21 nt, the total length of the antisense strand is 26–27 nt, region A1 is 1–4 nucleotides, and regions X2 and B2 are linked via phosphorothioate bonds.

[0268] In some embodiments, the total length of the sense strand is 17-21 nt, the total length of the antisense strand is 26-27 nt, region A1 is 1-4 nucleotides, Z and region N are linked via phosphorothioate bonds, and X2 and region B2 are linked via phosphorothioate bonds.

[0269] In some embodiments, the total length of the sense strand is 17–21 nt, the total length of the antisense strand is 26–27 nt, region A1 is 1–4 nucleotides, region N contains an inverse debasal deoxyribonucleotide (invAB), and regions Z and N are linked via a phosphorothioate bond.

[0270] In some embodiments, the total length of the sense strand is 17–21 nt, the total length of the antisense strand is 26–27 nt, region A1 is 1–4 nucleotides, region N contains an inverse debasal deoxyribonucleotide (invAB), and regions X2 and B2 are linked via phosphorothioate bonds.

[0271] In some embodiments, the total length of the sense strand is 17-21 nt, the total length of the antisense strand is 26-27 nt, region A1 is 1-4 nucleotides, region N contains an inverse debasal deoxyribonucleotide (invAB), Z and region N are linked via phosphorothioate bonds, and X2 and region B2 are linked via phosphorothioate bonds.

[0272] In some embodiments, the 1st and 2nd positions and the 2nd and 3rd positions, counted from the 5' and 3' ends of the sense and antisense strands, are independently and optionally linked via phosphorothioate bonds.

[0273] In some embodiments, the 1st and 2nd positions and the 2nd and 3rd positions, counted from the 5' and 3' ends of the sense and antisense strands, are linked via phosphorothioate bonds.

[0274] In one embodiment, this specification discloses a dsRNA-conjugate comprising a dsRNA and a delivery system, the delivery system being capable of delivering the dsRNA to a target RNA to achieve an RNA interference effect, the delivery system being coupled to the dsRNA, and the dsRNA-conjugate comprising one or more delivery systems.

[0275] In some embodiments, the delivery system is selected from the group consisting of liposomes, lipid nanoparticles (LNPs), lipid complexes, lipid polymer complexes (LPPs), mannose delivery systems, N-acetylgalactosamine (GalNAc)-coupled delivery systems, apelin receptor-targeted delivery systems, integrin receptor-targeted delivery systems, peptides, antibodies, and combinations thereof.

[0276] In some embodiments, the delivery system is conjugated at the 3' end of the sense strand.

[0277] In some embodiments, the delivery system is conjugated at the 5' end of the sense strand.

[0278] In some embodiments, the delivery system is conjugated at the 3' end of the antisense chain.

[0279] In some embodiments, the delivery system is conjugated at the 5' end of the antisense chain.

[0280] In some embodiments, the delivery system is coupled with a nucleotide that is not at the end of the dsRNA.

[0281] In one embodiment, this specification discloses a composition comprising the dsRNA disclosed herein and a pharmaceutically acceptable carrier or excipient.

[0282] In one embodiment, this specification discloses the use of the dsRNA, dsRNA conjugate, or composition disclosed herein in the manufacture of therapeutic agents for diseases or disorders related to RNA interference of gene expression.

[0283] In some embodiments, the gene is selected from the group consisting of AGT, complement factor B, DGAT2, DUX, ANGPTL8, APOC3, F12, INHBE, PNPLA3, Serpinc1, APP, SOD1, TMPRSS6, KHK, PCSK9, VEGFA, ANGPTL3, ANGPTL4, C3, C5, TTR, IGF-1R, VEGFR, ANG2, GIPR, GPR75, ActRII, NUDT21, PLN, HSD17b13, CNOT6L, PTP1B, CFHR, ATX, CIDEB, mARC1, TSHR, CB1, and LPA.

[0284] In some embodiments, the following are selected from the group consisting of AGT, complement factor B, DGAT2, SOD1, KHK, APP, DUX, ANGPTL3, ANGPTL4, and ANGPTL8.

[0285] In one embodiment, this specification relates to dsRNAs, dsRNA conjugates, or compositions disclosed herein for use in the treatment of diseases or disorders related to RNA interference of gene expression.

[0286] Embodiment I-1. Double-stranded RNA (dsRNA) is given by formula (I): [ka] It comprises a sense chain and an antisense chain having the structure shown, The sense chain consists of region A1, region A2, and X1 (5'~3'). The antisense chain consists of regions N, Z, Y, X2, B2, and B1 (5'~3'). Region A1 consists of 0 to 15 nucleotides. Region B1 consists of 0-15 nucleotides. Regions A2 and B2 are of the same length and consist of at least one nucleotide. Regions A2 and B2 contain double-stranded regions that form the reverse complement. Each of X1, X2, Y, and Z is independently a single nucleotide, and X1 and X2 can be associated with a hydrogen bond. Region N is at least one nucleotide, The total length of the sense chain is 17-23 nt. The total length of the antisense chain is at least 26 nt. dsRNA contains 23 or fewer complementary base pairs. Each nucleotide in dsRNA can be modified independently and arbitrarily.

[0287] Embodiment I-2. In the dsRNA of Embodiment I-1, X2 is A or U, and Z is G.

[0288] Embodiment I-3. In the dsRNA of Embodiment I-2, Y is either A or U.

[0289] Embodiment I-4. In the dsRNA of Embodiment I-3, Y is A.

[0290] Embodiment I-5. In the dsRNA of any one of Embodiments I-1 to I-4, region N consists of one nucleotide and group X, where group X is a nucleotide, a blocking group, or a combination of a nucleotide and a blocking group, the blocking group is a structure resistant to nuclease degradation, the blocking group is bound to region N at its 5' end, and the blocking group is independently and arbitrarily modified.

[0291] Embodiment I-6. In the dsRNA of Embodiment I-5, the blocking group is selected from inverted debasal deoxyribonucleotide or RX, where RX is [ka] That is the case.

[0292] Embodiment I-7. In the dsRNA of Embodiment I-6, region N consists of one nucleotide and one blocking group.

[0293] Embodiment I-8. In the dsRNA of Embodiment I-7, one nucleotide and one blocking group in region N are linked via a phosphorothioate bond.

[0294] Embodiment I-9. In any one of Embodiments I-1 to I-8, all nucleotides of the dsRNA are modified independently.

[0295] Embodiment I-10. In any one of Embodiments I-1 to I-8, the nucleotide modifications include one or more selected from the group consisting of 2'-OMe, 2'-F, 2'-deoxy, VP, 5'-MP, PS, PS2, MP, MOP, invAB, and modifications that increase the affinity between the dsRNA and the ARGO protein.

[0296] Embodiment I-11. In the dsRNA of Embodiment I-10, the modification that increases the affinity between the dsRNA and the ARGO protein is located on X2.

[0297] Embodiment I-12. In the dsRNA of any one of Embodiments I-1 to I-11, the total length of the sense strand is 17 to 21 nt, and furthermore, the total length of the sense strand is 18 to 21 nt.

[0298] Embodiment I-13. In the dsRNA of any one of Embodiments I-1 to I-11, the total length of the antisense strand is 26 to 35 nt, and furthermore, the total length of the antisense strand is 26 to 27 nt.

[0299] Embodiment I-14. In the dsRNA of any one of Embodiments I-1 to I-11, regions A1 and B1 include complementary double-stranded regions.

[0300] Embodiment I-15. In the dsRNA of Embodiment I-14, region A1 is 1 nucleotide and region B1 is 3 nucleotides.

[0301] Embodiment I-16. In the dsRNA of Embodiment I-15, the length of region A2 or B2 is 20 nt.

[0302] Embodiment I-17. In the dsRNA of any one of Embodiments I-1 to I-11, region A1 is 0 nucleotides and region B1 is 2 nucleotides.

[0303] Embodiment I-18. Provides an application for a dsRNA or a pharmaceutical composition thereof according to any one of Embodiments I-1 to I-17 in the manufacture of a therapeutic agent for a disease or disorder related to RNA interference of gene expression, further comprising the gene selected from the group consisting of AGT, complement factor B, DGAT2, DUX, ANGPTL8, APOC3, F12, INHBE, PNPLA3, Serpinc1, APP, SOD1, TMPRSS6, KHK, PCSK9, VEGFA, ANGPTL3, ANGPTL4, C3, C5, TTR, IGF-1R, VEGFR, ANG2, GIPR, GPR75, ActRII, NUDT21, PLN, HSD17b13, and LPA. Treatment method

[0304] This specification provides a method for modulating the expression of a target gene in a subject as required, comprising administering a pharmaceutically effective amount of a double-stranded oligonucleotide agent or a pharmaceutical composition disclosed herein to the subject.

[0305] This specification provides a method for inhibiting the expression of a target gene in a subject as required, comprising administering a pharmaceutically effective amount of a double-stranded oligonucleotide agent or a pharmaceutical composition disclosed herein to the subject.

