TW202519663A - Double-stranded oligonucleotide agents and uses thereof - Google Patents

Abstract

Disclosed herein is a double-stranded oligonucleotide agent and uses thereof, comprising a specific cleavage site for an endonuclease, wherein the cleaved double-stranded oligonucleotide agent is capable of inhibiting expression of the target gene via RNA interference (RNAi).

Description

Double-stranded oligonucleotide reagent and its use

Cross-reference to related applications

This application claims priority to Chinese Patent Application No. 202310900474.0 filed on July 21, 2023, Chinese Patent Application No. 202310904516.8 filed on July 21, 2023, Chinese Patent Application No. 202311532296.7 filed on November 16, 2023, and Chinese Patent Application No. 202410396475.0 filed on April 2, 2024, the contents of all of which are incorporated herein by reference in their entirety.

Disclosed herein is a double-stranded oligonucleotide reagent and its use, wherein the double-stranded oligonucleotide reagent can inhibit the expression of a target gene through RNA interference (RNAi).

RNA interference (RNAi) technology refers to the highly conserved, efficient and specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA) during the evolutionary process. dsRNA inhibits the expression of target genes by destroying target mRNA. RNAi technology can specifically eliminate or shut down the expression of target genes, and has therefore quickly become one of the most useful research tools for exploring gene functions and therapeutic agents for the treatment of many diseases, including metabolic diseases, infectious diseases and malignant tumors.

Provided herein is a double-stranded oligonucleotide reagent comprising a sense strand and an antisense strand, wherein: The sense strand and the antisense strand form a double-stranded portion having a length of 15 to 27 base pairs and a 5' extension in the antisense strand; The 5' extension has a length of at least three nucleotides and can be cleaved from the 3'-most nucleotide in the 5' extension, and the cleaved double-stranded oligonucleotide reagent can silence the target RNA or inhibit the expression of the target gene by RNA interference.

Provided herein is a double-stranded oligonucleotide reagent comprising a structure represented by formula (C): Right shares (5'→3'): Second segment Antisense strand (3'→5'): First Clip 5' extension Formula (C), wherein the first segment in the antisense strand forms a double-stranded portion with the second segment in the sense strand by base pairing, wherein the first segment and the second segment have equal lengths; wherein the length of the 5' extension comprises at least three nucleotides; wherein the antisense strand comprises a cleavage region, the cleavage region comprises the 5'-most nucleotide (X2) of the first segment and the 3'-most two nucleotides (YZ) of the 5' extension, and the cleavage region comprises a nucleotide sequence shown in Formula A: (3'-5') X2-YZ, which is capable of cleavage between X2 and Y, and wherein Formula A is as defined below; wherein the length of the sense strand is 15 to 35, 15 to 23, 15 to 22 or 15 to 21 nucleotides; and wherein the length of the antisense strand is 25 to 35, 26 to 35, 26 to 30, 25 to 27 or 26 to 27 nucleotides.

The double-stranded oligonucleotide reagent of formula (C) does not require the complementary region and the targeting region to form a blunt end. It is possible that the targeting region contains a 3' overhang, or the complementary region contains a 5' extension, or the complementary region and the targeting region can form a blunt end.

Provided herein is a double-stranded oligonucleotide reagent comprising a structure represented by formula (D): Right shares (5'→3'): Second segment Antisense strand (3'→5'): 3' extension First Clip 5' extension Formula (D), wherein the first segment in the antisense strand forms a double-stranded portion with the second segment in the sense strand by base pairing, wherein the first segment and the second segment have equal lengths; the length of the 5' extension comprises at least three nucleotides; the antisense strand comprises a cleavage region, the cleavage region comprises the 5'-most nucleotide (X2) of the first segment and the 3'-most two nucleotides (YZ) of the 5' extension, and the cleavage region comprises a nucleotide sequence shown in Formula A: (3'-5') X2-YZ, which is capable of cleavage between X2 and Y, and upon cleavage, the 5' extension is removed from its 3'-most nucleotide (Y), and Formula A is as defined below, wherein the sense strand is 15 to 35, 15 to 23, 15 to 22, 15 to 21, 16 to 25, 17 to 23, 18 to 23, 19 to 23, 19 to 21, 20 to 23, 20 to 21, 21 to 23, e.g., 17, 18, 19, 20, 21, 22, or 23 nucleotides in length; and wherein the antisense strand is 25 to 35, 25 to 30, 26 to 35, 26 to 30, 26 to 27, e.g., 25, 26, 27, 28, 29, or 30 nucleotides in length.

Provided herein is a double-stranded oligonucleotide reagent comprising a double-stranded oligonucleotide operably linked to a blocking group, wherein the blocking group comprises M03 or M06.

Provided herein is a double-stranded oligonucleotide reagent 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.

Provided herein is a pharmaceutical composition comprising a double-stranded oligonucleotide reagent disclosed herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

Provided herein is a method for inhibiting the expression of a target gene in an individual in need thereof, the method comprising administering to the individual a pharmaceutically effective amount of a double-stranded oligonucleotide reagent disclosed herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition disclosed herein.

Provided herein is a method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a pharmaceutically effective amount of a double-stranded oligonucleotide reagent disclosed herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition disclosed herein.

It should be understood that the above general description and the following detailed description are only exemplary and illustrative, rather than limiting, of the present invention. In addition, the drawings incorporated in this specification and constituting a part of this specification illustrate embodiments of the present invention and are used together with the specification to explain the principles of the present invention.

As will be apparent to one of ordinary skill in the art upon reading the present disclosure, each individual embodiment described and illustrated herein has discrete components and features that can be readily separated or combined with the features of any of the other several embodiments without departing from the scope or nature of the present disclosure.

The following description of the present disclosure is intended only to illustrate various embodiments of the present disclosure. As such, the specific modifications discussed should not be interpreted as limiting the scope of the present disclosure. It will be apparent to those of ordinary skill in the art that various equivalent forms, variations, and modifications may be made without departing from the scope of the present disclosure, and it should be understood that these equivalent embodiments are to be included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

In this application, the use of the singular includes the plural unless otherwise specifically stated. In this application, the use of "or" means "and/or" unless otherwise stated. In addition, the use of the term "including" and other forms such as "includes" and "included" are not limiting. In addition, unless otherwise specifically stated, terms such as "element" or "component" cover both elements and components that include one unit and elements and components that include more than one sub-unit. In addition, the section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

Definition

As used herein, unless otherwise indicated herein or clearly contradicted by context, the terms "a", "an", "the" and similar terms used in the context of the present invention (especially in the context of the claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

Reference herein to "about" a value or parameter includes (and describes) embodiments directed to the value or parameter itself. For example, description referring to "about X" includes description of "X". Numerical ranges include the numbers defining the range. In general, the term "about" refers to the indicated value of a variable and all values of the variable that are within the experimental error range of the indicated value (e.g., within the 95% confidence interval of the mean) or within 10% of the indicated value, whichever is greater.

In this application, the term "optional" or "optionally" means that the described event or situation may or may not occur, and includes situations where the event or situation occurs and situations where the event or situation does not occur. For example, "optionally modified" includes both unmodified and modified, and further, "optionally modified nucleotides" include both unmodified and modified nucleotides.

It should be noted that in the present disclosure, terms such as “comprises,” “comprised,” “comprising,” “contains,” “containing,” “has,” “having,” etc. are intended to be inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

As used herein, the term "disorder" refers to any disease, disorder, or condition that impairs the normal function of an individual (e.g., a human).

The term "effective amount" means the amount of an agent that produces some desired local or systemic therapeutic effect at a reasonable benefit/risk ratio that applies to any treatment, alone or in combination with another dose. In the case of treating a particular disorder, the desired local or systemic therapeutic effect preferably involves inhibiting the course of the disorder. This includes slowing the progression of the disorder and, especially, interrupting or reversing the progression of the disorder. When administered to prevent a disorder, the amount is sufficient to avoid or delay the onset of the disorder. An effective amount does not require curing or preventing constant occurrence of the disorder. The effective amount of the agents described herein will depend on the condition to be treated, the severity of the condition, the individual parameters of the patient (including age, physical condition, size and weight), the duration of treatment, the type of concomitant treatment (if any), the specific route of administration, and similar factors. Thus, the dosage of the agents described herein administered can depend on a variety of such parameters. In cases where the patient does not respond adequately to the initial dose, a higher dose may be used (or an effective higher dose achieved by a different, more localized route of administration). In certain embodiments, the effective amount of an agent will depend on its therapeutic index, solubility, etc.

As used herein, the term "inhibit" is used interchangeably with "reduce," "silence," "downregulate," "suppress," and other similar terms, and includes any amount of inhibition, specifically statistically significant or clinically significant inhibition.

As used herein, the term "inhibiting the expression of a target gene" refers to any significant reduction in the amount of target gene expression in cells, cell populations or tissues treated with the agent of interest compared to untreated cells, cell populations or tissues. Gene expression can be measured, for example, by the amount of mRNA transcripts of the target gene or the amount of protein expressed by the target gene. In certain embodiments, inhibition of target gene expression results in clinically relevant inhibition of target gene expression, such as being sufficiently inhibited to allow an effective therapeutic response.

As used herein, the term "pharmaceutically acceptable" indicates that a substance or composition is chemically and/or toxicologically compatible with the other ingredients that make up the formulation and/or the subject to be treated.

As used herein, unless otherwise indicated, the term "pharmaceutically acceptable salt" includes salts that retain the biological effectiveness of the free acids and bases of the specified compound and are not biologically or otherwise undesirable. Contemplated pharmaceutically acceptable salt forms include, but are not limited to, monosalts, disalts, trisalts, tetrasalts, and the like. Pharmaceutically acceptable salts are nontoxic in the amounts and concentrations in which they are administered. The preparation of such salts can facilitate pharmacological use by altering the physical properties of the compound without hindering it from exerting its physiological effects. Useful changes in physical properties include lowering the melting point to facilitate transmucosal administration and increasing solubility to facilitate administration of higher concentrations of the drug. Pharmaceutically acceptable salts may include acid addition salts, such as acid addition salts containing the following: sulfates, hydrochlorides, fumarates, maleates, phosphates, amidosulfonates, acetates, citrates, lactates, tartrates, malonic acids, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, cyclohexylaminosulfonates and quinates. Pharmaceutically acceptable salts may be obtained from acids such as sulfuric acid, hydrochloric acid, fumaric acid, maleic acid, phosphoric acid, amidosulfonates, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylaminosulfonates and quinic acid.

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

As used herein, the terms "treatment," "treat," or "treating" with respect to a disorder refer to managing, eliminating, reducing, or ameliorating the disorder and/or symptoms associated therewith. Although not exclusive, treatment of a disorder does not require complete elimination of the disorder or symptoms associated therewith. As used herein, the term "treatment" may include "preventive treatment," which is applied before any symptoms or manifestations of a disorder appear to reduce the likelihood of the occurrence or recurrence of the disorder, or to reduce the likelihood of recurrence of a previously controlled disorder, and which is applied to an individual who does not have the disorder but is at risk, or susceptible to recurrence of the disorder, or is at risk or susceptible to recurrence of the disorder. In the meaning of the present invention, "treatment" also includes the prevention of relapse or a preventive phase, as well as the treatment of acute or chronic signs, symptoms and/or functional impairments. Treatment can target symptoms, for example to suppress symptoms. It can act over a short period of time, over a medium period of time, or can be a long-term treatment, for example in the context of a maintenance therapy.

Nucleotides

As used herein, the term "nucleotide" means a base group linked to a sugar, wherein a phosphate group is covalently linked to the sugar moiety, and is intended to cover 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, a modified or unmodified nucleotide may be optionally further modified.

Natural nucleotides are composed of natural bases, natural sugar moieties, and phosphate moieties. As used herein, natural nucleotides refer to adenine ribonucleotides, adenine deoxyribonucleotides, guanine ribonucleotides, guanine deoxyribonucleotides, cytosine ribonucleotides, cytosine deoxyribonucleotides, uracil ribonucleotides, thymine ribonucleotides, or thymine deoxyribonucleotides. "Ribonucleotides" means nucleotides having a hydroxyl group at the 2' position of the sugar portion of the nucleotide. "Deoxyribonucleosides" means nucleotides having a hydrogen at the 2' position of the sugar portion of the nucleotide.

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

As used herein, unless otherwise specified, the nucleotides "G", "C", "A", "T" and "U" each refer to a natural or modified nucleotide containing guanine, cytosine, adenine, thymine and uracil as a base, respectively.

Nucleotides can be replaced by their analogs, including natural and non-natural analogs. Examples of guanosine analogs include 6-thioguanosine, 8-azaguanosine, 8-oxoguanosine, 2-aminopurine nucleoside, etc. Examples of adenosine analogs include cordycepin (3'-deoxyadenosine), N6-benzyladenosine, 2-chloroadenosine, etc. Examples of cytidine analogs include gemcitabine (2',2'-difluoro-2'-deoxycytidine), cytarabine (1-β-D-arabinofuranosylcytosine), decitabine (5-aza-2'-deoxycytidine), etc. Examples of uridine analogs include 5-fluorouridine, pseudouridine, 5-bromo-uridine, 4-thiouridine, 5-azido-uridine and the like.

As used herein, "ribonucleic acid" (RNA) is a carrier of genetic information found in cells and some viruses and viroids. RNA is composed of ribonucleotides linked to form strands by internucleotide bonds, including single-stranded RNA and double-stranded RNA. The natural internucleotide bonds are phosphodiester bonds.

As used herein, the term "modified nucleotide" refers to a nucleotide having at least a modified base, a modified sugar, or a modified phosphate group that can form a modified internucleotide bond. In some embodiments, a modified nucleotide can include one, two, three or more modifications. In some embodiments, a nucleotide can include one modification. In some embodiments, a nucleotide can include two modifications. In some embodiments, a nucleotide can include three modifications.

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).

Examples of modified sugars include, but are not limited to, 2'-modified sugars, 3'-modified sugars, and 5'-modified sugars, such as 2'-O-methyl (2'-OMe) modification, 2'-deoxy-2'-fluoro (2'-F) modification, 2'-O-methoxyethyl (2'-O-MOE) modification, 2'-deoxy (2'-d) modification, 5'-morpholine (5'-Mo) modification, unlocking nucleic acid (UNA) modification, glycol nucleic acid (GNA) modification, locking nucleic acid (LNA) modification, tricyclic-DNA (tcDNA) modification, (S)-constrained ethyl bridged nucleic acid ((S)-cEt-BNA) modification, 5'-(E)-vinylphosphonate modification, and 5'-(E)-vinylphosphonate modification. (VP) modification, 2'-O-C16 modification, 2'-C16 modification, conjugated to an inverted abasic nucleotide at the 5' or 3' end (invAB), replaced with an inverted abasic nucleotide (invAb), replaced with 2,4-difluoromethylphenyl ribonucleotide (rF), replaced with (S)-glycerol nucleic acid, replaced with inosine (I), conjugated to M03 at the 5' or 3' end, conjugated to M06 at the 5' or 3' end and conjugated to a ligand, such as a GalNAc ligand, a lipophilic ligand, or other ligands targeting a receptor that can promote endocytosis of the siRNA conjugate, such as a TfR targeting ligand, an LDL-R targeting ligand, or an integrin targeting ligand.

Examples of modified internucleotide bonds include, but are not limited to, methylphosphonate (MP), methoxypropylmethylphosphonate (MOP), phosphorothioate (PS), phosphorodithioate (PS2), phosphoroselenoate, phosphorodiselenoate, aniline phosphorothioate, aniline phosphonate, 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-, , , or etc., wherein R is independently selected from H, optionally substituted alkyl, optionally substituted cycloalkyl, and optionally substituted aryl. See, e.g., LaPlanche et al., Nucleic Acids Res. 14:9081 (1986); Stec et al., J. Am. Chem. Soc. 106:6077 (1984); Stein et al., Nucleic Acids Res. 16:3209 (1988); Zon et al., Anti-Cancer Drug Design 6:539 (1991); Zon et al., Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, ed., Oxford University Press, Oxford England (1991)); Stec et al., U.S. Pat. No. 5,151,510; Uhlmann and Peyman, Chemical Reviews. Reviews, 90:543 (1990); Handbook of RNA Biochemistry: Second, Completely Revised and Enlarged Edition (Roland K. Hartmann, Albrecht Bindereif, Astrid Schön, Eric Westhof, eds.; Wiley‐VCH Verlag GmbH & Co. KGaA (2014)); Nucleic Acids in Medicinal Chemistry and Chemical Biology: Drug Development and Clinical Applications (Lihe Zhang, Xinjiang Tang, Zhen Xi, Jyoti Chattopadhyaya, eds.; John Wiley & Sons, Inc. (2023)). In some embodiments, the modified internucleotide bond does not contain phosphorus but contains a peptide bond, such as in peptide nucleic acids (PNA), or a linker group comprising carbamate, amide, and linear and cyclic hydrocarbon groups. In some embodiments, the modified internucleotide bond is a phosphorothioate bond.

As used herein, the terms "small interfering RNA", "siRNA" and "iRNA reagent" are used interchangeably for reagents that can mediate silencing of a target RNA, such as an mRNA, such as a transcript of a gene encoding a protein. For convenience, such mRNA is also referred to herein as an mRNA to be silenced. Such a gene is also referred to as a target gene. Typically, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNAs other than mRNAs, such as tRNAs and viral RNAs, can also be targeted.

Typically, the siRNA comprises a double-stranded region of less than 60, 50, 40 or 30 complementary base pairs; preferably, the 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.

In some embodiments of the present application, the length of the sense strand and/or antisense strand of the siRNA is independently 15 to 35 nucleotides, thereby forming a complementary double-stranded region of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 base pairs in length. In some embodiments of the present application, the length of the sense strand of the siRNA is 15 to 35 nucleotides, and the length of the antisense strand of the siRNA is 25 to 35 nucleotides, thereby forming a complementary double-stranded region of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 base pairs in length. In some embodiments of the present application, the sense strand of the siRNA is 17 to 23 nucleotides in length, and the antisense strand of the siRNA is 25 to 30 nucleotides in length. In some embodiments of the present application, the sense strand of the siRNA is 21 to 23 nucleotides in length, and the antisense strand of the siRNA is 26 to 30 nucleotides in length. In some embodiments of the present application, the sense strand and antisense strand of the siRNA are completely complementary to each other and have a length of 15 to 30 base pairs. In some embodiments of the present application, the sense strand and antisense strand of the siRNA are completely complementary to each other and have a length of 17, 18, 19, 20, 21, 22 or 23 base pairs.

In some embodiments, the siRNA (e.g., antisense strand) is sufficiently complementary to the target RNA such that the siRNA silences the target RNA, e.g., inhibits the production of a protein encoded by the target RNA.

The term "sufficiently complementary" is used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between the siRNA (particularly its antisense strand) and the target RNA molecule. Such a degree of complementarity is sufficient to avoid non-specific binding of the siRNA to non-target sequences under conditions where specific binding is desired (i.e., under physiological conditions in the case of an assay or therapeutic treatment, or in the case of an assay performed in vitro). Non-target sequences typically differ by at least 4 nucleotides or more. In some embodiments, non-target sequences differ by at least 8 nucleotides or more. In addition, such a degree of complementarity is sufficient to allow the siRNA to silence the target RNA, e.g., to reduce the production of a protein encoded by the target mRNA.

The term "complementary" with respect to two nucleotide sequences means that the nucleotide sequences are paired in an antiparallel arrangement, thereby allowing specific hybridization of two single-stranded nucleotide sequences. The degree to which one oligonucleotide complements another oligonucleotide (referred to as complementarity) is measured by the percentage of bases in each strand that can form hydrogen bonds with each other, as indicated by established base pairing rules. Oligonucleotide sequences do not need to be completely complementary to their corresponding nucleic acid sequences (i.e., perfectly complementary). For example, if a first nucleotide sequence exhibits a certain degree of sequence complementarity, such as at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, then it can be considered to be complementary to a second nucleotide sequence. In an illustrative embodiment, 18 of the 20 nucleobases of the first nucleotide sequence match the corresponding region of the second nucleotide sequence, thereby achieving 90% complementarity. Non-complementary nucleobases are also called mismatches and can be grouped or interspersed among complementary nucleobases and need not be adjacent to each other or to complementary nucleobases.

As used herein, "mismatch" includes but is not limited to: 1) Pairing between two opposing (independently natural or non-natural) nucleotides other than A-T, A-U or G-C; 2) No hydrogen bond is formed between two opposing (independently natural or non-natural) nucleotides; 3) Lack of a base between two opposing (independently natural or non-natural) nucleotides.

In some embodiments, the mismatch comprises a dangling base pair and a Hoogstein base pair.

The term "completely complementary" is intended to mean that the first nucleotide sequence and the second nucleotide sequence form a hybrid consisting only of Watson-Crick base pairs in the completely complementary region.

A "fully complementary" oligonucleotide may include an internal region (e.g., having at least 7, 8, 9, or 10 nucleotides) that is completely complementary to the target RNA.

As used herein, the term "oligonucleotide" refers to a nucleic acid molecule (RNA or DNA) that is, for example, less than 100, 200, 300, or 400 nucleotides in length.

In the present application, "monomer" refers to a class of compounds that can be assembled into RNA chains and can perform certain functions. In the present application, "monomer" includes but is not limited to natural nucleotides, non-natural nucleotides (such as modified nucleotides, nucleotide analogs, abasic deoxyribonucleotides, GNA, LNA, etc.), capping groups, M03 as disclosed herein, and M06 as disclosed herein.

In the present application, "linkage" refers to the connection between the residues (e.g., nucleotide residues) of two monomers (e.g., nucleotides) through a single bond or a linking group (e.g., through a phosphodiester bond, a phosphorothioate bond, or a phosphorodithioate bond). In some embodiments, the connection refers to the connection between the residues of two monomers (e.g., nucleotides) through an internucleotide bond (e.g., a phosphodiester bond, a phosphorothioate bond, or a phosphorodithioate bond).

As used herein, the term "internucleotide bond" refers to a bond (e.g., a bond or linking group) between two parts of an oligonucleotide disclosed herein (e.g., between two monomers), including a bond between a nucleotide, between a nucleotide and a ligand, between a nucleotide and a blocking group, and between a nucleotide and an abasic nucleotide of an oligonucleotide disclosed herein.

As used herein, the term "alternating motif" or "alternating pattern" refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of a strand. Alternating nucleotides may refer to one every other nucleotide or one every three nucleotides, or a similar pattern. For example, if A, B, and C each represent a type of modification to a nucleotide, then the alternating motif may be "ABABABABABAB...", "AABBAABBAABB...", "AABAABAABAAB...", "AAABAAABAAAB...", "AAABBBAAABBB...", or "ABCABCABCABC...", etc.

The types of modifications contained in the alternating motifs can be the same or different. For example, if A, B, C, D each represent a type of modification on a nucleotide, then the alternating pattern, i.e., the modification on each other nucleotide, can be the same, but each of the sense strands or antisense strands can be selected from several possible modifications within the alternating motif, such as "ABABA ...", "ACACAC ...", "BDBDBD ...", or "CDCDCD ...", etc.

As used herein, the term "conjugate" or "conjugating" as a verb refers to the residues of two molecules (e.g., two nucleotides), such as the residues of two nucleotides connected by a bond (e.g., a single bond) or a linking group (e.g., a phosphodiester, a phosphorothioate, or a phosphorodithioate). Correspondingly, the term "conjugate" as a noun refers to a compound or complex formed by covalent linkage between chemical moieties. For example, "double-stranded RNA conjugate" means a compound or complex formed by covalent linkage to double-stranded RNA through one or more chemical moieties (e.g., a conjugating group, a ligand group, or a delivery system). As used herein, for the sake of brevity, the double-stranded RNA conjugate disclosed herein may also be referred to as a "conjugate", which should be understood as the full term "double-stranded RNA conjugate" based on the context. In some embodiments, the delivery system, ligand group or conjugate group can be linked to any available position of any nucleotide of the oligonucleotide reagent, including a phosphate group, a sugar ring (including a covalent connection of the delivery system, ligand group or conjugate group to an atom at the 3' or 5'' position of the sugar ring of the nucleotide through a phosphodiester bond), a 2'-hydroxyl group, a 5'-hydroxyl group, and a base group. In some embodiments, the ligand group or delivery system can be linked to the 3' position of the nucleotide, and unless otherwise specified, the nucleotides are linked through a 3'-5' phosphodiester bond. In some embodiments, the delivery system, ligand group, or conjugation group may also be linked to the 2'-position of the nucleotide, and unless otherwise specified, the nucleotides are linked via a 2'-5' phosphodiester bond.

Ligand

The double-stranded oligonucleotide reagents disclosed herein may be optionally conjugated to one or more ligands. The ligands may be attached to the sense strand, the antisense strand, or both strands at the 3' end, the 5' end, or both ends. In some embodiments, the ligand may be conjugated to the sense strand, specifically to the 3' end of the sense strand. In some embodiments, the ligand is conjugated to the antisense strand, specifically to the 5' end of the antisense strand.

A variety of entities can be coupled to the oligonucleotides disclosed herein. Preferred moieties are ligands, which are coupled directly or indirectly through an intermediate tether, preferably covalently.

In preferred embodiments, the ligand alters the distribution, targeting or lifetime of the molecule into which it is incorporated. In preferred embodiments, the ligand provides enhanced affinity for a selected target (e.g., a molecule, cell or cell type), compartment, receptor (e.g., a cell or organ compartment, tissue, organ or body region), for example, compared to a species in which such ligand is not present. Ligands that provide enhanced affinity for a selected target are also referred to as targeting ligands.

Some ligands may have endosomolytic properties. Endosomolytic ligands promote lysis of endosomes and/or transport of the compositions disclosed herein or components thereof from endosomes to the cytoplasm of cells.

Ligands can improve transport, hybridization and specificity properties, and can also improve the nuclease resistance of the resulting natural or modified oligoribonucleotide or polymer molecule comprising any combination of monomers and/or natural or modified ribonucleotides described herein.

Ligands can generally include therapeutic modulators, e.g., for enhancing uptake; diagnostic compounds or reporter groups, e.g., for monitoring distribution; crosslinkers; and moieties that confer nuclease resistance. Common examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptidomimetics. Ligands can also include targeting groups, e.g., cell or tissue targeting agents, e.g., lectins, glycoproteins, lipids, or proteins, e.g., antibodies that bind to specific cell types, e.g., kidney cells.

Other examples of ligands include dyes, intercalators (e.g., acridine), crosslinkers (e.g., pyrocatechol, mitomycin C), porphyrins (e.g., TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases or chelators (e.g., EDTA), lipophilic molecules (e.g., cholesterol, bile acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone), ketone, 1,3-bis-O-(hexadecyl)glycerol, geranyloxyhexyl, hexadecylglycerol, borneol, menthol, 1,3-propylene glycol, heptadecyl, palmitic acid, myristic acid, O3-(oleyl)cholestyric acid, O3-(oleyl)cholestyric acid, dimethoxytrityl or phenoxazine), and peptide conjugates (e.g., tripeptide, Tat peptide), alkylating agents, phosphates, amines, hydroxyls, PEGs (e.g., PEG-40K), MPEG, [MPEG] _2. Polyamines, alkyls, substituted alkyls, 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 clusters, acridine-imidazole conjugates, tetrazamacrocyclic compounds with Eu ³⁺ complexes), dinitrophenyl, HRP, and AP.

A ligand can be a protein (e.g., a glycoprotein) or a peptide (e.g., a molecule with a specific affinity for a co-ligand) or an antibody (e.g., an antibody that binds to a specific cell type (e.g., a cancer cell, an endothelial cell, or a bone cell). A ligand can be a substance, such as a drug, that can increase the uptake of an iRNA agent into a cell, for example, by disrupting the cell's cytoskeleton, for example, by disrupting the cell's microtubules, microfilaments, and/or interferons. For example, a ligand can increase the uptake of an oligonucleotide into a cell by activating an inflammatory response. Exemplary ligands that would have such an effect include tumor necrosis factor alpha (TNFα), interleukin-1 beta, or interferon gamma. In one aspect, the ligand is a lipid or lipid-based molecule. Lipid-based ligands can be used to regulate (e.g., control) the binding of the complex to the target tissue. In another aspect, the ligand is a moiety taken up by the target cell (e.g., a proliferative cell), such as a vitamin. In another aspect, the ligand is a cell permeabilizer, preferably a helical cell permeabilizer. The ligand can be a peptide or a peptide mimetic. In one embodiment, the targeting peptide can be an amphipathic α-helical peptide.

The targeting ligand can be any ligand capable of targeting a specific receptor. Examples are: folic acid, GalNAc, galactose, mannose, mannose-6P, sugar clusters (e.g., GalNAc clusters, mannose clusters, galactose clusters, etc.), or aptamers. A cluster is a combination of two or more sugar units. Targeting ligands also include integrin receptor ligands, tactin receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDL ligands. The ligand can also be based on nucleic acids, such as aptamers. The aptamer can be unmodified or have any combination of modifications disclosed herein.

Other ligand conjugates suitable for use in the present invention are described in U.S. Patent Application Nos. 10/916,185; 10/946,873; 10/833,934; 11/115,989 and 11/944,227, which are incorporated by reference in their entirety for all purposes.