[0306] This specification provides a method for treating a disease or disorder in a subject that requires modulation (e.g., inhibition) of the expression of a target gene, comprising, for example, administering to the subject a pharmaceutically effective amount of a double-stranded oligonucleotide agent or a pharmaceutical composition disclosed herein.

[0307] This specification provides a method for treating a subject disease or disorder as required, comprising administering to the subject a pharmaceutically effective amount of a double-stranded oligonucleotide agent or a pharmaceutical composition disclosed herein. In some embodiments, the disease or disorder is related to a target gene. In some embodiments, the disease or disorder is related to the overexpression or activation of a target gene.

[0308] In some embodiments, target genes include, but are not limited to, AGT, CFB, DGAT2, DUX, ANGPTL8, APOC3, F12, INHBE, PNPLA3, Serpinc1, APP, SOD1, TMPRSS6, KHK, PCSK9, VEGFA, ANGPTL3, ANGPTL4, C3, C5, TTR, IGF-1R, VEGFR, ANG2, GIPR, GPR75, ActRII, NUDT21, PLN, HSD17b13, CNOT6L, PTP1B, CFHR, ATX, CIDEB, mARC1, TSHR, CB1, and LPA.

[0309] The double-stranded oligonucleotide agents or pharmaceutical compositions disclosed herein may be administered to subjects by various routes depending on whether topical or systemic treatment is preferred and the area of ​​treatment. Administration may be topical (ophthalmic, vaginal, rectal, intranasal, transdermal), oral, or parenteral. Parenteral administration includes intravenous drip, subcutaneous injection, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.

[0310] The route and site of administration may be selected to enhance targeting. For example, when targeting muscle cells, intramuscular injection into the muscle is an unavoidable option. Lung cells may be targeted by administering oligonucleotides in aerosol form.

[0311] The prescribed dose may be an effective amount for treating or preventing diseases or disorders, such as those related to target RNA. The unit dose may be administered by means of injection (intravenous, subcutaneous, intramuscular, etc.), inhalation, topical application, etc.

[0312] In some embodiments, the unit dose is administered less frequently than once a day, for example, less frequently than once every 2, 4, 8, or 30 days. In other embodiments, the unit dose is not administered at a constant frequency (e.g., not at a regular frequency). For example, the unit dose may be administered as a single dose.

[0313] In one embodiment, the effective dose is administered in combination with other conventional therapeutic modalities. The effective dose may be administered as a single dose or in two or more divided doses, as desired or as deemed appropriate under specific circumstances. Furthermore, if it is necessary to facilitate repeated or frequent infusions, the implantation of a delivery device such as a pump, semi-permanent stent (e.g., intravenous, intraperitoneal, intracisional, intrasacral, etc.), or reservoir is advisable. Pharmaceutical composition

[0314] The oligonucleotide agents disclosed herein may be formulated for pharmaceutical use. A pharmaceutically acceptable composition comprises the oligonucleotide agent disclosed herein (e.g., a pharmaceutically effective amount of the oligonucleotide agent) alone or in combination with one or more pharmaceutically acceptable carriers (additives), excipients and / or diluents.

[0315] Pharmaceutical compositions may be specifically formulated for administration in solid or liquid form, and include the following: (1) oral administration such as drench preparations (aqueous solutions, non-aqueous solutions, or suspensions), tablets, boluses, powders, granules, and pastes applied to the tongue for oral, sublingual, and systemic absorption; (2) parenteral administration such as subcutaneous, intramuscular, intravenous, or epidural injection of sterile solutions or suspensions or sustained-release preparations; (3) topical application such as creams, ointments, sustained-release patches, and sprays applied to the skin; (4) vaginal or rectal administration such as pessaries, creams, and foams; (5) sublingual administration; (6) intraocular administration; (7) transdermal administration; and (8) nasal administration. Subcutaneous or intravenous delivery is particularly advantageous.

[0316] A pharmaceutically acceptable carrier may be a composition or medium such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium, zinc stearate, steric acid), or solvent encapsulant, which is involved in the transport or delivery of the target compound from one organ or part of the body to another. Each carrier must be compatible with the other components of the formulation and be "approved" in the sense that it is not harmful to the patient. Materials that can be used as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and acetylcellulose; (4) tragacanth powder; (5) malt; (6) gelatin; (7) lubricants such as magnesium sulfate, sodium lauryl sulfate, and talc; (8) excipients such as cocoa butter and suppository wax; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols such as propylene glycol; (11) glycerides Examples include polyols such as sorbitol, mannitol, and polyethylene glycol, (12) esters such as ethyl oleate and ethyl laurate, (13) agar, (14) buffering agents such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) water free of pyrogenic substances, (17) isotonic physiological saline, (18) Ringer's solution, (19) ethyl alcohol, (20) pH buffer solution, (21) polyester, polycarbonate and / or polyacid anhydride, (22) bulking agents such as polypeptides and amino acids, (23) serum components such as serum albumin, HDL and LDL, and (22) other non-toxic compatible substances used in pharmaceutical preparations.

[0317] The formulation may be presented simply in unit dosage forms and may be prepared by any method well known in the field of pharmacy. The amount of active ingredient that can be combined with a carrier material to prepare a single dosage form varies depending on the host being treated and the specific form of administration. Generally, the amount of active ingredient that can be combined with a carrier material to prepare a single dosage form is the amount of the compound that exerts a therapeutic effect. Generally, this amount is in the range of about 0.1% to about 99% of the active ingredient out of 100%, preferably about 5% to about 70%, and most preferably about 10% to about 30%.

[0318] In one embodiment, the formulation of the present invention comprises an excipient selected from the group consisting of cyclodextrin, cellulose, liposomes, micellars such as bile acids, polymer carriers such as polyesters and polyanhydrides, and an oligonucleotide disclosed herein. In one embodiment, the formulation enables oral bioavailability of the oligonucleotide formulation disclosed herein.

[0319] Double-stranded oligonucleotide agents can be formulated in combination with other agents, such as other therapeutic agents, or stabilizers, such as proteins compounded with double-stranded oligonucleotide agents. Further examples of other agents include EDTA (e.g., Mg 2+ Examples include chelating agents (such as those that remove divalent cations), salts, and RNAse inhibitors (for example, broad-specific RNAse inhibitors such as RNAsin).

[0320] Methods for preparing these formulations or compositions include the step of mixing the oligonucleotide agent disclosed herein with a carrier and optionally one or more auxiliary components. Generally, formulations are prepared by homogeneously and closely mixing the oligonucleotide disclosed herein with a liquid carrier or a micronized solid carrier or both, and then shaping the product as necessary.

[0321] To prolong the effects of a drug, it may be desirable to slow down its absorption from subcutaneous or intramuscular injection. This can be achieved by using suspensions of poorly soluble crystalline or amorphous materials. The absorption rate of a drug depends on its dissolution rate, which in turn depends on the crystal size and morphology. Alternatively, delayed absorption of parenterally administered drug forms can be achieved by dissolving or suspending the drug in an oil-based medium.

[0322] The oligonucleotide agents disclosed herein may be formulated, like other pharmaceuticals, for administration in any convenient manner for human or veterinary use. Examples

[0323] Abbreviation

[0324] In this specification, the term “nucleotide” includes ribonucleotides and deoxyribonucleotides. In this specification, “G,” “C,” “A,” “T,” and “U” refer to nucleotides containing guanine, cytosine, adenine, thymine, and uracil, respectively. When “d” precedes A, U, C, G, or T, it represents 2'-deoxyribonucleotide (DNA), and when “d” is not present, it represents ribonucleotide (RNA). For example, "dA" represents deoxyadenosine, "dG" represents deoxyguanosine, "dT" represents deoxythymidine, "dU" represents deoxyuridine, "dC" represents deoxycytidine, "A" represents adenosine, "G" represents guanosine, "T" represents ribothymidine or 5-methyluridine, "U" represents uridine, and "C" represents cytidine.

[0325] In this specification, when “f” follows A, U, C, G, and T, it represents a 2'-fluoromodified nucleotide. For example, “Af” represents 2'-fluoroadenosine, “Gf” represents 2'-fluoroguanosine, “Tf” represents 2'-fluororibothymidine or 5-methyl-2'-fluorouridine, “Uf” represents 2'-fluorouridine, and “Cf” represents 2'-fluorocytidine.

[0326] In this specification, lowercase letters such as "a", "u", "c", "g", "t", and "i" represent 2'-methoxy-modified A, U, C, G, T, and I, respectively. For example, "a" represents 2'-methoxyadenosine, "g" represents 2'-methoxyguanosine, "t" represents 2'-methoxyribothymidine or 5-methyl-2'-methoxyuridine, "u" represents 2'-methoxyuridine, and "c" represents 2'-methoxycytidine.