When two or more ligands are present, the ligands may all have the same properties, all have different properties, or some ligands may have the same properties while others may have different properties. For example, a ligand may have a targeting property, have endosomolytic activity, or have a PK modulating property. In some embodiments, all ligands have different properties.

The ligand can be coupled to the oligonucleotide at various positions, such as the 3' end, the 5' end and/or an internal position. In preferred embodiments, the ligand is linked to the oligonucleotide via an intermediate tether. The ligand or tethered ligand can be present on the monomer when the monomer is incorporated into the growing strand. In some embodiments, the ligand can be incorporated by coupling to the "prodriver" monomer after it has been incorporated into the growing strand. For example, a monomer having, for example, an amine-terminated tether (i.e., without an associated ligand), such as TAP-(CH ₂ ) _n NH ₂ , can be incorporated into the growing oligonucleotide strand. In a subsequent operation, i.e., after incorporation of the prodriver monomer into the strand, a ligand having an electrophilic group (e.g., a pentafluorophenyl ester or aldehyde group) can then be attached to the prodriver monomer by coupling the electrophilic group of the ligand to the terminal nucleophilic group of the tether of the prodriver monomer.

In another example, a monomer having a chemical group suitable for participating in a click chemistry reaction, such as an azide or alkyne-terminated tether/linker, can be incorporated. In a subsequent operation, i.e., after the prodriver monomer is incorporated into the strand, a ligand having a complementary chemical group (e.g., an alkyne or an azide) can be attached to the prodriver monomer by coupling the alkyne and the azide together.

For double-stranded oligonucleotides, the ligand can be attached to one or both strands. In some embodiments, the double-stranded iRNA reagent contains a ligand attached to the sense strand. In other embodiments, the double-stranded iRNA reagent contains a ligand attached to the antisense strand.

In some embodiments, the ligand can be conjugated to the base, sugar or internucleotide bond of the nucleic acid molecule. The conjugation to the purine base or its derivatives can occur at any position including the atoms inside and outside the ring. In some embodiments, the 2, 6, 7 or 8 position of the purine base is connected to the conjugation part. The conjugation to the pyrimidine base or its derivatives can also occur at any position. In some embodiments, the 2, 5 and 6 positions of the pyrimidine base can be replaced by the conjugation part. The conjugation to the sugar of the nucleoside can occur at any carbon atom. Exemplary carbon atoms of the sugar part that can be connected to the conjugation part include 2', 3' and 5' carbon atoms. The 1' position can also be connected to the conjugation part, for example in a base-free residue. The internucleotide bond can also carry the conjugation part. For phosphorus-containing bonds (e.g., phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, etc.), the conjugate can be directly attached to the phosphorus atom or to an O, N, or S atom bound to the phosphorus atom. For amine- or amide-containing internucleotide bonds (e.g., PNA), the conjugate can be attached to the nitrogen atom of the amine or amide or to an adjacent carbon atom.

Any suitable ligand in the field of RNA interference can be used, but the ligand is typically a carbohydrate, such as a monosaccharide (e.g., GalNAc), a disaccharide, a trisaccharide, a tetrasaccharide, or a polysaccharide.

Linkers that bind the ligand to the nucleic acid include those discussed above. For example, the ligand can be one or more GalNAc (N-acetylglucosamine) derivatives attached via a bivalent or trivalent branched linker.

In some embodiments, the ligand is ligated 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 ligand is a GalNAc ligand, a lipophilic ligand, or other ligand that targets a receptor capable of promoting endocytosis of siRNA conjugates, such as a TfR targeting ligand, an LDL-R targeting ligand, or an integrin targeting ligand.

Capping Group

The double-stranded oligonucleotide reagents disclosed herein may be optionally conjugated to one or more capping groups. The capping group may be attached to the sense strand, the antisense strand, or both strands at the 3' end, the 5' end, or both ends. In some embodiments, the capping group is conjugated to the antisense strand, specifically to the 5' end of the antisense strand.

In some embodiments, the capping group is linked 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 capping group is linked to the double-stranded oligonucleotide reagent via a phosphorothioate. In some embodiments, the capping group is linked to the double-stranded oligonucleotide reagent via a phosphodiester.

As used herein, a "capping group" refers to a group that can be conjugated to an oligonucleotide disclosed herein, for example, at the 5' end of the antisense strand, which can reduce or inhibit exonuclease degradation metabolism. In some embodiments, the capping group can reduce or inhibit the RNA interference effect of the oligonucleotide. In some embodiments, the capping group is cleaved from the oligonucleotide before providing an RNA interference effect.

Examples of the capping group include, but are not limited to, abatic residues, reverse abatic residues, M03, and M06.

I. Double stranded oligonucleotide reagent

The present disclosure provides a double-stranded oligonucleotide reagent comprising a sense strand and an antisense strand. The sense strand and the antisense strand form a double-stranded portion and a 5' extension in the antisense strand. In certain embodiments, the length of the 5' extension is at least three nucleotides.

The inventors were surprised to find that the 5' extension in the antisense strand can be designed so that it can be cleaved at a specific cleavage site. Specifically, the double-stranded oligonucleotide reagents provided herein are particularly advantageous because the 5' extension can be cleaved from the 3'-most nucleotide in the 5' extension. In other words, when the double-stranded oligonucleotide reagent reaches the target tissue, it can be converted into a cleavage product. Such cleaved double-stranded oligonucleotide reagents can inhibit the expression of the target gene by RNA interference.

As used herein, the phrase "RNA interference" refers to the ability of double-stranded short interfering RNA (siRNA) to silence target RNA in a sequence-specific manner. The first step in RNA interference (RNAi) involves activation of the RNA-induced silencing complex (RISC), which necessitates degradation of the sense strand of the double-stranded RNA (dsRNA) duplex. The sense strand serves as the initial substrate for RISC and is cleaved by Argonaute 2 (Ago2) in the middle of the duplex region. Once the cleaved 5' and 3' fragments of the sense strand are removed by Ago2, RISC is activated by the antisense strand (Rand et al. (2005) Cell 123, 621).

In some embodiments, the double-stranded oligonucleotide reagents provided herein have enhanced activity in inhibiting target gene expression compared to a reference siRNA having an otherwise equivalent antisense sequence except for the 5' extension or cleavage region provided herein.

In some embodiments, the double-stranded oligonucleotide reagents provided herein comprise ribonucleic acid (RNA), consist essentially of ribonucleic acid, or consist of ribonucleic acid. In some embodiments, the sense strand of the double-stranded oligonucleotide reagents provided herein comprises RNA, consists essentially of RNA, or consists of RNA. In some embodiments, the antisense strand of the double-stranded oligonucleotide reagents provided herein comprises RNA, consists essentially of RNA, or consists of RNA.

In some embodiments, the double-stranded oligonucleotide reagent provided herein can be optionally converted into siRNA after being delivered to target tissue or target cell. In some embodiments, the double-stranded oligonucleotide reagent provided herein can be converted into siRNA by enzyme or optionally by RNase III (RNAase III) or optionally by dicer enzyme. In some embodiments, the double-stranded oligonucleotide reagent of cracking provided herein is siRNA.

a. Double stranded part and 5 ' extension

In certain embodiments, the double-stranded portion is formed by base pairing between a first segment in the antisense strand and a second segment in the sense strand, wherein the first segment and the second segment have equal lengths.

In some embodiments, the base pairing between the first fragment and the second fragment can include base pairing in a Watson-Crick manner and/or in any other manner that allows the formation of a stable double strand. In Watson-Crick base pairing, adenine (A) pairs with thymine (T) in DNA and uracil (U) in RNA; guanine (G) pairs with cytosine (C). Base pairs can also be formed by non-Watson-Crick base pairs, including but not limited to G-U wiggling base pairs and Hoogstein base pairs. In some embodiments, a nucleotide containing hypoxanthine as its base can be base-paired with a nucleotide containing adenine, cytosine or uracil. In some embodiments, nucleotides containing uracil, guanine, or adenine can be replaced with nucleotides containing, for example, inosine (as used herein, "I" can represent hypoxanthine base, inosine, or an inosine-containing nucleotide, depending on the context). Such substitutions are referred to as "I-modifications." In some embodiments, adenine and cytosine anywhere in an oligonucleotide can be replaced with guanine and uracil, respectively, to form G-U diagonal base pairs with the target mRNA.

In some embodiments, the base pairs formed in the double-stranded portion may comprise or consist essentially of Watson-Crick base pairs and/or non-Watson-Crick base pairs (e.g., G-U dangling base pairs and Hoogstein base pairs) or a combination thereof.

In some embodiments, the first segment in the sense strand and the second segment in the antisense strand are of equal length and complement each other to form a double-stranded portion. In some embodiments, the first segment and the second segment are at least 80%, 85%, 90%, 95% or 100% complementary.

In certain embodiments, the length of the double-stranded portion is 15 to 27 nucleotide pairs. In certain embodiments, 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, 19 to 21 nucleotide pairs. In certain embodiments, the double-stranded portion is 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 in length.

In certain embodiments, the length of the sense strand of the double-stranded oligonucleotide reagent is 15 to 35 nucleotides (nt), 16 to 35, 16 to 30, 16 to 27, 16 to 26, 16 to 25, 16 to 24, 16 to 23, 16 to 22, 16 to 21, 17 to 35, 17 to 30, 17 to 25, 17 to 23, 17 to 22, 17 to 21, 18 to 35, 18 to 30, 18 to 25, 18 to 23, 18 to 22, 21, 20, 19, 18, 17, 16, or 15 nucleotides (nt).

In certain embodiments, the sense strand of the double-stranded oligonucleotide reagent is 15 to 23, 15 to 22, or 15 to 21 nucleotides (nt) in length.

In certain embodiments, the 5' extension is at least 3, 4, 5, 6, 7, or more nucleotides in length.

In some embodiments, the double-stranded oligonucleotide reagent further comprises a 3' extension in the antisense strand. In some embodiments, the 3' extension is at least 1, 2 or more nucleotides in length. In some embodiments, the 3' extension can be an overhang of at least 1, 2, 3, 4 or 5 nucleotides in length.

The length of the extension (e.g., 3' extension) can be 1 to 6 nucleotides, such as 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., 5' extension or 3' extension) can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered.

In one embodiment, the nucleotides in the extension region can each independently be a modified or unmodified nucleotide, including but not limited to a 2'-sugar modification, such as 2'-F or 2'-OMe.

The 5' extension or 3' extension at the sense strand, the antisense strand, or both strands may be phosphorylated. In some embodiments, the extension region contains two nucleotides with a phosphorothioate bond between the two nucleotides, wherein the two nucleotides may be the same or different. In one embodiment, such a 3' extension is present in the antisense strand.

In certain embodiments, the length of the antisense strand of a double-stranded oligonucleotide reagent is at least 25 to 35, 25 to 34, 25 to 33, 25 to 32, 25 to 31, 25 to 30, 25 to 29, 25 to 28, 25 to 27, 25 to 26, 26 to 35, 26 to 34, 26 to 33, 26 to 32, 26 to 31, 26 to 30, 26 to 29, 26 to 28, 26 to 27 nt. In certain embodiments, the length of the antisense strand of a double-stranded oligonucleotide reagent is 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt.

In certain embodiments, the first segment comprises a targeting region that is substantially complementary to a portion of a target RNA, optionally an mRNA, encoding a target gene.

In one embodiment, the targeting region disclosed herein is at least 90%, at least 95% or 100% complementary to a portion of the target RNA. In some embodiments, the targeting region binds to the target RNA and forms a double helix consisting only of Watson-Crick base pairs in the complementary region. In one embodiment, the targeting region can include an internal region (e.g., having 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 can be a seed region located in the antisense strand from position 2 to position 8 starting from the 5' end of the targeting region. In another embodiment, the targeted region is 100% complementary to the target RNA, e.g., the target RNA and dsRNA duplex reagent anneal, e.g., to form a hybrid consisting of only Watson-Crick base pairs in the region of complete complementarity. In addition, in some embodiments, the dsRNA reagents of the present invention specifically distinguish single nucleotide differences. In this case, if complete complementarity is found in the region of single nucleotide differences (e.g., within 7 nucleotides), then the dsRNA reagent only mediates RNAi.

b. Cracking Zone

In certain embodiments, the antisense strand of a double-stranded oligonucleotide reagent comprises a cleavage region that allows for specific cleavage from the 3'-most nucleotide in the 5' extension.

In some embodiments, the double-stranded oligonucleotide reagents disclosed herein (e.g., 5' extensions) undergo enzyme (e.g., endonuclease) mediated cleavage prior to mediating RNAi. In some embodiments, the cleavage is mediated by ribonucleases (RNases). In some embodiments, the cleavage is mediated by endonucleases. In some embodiments, the cleavage is mediated by RNase III. RNase III represents a class of endoribonucleases that produce small RNAs for RNA silencing pathways. In some embodiments, RNase III is dicer. In some embodiments, the cleavage is specific cleavage.

As used herein, the term "specific" or "specifically" with respect to the cleavage of an oligonucleotide reagent (e.g., an siRNA reagent) refers to controlled or selective cleavage at a specific position (i.e., between two desired nucleotides of an oligonucleotide strand undergoing specific cleavage). The product of specific cleavage can include a variety of cleavage products, wherein the desired cleavage product (i.e., the product obtained by cleavage at a specific or desired position) is the most abundant cleavage product. In some embodiments, the desired cleavage product accounts for at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% (by mole or weight) of all cleavage products. In some embodiments, the amount of a desired cleavage product (by mole or weight) is at least 2, 3, 4, 5, 6, 7, 8, 9, 10 times greater than any other cleavage product. While not wishing to be bound by theory, specific cleavage is based on the recognition of a certain sequence, feature, motif, or combination thereof, of the oligonucleotide reagent undergoing cleavage by the enzyme mediating cleavage.

In certain embodiments, the cleavage region comprises a nucleotide sequence shown in Formula A: (3'-5') X2-Y-Z, which is capable of cleaving between X2 and Y, wherein X2 is the 5'-most nucleotide of the first fragment and Y-Z are the two 3'-most nucleotides of the 5' extension.

In certain embodiments, the cleavage region further comprises a fourth nucleotide (N1), which is the third nucleotide from the 3' end of the 5' extension, and the cleavage region comprises a nucleotide sequence shown in Formula B: (3'-5') X2-Y-Z-N1, which is capable of cleaving between X2 and Y.

In certain embodiments, the cleavage region further comprises a third fragment (N), wherein the third fragment comprises at least one nucleotide, wherein N1 is the 3'-most nucleotide of the third fragment, and the cleavage region comprises a nucleotide sequence shown in formula B': (3'-5') X2-Y-Z-N, which is capable of cleaving between X2 and Y, and the length of N is 1 to 10 nucleotides, preferably the length of N is 1 to 5 nucleotides, and further preferably the length of N is 1 nucleotide.

Without wishing to be bound by any theory, it is believed that enzymatic cleavage can be determined using a target tissue (e.g., liver, eye, lung, kidney, brain, spinal cord, muscle, fat, etc.) homogenization treatment reaction assay (see, e.g., the method described in Example 4.6) or using a dicer endonuclease treatment assay.

Without wishing to be bound by any theory, it is believed that certain nucleotides at certain positions at the cleavage zone provide superior effects.

In certain embodiments, Z is selected from G or A or a natural or non-natural analog thereof. In certain embodiments, Z is selected from G or a natural or non-natural analog thereof.

In certain embodiments, X2 is selected from A or U, or a natural or non-natural analogue thereof.

Without wishing to be bound by any theory, the double-stranded oligonucleotide reagent disclosed herein can be specifically cleaved between X2 and Y to remove the 5' extension, thereby generating a cleaved double-stranded oligonucleotide reagent. The cleaved double-stranded oligonucleotide is believed to be able to mediate RNAi.

The cleaved double-stranded oligonucleotide reagent has a blunt end at the 5' end of the antisense strand, wherein the first base pair at the 5' end of the antisense strand is composed of X2 and its complementary nucleotide in the sense strand. In some embodiments, such first base pair at the 5' end of the antisense strand is an AU base pair.

In certain embodiments, Formula A has a nucleotide sequence from 3' to 5' selected from the group consisting of UUG, UAG, AUG, AAG, UUA, UAA, AUA, AAA, UCG, UGG, ACG, AGG, UCA, UGA, ACA and AGA, or a natural or non-natural analogue thereof.

In certain embodiments, the nucleotide Y is selected from A or U, or a natural or non-natural analogue thereof.

In certain embodiments, Formula A has a nucleotide sequence from 3' to 5' selected from the group consisting of: UUG, UAG, AUG, AAG, UUA, UAA, AUA and AAA.

In certain embodiments, at least one nucleotide in the cleavage zone is a modified nucleotide, and preferably all nucleotides in the cleavage zone are modified nucleotides.

In certain embodiments, the modified nucleotide has a modified base, a modified sugar, or a modified internucleotide bond. In certain embodiments, the modified nucleotide has a modified base, and optionally the modified base comprises 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).

In certain embodiments, the modified nucleotide has a modified sugar, and optionally the modified sugar includes a 2'-sugar modification, a 3'-sugar modification, and a 5'-sugar modification, such as a 2'-O-methyl (2'-OMe) modification, a 2'-deoxy-2'-fluoro (2'-F) modification, a 2'-O-methoxyethyl (2'-O-MOE) modification, a 2'-deoxy (2'-d) modification, a 5'-morpholine (5'-Mo) modification, an unblocked nucleic acid (UNA) modification, a glycol nucleic acid (GNA) modification, a locked nucleic acid (LNA) modification, a tricyclic-DNA (tcDNA) modification, a (S)-constrained ethyl bridged nucleic acid ((S)-cEt-BNA) modification, 5'-(E)-vinylphosphonate (VP) modification, 2'-O-C16 modification, conjugated to an inverted abasic nucleotide at the 5' or 3' end (invAB), replaced with an inverted abasic nucleotide (invAb), replaced with 2,4-difluoromethylphenyl ribonucleotide (rF), replaced with (S)-glycerol nucleic acid, replaced with inosine (I), conjugated to M03 at the 5' or 3' end, conjugated to M06 at the 5' or 3' end and conjugated to a ligand, such as a GalNAc ligand, a lipophilic ligand, or other ligands targeting a receptor that can promote endocytosis of the siRNA conjugate, such as a TfR targeting ligand, an LDL-R targeting ligand, or an integrin targeting ligand.

In certain embodiments, the modified nucleotide has a modified internucleotide bond, and optionally the modified internucleotide bond comprises methylphosphonate (MP), methoxypropylmethylphosphonate (MOP), phosphorothioate (PS), phosphorodithioate (PS2), phosphoroselenate, phosphorodiselenoate, aniline phosphorothioate, aniline phosphate, phosphoamidate, and PNA.

In certain embodiments, at least one nucleotide in the cleavage zone is 2'-OMe modified or 2'-F modified. In certain embodiments, all nucleotides in the cleavage zone are modified nucleotides selected from 2'-OMe modified and 2'-F modified. In certain embodiments, at least one nucleotide in the cleavage zone is 2'-F modified. In certain embodiments, no more than two nucleotides in the cleavage zone are 2'-F modified.

In certain embodiments, Z in Formula A or B is modified with 2'-F, and optionally X2 in Formula A or B is modified with 2'-F. In certain embodiments, N1 in Formula A or B is modified with 2'-F. In certain embodiments, both X2 and Y in Formula A or B are modified with 2'-OMe, and both Z and N1 in Formula A or B are modified with 2'-F. In certain embodiments, all X2, Y, and Z in Formula A or B are modified with 2'-OMe, and N1 in Formula A or B is modified with 2'-F.

Without wishing to be bound by any theory, it is believed that certain internucleotide bonds at certain positions at the cleavage zone provide superior effects.

In certain embodiments, at least one of the internucleotide bonds between nucleotides in the cleavage zone is not a phosphorothioate bond. In certain embodiments, the internucleotide bond between X2 and Y is not a phosphorothioate bond. In certain embodiments, the internucleotide bond between Y and Z is not a phosphorothioate bond. In certain embodiments, none of the internucleotide bonds between nucleotides in the cleavage zone are phosphorothioate bonds. In certain embodiments, at least one of the internucleotide bonds between nucleotides in the cleavage zone is a phosphodiester bond. In certain embodiments, the internucleotide bond between X2 and Y is a phosphodiester bond. In certain embodiments, the internucleotide bond between Y and Z is a phosphodiester bond. In certain embodiments, each of the internucleotide bonds between nucleotides in the cleavage zone is a phosphodiester bond.

c. Modification of double stranded oligonucleotide reagents

In certain embodiments, the double-stranded oligonucleotide reagent further comprises at least one phosphorothioate or methylphosphonate internucleotide bond.

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

In certain embodiments, the double-stranded oligonucleotide reagent provided herein comprises 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 its 5' end) of the second fragment. In certain embodiments, the double-stranded oligonucleotide reagent provided herein comprises 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 its 5' end) of the second fragment.

In certain embodiments, every nucleotide of the first segment of the antisense strand is modified. In certain embodiments, every nucleotide of the antisense strand is modified. In certain embodiments, every nucleotide of the second segment of the sense strand is modified. In certain embodiments, every nucleotide of the sense strand is modified.

In certain embodiments, every nucleotide of the antisense strand is 2'-F modified or 2'-OMe modified; and/or every nucleotide of the sense strand is 2'-F modified or 2'-OMe modified.

In certain embodiments, the sense strand further comprises a) a motif of three consecutive 2'-F modified nucleotides, optionally the motif occurs at positions 9, 10 and 11, counting from the 5'-terminal nucleotide of the sense strand; and/or b) a 2'-F modified nucleotide at position 7 and/or 18, counting from the 5'-terminal nucleotide of the sense strand. In certain embodiments, the sense strand further comprises an alternating motif at positions 9 to 13, counting from the 5' end, optionally the 2'-F modification and the 2'-OMe modification occur at alternating nucleotides in the motif.

In certain embodiments, the first segment of the antisense strand and/or the second segment of the sense strand further comprises an alternating motif, wherein modifications occur at alternating nucleotides in the motif, optionally 2'-F modifications occur at alternating nucleotides in the motif and/or 2'-OMe modifications occur at alternating nucleotides in the motif.

In certain embodiments, the first segment of the antisense strand and/or the second segment of the sense strand comprises a modification selected from the group consisting of: a 2'-O-methyl (2'-OMe) modification, a 2'-deoxy-2'-fluoro (2'-F) modification, a 2'-O-methoxyethyl (2'-O-MOE) modification, a 2'-deoxy (2'-d) modification, a 5'-morpholino (5'-Mo) modification, an unblocked nucleic acid (UNA) modification, a glycol nucleic acid (GNA) modification, a locked nucleic acid (LNA) modification, modification, tricyclic-DNA (tcDNA) modification, (S)-constrained ethyl bridged nucleic acid ((S)-cEt-BNA) modification, 5'-(E)-vinylphosphonate (VP) modification, 2'-O-C16 modification, ligation to an inverted abasic nucleotide at the 5' or 3' end (invAB), replacement with an inverted abasic nucleotide (invAb), replacement with 2,4-difluorotolyl ribonucleotide (rF), replacement with (S)-glycerol nucleic acid, and replacement with inosine (I).

d. Capping Group

The double-stranded oligonucleotide reagent disclosed herein can be optionally linked to one or more capping groups. The capping group can be attached to the sense strand, the antisense strand, or both strands at the 3' end, the 5' end, or both ends. In some embodiments, the capping group is linked to the antisense strand, specifically to the 5' end of the antisense strand.

In some embodiments, the capping group is linked 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 capping group is linked to the double-stranded oligonucleotide reagent via a phosphorothioate. In some embodiments, the capping group is linked to the double-stranded oligonucleotide reagent via a phosphodiester bond.

For example, a nucleotide attached to a reverse abatic deoxyribonucleotide (e.g., attached at the 5' end or the 3' end) has the structure: or .

For example, a nucleotide attached to M03 (e.g., attached at the 5' end or the 3' end) has the structure: or In some embodiments, the conjugation to M03 is by nucleotides and reaction.

For example, a nucleotide attached to M06 (e.g., attached at the 5' end or the 3' end) has the structure: or In some embodiments, the bond to M06 is through a nucleotide reaction.

Also provided herein is a method for reducing or inhibiting exonuclease degradation metabolism of an oligonucleotide (e.g., a double-stranded oligonucleotide, such as siRNA) by conjugating or linking a capping group to an oligonucleotide. In some embodiments, the capping group is an abatic residue, a reverse abatic residue, M03 or M06. In some embodiments, the capping group is M03 or M06. In some embodiments, the capping group (e.g., M03 or M06) is linked to the antisense strand of the oligonucleotide, specifically to the 5' end or 3' end of the antisense strand. In some embodiments, the capping group (e.g., M03 or M06) is linked to the 5' end of the antisense strand. In some embodiments, the capping group can reduce or inhibit the RNA interference effect of the oligonucleotide.

Also provided herein is the use of a capping group in reducing or inhibiting exonuclease degradation metabolism of an oligonucleotide (e.g., a double-stranded oligonucleotide, such as siRNA), wherein the capping group is conjugated or linked to the 5' end or 3' end of the oligonucleotide, specifically to the antisense strand. In some embodiments, the capping group is an abatic residue, a reverse abatic residue, M03 or M06. In some embodiments, the capping group is M03 or M06. In some embodiments, the capping group (e.g., M03 or M06) is linked to the 5' end of the antisense strand. In some embodiments, the capping group can reduce or inhibit the RNA interference effect of the oligonucleotide.

e. Specific Examples of Double-Stranded Oligonucleotide Reagents

In certain embodiments, the double-stranded oligonucleotide reagent provided herein comprises a structure represented by formula (C): Right shares (5'→3'): Second segment Antisense strand (3'→5'): First Clip 5' extension Formula (C), wherein the first segment in the antisense strand forms a double-stranded portion with the second segment in the sense strand by base pairing, wherein the first segment and the second segment have equal lengths; wherein the length of the 5' extension comprises at least three nucleotides; wherein the antisense strand comprises a cleavage region, the cleavage region comprises the 5'-most nucleotide (X2) of the first segment and the 3'-most two nucleotides (YZ) of the 5' extension, and the cleavage region comprises a nucleotide sequence shown in Formula A: (3'-5') X2-YZ, which is capable of cleavage between X2 and Y, and the formula A is as defined herein; wherein the length of the sense strand is 15 to 35, 15 to 23, 15 to 22 or 15 to 21 nucleotides; and wherein the length of the antisense strand is 25 to 35, 26 to 35, 26 to 30, 25 to 27 or 26 to 27 nucleotides.

In some embodiments, the sense strand of the double-stranded oligonucleotide reagent of formula (C) has a length of 17 to 23, 17 to 22, or 17 to 21 nucleotides; and the antisense strand of the double-stranded oligonucleotide reagent of formula (C) has a length of 20 to 26, 20 to 25, or 20 to 24 nucleotides.

In some embodiments, the sense strand of the double-stranded oligonucleotide reagent of formula (C) and the antisense strand of the double-stranded oligonucleotide reagent of formula (C) have the following lengths: 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.

In some embodiments, the double stranded oligonucleotide reagent disclosed herein can be optionally linked to one or more capping groups. Any suitable capping group provided in the present disclosure can be used. The capping group can be attached to the sense strand, the antisense strand, or both strands at the 3' end, the 5' end, or both ends. In some embodiments, the capping group is linked to the antisense strand, specifically to the 5' end of the antisense strand. In some embodiments, the capping group is linked to the 5' end of the 5' extension.

Also provided herein is a double-stranded oligonucleotide reagent comprising a structure represented by formula (D): Right shares (5'→3'): Second segment Antisense strand (3'→5'): 3' extension First Clip 5' extension Formula (D), wherein the first segment in the antisense strand forms a double-stranded portion with the second segment in the sense strand by base pairing, wherein the first segment and the second segment have equal lengths; the length of the 5' extension comprises at least three nucleotides; the antisense strand comprises a cleavage region, the cleavage region comprises the 5'-most nucleotide (X2) of the first segment and the 3'-most two nucleotides (YZ) of the 5' extension, and the cleavage region comprises a nucleotide sequence shown in Formula A: (3'-5') X2-YZ, which is capable of cleavage between X2 and Y, and upon cleavage, the 5' extension is removed from its 3'-most nucleotide (Y), and the Formula A is as defined herein, wherein the sense strand is 15 to 35, 15 to 23, 15 to 22, 15 to 21, 16 to 25, 17 to 23, 18 to 23, 19 to 23, 19 to 21, 20 to 23, 20 to 21, 21 to 23, e.g., 17, 18, 19, 20, 21, 22, or 23 nucleotides in length; and wherein the antisense strand is 25 to 35, 25 to 30, 26 to 35, 26 to 30, 26 to 27, e.g., 25, 26, 27, 28, 29, or 30 nucleotides in length.

In some embodiments, the sense strand of the double-stranded oligonucleotide reagent of formula (D) has a length of 17 to 23, 17 to 22, or 17 to 21 nucleotides; and the antisense strand of the double-stranded oligonucleotide reagent of formula (D) has a length of 22 to 28, 22 to 27, or 22 to 26 nucleotides.

In some embodiments, the sense strand of the double-stranded oligonucleotide reagent of formula (D) and the antisense strand of the double-stranded oligonucleotide reagent of formula (D) have the following lengths: 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.