[0327] In this specification, “C16” or “C16 modification” refers to a 2'-O-C16 modification or a 2'-C16 modification. When “C16” precedes A, U, C, G, or T, it represents a 2'-hexadecyloxy modified nucleotide. For example, “C16U” means [ka] This represents, for example, "C16(dU)" [ka] It represents.

[0328] In this specification, when "GNA-" precedes A, U, C, G, and T, it represents a nucleotide modified with glycol nucleic acid. "Tgn" is another name for "GNA-T," and "Tgn" is synonymous with "GNA-T." For example, GNA-G, GNA-A, GNA-C, GNA-U, and GNA-T have the following structures. [ka]

[0329] In this specification, the asterisk (*) between two nucleotides such as A, U, C, G, and T represents a phosphorothioate nucleotide bond, i.e., the two nucleotides are joined via a phosphorothioate bond. In this specification, the absence of the asterisk (*) between two nucleotides such as A, U, C, G, and T represents an unmodified nucleotide bond (i.e., a phosphodiester bond).

[0330] For example, the sequence "5'-AdUgCf*T-3" represents a sequence in which, starting from the 5' end, adenosine is at position 1, deoxyuridine at position 2, methoxyguanosine at position 3, 2'-fluorocytidine at position 4, ribothymidine at position 5, with positions 4 and 5 linked via phosphorothioate bonds, and other adjacent positions linked via phosphodiester bonds.

[0331] In this specification, "invAB" or "invAB modification" refers to a nucleotide that is conjugated with an inverse debasal deoxyribonucleotide (for example, conjugated at the 5' or 3' end). For example, a nucleotide [ka] If it has the structure, the corresponding invAB modified nucleotide is [ka] It has the structure of [the object].

[0332] In this specification, "invAb" or "invAb modification" refers to the substitution of a nucleotide with an inverse debasal deoxyribonucleotide. For example, a nucleotide [ka] If it has the structure, the corresponding invAb modified nucleotide is [ka] It has the structure of [the object].

[0333] In this specification, "VP" or "VP modification" refers to a nucleotide that has undergone (E)-vinyl phosphate modification (e.g., 5'-(E)-vinyl phosphate modification). For example, VP-modified U, u, dU, and Uf have the following structures. [ka]

[0334] In this specification, "M03" or "M03 modification" means [ka] This refers to a nucleotide that is coupled to a molecule having the structure (for example, coupled at the 5' or 3' end). For example, a nucleotide is [ka] If the structure is such that the corresponding M03 modified nucleotide is, [ka] It has the structure. In some embodiments, conjugation with M03 is nucleotide and [ka] This is due to the reaction.

[0335] In this specification, "M06" or "M06 modification" means [ka] This refers to a nucleotide that is coupled to a molecule having the structure (for example, coupled at the 5' or 3' end). For example, a nucleotide is [ka] If the structure is such that the corresponding M06 modified nucleotide is, [ka] It has the structure. In some embodiments, conjugation with M06 is nucleotide and [ka] This is due to the reaction.

[0336] For example, the sequence "5'-(M06)*AdTgCf*(invAB)-3" is, [ka] This represents an oligonucleotide having the structure.

[0337] For example, the sequence "5'-(M06)*AdTgCf*[L96]-3" is, [ka] This represents an oligonucleotide having the structure.

[0338] Example 1 Synthesis of ligand precursor

[0339] Example 1.1 Synthesis of a precursor of VSDL-01 (2-cyanoethyldocosyldiisopropylphosphoramidite) [ka]

[0340] To a solution of compound 1-1 (10.00 g, 30.62 mmol) in anhydrous DCM (100 mL), 1H-imidazole-4,5-dicarbonitride (1.81 g, 15.31 mmol) was added, and then a solution of compound 1-2 (9.23 g, 30.62 mmol) in anhydrous DCM (20 mL) was added dropwise to the mixture. The reaction mixture was stirred under N2 at 25°C for 16 hours. The mixture was concentrated under vacuum to remove the DCM. The residue was purified by silica gel chromatography (eluted with hexane:Â=5:1, containing 1% TEA) to obtain the precursor of VSDL-01.

[0341] 1 H NMR (400MHz, CDCl3)δ 3.91-3.72(m, 2H), 3.68-3.51(m, 4H), 2.63(t, J=6.8Hz, 2H), 1.58(p, J=6.8Hz , 2H), 1.30-1.23(m, 38H), 1.17(dd, J=6.8, 4.2Hz, 12H), 0.87(t, J=6.8Hz, 3H).

[0342] 31 P NMR: (400MHz, CDCl3)δ 147.17.

[0343] Example 1.2 Synthesis of a precursor of VSDL-02 (2-cyanoethylicosyldiisopropylphosphoramidite) [ka]

[0344] The synthesis method is the same as in Example 1.1.

[0345] 1 H NMR (400MHz, CDCl3)δ 3.92-3.72(m, 2H), 3.66-3.50(m, 4H), 2.62(t, J=6.8Hz, 2H), 1.60(p, J=6.8Hz , 2H), 1.28-1.24(m, 34H), 1.17(dd, J=6.8, 4.2Hz, 12H), 0.88(t, J=6.8Hz, 3H).

[0346] 31 P NMR: (400MHz, CDCl3)δ 147.32.

[0347] Example 1.3 Synthesis of a precursor of VSDL-03 (2-cyanoethylhexadecyldiisopropylphosphoramidite) [ka]

[0348] The synthesis method is the same as in Example 1.1.

[0349] 1 1H NMR (400 MHz, CDCl3) δ 3.90 - 3.72 (m, 2H), 3.68 - 3.52 (m, 4H), 2.63 (t, J = 6.8 Hz, 2H), 1.59 (p, J = 6.8 Hz, 2H), 1.28 - 1.24 (m, 26H), 1.17 (d, J = 6.8, 4.2 Hz, 12H), 0.87 (t, J = 6.8 Hz, 3H).

[0350] 31 31P NMR: (400 MHz, CDCl3) δ 147.24.

[0351] Example 1.4 Synthesis of the Precursor of VSDL-04 (2-Cyanoethyl (6-Icosanamidohexyl) Diisopropyl Phosphoramidite) [Chemical Formula] [[ID=2D0]]

[0352] To a solution of compound 4-1 (3.12 g, 10 mmol) and TEA (3.04 g, 30 mmol) in anhydrous DCM (200 mL) was added dropwise pentafluorophenyl trifluoroacetate (4.2 g, 15 mmol). After stirring for 15 minutes, compound 4-2 (1.4 g, 12 mmol) was added to the mixture. The reaction mixture was stirred at 25 °C for 16 hours. The mixture was concentrated in vacuo to remove DCM. The residue was purified by silica gel chromatography (eluting with hexane:EtOAc = 5:1 containing 1% TEA) to give compound 4-3 (3.82 g).

[0353] To a solution of compound 4-3 (3.63 g, 8.82 mmol) in anhydrous DMF (60 mL), 1H-imidazole-4,5-dicarbonitride (0.52 g, 4.41 mmol) was added, and then a solution of compound 1-2 (2.65 g, 8.82 mmol) in anhydrous DMF (5 mL) was added dropwise to the mixture. The reaction mixture was stirred under N2 at 25°C for 16 hours. The mixture was diluted with saturated aqueous NaHCO3 (200 mL), extracted with HCl (3 × 50 mL), the organic phases were combined, dried over anhydrous Na2SO4, and concentrated under vacuum. The residue was purified by silica gel chromatography (eluted with hexane:HCl = 5:1, containing 1% TEA) to obtain the precursor of VSDL-04.

[0354] 1 H NMR (400MHz, CDCl3)δ 5.45(t, J=5.6Hz, 1H), 3.92-3.75(m, 2H), 3.70-3.54(m, 4H), 3.23(t, J=6.8Hz, 2H), 2.64(t, J=6.4Hz, 2H), 2.14(t, J=7. 6Hz, 2H), 1.63-1.57(m, 4H), 1.53-1.56(m, 2H), 1.28-1.24(m, 36H), 1.17(dd, J=6.8, 4.2Hz, 12H), 0.87(t, J=6.8Hz, 3H).

[0355] 31 P NMR: (400MHz, CDCl3)δ 147.32.

[0356] Example 1.5 Synthesis of a precursor of VSDL-05 (2-cyanoethyl(6-stearamidohexyl)diisopropylphosphoramidite) [ka]

[0357] The synthesis method is the same as in Example 1.4.

[0358] 1H NMR (400MHz, CDCl3)δ 5.47(t, J=5.6Hz, 1H), 3.89-3.75(m, 2H), 3.63-3.67(m, 4H), 3.23(t, J=6.8Hz, 2H), 2.64(t, J=6.4Hz, 2H), 2.14(t, J=7. 6Hz, 2H), 1.66-1.57(m, 4H), 1.51-1.46(m, 2H), 1.27-1.23(m, 32H), 1.17(dd, J=6.8, 4.2Hz, 12H), 0.87(t, J=6.8Hz, 3H).