In some embodiments, the double stranded oligonucleotide reagent disclosed herein can be optionally linked to one or more capping groups. Any suitable capping group provided in the present disclosure can be used. The capping group can be attached to the sense strand, the antisense strand, or both strands at the 3' end, the 5' end, or both ends. In some embodiments, the capping group is linked to the antisense strand, specifically to the 5' end of the antisense strand. In some embodiments, the capping group is linked to the 5' end of the 5' extension.

In some embodiments, the sense strand of the oligonucleotide reagent disclosed herein comprises at least 19, 20, 21, or 22 consecutive nucleotides of any one of SEQ ID NOs: 1-97. In some embodiments, the sense strand of the oligonucleotide reagent provided herein is any one of SEQ ID NOs: 1-97.

In some embodiments, the antisense strand of the oligonucleotide reagent disclosed herein comprises at least 19, 20, 21 or 22 consecutive nucleotides of any one of SEQ ID NOs: 98-194. In some embodiments, the antisense strand of the oligonucleotide reagent disclosed herein is any one of SEQ ID NOs: 98-194.

In some embodiments, the antisense strand of the oligonucleotide reagent disclosed herein comprises a pair of sense strands or antisense strands as shown in Table 1.

surface 1 : Exemplary oligonucleotides SEQ ID NO SS (5'-3') SEQ ID NO AS(5'-3') 1 ACGGGACAGUAUUCUCAGUGA 98 ACGAUCACUGAGAAUACUGUCCCGUUU 2 ACCAGUCGUCCCAGAAUAACU 99 CUGAAGUUAUUCUGGGACGACUGGUCU 3 UGAGCAAGAUUUGCAACUUGA 100 ACGAUCAAGUUGCAAAUCUUGCUCAUG 4 GGUUAACACCAUUUACUUCAA 101 CCGAUUGAAGUAAAUGGUUAACCAG 5 GGCUACGAAAAUCCAACCUAA 102 UCGAUUAGGUUGGAUUUUCGUAGCCGU 6 CACUUUAAUCCUCUAUCCAGA 103 AGGAUCUGGAUAGAGGAUUAAAGUGAG 7 CCUUUGGAAUAAAGCUGCCUU 104 CCGUAAGGCAGCUUUAUUCCAAAGGGC 8 GCAGGAAGCACUGAGAUUCGU 105 CCGAACGAAUCUCAGUGCUUCCUGCAC 9 CUAGACCUGUUUUGCUUUUGU 106 UGGAACAAAAGCAAAACAGGUCUAGAA 10 AUGCAGAUUAUGCGGAUCAAA 107 ACGAUUUGAUCCGCAUAAUCUGCAUGG 11 GCUCACAUAUUUGAUCAGUA 108 CCGAUACUGAUCAAAUAUGUUGAGCUU 12 GAGCCGUUCUCUACAAUUACU 109 GCGAAGUAAUUGUAGAGAACGGCUCGG 13 AAGCAAGAUAUUUUUAUAAUA 110 ACGUUAUUAUAAAAAUAUCUUGCUUUU 14 UGGGAUUUCAUGUAACCAAGA 111 CCGAUCUUGGUUACAUGAAAUCCCAUC 15 GUCAUCCACAAUGAGAGUACA 112 CUUUGUACGCUCAUUGUGGAUGACGA 16 GUCAUCCACAAUGAGAGUACA 113 CAUUGUACGCUCAUUGUGGAUGACGA 17 GUCAUCCACAAUGAGAGUACA 114 CCUUGUACGCUCAUUGUGGAUGACGA 18 GUCAUCCACAAUGAGAGUACA 115 CGUUGUACGCUCAUUGUGGAUGACGA 19 GUCAUCCACAAUGAGAGUACA 116 CUAUGUACGCUCAUUGUGGAUGACGA 20 GUCAUCCACAAUGAGAGUACA 117 CAAUGUACGCUCAUUGUGGAUGACGA twenty one GUCAUCCACAAUGAGAGUACA 118 CCAUGUACGCUCAUUGUGGAUGACGA twenty two GUCAUCCACAAUGAGAGUACA 119 CGAUGUACGCUCAUUGUGGAUGACGA twenty three GUCAUCCACAAUGAGAGUACU 120 CUUAGUACGCUCAUUGUGGAUGACGA twenty four GUCAUCCACAAUGAGAGUACU 121 CAUAGUACGCUCAUUGUGGAUGACGA 25 GUCAUCCACAAUGAGAGUACU 122 CCUAGUACGCUCAUUGUGGAUGACGA 26 GUCAUCCACAAUGAGAGUACU 123 CGUAGUACGCUCAUUGUGGAUGACGA 27 GUCAUCCACAAUGAGAGUACU 124 CUAAGUACGCUCAUUGUGGAUGACGA 28 GUCAUCCACAAUGAGAGUACU 125 CAAAGUACGCUCAUUGUGGAUGACGA 29 GUCAUCCACAAUGAGAGUACU 126 CCAAGUACGCUCAUUGUGGAUGACGA 30 GUCAUCCACAAUGAGAGUACU 127 CGAAGUACGCUCAUUGUGGAUGACGA 31 GCUCACAUAUUUGAUCAGUA 128 CUUUACUGAUCAAAUAUGUUGAGCAA 32 GCUCACAUAUUUGAUCAGUA 129 CAUUACUGAUCAAAUAUGUUGAGCAA 33 GCUCACAUAUUUGAUCAGUA 130 CCUUACUGAUCAAAUAUGUUGAGCAA 34 GCUCACAUAUUUGAUCAGUA 131 CGUUACUGAUCAAAUAUGUUGAGCAA 35 GCUCACAUAUUUGAUCAGUA 132 CUAUACUGAUCAAAUAUGUUGAGCAA 36 GCUCACAUAUUUGAUCAGUA 133 CAAUACUGAUCAAAUAUGUUGAGCAA 37 GCUCACAUAUUUGAUCAGUA 134 CCAUACUGAUCAAAUAUGUUGAGCAA 38 GCUCACAUAUUUGAUCAGUA 135 CGAUACUGAUCAAAUAUGUUGAGCAA 39 GCUCAACAUAUUUGAUCAGUU 136 CUUAACUGAUCAAAUAUGUUGAGCAA 40 GCUCAACAUAUUUGAUCAGUU 137 CAUAACUGAUCAAAUAUGUUGAGCAA 41 GCUCAACAUAUUUGAUCAGUU 138 CCUAACUGAUCAAAUAUGUUGAGCAA 42 GCUCAACAUAUUUGAUCAGUU 139 CGUAACUGAUCAAAUAUGUUGAGCAA 43 GCUCAACAUAUUUGAUCAGUU 140 CUAAACUGAUCAAAUAUGUUGAGCAA 44 GCUCAACAUAUUUGAUCAGUU 141 CAAAACUGAUCAAAUAUGUUGAGCAA 45 GCUCAACAUAUUUGAUCAGUU 142 CCAAACUGAUCAAAUAUGUUGAGCAA 46 GCUCAACAUAUUUGAUCAGUU 143 CGAAACUGAUCAAAUAUGUUGAGCAA 47 GUCAUCCACAAUGAGAGUACU 144 CGAAGUACGCUCAUUGUGGAUGACGA 48 GUCAUCCACAAUGAGAGUACU 145 CGUAGUACGCUCAUUGUGGAUGACGA 49 GUCAUCCACAAUGAGAGUACU 146 CGGAGUACGCUCAUUGUGGAUGACGA 50 GUCAUCCACAAUGAGAGUACU 147 CGCAGUACGCUCAUUGUGGAUGACGA 51 GUCAUCCACAAUGAGAGUACU 148 GGAAGUACGCUCAUUGUGGAUGACGA 52 GUCAUCCACAAUGAGAGUACU 149 UGAAGUACGCUCAUUGUGGAUGACGA 53 GUCAUCCACAAUGAGAGUACG 150 CGACGUACGCUCAUUGUGGAUGACGA 54 GUCAUCCACAAUGAGAGUACC 151 CGAGGUACGCUCAUUGUGGAUGACGA 55 GUCAUCCACAAUGAGAGUACU 152 CCGCCGAAGUACGCUCAUUGUGGAUGACGA 56 CUAGACCUGUTUUGCUUUUGU 153 CGAACAAAAGCAAAACAGGUCUAGAA 57 CUAGACCUGUTUUGCUUUUGU 154 CGUACAAAAGCAAAACAGGUCUAGAA 58 CUAGACCUGUTUUGCUUUUGU 155 CGGACAAAAGCAAAACAGGUCUAGAA 59 CUAGACCUGUTUUGCUUUUGU 156 CGCACAAAAGCAAAACAGGUCUAGAA 60 CUAGACCUGUTUUGCUUUUGU 157 AGAACAAAAGCAAAACAGGUCUAGAA 61 CUAGACCUGUTUUGCUUUUGU 158 GGUACAAAAGCAAAACAGGUCUAGAA 62 CUAGACCUGUTUUGCUUUUGG 159 CGACCAAAAGCAAAACAGGUCUAGAA 63 CUAGACCUGUTUUGCUUUUGC 160 CGAGCAAAAGCAAAACAGGUCUAGAA 64 CUAGACCUGUTUUGCUUUUGU 161 GCUUCGAACAAAAGCAAAACAGGUCUAGAA 65 GUCAUCCACAAUGAGAGUACU 162 CGAAGUACGCUCAUUGUGGAUGACGA 66 GUCAUCCACAAUGAGAGUACU 163 CGAAGUACGCUCAUUGUGGAUGACGA 67 GUCAUCCACAAUGAGAGUACU 164 CGAAGUACGCUCAUUGUGGAUGACGA 68 GUCAUCCACAAUGAGAGUACU 165 CGAAGUACGCUCAUUGUGGAUGACGA 69 GUCAUCCACAAUGAGAGUACU 166 CGAAGUACGCUCAUUGUGGAUGACGA 70 GUCAUCCACAAUGAGAGUACU 167 CGAAGUACGCUCAUUGUGGAUGACGA 71 GUCAUCCACAAUGAGAGUACU 168 CGAAGUACGCUCAUUGUGGAUGACGA 72 GUCAUCCACAAUGAGAGUACU 169 CGAAGUACGCUCAUUGUGGAUGACGA 73 GUCAUCCACAAUGAGAGUACU 170 CGAAGUACGCUCAUUGUGGAUGACGA 74 GUCAUCCACAAUGAGAGUACU 171 CGAAGUACGCUCAUUGUGGAUGACGA 75 GUCAUCCACAAUGAGAGUACU 172 CGAAGUACGCUCAUUGUGGAUGACGA 76 GUCAUCCACAAUGAGAGUACU 173 CGAAGUACGCUCAUUGUGGAUGACGA 77 GUCAUCCACAAUGAGAGUACU 174 CGAAGUACGCUCAUUGUGGAUGACGA 78 GUCAUCCACAAUGAGAGUACU 175 CGAAGUACGCUCAUUGUGGAUGACGA 79 GUCAUCCACAAUGAGAGUACA 176 UGUACGCUCAUUGUGGAUGACGA 80 ACCAGUCGUCCCAGAAUAACU 177 AGUUAUTCUGGGACGACUGGUCU 81 ACCAGUCGUCCCAGAAUAACU 178 CGAAGUUAUTCUGGGACGACUGGUCU 82 AAGCAAGAUAUUUUUAUAAUA 179 UAUUAUAAAAAUAUCUUGCUUUU 83 AAGCAAGAUAUUUUUAUAAUA 180 CGAUAUUAUAAAAAUAUCUUGCUUUU 84 GGUUAACACCAUUUACUUCAA 181 UUGAAGUAAAUGGUGUUAACCAG 85 GGUUAACACCAUUUACUUCAA 182 CGAUUGAAGUAAAUGGUGUUAACCAG 86 GAGCCGUUCUCUACAAUUACU 183 AGUAAUUGUAGAGAACGGCUCGG 87 GAGCCGUUCUCUACAAUUACU 184 CGAAGUAAUUGUAGAGAACGGCUCGG 88 CAUUUUAAUCCUCACUCUAAA 185 UUUAGAGUGAGGAUUAAAAUGAG 89 CAUUUUAAUCCUCACUCUAAU 186 CGAAUUAGAGUGAGGAUUAAAAUGAG 90 CAUUUUAAUCCUCACUCUAAU 187 CGAAUUAGAGUGAGGAUUAAAAUGAG 91 CAUUUUAAUCCUCACUCUAAU 188 CGAAUUAGAGUGAGGAUUAAAAUGAG 92 CAUUUUAAUCCUCACUCUAAA 189 UUUAGAGUGAGGAUUAAAAUGAG 93 CAUUUUAAUCCUCACUCUAAU 190 CGAAUUAGAGUGAGGAUUAAAAUGAG 94 GGCUACGAAAAUCCAACCUAA 191 UUAGGUGGGAUTUUCGUAGCCGU 95 GGCUACGAAAAUCCAACCUAA 192 CGAUUAGGUGGGAUTUUCGUAGCCGU 96 AACAGUGUUCUUGCUCUAUAA 193 UUUAAGAGCAAGAACACUGUUUU 97 AACAGUGUUCUUGCUCUAUAA 194 CGAUUUAGAGCAAGAACACUGUUUU

In some embodiments, the sense strand of the oligonucleotide reagent provided herein is any one of SEQ ID NO: 1-97, the antisense strand of the oligonucleotide reagent provided herein is any one of SEQ ID NO: 98-194, and a)   at least one of the internucleotide bonds between the nucleotides in the cleavage zone 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)   none of the internucleotide bonds between the nucleotides in the cleavage zone are phosphorothioate bonds; e)   at least one of the internucleotide bonds between the nucleotides in the cleavage zone 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 the nucleotides in the cleavage region is a phosphodiester bond.

In some embodiments, the sense strand of the oligonucleotide reagent provided herein is any one of SEQ ID NO: 1-97, the antisense strand of the oligonucleotide reagent provided herein is any one of SEQ ID NO: 98-194, and a)   at least one nucleotide in the cleavage region is modified with 2'-OMe or 2'-F; b)   each nucleotide in the cleavage region is modified with 2'-OMe or 2'-F; c)   at least one nucleotide in the cleavage region is modified with 2'-F; d)   no more than two nucleotides in the cleavage region are modified with 2'-F; e)   Z in the formula A or the formula B is modified with 2'-F, and optionally X2 in the formula A or the formula B is modified with 2'-F; f)   N1 in the formula A or the formula B is modified with 2'-F; g) Both X2 and Y in the formula A or the formula B are modified with 2'-OMe, and both Z and N1 in the formula A or the formula B are modified with 2'-F; and/or h) X2, Y and Z in the formula A or the formula B are all modified with 2'-OMe, and N1 in the formula A or the formula B is modified with 2'-F.

In some embodiments, the oligonucleotide reagent disclosed herein is any one of ds1-ds121.

In some embodiments, the double stranded oligonucleotide reagents disclosed herein can be optionally linked to one or more capping groups. Any suitable capping group provided in the disclosure can be used. The capping group can be attached to the sense strand, the antisense strand, or both strands at the 3' end, the 5' end, or both ends. In some embodiments, the capping group is linked to the antisense strand, specifically to the 5' end of the antisense strand.

f. Cleavage of dsOligonucleotides Reagent

In some embodiments, the cleaved double-stranded oligonucleotide reagent comprises a 3' extension on the antisense strand.

As described above, each nucleotide in the cleaved double-stranded oligonucleotide reagent can be independently modified or unmodified. In some embodiments, all nucleotides in the cleaved double-stranded oligonucleotide reagent are independently modified. In some embodiments, all nucleotides in the cleaved double-stranded oligonucleotide reagent are independently modified with 2'-F or 2'-OMe.

As described above, each internucleotide bond in the cleaved double-stranded oligonucleotide reagent can be independently modified or unmodified. In some embodiments, the cleaved double-stranded oligonucleotide reagent comprises at least one modified internucleotide bond. In some embodiments, the modified internucleotide bond is phosphorothioate (PS) or phosphorodithioate (PS2).

In some embodiments, the cleaved double-stranded oligonucleotide reagent comprises an alternating pattern of modifications as described above.

In some embodiments, the cleaved double-stranded oligonucleotide reagent comprises a phosphorothioate or methylphosphonate internucleotide bond. The phosphorothioate or methylphosphonate internucleotide bond modification can occur on any nucleotide of the sense strand or the antisense strand or both strands in any position of the strand. For example, the internucleotide bond modification can occur on every nucleotide on the sense strand and/or the antisense strand; each internucleotide bond modification can occur in an alternating pattern on the sense strand or the antisense strand; or the sense strand or the antisense strand comprises two internucleotide bond modifications in an alternating pattern. The alternating pattern of the internucleotide bond modification on the sense strand can be the same or different from the antisense strand, and the alternating pattern of the internucleotide bond modification on the sense strand can have an offset relative to the alternating pattern of the internucleotide bond on the antisense strand.

In one embodiment, the double-stranded oligonucleotide reagent of cleavage comprises a phosphorothioate or methylphosphonate internucleotide bond modification in the extension (e.g., 3' extension) region. For example, the extension (e.g., 3' extension) region comprises two nucleotides with a phosphorothioate or methylphosphonate internucleotide bond between the two nucleotides. Internucleotide bond modifications can also be performed to connect the extension nucleotides to the terminal paired nucleotides in the duplex region. For example, at least 2, 3, 4 or all of the extension (e.g., 3' extension) nucleotides can be connected by phosphorothioate or methylphosphonate internucleotide bonds, and optionally, additional phosphorothioate or methylphosphonate internucleotide bonds can be present to connect the extension nucleotides to the paired nucleotides next to the extension nucleotides. For example, there may be at least two phosphorothioate internucleotide bonds between the terminal three nucleotides, two of which are extension nucleotides and the third is a paired nucleotide next to the extension nucleotide. Preferably, these terminal three nucleotides may be located at the 3' end of the antisense strand.

In one embodiment, the sense strand of the cleaved double-stranded oligonucleotide reagent comprises 1 to 10 blocks of two to ten 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, wherein one of the phosphorothioate or methylphosphonate internucleotide bonds is located at any position in the oligonucleotide sequence, and the sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide bonds, or an antisense strand comprising phosphorothioate or methylphosphonate or phosphate bonds.

In one embodiment, the antisense strand of the cleaved double-stranded oligonucleotide reagent 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, wherein one of the phosphorothioate or methylphosphonate internucleotide bonds is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide bonds or a sense strand comprising phosphorothioate or methylphosphonate or phosphate bonds.

In one embodiment, the cleaved double-stranded oligonucleotide reagent molecule further comprises one to five phosphorothioate or methylphosphonate internucleotide bond modifications within positions 1 to 5 and one to five phosphorothioate or methylphosphonate internucleotide bond modifications within positions 18 to 23 (counting from the 5' end) of the sense strand, and one to five phosphorothioate or methylphosphonate internucleotide bond modifications at positions 1 and 2 and one to five within positions 18 to 23 (counting from the 5' end) of the antisense strand.

In one embodiment, the cleaved double-stranded oligonucleotide reagent comprises a pattern of alternating motifs of 2'-O-methyl modifications and 2'-F modifications on the sense strand that is initially shifted relative to the pattern of alternating motifs of 2'-O-methyl modifications and 2'-F modifications on the antisense strand, i.e., nucleotides with 2'-O-methyl modifications on the base of the sense strand are paired with nucleotides with 2'-F modifications on the antisense strand, and vice versa. For example, the 1 position of the sense strand may start with a 2'-F modification, and the 1 position of the antisense strand may start with a 2'-O-methyl modification. Introducing one or more motifs having three identical modifications on three consecutive nucleotides into the sense strand and/or the antisense strand may interrupt the initial modification pattern present in the sense strand and/or the antisense strand. Disrupting the modification pattern of the sense strand and/or antisense strand by introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense strand and/or antisense strand unexpectedly enhances the gene silencing activity against the target gene.

g. Ligand

In certain embodiments, the double-stranded oligonucleotide reagents disclosed herein can be optionally conjugated to one or more ligands. The ligand can be attached to the sense strand, antisense strand, or both strands at the 3' end, the 5' end, or both ends, or at any available nucleotide (e.g., at the sugar ring). In some embodiments, the ligand can be conjugated to the sense strand, specifically to the 3' end of the sense strand. In some embodiments, the ligand is conjugated to the antisense strand, specifically to the 5' end of the antisense strand.

In some embodiments, a nucleotide (e.g., of the sense strand or antisense strand) is replaced by a nucleotide that binds to a ligand. In this case, the nucleotide that binds to a ligand is also included in the scope of "ligand".

In some embodiments, the ligand is a GalNAc ligand, a lipophilic ligand, or other ligand that targets a receptor capable of promoting endocytosis of siRNA conjugates, such as a TfR targeting ligand, an LDL-R targeting ligand, or an integrin targeting ligand.

In some embodiments, the ligand is conjugated 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 ligand is linked to the double-stranded oligonucleotide reagent via a phosphorothioate.

In some embodiments, the ligand is L96, having the structure: .

In some embodiments, the ligand is a hexadecyloxy group. For example, a C16 ligand can be conjugated to a uridine nucleotide at the 2' position, which is abbreviated herein as "C16U" and has the structure: .

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, having the following structure: , where X is S or O.

For example, "5'-(VSDL-03A)*A-3'" having the following structure .

h. Target Gene

In some embodiments, the double-stranded oligonucleotide reagent provided herein can inhibit the expression of a target gene. In certain 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, mAcl, TSHR, CB1 and LPA.

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

II. Novel end-capping groups and novel ligands

Also provided herein is a double-stranded oligonucleotide reagent comprising a double-stranded oligonucleotide operably linked to a blocking group, wherein the blocking group comprises M03 or M06.

In certain embodiments, the double-stranded oligonucleotide can be any suitable siRNA or RNAi agent.

Also provided herein is a double-stranded oligonucleotide reagent 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. In certain embodiments, the double-stranded oligonucleotide can be any suitable siRNA or RNAi reagent.

In some embodiments, a double-stranded oligonucleotide reagent operably linked to a capping group or a ligand comprises a sense strand and an antisense strand. In some embodiments, a double-stranded oligonucleotide reagent operably linked to a capping group or a ligand comprises a capping group bound to the 5' end of the antisense strand. In some embodiments, a double-stranded oligonucleotide reagent operably linked to a capping group or a ligand comprises a ligand bound 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, a double-stranded oligonucleotide reagent operably linked to a capping group or a ligand is operably linked to a capping group at one end and to a ligand at the other end. In some embodiments, a double-stranded oligonucleotide reagent operably linked to a capping group or a ligand is capable of inhibiting the expression of a target gene by RNA interference.

In some embodiments, a double-stranded oligonucleotide reagent operably linked to a capping group or ligand comprises a sense strand and an antisense strand, wherein each strand has 14 to 30 nucleotides.

In some embodiments, the antisense strand of the double-stranded oligonucleotide reagent operably linked to the capping group or ligand is substantially complementary to a portion of a target RNA or mRNA encoding a target gene, and the sense strand of the double-stranded oligonucleotide reagent operably linked to the capping group or ligand is substantially complementary to the targeted region of the antisense strand.

In some embodiments, a blocking group is ligated to the 5' end of the antisense strand.

In some embodiments, the ligand is ligated 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 antisense strand of a double-stranded oligonucleotide reagent operably linked to a capping group or a ligand is operably linked to a capping group at one end and to a ligand at the other end, and/or

In some embodiments, the antisense strand of a double-stranded oligonucleotide reagent operably linked to a capping group or ligand is capable of silencing a target RNA or inhibiting the expression of a target gene by RNA interference.

In one aspect, disclosed herein is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand having a structure represented by formula (I): , wherein the sense strand consists of region A1, region A2 and X1 (from 5' to 3'), the antisense strand consists of region N, Z, Y, X2, region B2 and region B1 (from 5' to 3'), the region A1 is 0-15 nucleotides, the region B1 is 0-15 nucleotides, the region A2 and region B2 have the same length and are at least 1 nucleotide, the region A2 and region B2 comprise complementary double-stranded regions, each of X1, X2, Y and Z is independently 1 nucleotide, wherein X1 and X2 can form a hydrogen bond, the region N is at least 1 nucleotide, the total length of the sense strand is 17-23 nt, the total length of the antisense strand is at least 26 nt, The dsRNA comprises no more than 23 complementary base pairs, and each of the nucleotides of the dsRNA is independently optionally modified.

In some embodiments, the sense strand consists of region A1, region A2 and X1 (from 5' to 3'), the antisense strand consists of region N, Z, Y, X2, region B2 and region B1 (from 5' to 3'), region A1 is 1 to 6 nucleotides, region B1 is 0 to 6 nucleotides, region A2 and region B2 have the same length and are at least 1 nucleotide, region A2 and region B2 contain complementary double-stranded regions, each of X1, X2, Y and Z is independently 1 nucleotide, wherein X1 and X2 are complementary, region N is at least 1 nucleotide, the total length of the sense strand is 17 to 23 nt, and the total length of the antisense strand is at least 26 nt, the dsRNA comprises no more than 23 complementary base pairs, and each of the nucleotides of the dsRNA is independently optionally modified.

In some embodiments, each of X2 and Y is independently A, dA, U or dU, and Z is G or dG.

In some embodiments, each of X2 and Y is independently A, dA, U or dU, Z is G or dG, and each of the nucleotides of the dsRNA is independently modified.

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

In some embodiments, X2 is A or U, Z is G, and each of the nucleotides of the dsRNA is independently modified.

In some embodiments, Y is A or U.

In some embodiments, Y is A or U, and each of the nucleotides of the dsRNA is independently modified.

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

In some embodiments, X2 is A or U, Y is A or U, and each of the nucleotides of the dsRNA is independently modified.

In some embodiments, Y is A.

In some embodiments, Y is A and each of the nucleotides of the dsRNA is independently modified.

In some embodiments, X1 is A or U.

In some embodiments, X1 is A, U or I.

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

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

In some embodiments, at least one nucleotide in Y, Z, and region N is modified.

In some embodiments, region N consists of 1 nucleotide and group X, group X is a nucleotide, a blocking group, or a combination of a nucleotide and a blocking group, wherein the blocking group is a structure that resists nuclease degradation, the blocking group is linked to region N at the 5' end, and the blocking group is independently optionally modified.

In some embodiments, the capping group is selected from reverse abasic deoxyribonucleotides or RX, wherein RX is or .

In some embodiments, region N consists of 1 nucleotide and 1 blocking group.

In some embodiments, one nucleotide and one blocking group in region N are linked via a phosphorothioate bond.

In some embodiments, region N consists of 2 nucleotides and 1 blocking group.

In some embodiments, the two nucleotides and one blocking group of region N are linked by a phosphorothioate bond.

In some embodiments, region N consists of 1 nucleotide and 2 capping groups.

In some embodiments, one nucleotide and two blocking groups in region N are linked by phosphorothioate bonds.

In some embodiments, region N consists of 1 nucleotide and 2 capping groups.

In some embodiments, 1 nucleotide and 2 capping groups of region N are connected by a bond, wherein the bond is connected by a single bond.

In some embodiments, one nucleotide and two capping groups of region N are linked by a bond, wherein the bond is linked by a phosphodiester, phosphorothioate, or phosphorodithioate bond.

In some embodiments, all nucleotides of a dsRNA are independently modified.

In some embodiments, nucleotide modifications include 1, 2 or more selected from the group consisting of: base modification, sugar ring modification, phosphate backbone modification and terminal modification. Base modification includes substitution with a stable base, a destabilizing base or a base paired with an expanded complementary library; base removal (abasic nucleotide) or conjugated base. Sugar modification includes modification or sugar substitution at the 2' position, 3' position or 4' position. Phosphate backbone modification includes modification or replacement of phosphodiester bonds. Terminal modifications include 5' terminal modifications (phosphorylation, conjugation, reverse bonding, etc.) and 3' terminal modifications (conjugation, DNA nucleotides, reverse bonding, etc.).

In some embodiments, the nucleotide modifications include 1, 2 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 and ARGO protein.

In some embodiments, the modification that enhances the affinity of dsRNA and ARGO protein comprises using , or Replace nucleotides.

In some embodiments, the nucleotide modifications include 1, 2 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 enhance the affinity of dsRNA and ARGO protein.

In some embodiments, the modification that enhances the affinity of the dsRNA to the ARGO protein is on X2.

In some embodiments, region A1 and region B1 comprise complementary double-stranded regions.

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

In some embodiments, region A2 or B2 is 20 nt in length.

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

In some embodiments, the total length of the sense strand is 17 to 21 nt. In some embodiments, the total length of the sense strand is 18 to 21 nt.

In some embodiments, the total length of the antisense strand is 26-35 nt. In some embodiments, the total length of the antisense strand is 26-32 nt. In some embodiments, the total length of the antisense strand is 26-28 nt. In some embodiments, the total length of the antisense strand is 26-27 nt. In some embodiments, the total length of the antisense strand is 26 nt, 27 nt, 28 nt, 29 nt, or 30 nt.

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

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

In some embodiments, region N comprises inverted abasic deoxyribonucleotides (invAB).

In some embodiments, the total length of the antisense strand is 26-27 nt, region A1 is 1-4 nucleotides, and region N comprises inverted abasic deoxyribonucleotides (invAB).

In some embodiments, Z and region N are linked via a phosphorothioate bond.

In some embodiments, X2 and region B2 are linked via a phosphorothioate bond.

In some embodiments, Z and region N are linked by a phosphorothioate bond, and X2 and region B2 are linked by a phosphorothioate bond.