[0359] 31 P NMR: (400MHz, CDCl3)δ 147.20.

[0360] Example 1.6 Synthesis of a precursor of VSDL-06 (2-cyanoethyl(6-palmitamidehexyl)diisopropylphosphoramidite) [ka]

[0361] The synthesis method is the same as in Example 1.4.

[0362] 1 H NMR (400MHz, CDCl3)δ 5.69(t, J=5.6Hz, 1H), 3.90-3.75(m, 2H), 3.69-3.54(m, 4H), 3.26-3.21(m, 2H), 2.65(t, J=6.4Hz, 2H), 2.15(t, J=7.6H) z, 2H), 1.65-1.58(m, 4H), 1.54-1.47(m, 2H), 1.28-1.24(m, 28H), 1.18(dd, J=6.8, 4.2Hz, 12H), 0.88(t, J=6.8Hz, 3H).

[0363] 31 P NMR: (400MHz, CDCl3)δ 147.25.

[0364] Example 1.7 Synthesis of a precursor of VSDL-07 (2-cyanoethyl(6-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentanamide)hexyl)diisopropylphosphoramidite) [ka]

[0365] The synthesis method is the same as in Example 1.4.

[0366] 1 H NMR (400MHz, CDCl3)δ 5.52-5.24(m, 11H), 3.89-3.74(m, 2H), 3.69-3.53(m, 4H), 3.25-3.20(m, 2H), 2.85-2.78(m, 8H), 2.63(t, J=6.5Hz, 2H), 2.17-2.03(m, 6H), 1.74-1.68(m, 2H), 1.64-1.57(m, 2H), 1.53-1.46(m, 2H), 1.42-1.31(m, 4H), 1.17(dd, J=6.8, 4.2Hz, 12H), 0.97(t, J=7.6Hz, 3H).

[0367] 31 P NMR: (400MHz, CDCl3)δ 147.32.

[0368] Example 1.9 Synthesis of a precursor of VSDL-09 (dihexadecyldiisopropylphosphoramidite) [ka]

[0369] To a solution of compound 9-1 (1 g, 4.949 mmol) in anhydrous THF (5 mL), TEA (1.376 mL, 9.898 mmol) was added. The reaction mixture was cooled to 0°C in an ice bath, and then compound 3-1 (2.934 mL, 9.898 mmol) was added dropwise under nitrogen protection. The system temperature was maintained around 0°C during the addition. After addition, a suspension containing a white solid was obtained, the ice bath was removed, and the mixture was allowed to return to room temperature and stirred continuously for 3 hours at room temperature. The mixture was filtered, the filter was washed with THF, the filtrate was collected, and concentrated under vacuum to obtain the precursor of VSDL-09.

[0370] 1 H NMR (400MHz, CDCl3)δ 3.68-3.52(m, 6H), 1.64-1.57(m, 4H), 1.36-1.25(m, 52H), 1.18(d, J=6.8Hz, 12H), 0.88(t, J=6.8Hz, 6H).

[0371] 31 P NMR: (400MHz, CDCl3)δ 144.91.

[0372] Example 1.10 Synthesis of a precursor of VSDL-10 (butyl hexadecyl diisopropyl phosphoramidite) [ka]

[0373] To a solution of compound 10-2 (4 g, 15 mmol) in anhydrous THF (10 mL), TEA (3.13 mL, 22.49 mmol) was added. After cooling the reaction mixture to 0°C in an ice bath, compound 10-1 (1.11 g, 15 mmol) was added dropwise under nitrogen protection. The system temperature was maintained around 0°C during the addition. After addition, the mixture was stirred continuously at room temperature for 3 hours. The mixture was concentrated under vacuum to obtain compound 10-3, which was used directly in the next step without purification.

[0374] To a 10 mL solution of compound 3-1 (1.33 g, 5.47 mmol) in anhydrous DCM, 1H-imidazole-4,5-dicarbonitride (0.32 g, 2.74 mmol) was added, and then a 5 mL solution of compound 10-3 (2.5 g, 8.21 mmol) in anhydrous DCM was added dropwise to the mixture. The reaction mixture was stirred under N2 at 25°C for 16 hours. The mixture was concentrated under vacuum. The residue was purified by silica gel chromatography (eluted with hexane:Â=5:1, containing 1% TEA) to obtain a precursor of VSDL-10.

[0375] 1 H NMR (400MHz, CDCl3)δ 3.69-3.51(m, 6H), 1.64-1.55(m, 4H), 1.44-1.22(m, 28H), 1.17(d, J=6.8Hz, 12H), 0.90(dt, J=14.0, 7.2Hz, 6H).

[0376] 31 P NMR: (400MHz, CDCl3)δ 145.00.

[0377] Example 1.12 Synthesis of a precursor of VSDL-12 (hexadecyloctyldiisopropylphosphoramidite) [ka]

[0378] To a solution of compound 10-2 (4 g, 15 mmol) in anhydrous THF (10 mL), TEA (3.13 mL, 22.49 mmol) was added. After cooling the reaction mixture to 0°C in an ice bath, compound 12-1 (1.95 g, 15 mmol) was added dropwise under nitrogen protection. The system temperature was maintained around 0°C during the addition. After addition, the mixture was stirred continuously at room temperature for 3 hours. The mixture was concentrated under vacuum to obtain compound 12-2, which was used directly in the next step without purification.

[0379] To a 10 mL solution of compound 3-1 (1.61 g, 6.66 mmol) in anhydrous DCM, 1H-imidazole-4,5-dicarbonitride (0.39 g, 3.33 mmol) was added, and then a 5 mL solution of compound 12-2 (3.6 g, 9.98 mmol) in anhydrous DCM was added dropwise to the mixture. The reaction mixture was stirred under N2 at 25°C for 16 hours. The mixture was concentrated under vacuum. The residue was purified by silica gel chromatography (eluted with hexane:Â=5:1, containing 1% TEA) to obtain a precursor of VSDL-12.

[0380] 1 H NMR (400MHz, CDCl3)δ 3.69-3.52(m, 6H), 1.64-1.56(m, 4H), 1.37-1.25(m, 36H), 1.17(d, J=6.8Hz, 12H), 0.90-0.86(m, 6H).

[0381] 31 P NMR: (400MHz, CDCl3)δ 144.97.

[0382] Example 1.13 Synthesis of a precursor of VSDL-13 (dioctyl diisopropyl phosphoramidite) [ka]

[0383] The synthesis method is the same as in Example 1.9.

[0384] 1 H NMR (400MHz, CDCl3)δ 3.67-3.51(m, 6H), 1.64-1.57(m, 4H), 1.37-1.26(m, 20H), 1.18(d, J=6.8Hz, 12H), 0.89-0.85(m, 6H).

[0385] 31 P NMR: (400MHz, CDCl3)δ 145.00.

[0386] Example 2 Synthesis of blocking group precursors

[0387] Example 2.1 Synthesis of a precursor of M06 (2-cyanoethyl(2-morpholinoethyl)diisopropylphosphoramidite) [ka]

[0388] To a solution of compound 15-1 (10.88 g, 83.05 mmol) in anhydrous DCM (100 mL), 1H-imidazole-4,5-dicarbonitride (4.9 g, 41.53 mmol) was added. After cooling the reaction mixture to 0°C, compound 1-2 (25 g, 83.05 mmol) in DCM (30 mL) was added to the mixture under nitrogen protection. The mixture was stirred at 25°C for 16 hours under N2. The mixture was filtered, concentrated, and the DCM was removed. The residue was diluted with hexane and purified by silica gel chromatography (eluting with hexane) to obtain the precursor of M06.

[0389] 1 H NMR:(400MHz, CDCl3)δ 3.87-3.75(m, 3H), 3.71-3.65(m, 5H), 3.62-3.53(m, 2H), 2.65-2.57(m, 4H), 2.54-2.45(m, 4H), 1.17(dd, J=8.0, 4.0Hz, 12H).

[0390] 31 P NMR: (160MHz, CDCl3)δ 148.02.

[0391] Example 2.2 Synthesis of a precursor of M03 (2-cyanoethyl((tetrahydro-2H-pyran-4-yl)methyl)diisopropylphosphoramidite)

[0392] [ka]

[0393] To a solution of compound 16-1 (1.16 g, 10 mmol) in anhydrous DCM (20 mL), 1H-imidazole-4,5-dicarbonitride (0.6 g, 5 mmol) was added. After cooling the reaction mixture to 0°C, compound 1-2 (3 g, 10 mmol) in DCM (5 mL) was added to the mixture under nitrogen protection. The mixture was stirred at 25°C for 16 hours under N2. The mixture was filtered, concentrated, and the DCM was removed. The residue was diluted with hexane and purified by silica gel chromatography (eluting with hexane) to obtain the precursor of M03.