In some embodiments, the total length of the antisense strand is 26-27 nt, region A1 is 1-4 nucleotides, and Z and region N are linked by a phosphorothioate bond.

In some embodiments, the total length of the antisense strand is 26-27 nt, region A1 is 1-4 nucleotides, and X2 and region B2 are linked by a phosphorothioate bond.

In some embodiments, the total length of the antisense strand is 26-27 nt, region A1 is 1-4 nucleotides, Z and region N are linked by a phosphorothioate bond, and X2 and region B2 are linked by a phosphorothioate bond.

In some embodiments, the total length of the antisense strand is 26-27 nt, region A1 is 1-4 nucleotides, region N comprises inverted abasic deoxyribonucleotides (invAB), and Z and region N are linked by a phosphorothioate bond.

In some embodiments, the total length of the antisense strand is 26-27 nt, region A1 is 1-4 nucleotides, region N comprises inverted abasic deoxyribonucleotides (invAB), and X2 and region B2 are linked by a phosphorothioate bond.

In some embodiments, the total length of the antisense strand is 26-27 nt, region A1 is 1-4 nucleotides, region N comprises inverted abasic deoxyribonucleotides (invAB), Z and region N are linked by a phosphorothioate bond, and X2 and region B2 are linked by a phosphorothioate bond.

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, and region A1 is 1-4 nucleotides.

In some embodiments, the total length of the sense strand is 17-21 nt, and region N comprises inverted abasic deoxyribonucleotides (invAB).

In some embodiments, the total length of the sense strand is 17-21 nt, region A1 is 1-4 nucleotides, and region N comprises inverted abasic deoxyribonucleotides (invAB).

In some embodiments, the total length of the antisense strand is 26-27 nt, region A1 is 1-4 nucleotides, and position 1 of region N, counting from its 5' end, is replaced by RX, wherein RX is or , and Z and region N are connected via a phosphorothioate bond.

In some embodiments, the total length of the antisense strand is 26-27 nt, region A1 is 1-4 nucleotides, and position 1 of region N, counting from its 5' end, is replaced by RX, wherein RX is or , and X2 and region B2 are connected via a phosphorothioate bond.

In some embodiments, the total length of the antisense strand is 26-27 nt, region A1 is 1-4 nucleotides, and position 1 of region N, counting from its 5' end, is replaced by RX, wherein RX is or , Z and region N are connected via a phosphorothioate bond, and X2 and region B2 are connected via a phosphorothioate bond.

In some embodiments, the total length of the sense strand is 17-21 nt, region A1 is 1-4 nucleotides, and position 1 of region N, counting from its 5' end, is replaced by RX, wherein RX is or .

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 position 1 of region N, counting from its 5' end, is replaced by RX, wherein RX is or .

In some embodiments, the total length of the sense strand is 17-21 nt, and Z and region N are linked by a phosphorothioate bond.

In some embodiments, the total length of the sense strand is 17-21 nt, and X2 and region B2 are linked by a phosphorothioate bond.

In some embodiments, the total length of the sense strand is 17-21 nt, Z and region N are linked by a phosphorothioate bond, and X2 and region B2 are linked by a phosphorothioate bond.

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 Z and region N are linked by a phosphorothioate bond.

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 X2 and region B2 are linked by a phosphorothioate bond.

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 by a phosphorothioate bond, and X2 and region B2 are linked by a phosphorothioate bond.

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 comprises inverted abasic deoxyribonucleotides (invAB), and Z and region N are linked by a phosphorothioate bond.

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 comprises inverted abasic deoxyribonucleotides (invAB), and X2 and region B2 are linked by a phosphorothioate bond.

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 comprises inverted abasic deoxyribonucleotides (invAB), Z and region N are linked by a phosphorothioate bond, and X2 and region B2 are linked by a phosphorothioate bond.

In some embodiments, positions 1 and 2 and positions 2 and 3, counted from the 5' and 3' ends of the sense and antisense strands, are independently optionally linked by phosphorothioate bonds.

In some embodiments, positions 1 and 2 and positions 2 and 3, counting from the 5' and 3' ends of the sense and antisense strands, are linked by phosphorothioate bonds.

In one aspect, disclosed herein is a dsRNA conjugate comprising a dsRNA disclosed herein and a delivery system, wherein the delivery system is capable of delivering the dsRNA to a target RNA and thereby providing an RNA interference effect, the delivery system is conjugated to the dsRNA, and the dsRNA conjugate comprises one or more delivery systems.

In some embodiments, the delivery system is selected from the group consisting of liposomes, lipid nanoparticles (LNPs), lipoplexes, lipid polymer complexes (LPPs), mannose delivery systems, N-acetylgalactosamine (GalNAc) conjugated delivery systems, Apelin receptor targeted delivery systems, integrin receptor targeted delivery systems, peptides, antibodies, and any combination thereof.

In some embodiments, the delivery system is ligated at the 3' end of the sense strand.

In some embodiments, the delivery system is ligated at the 5' end of the sense strand.

In some embodiments, the delivery system is ligated at the 3' end of the antisense strand.

In some embodiments, the delivery system is ligated at the 5' end of the antisense strand.

In some embodiments, the delivery system is ligated at a nucleotide that is not at the terminus of the dsRNA.

In one aspect, disclosed herein is a composition comprising a dsRNA disclosed herein and a pharmaceutically acceptable carrier or formulation.

In one aspect, disclosed herein is the use of a dsRNA, dsRNA conjugate or composition disclosed herein in the preparation of a medicament for treating a disease or disorder associated with RNA interference gene expression.

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, mACl, TSHR, CB1, and LPA.

In some embodiments, the gene is selected from the group consisting of AGT, complement factor B, DGAT2, SOD1, KHK, APP, DUX, ANGPTL3, ANGPTL4 and ANGPTL8.

In one aspect, disclosed herein is a dsRNA, dsRNA conjugate or composition disclosed herein for use in treating a disease or disorder associated with RNA interference gene expression.

Example I-1. A double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand having a structure represented by formula (I): , wherein the sense strand consists of region A1, region A2 and X1 (from 5' to 3'), the antisense strand consists of region N, Z, Y, X2, region B2 and region B1 (from 5' to 3'), the region A1 is 0 to 15 nucleotides, the region B1 is 0 to 15 nucleotides, the region A2 and region B2 have the same length of at least 1 nucleotide, the region A2 and region B2 comprise double-stranded regions that form reverse complements, each of X1, X2, Y and Z is independently 1 nucleotide, wherein X1 and X2 can be hydrogen bonded, the 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 comprises no more than 23 complementary base pairs, and each of the nucleotides of the dsRNA is independently optionally modified.

Embodiment I-2. The dsRNA according to Embodiment I-1, wherein X2 is A or U, and Z is G.

Embodiment I-3. The dsRNA according to Embodiment I-2, wherein Y is A or U.

Embodiment I-4. The dsRNA according to Embodiment I-3, wherein Y is A.

Embodiment I-5. The dsRNA according to any one of Embodiments I-1 to I-4, wherein the region N consists of 1 nucleotide and a group X, wherein the group X is a nucleotide, a blocking group, or a combination of a nucleotide and a blocking group, wherein the blocking group is a structure that resists nuclease decomposition, the blocking group is linked to the region N at the 5' end, and the blocking group is independently and optionally modified.

Embodiment 1-6. The dsRNA according to embodiment 1-5, wherein the blocking group is selected from reverse abasic deoxyribonucleotides or RX, wherein RX is or .

Embodiment I-7. The dsRNA according to Embodiment I-6, wherein the region N consists of 1 nucleotide and 1 blocking group.

Embodiment I-8. The dsRNA according to Embodiment I-7, wherein the 1 nucleotide of the region N and the 1 blocking group are linked via a phosphorothioate bond.

Embodiment I-9. The dsRNA according to any one of Embodiments I-1 to I-8, wherein all nucleotides of the dsRNA are independently modified.

Embodiment I-10. The dsRNA according to any one of Embodiments I-1 to I-8, wherein the nucleotide modifications include 1, 2 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 enhance the affinity of dsRNA and ARGO protein.

Embodiment I-11. The dsRNA according to Embodiment I-10, wherein the modification that enhances the affinity of the dsRNA and the ARGO protein is on X2.

Embodiment I-12. The dsRNA according to any one of Embodiments I-1 to I-11, wherein the total length of the sense strand is 17 to 21 nt; further, the total length of the sense strand is 18 to 21 nt.

Embodiment I-13. The dsRNA according to any one of Embodiments I-1 to I-11, wherein the total length of the antisense strand is 26 to 35 nt; further, the total length of the antisense strand is 26 to 27 nt.

Embodiment I-14. The dsRNA according to any one of Embodiments I-1 to I-11, wherein the region A1 and the region B1 comprise complementary double-stranded regions.

Embodiment I-15. The dsRNA according to Embodiment I-14, wherein the region A1 is 1 nucleotide and the region B1 is 3 nucleotides.

Embodiment I-16. The dsRNA according to Embodiment I-15, wherein the length of region A2 or B2 is 20 nt.

Embodiment I-17. The dsRNA according to any one of Embodiments I-1 to I-11, wherein the region A1 is 0 nucleotides, and the region B1 is 2 nucleotides.

Embodiment I-18. Use of the dsRNA or pharmaceutical composition thereof according to any one of Embodiments I-1 to I-17 for the preparation of a medicament for treating a disease or disorder associated with RNA interference gene expression; further, 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 and LPA.

Treatment

Provided herein is a method for regulating the expression of a target gene in an individual in need thereof, the method comprising administering to the individual a pharmaceutically effective amount of a double-stranded oligonucleotide reagent disclosed herein or a pharmaceutical composition disclosed herein.

Provided herein is a method for inhibiting the expression of a target gene in an individual in need thereof, the method comprising administering to the individual a pharmaceutically effective amount of a double-stranded oligonucleotide reagent disclosed herein or a pharmaceutical composition disclosed herein.

Provided herein is a method for treating a disease or condition in an individual in need thereof by modulating (e.g., inhibiting) the expression of a target gene, for example comprising administering to the individual a pharmaceutically effective amount of a double-stranded oligonucleotide reagent disclosed herein or a pharmaceutical composition disclosed herein.

Provided herein is a method for treating a disease or condition in an individual in need thereof, the method comprising administering to the individual a pharmaceutically effective amount of a double-stranded oligonucleotide reagent disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the disease or condition is associated with a target gene. In some embodiments, the disease or condition is associated with overexpression or activation of a target gene.

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, mACl, TSHR, CB1, and LPA.

The double-stranded oligonucleotide reagent disclosed herein or the pharmaceutical composition disclosed herein can be administered to a subject by various routes, depending on whether local or systemic treatment is desired and the area to be treated. Administration can be topical (including ocular, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous instillation, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.

The route and site of administration can be selected to enhance targeting. For example, to target muscle cells, intramuscular injection into the target muscle is a logical choice. Lung cells can be targeted by administering oligonucleotides in the form of an aerosol.

The defined amount can be an amount effective for treating or preventing a disease or condition (e.g., a disease or condition associated with the target RNA). For example, a unit dose can be administered by injection (e.g., intravenous, subcutaneous or intramuscular), inhalation administration or topical application.

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 another embodiment, the unit dose is not administered frequently (e.g., not at a regular frequency). For example, the unit dose can be administered once.

In one embodiment, an effective dose is administered in conjunction with other conventional treatment modalities. The effective dose may be administered in a single dose or in two or more doses, as desired or deemed appropriate under the particular circumstances. If necessary to facilitate repeated or frequent infusions, implantation of a delivery device such as a pump, a semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal or intracapsular), or a reservoir is desirable.

Pharmaceutical ingredients

Oligonucleotide reagent disclosed herein can be formulated for pharmaceutical use. A pharmaceutically acceptable composition comprises an oligonucleotide reagent disclosed herein (e.g., a pharmaceutically effective amount of an oligonucleotide reagent), which is taken alone or formulated with one or more pharmaceutically acceptable carriers (additives), excipients and/or diluents.

The pharmaceutical compositions may be formulated for administration in solid or liquid form, including those suitable for: (1) oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets, such as those intended for buccal, sublingual, and systemic absorption, pills, powders, granules, pastes for application to the tongue; (2) parenteral administration, For example, as a sterile solution or suspension or as a sustained release formulation, by subcutaneous, intramuscular, intravenous, or epidural injection; (3) topically, for example, as a cream, ointment, or controlled release patch or spray applied to the skin; (4) intravaginally, intrarectally, for example, as a pessary, cream, or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally. Delivery using subcutaneous or intravenous methods may be particularly advantageous.

A pharmaceutically acceptable carrier can be a composition or vehicle that participates in carrying or transporting the target compound from one organ or part of the body to another, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium, calcium or zinc stearate or stearic acid), or solvent encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve 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, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients such as cocoa butter and suppository waxes; (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) polyols, Such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic water; (18) Ringer's solution; (19) ethanol; (20) pH buffer solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) fillers, such as peptides and amino acids; (23) serum components, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances used in drug formulations.

The formulation can be conveniently presented in unit dosage form and can be prepared by any method well known in the pharmaceutical field. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated, the specific mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the compound that produces a therapeutic effect. This amount is generally (in percentage) in the range of about 0.1% to about 99% of the active ingredient, preferably about 5% to about 70%, and most preferably about 10% to about 30%.

In certain embodiments, the formulation of the present invention comprises a formulation selected from the group consisting of cyclodextrin, cellulose, liposome, micelle forming agent (e.g., bile acid) and polymer carrier (e.g., polyester and polyanhydride); and the oligonucleotide disclosed herein. In certain embodiments, the formulation makes the oligonucleotide reagent disclosed herein orally bioavailable.

The double-stranded oligonucleotide reagent can be formulated in combination with another reagent (e.g., another therapeutic agent) or a stabilizer (e.g., a protein complexed with the double-stranded oligonucleotide reagent). Other reagents include chelating agents such as EDTA (e.g., for removing divalent cations such as Mg2+), salts, RNase inhibitors (e.g., broad specificity RNase inhibitors such as RNAsin), etc.

The method for preparing these formulations or compositions includes the step of combining the oligonucleotide reagent disclosed herein with a carrier and optionally one or more auxiliary components. Generally, the formulation is prepared by uniformly and closely combining the oligonucleotide disclosed herein with a liquid carrier or a finely powdered solid carrier or both, and then shaping the product if necessary.

In some cases, in order to prolong the effect of the drug, it is desirable to slow the absorption of a subcutaneously or intramuscularly injected drug. This can be accomplished by using a liquid suspension of a poorly water-soluble crystalline or amorphous material. The rate of absorption of the drug then depends on its dissolution rate, which in turn may depend on the crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Similar to other drugs, the oligonucleotide reagents disclosed herein can be formulated for administration in any convenient manner for use in human or veterinary medicine.