[0394] 1 H NMR:(400MHz, CDCl3)δ 3.98-3.94(m, 2H), 3.88-3.73(m, 2H), 3.61-3.34(m, 6H), 2.62(t, J=8.0Hz, 2H), 1.8 8-1.78(m, 1H), 1.66-1.59(m, 2H), 1.39-1.31(m, 2H), 1.17(dd, J=8.0, 4.0Hz, 12H).

[0395] 31 P NMR: (160MHz, CDCl3)δ 147.82.

[0396] Example 3: Preparation of double-stranded oligonucleotides

[0397] Oligonucleotide synthesis was performed using a MerMade 12 synthesizer with a standard solid-phase synthesis protocol. All phosphoramidites, including 2'-OMe-rA(Bz), 2'-OMe-rC(Ac), 2'-OMe-rU, 2'-OMe-rG(iBu), 2'-F-rA(Bz), 2'-F-rC(Ac), 2'-F-rU, and 2'-F-rG(iBu), were purchased from Honjin Biotech. 2'-OMe-rU was dissolved in a DMF / acetonitrile (1:4, v / v) mixed solvent, all other phosphoramidites were dissolved in acetonitrile, and molecular sieves (3 Å) were added. Unless otherwise specified, Unilinker CPG was used as the solid support. The synthesis cycle consisted of four separate steps: detritylation, bonding, oxidation (or sulfidation), and capping. 5-ethylthio-1H-tetrazole (ETT, 0.25 M acetonitrile solution) was used as an activator. A phosphorothioate bond was introduced into a 0.05 M pyridine solution of 3-((N,N-dimethylaminomethylidene)amino)-3H-1,2,4-dithiazol-5-thione (DDTT). After solid-phase synthesis, the phosphate protecting group (2-cyanoethyl group) was deprotected for 1 hour with a 20% diethylamine (DEA) solution in acetonitrile. Cleavage from the solid support and deprotection of nucleic acid bases (C&D) were performed in NH4OH at 65°C for 5 hours. The crude oligonucleotide solution was concentrated by centrifugation at high temperature (45°C) and reduced pressure (5.6 Torr) for 8 hours to obtain the crude oligonucleotide as a solid. The obtained solid was purified by preparative HPLC. Appropriate fractions were pooled and lyophilized to obtain the purified product.

[0398] Double-stranded oligonucleotides are prepared by annealing single-stranded oligonucleotides.

[0399] Annealed single-stranded oligonucleotides were prepared in sterile RNase-free H2O.

[0400] In the kneeling reaction system, the mixture was placed in a 95°C water bath for 10 minutes (20 minutes if ≥100 nmol), then rapidly transferred to a 60°C water bath for cooling, and freeze-dried to obtain double-stranded oligonucleotides, which were then stored at low temperatures.

[0401] Complementary double-stranded oligonucleotides were prepared by combining them with an equimolar solution of single-stranded oligonucleotides.

[0402] Example 4: Bioactivity assay of double-stranded oligonucleotides

[0403] Example 4.1 Huh 7 cell transfection assay

[0404] The cell suspension was diluted with 10% FBS DMEM, and the final cell density was 2 × 10⁶. 5 Cells / mL were obtained. 20 μL / well of siRNA-RNAiMAX complex was added to a 96-well plate, followed by the addition of cell suspension (100 μL / well). Cells were cultured at 37°C in 5% CO2 for 24 hours.

[0405] Intracellular RNA was isolated using the RNeasy kit (Qiagen-74182) according to the instructions. The RNA was then processed using HiScript according to the instructions. (R) Reverse transcription was performed using III RT SuperMix for qPCR (+gDNA wiper). The expression levels of target genes were quantified using qPCR with gene-specific primers (e.g., commercially available TaqMan(R) assay or the primers listed below). GAPDH was measured in parallel as a housekeeping gene. Samples were treated for TaqMan cycling using Applied Biosystems' Fast Real-Time PCR system, and 40 cycles were performed according to the thermal profile, with 10 minutes at 95°C, followed by 15 seconds at 95°C and 1 minute at 60°C.

[0406] Commercially available TaqMan(R) assay: [Table 2]

[0407] Other primers: [Table 3] [Table 4-1] [Table 4-2] [Table 5] [Table 6] [Table 7] Relative IC 50 = (ds X) / ds67 [Table 8] Relative IC 50 =(ds X) / ds74

[0408] As shown in the data above, the dsRNA agents disclosed herein exhibited potent inhibitory activity against target gene expression. Among dsRNA agents targeting different genes, dsRNA agents having purine at Z, more preferably guanine at Z, exhibited better inhibitory activity than pyrimidines. Furthermore, dsRNA agents having adenine or uracil at X1 / X2 showed potent inhibitory activity, while dsRNA agents having cytosine or guanine at X1 / X2 showed relatively weak activity.

[0409] Surprisingly, dsRNA agents having a blocking group (e.g., M03, M06, or invAB) or ligand (e.g., VSDL-03A) at the 5' end of the antisense strand exhibited activity that inhibited the expression of the target gene, whereas other reported dsRNA agents significantly or completely lost their activity when the 5' end of the antisense strand was bound to the blocking group or ligand. While we do not wish to be bound by theory, it is reasonable that the blocking group or ligand is cleaved along with the cleavage region of the dsRNA disclosed herein before achieving the RNAi effect.

[0410] Example 4.2 PHH-free KD uptake assay

[0411] After mixing primary human hepatocytes (PHH) with the appropriate culture medium, the cell suspension was raised to a final cell density of 6 × 10⁶. 5 The concentration was adjusted to cells / mL. The dsRNA was diluted and added to a collagen-I coated 96-well plate at 10 μL / well, followed by the addition of the cell suspension (90 μL / well). The cells were cultured at 37°C in 5% CO2 for 48 hours.

[0412] Intracellular RNA was isolated using the RNeasy kit (Qiagen-74182) according to the instructions for use. The RNA was reverse transcribed using HiScript(R)III RT SuperMix for qPCR (+gDNA wiper) according to the instructions for use. The expression levels of target genes were quantified using qPCR with gene-specific primers (e.g., commercially available TaqMan(R) assay or the primers listed below). GAPDH was measured in parallel as a housekeeping gene. Samples were treated for TaqMan cycling using Applied Biosystems' Fast Real-Time PCR system, and 40 cycles were performed according to the thermal profile, with 10 minutes at 95°C, followed by 15 seconds at 95°C and 1 minute at 60°C.

[0413] Commercially available TaqMan(R) assay: [Table 9]

[0414] Other primers: [Table 10] [Table 11-1] [Table 11-2]

[0415] As shown in the table above, cleavage regions containing both 2'-F and 2'-OMe exhibited potent inhibitory activity. In a preferred embodiment, X2 and Y are modified with 2'-OMe, and Z and N1 are modified with 2'-F.

[0416] It has been found that nucleotide bond modifications between X2 and Y, or between Y and Z, reduce the inhibitory activity of dsRNA. [Table 12] Table 8: Primary human hepatocyte free uptake assay

[0417] As shown in the table above, the dsRNA agents disclosed herein, having a 5' extension and cleavage region on the antisense strand, showed relatively potent inhibitory activity against different genes and targets compared to parental dsRNA agents without a 5' extension.

[0418] Example 4.3 In vivo mouse HDI model

[0419] Mice (BALB / C, 6-7 weeks old, female) were administered either the vector or test siRNA (5 mg / kg) on ​​day 0. On day 3, all mice were injected with a plasmid DNA solution equivalent to 8% of their body weight via the tail vein within 5 seconds (injection volume (mL) = mouse body weight (g) × 8%). The mass of plasmid injected into each mouse was 10 μg. All animals were sacrificed on day 4. Liver tissue was collected from all groups and targeted mRNA analysis was performed using QPCR. RNA was reverse transcribed to cDNA using HiScript(R)III RT SuperMix for qPCR (+gDNA wiper) (Vazyme-R323) according to the instructions. cDNA was quantified using QPCR. NEO (sequence information shown in the table) mRNA was detected in parallel as an internal control. [Table 13]

[0420] Test compound: [Table 14]

[0421] The results are shown in Figure 1.

[0422] As shown in Figure 1, the dsRNA agents disclosed herein, which have a 5' extension and a cleavage region on the antisense strand, showed a significantly relatively potent inhibitory effect on the target gene compared to parental dsRNA agents that do not have a 5' extension.

[0423] Example 4.4 In vivo rat CNS target knockdown model (IT injection)

[0424] The test compound was formulated in 10 mM PBS (pH 7.4) up to 20 mg / mL, and 50 μL of IT injection (0.9 mg / dose) was administered via lumbar puncture. Tail flapping or tail tip wagging was used as a marker of successful manipulation. After administration, the anesthesia machine was removed and the animals were returned to their cages. On day 14, the animals were sacrificed, tissue samples were collected, and qPCR analysis was performed.

[0425] Measurement of SOD1 mRNA levels by qPCR

[0426] RNA was extracted from tissue samples using an automated nucleic acid extraction system. The samples were transferred to new RNase-free tubes, and cDNA synthesis and qPCR were performed. [Table 15]

[0427] Test compound: [Table 16]

[0428] The results are shown in Figures 2 and 3.

[0429] As shown in Figures 2 and 3, the dsRNA agents disclosed herein, having a 5' extension, cleavage region, and VSDL ligand on the antisense strand, showed similar inhibitory effects on target genes compared to parental dsRNA agents having 5'VPu modification and C16U. However, the dsRNA agents disclosed herein showed more tissue-specific target knockdown compared to dsRNA agents having 5'VPu modification and C16U.

[0430] Example 4.5 In vivo mouse IVT model

[0431] On the day before and the day after IVT injection, a topical antibiotic (tobramycin) was applied twice to both eyes of the mice. On day 0, either 1×PBS or siRNA formulated in 1×PBS was administered to both eyes via IVT injection at a dose of 3 μg per dose. On day 7, all animals were sacrificed, and the entire eye was collected for qPCR analysis.

[0432] Test compound: [Table 17]

[0433] The results are shown in Figure 4.

[0434] As shown in Figure 4, the dsRNA agent disclosed herein, having a 5' extension, cleavage region, and VSDL ligand on the antisense strand, showed a stronger inhibitory effect on the target gene compared to the parental dsRNA agent having a 5'VPu modification and C16U.

[0435] Example 4.6 Reaction of rat liver homogenate treatment

[0436] 1.00 mg of the test compound was precisely dissolved in 0.961 mL of water to obtain a working solution with a corrected concentration of 1,000,000 ng / mL. The working solution was mixed with 20% rat liver homogenate and incubated at 37°C for 48 hours to obtain a final sample with a concentration of 10,000 ng / mL. 50.0 μL of extraction IS working solution (phenol / chloroform / isoamyl alcohol = 25 / 24 / 1, V / V / V) was added to the incubated sample, and liquid-liquid extraction was performed. After centrifugation, 300 μL of the supernatant was extracted using solid-phase extraction. The sample was then transferred to an equilibrated SPE plate, washed, and the SPE plate was eluted. The recovered eluate was evaporated. (LC-HRMA: Liquid Chromatography High Resolution Mass Spectrometer, LC: Shimadzu Corporation, LC-30AD, HRMA: Q Exactive Plus or Q Exactive Focus (Thermo San Jose, CA))

[0437] Test compound: [Table 18]

[0438] The results are shown in Figure 5. After incubation for 48 hours, the observed product included the unused test compound (31%), the test compound with one nucleotide cleaved at the 3' end (15%), the test compound with two nucleotides cleaved at the 3' end and a cleaved M06 (18%), the test compound with three nucleotides cleaved at the 5' end (15% + 5% + 10%), and other trace metabolites (5%).

[0439] In summary, (1) only the 5' cleavage product observed was from the test compound having 3 nucleotides cleaved at the 5' end, (2) the 3' cleavage product observed included the test compound having 1 or 2 nucleotides cleaved at the 3' end, and (3) the cleavage product of the test compound having 3 nucleotides cleaved at the 5' end was the major product among all cleavage products.

[0440] Example 4.7 Long-term plasma-targeted knockdown model of NHP

[0441] The in vivo targeted knockdown effect of modified siRNA was evaluated in cynomolgus monkeys. Animals (N=2 per group) were administered a single subcutaneous dose of 2 mg / kg on day 1. Blood samples were collected on days 1, 2, 7, 14, 28, 42, 56, and 70 after administration. Circulating AGT levels were quantified using a human angiotensinogen-specific ELISA method (and cross-reactivity with cynomolgus monkeys) according to the manufacturer's protocol (Sinobiological Corporation KIT10994). Data were expressed as a percentage of the baseline value, expressed as mean ± standard error.

[0442] Test compound: [Table 19]

[0443] The results are shown in Figure 6.

[0444] Example 4.8 Repeated dose tolerance study in rats A repeated-dose tolerance study was conducted in rats. The test compounds (ds86, ds99) were administered subcutaneously once a week for 3 weeks (total of 3 doses) at doses of 250 mg / kg or higher (300 mg / kg*1, 250 mg / kg*2). [Table 20]

[0445] Results: No abnormalities were found in clinical observation, pathology, or gross anatomy.

[0446] Histopathology:

[0447] Kidney: Both groups showed mild basophilic granules and tubular vacuolation.

[0448] Liver: Both groups showed mild to moderate vacuolation, pigmented hepatocytes, and Kupffer cells.

[0449] Injection site: Mild to moderate mixed cell inflammation.

[0450] Conclusion: The results were consistent with the uptake and removal of GalNAc siRNA and were not considered harmful.

Claims

1. A double-stranded oligonucleotide agent comprising a sense strand and an antisense strand, The sense strand and the antisense strand form a double-stranded portion of 15 to 27 base pairs in length and a 5' extension in the antisense strand. The 5' extension is at least 3 nucleotides in length and is cleavable from the 3' nucleotide of the 5' extension, and the cleaved double-stranded oligonucleotide agent can silence target RNA or inhibit the expression of a target gene via RNA interference.

2. The double-stranded oligonucleotide agent according to claim 1, further comprising a 3' extension in the antisense chain.

3. The double-stranded oligonucleotide agent according to any one of the claims, wherein the double-stranded portion is formed by base pairing of a first fragment in the antisense strand and a second fragment in the sense strand, and the first and second fragments are of equal length.

4. The length of the double-stranded portion is, a) 15-25 nucleotide pairs, 15-24 nucleotide pairs, 15-23 nucleotide pairs, 16-24 nucleotide pairs, 16-23 nucleotide pairs, 16-22 nucleotide pairs, 16-21 nucleotide pairs, 16-20 nucleotide pairs, 17-23 nucleotide pairs, 17-22 nucleotide pairs, 17-21 nucleotide pairs, 17-20 nucleotide pairs, 18-23 nucleotide pairs, 18-22 nucleotide pairs, 18-21 nucleotide pairs, 18-20 nucleotide pairs, 19-23 nucleotide pairs, 19-22 nucleotide pairs, 19-21 nucleotide pairs, or, b) A double-stranded nucleotide agent according to any one of the claims, wherein the nucleotides are 15 nucleotide pairs, 16 nucleotide pairs, 17 nucleotide pairs, 18 nucleotide pairs, 19 nucleotide pairs, 20 nucleotide pairs, 21 nucleotide pairs, 22 nucleotide pairs, or 23 nucleotide pairs.

5. The double-stranded oligonucleotide agent according to any one of the claims, wherein the length of the sense chain is 15-35, 16-35, 16-30, 16-27, 16-26, 16-25, 16-21, 17-35, 17-30, 17-25, 17-21, 18-35, 18-30, 18-25, 18-23, 18-21, 19-35, 19-30, 19-25, 19-21, 20-35, 20-30, 20-25, 20-23, 21-35, 21-30, 21-25, 21-23, for example, 25, 24, 23, 22 or 21nt.

6. The double-stranded oligonucleotide agent according to any one of the claims, wherein the length of the 5' extension is at least 3, 4, 5, 6, or 7 nucleotides, and / or the length of the 3' extension is at least 1 or 2 nucleotides.

7. The double-stranded oligonucleotide agent according to any one of the claims, wherein the first fragment comprises a target region sufficiently complementary to a portion of the target mRNA encoding the target RNA or the target gene.

8. The double-stranded oligonucleotide agent according to any one of the claims, wherein the 5' extension is enzymatically cleavable from the most 3' nucleotide of the 5' extension, and optionally the 5' extension is specifically cleavable by an endonuclease or optionally a ribonuclease (RNase).

9. The double-stranded oligonucleotide agent according to any one of the claims, wherein the antisense strand comprises a cleavage region having a nucleotide sequence represented by formula A: (3'-5')X2-Y-Z, and is cleavable between X2 and Y, where X2 is the furthest 5' nucleotide of the first fragment and Y-Z are the two furthest 3' nucleotides of the 5' extension.

10. The double-stranded oligonucleotide agent according to any one of the claims, wherein the cleavage region further comprises a fourth nucleotide (N1) which is a third nucleotide from the 3' end of the 5' extension, and the cleavage region comprises a nucleotide sequence represented by formula B: (3'-5')X2-Y-Z-N1 and is cleavable between X2 and Y.

11. The double-stranded oligonucleotide agent according to any one of claims 1 to 9, wherein the cleavage region further comprises a third fragment (N), the third fragment comprising at least one nucleotide, N1 being the most 3' nucleotide of the third fragment, the cleavage region comprising a nucleotide sequence represented by formula B': (3'-5')X2-Y-Z-N, cleavable between X2 and Y, and the length of N being 1 to 10 nucleotides, preferably 1 to 5 nucleotides, and more preferably 1 nucleotide.

12. Z is selected from G or A or natural or unnatural analogs thereof, the double-stranded oligonucleotide agent according to any one of claims 9 to 11.

13. Z is selected from G or a natural or unnatural analog thereof, the double-stranded oligonucleotide agent according to any one of claims 9 to 12.

14. X2 is selected from A or U or natural or unnatural analogs thereof, a double-stranded oligonucleotide agent according to any one of claims 9 to 13.

15. Formula A is a double-stranded oligonucleotide agent according to any one of claims 9 to 11, having a nucleotide sequence from 3'–5' selected from the group consisting of UUG, UAG, AUG, AAG, UUA, UAA, AUA, AAA, UCG, UGG, ACG, AGG, UCA, UGA, ACA, AGA, or natural or unnatural analogs thereof.

16. The double-stranded oligonucleotide agent according to any one of claims 9 to 15, wherein the nucleotide Y is selected from A or U or natural or unnatural analogs thereof.

17. Formula A is a double-stranded oligonucleotide agent according to any one of claims 9 to 16, having a nucleotide sequence from 3'-5' selected from the group consisting of UUG, UAG, AUG, AAG, UUA, UAA, AUA, and AAA.

18. The double-stranded oligonucleotide agent according to any one of the claims, wherein at least one nucleotide in the cleavage region is a modified nucleotide, and preferably all nucleotides in the cleavage region are modified nucleotides.

19. The double-stranded oligonucleotide agent according to claim 18, wherein the modified nucleotide has a modified base, a modified sugar, or an inter-modified nucleotide bond.

20. a) The modified bases include hypoxanthine (I), xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine (m5C), 5-hydroxymethylcytosine, N6-methyladenosine (m6A), 3-methyluridine (m3U), 5-methyluridine (m5U), pseudouridine, 2-thiouridine (s2U), and 5-propyluridine (5-pU). b) The modified sugars include 2'-sugar modifications, 3'-sugar modifications, 5'-sugar modifications, for example, 2'-OMe (2'-O-methyl) modifications, 2'-F (2'-deoxy-2'-fluoro) modifications, 2'-O-MOE (2'-O-methoxyethyl) modifications, 2'-deoxy(2'-d) modifications, 5'-morpholine (5'-Mo) modifications, unlocked nucleic acid (UNA) modifications, glycol nucleic acid (GNA) modifications, locked nucleic acid (LNA) modifications, tricyclo-DNA (tcDNA) modifications, (S)-restricted ethyl cross-linked nucleic acid ((S)-cEt-BNA) modifications, 5'-(E)-vinyl phosphate (VP) modifications, 2'-O-C16 modifications, and 5'-terminus or 3'-terminus. This includes conjugation with inverted debasic nucleotides (invAB), substitution with inverted debasic nucleotides (invAb), substitution with 2,4-difluorotolyl ribonucleotide (rF), substitution with (S)-glycerol nucleic acid, substitution with inosine (I), conjugation with M03 at the 5' or 3' end, conjugation with M06 at the 5' or 3' end, and conjugation with ligands such as GalNAc ligands, lipophilic ligands, or other receptor-targeting ligands that may promote endocytosis of siRNA-conjugates (e.g., TfR-targeting ligands, LDL-R-targeting ligands, integrin-targeting ligands, etc.), and / or c) The double-stranded oligonucleotide agent according to claim 18 or 19, wherein the modified nucleotide bond comprises methylphosphonate (MP), methoxypropylmethylphosphonate (MOP), phosphorothioate (PS), phosphorodithioate (PS2), phosphoroselenoate, phosphorodiselenoate, phosphoranilothioate, phosphoraniladete, phosphoramidate, and PNA.

21. a) At least one of the internucleotide bonds between the nucleotides within the cleavage region is not a phosphorothioate bond, b) The internucleotide bond between X2 and Y is not a phosphorothioate bond, c) The internucleotide bond between Y and Z is not a phosphorothioate bond, d) The internucleotide bonds between the nucleotides within the cleavage region are not phosphorothioate bonds, e) At least one of the internucleotide bonds between the nucleotides within the cleavage region is a phosphodiester bond, f) The nucleotide bond between X2 and Y is a phosphodiester bond, g) The nucleotide bond between Y and Z is a phosphodiester bond, and / or h) The double-stranded oligonucleotide agent according to any one of claims 4 to 20, wherein each of the internucleotide bonds between the nucleotides within the cleavage region is a phosphodiester bond.

22. a) At least one nucleotide within the cleavage region is 2'-OMe modified or 2'-F modified, b) Each nucleotide within the cleavage region is modified with 2'-OMe or 2'-F, c) At least one nucleotide within the cleavage region is 2'-F modified, d) Two or fewer nucleotides within the cleavage region are 2'-F modified, e) Z in formula A or formula B is modified by 2'-F, and optionally X2 in formula A or formula B is modified by 2'-F, f) N1 in formula A or formula B is modified by 2'-F, g) Both X2 and Y in formula A or formula B are modified by 2'-OMe, both Z and N1 in formula A or formula B are modified by 2'-F, and / or h) A double-stranded oligonucleotide agent according to any one of claims 4 to 21, wherein all of X2, Y and Z in formula A or formula B are 2'-OMe modified, and N1 in formula A or formula B is 2'-F modified.

23. A double-stranded oligonucleotide agent according to any one of the claims, further comprising at least one phosphorothioate or methylphosphonate internucleotide bond.

24. a) At least one phosphorothioate or methylphosphonate internucleotide bond at position 1 and / or position 2 (counting from its 5'-terminus) of the first fragment, and / or b) The double-stranded oligonucleotide agent according to any one of the claims, further comprising at least one phosphorothioate or methylphosphonate internucleotide bond at the 1 and / or 2 positions (counting from the 3'-terminus) of the first fragment.

25. a) At least one phosphorothioate or methylphosphonate internucleotide bond at positions 1 to 8, or 1 to 6, or 1 and / or 2 (counting from the 5'-terminus) of the second fragment, and / or b) The double-stranded oligonucleotide agent according to any one of the claims, further comprising at least one phosphorothioate or methylphosphonate internucleotide bond at positions 1 to 8, or positions 1 to 6, or positions 1 and / or 2 (counting from the 3'-terminus) of the second fragment.

26. The double-stranded oligonucleotide agent according to any one of the claims, wherein the antisense chain further comprises a blocking group at its 5' end, and optionally the blocking group is bonded to the 5' end of the 5' extension.

27. The double-stranded oligonucleotide agent according to claim 26, wherein the blocking group comprises a debasic residue, an inverted debasic residue, M03, or M06.

28. The double-stranded oligonucleotide agent according to claim 26 or 27, wherein the blocking group is bound to the 5' end of the 5' extension via optionally modified or unmodified internucleotide bonds.

29. The double-stranded oligonucleotide agent according to claim 28, wherein the blocking group is bonded to the 5' end of the 5' extension via a phosphorothioate bond.

30. The double-stranded oligonucleotide agent according to any one of the claims, wherein the sense strand is complementary to the antisense strand over its entire length.

31. A double-stranded oligonucleotide agent according to any one of claims 9 to 30, wherein cleavage in the cleavage region generates a blunt end at the 5' end of the first fragment.

32. a) Each nucleotide of the first fragment of the antisense strand is modified, b) Each nucleotide of the antisense strand is modified, c) Each nucleotide of the second fragment of the sense strand is modified and / or d) The double-stranded oligonucleotide agent according to any one of the claims, wherein each nucleotide of the sense strand is modified.

33. The double-stranded oligonucleotide agent according to any one of the claims, wherein each nucleotide of the antisense strand is 2'-F modified or 2'-OMe modified, and / or each nucleotide of the sense strand is 2'-F modified or 2'-OMe modified.

34. The aforementioned sense chain is a) Optionally, a motif of three consecutive 2'-F modified nucleotides located at positions 9, 10, and 11, counting from the 5' end nucleotide of the sense strand, and / or b) A 2'-F modified nucleotide at the 7th and / or 18th position, counted from the 5' end of the sense strand, and / or c) The double-stranded oligonucleotide agent according to any one of the claims, further comprising the alternating motif at positions 9 to 13, counted from the 5' end, wherein the 2'-F modification and the 2'-OMe modification occur on the alternating nucleotides within the alternating motif.

35. The double-stranded oligonucleotide agent according to any one of the claims, wherein the first fragment of the antisense strand and / or the second fragment of the sense strand further comprises an alternating motif in which modifications occur on alternating nucleotides within the alternating motif, and optionally, 2'-F modifications occur on alternating nucleotides within the alternating motif, and / or 2'-OMe modifications occur on alternating nucleotides within the alternating motif.

36. The first fragment of the antisense strand and / or the second fragment of the sense strand are modified with 2'-OMe (2'-O-methyl), 2'-F (2'-deoxy-2'-fluoro), 2'-O-MOE (2'-O-methoxyethyl), 2'-deoxy (2'-d), 5'-morpholine (5'-Mo), unlocked nucleic acid (UNA), glycol nucleic acid (GNA), locked nucleic acid (LNA), tricyclo-DNA (tcDNA), and (S)-restricted ethyl frame. A double-stranded oligonucleotide agent according to any one of the claims, comprising modifications selected from the group consisting of bridge nucleic acid ((S)-cEt-BNA) modification, 5'-(E)-vinyl phosphate (VP) modification, 2'-O-C16 modification, conjugation with an inverted debasic nucleotide (invAB) at the 5' or 3' end, substitution with an inverted debasic nucleotide (invAb), substitution with 2,4-difluorotolyl ribonucleotide (rF), substitution with (S)-glycerol nucleic acid, and substitution with inosine (I).

37. Formula (C): 【Chemistry 1】 A double-stranded oligonucleotide agent comprising the structure shown in formula (C), The first fragment in the antisense strand and the second fragment in the sense strand form a double-stranded portion by base pairing, and the first fragment and the second fragment are of equal length. The length of the 5' extension is at least 3 nucleotides. The antisense strand includes a cleavage region comprising the most 5' nucleotide (X2) of the first fragment and the two most 3' nucleotides (Y-Z) of the 5' extension, wherein the cleavage region comprises a nucleotide sequence represented by formula A: (3'-5')X2-Y-Z, which is cleavable between X2 and Y, and formula A is as defined in any one of claims 9 to 36. The length of the sense strand is 15-35, 15-23, 15-22, or 15-21 nucleotides. The length of the antisense strand is 25-35, 26-35, 26-30, 25-27, or 26-27 nucleotides, and optionally the antisense strand further includes a blocking group at its 5' end, and optionally the blocking group is bound to the 5' end of the 5' extension, wherein the double-stranded oligonucleotide agent.

38. The double-stranded oligonucleotide agent according to claim 37, wherein the length of the sense strand is 17-23, 17-22, 21-23, or 17-21 nucleotides, and the length of the antisense strand is 25-30, 25-27, or 26-27 nucleotides.

39. The double-stranded oligonucleotide agent according to claim 37, wherein the lengths of the sense strand and the antisense strand are a) 17 nt and 20 nt, respectively; b) 18 nt and 21 nt, respectively; c) 19 nt and 22 nt, respectively; d) 20 nt and 23 nt, respectively; or e) 21 nt and 24 nt, respectively.

40. Formula (D): 【Chemistry 2】 A double-stranded oligonucleotide agent comprising the structure shown in formula (D), The first fragment in the antisense strand and the second fragment in the sense strand form a double-stranded portion by base pairing, and the first fragment and the second fragment are of equal length. The length of the 5' extension is at least 3 nucleotides. The antisense strand comprises a cleavage region including the furthest 5' nucleotide (X2) of the first fragment and the two furthest 3' nucleotides (Y-Z) of the 5' extension, wherein the cleavage region comprises a nucleotide sequence represented by formula A: (3'-5')X2-Y-Z, which is enzymatically cleavable between X2 and Y, and upon cleavage, removes the furthest 3' nucleotide (Y) of the 5' extension, where formula A is as defined in any one of claims 9 to 36. The length of the sense strand is 15-35, 15-23, 15-22, 15-21, 16-25, 17-23, 18-23, 19-23, 19-21, 20-23, 20-21, 21-23, for example, 17, 18, 19, 20, 21, 22, or 23 nucleotides. The length of the antisense strand is 25-35, 25-30, 26-35, 26-30, 26-27, for example, 25, 26, 27, 28, 29, or 30 nucleotides, and optionally the antisense strand further includes a blocking group at its 5' end, and optionally the blocking group is bound to the 5' end of the 5' extension, wherein the double-stranded oligonucleotide agent.

41. The double-stranded nucleotide agent according to claim 40, wherein the length of the sense strand is 17 to 23, 17 to 22, or 17 to 21 nucleotides, and the length of the antisense strand is 22 to 28, 22 to 27, or 22 to 26 nucleotides.

42. The double-stranded oligonucleotide agent according to claim 40, wherein the lengths of the sense strand and the antisense strand are a) 19 nt and 25 nt, b) 20 nt and 25 nt, c) 21 nt and 25 nt, d) 19 nt and 26 nt, e) 20 nt and 26 nt, f) 21 nt and 26 nt, or g) 21 nt and 27 nt, respectively.

43. A double-stranded oligonucleotide agent according to any one of the claims, further comprising a ligand operably bound to the sense strand or the antisense strand.

44. The double-stranded oligonucleotide agent according to claim 43, wherein the ligand is operably bound to the 5' end of the sense strand or the 5' end of the antisense strand.

45. The double-stranded oligonucleotide agent according to claim 43 or 44, wherein the ligand is bound to the double-stranded oligonucleotide agent via a phosphorothioate.

46. The double-stranded oligonucleotide agent according to any one of claims 43 to 45, wherein the ligand is a GalNAc ligand, a lipophilic ligand, or another ligand that targets a receptor that may promote endocytosis of an siRNA-coupled organism (such as a TfR-targeting ligand, an LDL-R-targeting ligand, or an integrin-targeting ligand).

47. The ligand is selected from the group consisting of L96, C16U, C16G, C16A, C16C, VSDL-01, VSDL-01A, VSDL-02, VSDL-02A, VSDL-03, VSDL-03A, VSDL-04, VSDL-04A, VSDL-05, VSDL-05A, VSDL-06, VSDL-06A, VSDL-07, VSDL-07A, VSDL-08, VSDL-08A, VSDL-09, VSDL-10, VSDL-11, VSDL-12, VSDL-13, and VSDL-14, as described in any one of claims 44 to 46.

48. The double-stranded oligonucleotide agent according to any one of the claims, wherein the target RNA encodes a target gene selected from the group consisting of AGT, CFB, DGAT2, DUX, ANGPTL8, APOC3, F12, INHBE, PNPLA3, Serpinc1, APP, SOD1, TMPRSS6, KHK, PCSK9, VEGFA, ANGPTL3, ANGPTL4, C3, C5, TTR, IGF-1R, VEGFR, ANG2, GIPR, GPR75, ActRII, NUDT21, PLN, HSD17b13, CNOT6L, PTP1B, CFHR, ATX, CIDEB, mARC1, TSHR, CB1, and LPA.

49. A double-stranded oligonucleotide agent comprising a double-stranded oligonucleotide operably bound to a blocking group, wherein the blocking group comprises M03 or M06.

50. A double-stranded oligonucleotide agent comprising a double-stranded oligonucleotide operably bound to a ligand, wherein the ligand comprises a chemical structure selected from the group consisting of VSDL-01, VSDL-01A, VSDL-02, VSDL-02A, VSDL-03, VSDL-03A, VSDL-04, VSDL-04A, VSDL-05, VSDL-05A, VSDL-06, VSDL-06A, VSDL-07, VSDL-07A, VSDL-08, VSDL-08A, VSDL-09, VSDL-10, VSDL-11, VSDL-12, VSDL-13, and VSDL-14.

51. a) The double-stranded oligonucleotide comprises a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and / or b) The antisense strand is sufficiently complementary to a portion of the mRNA encoding the target RNA or target gene, and the sense strand is sufficiently complementary to the target region of the antisense strand. c) The blocking group is conjugated to the 5' end of the antisense chain, and / or d) The ligand is conjugated to the 5' end of the antisense chain, the 5' end of the sense chain, or the 3' end of the sense chain, and / or e) The double-stranded oligonucleotide has one end operably bound to the blocking group, the other end operably bound to the ligand, and / or f) The double-stranded oligonucleotide agent according to claim 49 or 50, wherein the double-stranded oligonucleotide agent can silence a target RNA or inhibit the expression of a target gene via RNA interference.

52. A pharmaceutical composition comprising a double-stranded oligonucleotide agent according to any one of the above claims and a pharmaceutically acceptable carrier.

53. A method for inhibiting the expression of a target gene in a subject requiring it, comprising administering to the subject a pharmaceutically effective amount of a double-stranded oligonucleotide agent according to any one of claims 1 to 51 or a pharmaceutical composition according to claim 52.

54. The method according to claim 53, wherein the target gene is selected from the group consisting of AGT, CFB, DGAT2, DUX, ANGPTL8, APOC3, F12, INHBE, PNPLA3, Serpinc1, APP, SOD1, TMPRSS6, KHK, PCSK9, VEGFA, ANGPTL3, ANGPTL4, C3, C5, TTR, IGF-1R, VEGFR, ANG2, GIPR, GPR75, ActRII, NUDT21, PLN, HSD17b13, CNOT6L, PTP1B, CFHR, ATX, CIDEB, mARC1, TSHR, CB1, and LPA.

55. A method for treating a disease or disorder of a subject requiring treatment, comprising administering to the subject a pharmaceutically effective amount of a double-stranded oligonucleotide agent according to any one of claims 1 to 51 or a pharmaceutical composition according to claim 52.

56. The method according to claim 55, wherein the disease or disorder is related to the target gene.

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