Examples

Abbreviation

As used herein, the term "nucleotide" encompasses both ribonucleotides and deoxyribonucleotides. As used herein, "G", "C", "A", "T", and "U" refer to nucleotides containing guanine, cytosine, adenine, thymine, and uracil, respectively. When "d" is marked before A, U, C, G, or T, it represents a 2'-deoxyribonucleotide (DNA), and when "d" is not marked, it represents a ribonucleotide (RNA). For example, "dA" represents deoxyadenosine, "dG" represents deoxyguanosine, "dT" represents deoxythymidine, "dU" represents deoxyuridine, and "dC" represents deoxycytidine; "A" represents adenosine, "G" represents guanosine, "T" represents ribothymidine or 5-methyluridine, "U" represents uridine, and "C" represents cytidine.

As used herein, "f" represents a 2'-fluorine-modified nucleotide when it is labeled after A, U, C, G, and T. 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.

As used herein, 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.

As used herein, "C16" or "C16 modification" refers to a 2'-O-C16 modification or a 2'-C16 modification. When "C16" is marked before A, U, C, G, or T, it represents a 2'-hexadecyloxy modified nucleotide. For example, "C16U" represents For example, "C16(dU)" means .

As used herein, when "GNA-" is marked before A, U, C, G, and T, it represents a nucleotide modified with ethylene glycol nucleic acid. "Tgn" is another name for "GNA-T", and "Tgn" is equivalent to "GNA-T". For example, GNA-G, GNA-A, GNA-C, GNA-U, and GNA-T have the following structures.

As used herein, "*" between two nucleotides such as A, U, C, G, and T represents a phosphorothioate internucleotide bond, i.e., the two nucleotides are linked by a phosphorothioate bond. As used herein, the absence of "*" between two nucleotides (e.g., A, U, C, G, and T) represents an unmodified internucleotide bond (i.e., a phosphodiester bond).

For example, the sequence "5'-AdUgCf*T-3'" represents a sequence in which, starting from the 5' end, position 1 is adenosine, position 2 is deoxyuridine, position 3 is 2'-methoxyguanosine, position 4 is 2'-fluorocytidine, position 5 is ribothymidine, position 4 and position 5 are linked by a phosphorothioate bond, and other adjacent positions are linked by a phosphodiester bond.

As used herein, "invAB" or "invAB modification" refers to a nucleotide fused to an inverted abasic deoxyribonucleotide (e.g., fused at the 5' end or the 3' end). For example, if the nucleotide has the structure: （ ), then the corresponding invAB-modified nucleotide has the structure: （ )or （ ).

As used herein, "invAb" or "invAb modification" refers to the replacement of a nucleotide with an inverted abasic deoxyribonucleotide. For example, if the nucleotide has the structure: （ ), then the corresponding invAb-modified nucleotide has the structure: （ ).

As used herein, "VP" or "VP modification" refers to a nucleotide having an (E)-vinylphosphonate modification (e.g., a 5'-(E)-vinylphosphonate modification). For example, VP-modified U, u, dU, and Uf have the following structures.

As used herein, "M03" or "M03 modification" refers to a nucleotide conjugated (e.g., conjugated at the 5' end or the 3' end) to a molecule having the following structure: For example, if a nucleotide has the structure: , then the corresponding M03-modified nucleotide has the structure: or In some embodiments, the conjugation to M03 is by nucleotides and reaction.

As used herein, "M06" or "M06 modification" refers to a nucleotide conjugated (e.g., conjugated at the 5' end or the 3' end) to a molecule having the following structure: For example, if a nucleotide has the structure: , the corresponding M06-modified nucleotide has the structure: or In some embodiments, the conjugation to M06 is by nucleotides and reaction.

For example, the sequence "5'-(M06)*AdTgCf*(invAB)-3'" represents an oligonucleotide having the following structure: .

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

Examples 1 Synthesis of ligand promotors

Example 1.1 Synthesis of VSDL-01 precursor ( 2- cyanoethyl behenyl diisopropylaminophosphite)

To a solution of compound 1-1 (10.00 g, 30.62 mmol) in anhydrous DCM (100 mL) was added 1H-imidazole-4,5-dicarbonitrile (1.81 g, 15.31 mmol), 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 at 25 ° C for 16 hours under N _2. The mixture was concentrated in vacuo to remove DCM. The residue was purified by silica gel chromatography (eluted with hexane containing 1% TEA: EtOAc = 5: 1) to obtain the prodrug of VSDL-01 .

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

³¹ P NMR: (400 MHz, CDCl ₃ ) δ 147.17.

Example 1.2 Synthesis of VSDL- 02 precursor ( 2- cyanoethyl eicosyl diisopropylaminophosphite)

The synthesis method is similar to that of Example 1.1.

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

³¹ P NMR: (400 MHz, CDCl ₃ ) δ 147.32.

Example 1.3 Synthesis of VSDL- 03 precursor ( 2- cyanoethyl hexadecyl diisopropylaminophosphite)

The synthesis method is similar to that of Example 1.1.

¹ H NMR (400 MHz, CDCl ₃ ) 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).

³¹ P NMR: (400 MHz, CDCl ₃ ) δ 147.24.

Example 1.4 Synthesis of VSDL- 04 precursor ( 2- cyanoethyl (6- eicosylamidohexyl ) diisopropylamidophosphite)

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 pentafluorophenyl trifluoroacetate (4.2 g, 15 mmol) dropwise. 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 under vacuum to remove DCM. The residue was purified by silica gel chromatography (eluted with hexane: EtOAc = 5: 1) to give compound 4-3 (3.82 g).

To a solution of compound 4-3 (3.63 g, 8.82 mmol) in anhydrous DMF (60 mL) was added 1H-imidazole-4,5-dicarbonitrile (0.52 g, 4.41 mmol), 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 at 25 °C under _N2 for 16 hours. The mixture was washed with saturated aqueous _NaHCO3 solution (200 mL), extracted with EtOAc (3 × 50 mL), and _the organic phases were combined, dried over anhydrous _Na2SO4 , and concentrated under vacuum. The residue was purified by silica gel chromatography (eluted with hexane containing 1% TEA:EtOAc = 5:1) to obtain the pro-form of VSDL-04 .

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.45 (t, J = 5.6 Hz, 1H), 3.92-3.75 (m, 2H), 3.70-3.54 (m, 4H), 3.23 (q, J = 6.8 Hz, 2H), 2.64 (t, J = 6.4 Hz, 2H), 2.14 (t, J = 7.6 Hz, 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, 4.2 Hz, 12H), 0.87 (t, J = 6.8 Hz, 3H).

³¹ P NMR: (400 MHz, CDCl ₃ ) δ 147.32.

Example 1.5 Synthesis of VSDL- 05 Promotor ( 2- cyanoethyl (6- stearylamidohexyl ) diisopropylamidophosphite)

Similar to the synthesis method in Example 1.4.

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.47 (t, J = 5.6 Hz, 1H), 3.89-3.75 (m, 2H), 3.63-3.67 (m, 4H), 3.23 (q, J = 6.8 Hz, 2H), 2.64 (t, J = 6.4 Hz, 2H), 2.14 (t, J = 7.6 Hz, 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, 4.2 Hz, 12H), 0.87 (t, J = 6.8 Hz, 3H).

³¹ P NMR: (400 MHz, CDCl ₃ ) δ 147.20.

Example 1.6 Synthesis of VSDL- 06 precursor ( 2- cyanoethyl (6- palmitoylaminohexyl ) diisopropylamidophosphite)

Similar to the synthesis method in Example 1.4.

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.69 (t, J = 5.6 Hz, 1H), 3.90-3.75 (m, 2H), 3.69-3.54 (m, 4H), 3.26-3.21 (m, 2H), 2.65 (t, J = 6.4 Hz, 2H), 2.15 (t, J = 7.6 Hz, 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, 4.2 Hz, 12H), 0.88 (t, J = 6.8 Hz, 3H).

³¹ P NMR: (400 MHz, CDCl ₃ ) δ 147.25.

Example 1.7 Synthesis of VSDL-07 promotors ( 2- cyanoethyl ( 6-((5Z,8Z,11Z,14Z,17Z) -eicosyl -5,8,11,14,17- pentaenylamido ) hexyl ) diisopropylamidophosphite )

Similar to the synthesis method in Example 1.4.

¹ H NMR (400 MHz, CDCl ₃ ) 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 = 6.5 Hz, 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, 4.2 Hz, 12H), 0.97 (t, J = 7.6 Hz, 3H).

³¹ P NMR: (400 MHz, CDCl ₃ ) δ 147.32.

Example 1.9 Synthesis of VSDL -09 Prodromal ( Diisopropylamidophosphite )

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

¹ H NMR (400 MHz, CDCl ₃ ) δ 3.68-3.52 (m, 6H), 1.64-1.57 (m, 4H), 1.36-1.25 (m, 52H), 1.18 (d, J = 6.8 Hz, 12H), 0.88 (t, J = 6.8 Hz, 6H).

³¹ P NMR: (400 MHz, CDCl ₃ ) δ 144.91.

Example 1.10 Synthesis of VSDL -10 promotors (butyl hexadecyl diisopropylamino phosphite )

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

To a solution of compound 3-1 (1.33 g, 5.47 mmol) in anhydrous DCM (10 mL) was added 1H-imidazole-4,5-dicarbonitrile (0.32 g, 2.74 mmol), and then a solution of compound 10-3 (2.5 g, 8.21 mmol) in anhydrous DCM (5 mL) was added dropwise to the mixture. The reaction mixture was stirred at 25 ° C. under N ₂ for 16 hours. The mixture was concentrated under vacuum. The residue was purified by silica gel chromatography (eluted with hexane containing 1% TEA: EtOAc = 5: 1) to obtain the prodrug of VSDL-10 .

¹ H NMR (400 MHz, CDCl ₃ ) δ 3.69-3.51 (m, 6H), 1.64-1.55 (m, 4H), 1.44-1.22 (m, 28H), 1.17 (d, J = 6.8 Hz, 12H), 0.90 (dt, J = 14.0, 7.2 Hz, 6H).

³¹ P NMR: (400 MHz, CDCl ₃ ) δ 145.00.

Example 1.12 Synthesis of VSDL -12 Prodromal (Diisopropylamino Phosphite Hexadecyl Octyl Ester)

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

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

¹ H NMR (400 MHz, CDCl ₃ ) δ 3.69-3.52(m, 6H), 1.64-1.56 (m, 4H), 1.37-1.25 (m, 36H), 1.17 (d, J = 6.8 Hz, 12H), 0.90-0.86 (m, 6H).

³¹ P NMR: (400 MHz, CDCl ₃ ) δ 144.97.

Example 1.13 Synthesis of VSDL- 13 prodrug (dioctyl diisopropylaminophosphite)

Similar to the synthesis method in Example 1.9.

¹ H NMR (400 MHz, CDCl ₃ ) δ 3.67-3.51 (m, 6H), 1.64-1.57 (m, 4H), 1.37-1.26 (m, 20H), 1.18 (d, J = 6.8 Hz, 12H), 0.89-0.85 (m, 6H).

³¹ P NMR: (400 MHz, CDCl ₃ ) δ 145.00.

Examples 2 Synthesis of end-group precursors

Example 2.1 Synthesis of the precursor of M06 ( 2- cyanoethyl ( 2- morpholinoethyl ) diisopropylaminophosphite)

To a solution of compound 15-1 (10.88 g, 83.05 mmol) in anhydrous DCM (100 mL) was added 1H-imidazole-4,5-dicarbonitrile (4.9 g, 41.53 mmol). The reaction mixture was cooled to 0 °C, and then a solution of compound 1-2 (25 g, 83.05 mmol) in DCM (30 mL) was added to the mixture. The mixture was stirred at 25 °C under _N2 for 16 hours. The mixture was filtered and concentrated to remove DCM. The residue was diluted with hexane and purified by silica gel chromatography (eluted with hexane) to obtain the prodrug of M06 .

¹ H NMR: (400 MHz, 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, 4.0 Hz, 12H).

³¹ P NMR: (160 MHz, CDCl3) δ 148.02.

Example 2.2 Synthesis of the precursor of M03 ( 2 - cyanoethyl (( tetrahydro -2H -pyran - 4- yl ) methyl ) diisopropylamidophosphite )

To a solution of compound 16-1 (1.16 g, 10 mmol) in anhydrous DCM (20 mL) was added 1H-imidazole-4,5-dicarbonitrile (0.6 g, 5 mmol). The reaction mixture was cooled to 0 °C, and then a solution of compound 1-2 (3 g, 10 mmol) in DCM (5 mL) was added to the mixture. The mixture was stirred at 25 °C under _N2 for 16 hours. The mixture was filtered and concentrated to remove DCM. The residue was diluted with hexane and purified by silica gel chromatography (eluted with hexane) to obtain the prodrug of M03 .

¹ H NMR: (400 MHz, CDCl3) δ 3.98-3.94 (m, 2H), 3.88-3.73 (m, 2H), 3.61-3.34 (m, 6H), 2.62 (t, J = 8.0 Hz, 2H), 1.88-1.78 (m, 1H), 1.66-1.59 (m, 2H), 1.39-1.31 (m, 2H), 1.17 (dd, J = 8.0, 4.0Hz, 12H).

³¹ P NMR: (160 MHz, CDCl3) δ 147.82.

Examples 3 Preparation of double-stranded oligonucleotides

Oligonucleotide synthesis was performed on a MerMade 12 synthesizer using a standard solid phase synthesis protocol. All phosphoamidates were purchased from Hongene Biotech, 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). 2'-OMe-rU was dissolved in a mixed solvent of DMF/acetonitrile (1:4, v/v), while all other phosphoramidites were dissolved in acetonitrile and a molecular sieve (3 Å) was added. Unylinker CPG was used as a solid support unless otherwise stated. The synthetic cycle consists of four separate steps, namely detritylation, coupling, oxidation (or sulfidation), and capping. 5-Ethylthio-1H-tetrazole (ETT, 0.25 M in acetonitrile) was used as an activator solution. The phosphorothioate bond was introduced using a 0.05 M solution of 3-((N,N-dimethylaminomethylene)amino)-3H-1,2,4-dithiazole-5-thione (DDTT) in pyridine. After the solid phase synthesis was completed, the phosphate protecting group (2-cyanoethyl) was removed using 20% diethylamine (DEA) in ethyl solution for 1 hour. Cleavage from the solid support and nucleobase deprotection (C&D) were performed in NH ₄ OH at 65 °C for 5 hours. The crude oligonucleotide solution was concentrated by centrifugal evaporation 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. The appropriate fractions were combined and freeze-dried to obtain the purified product.

Single-stranded oligonucleotides are ligated to produce double-stranded oligonucleotides.

Prepare single-stranded oligonucleotides to be annealed in sterile RNase-free _H2O .

Annealing reaction system: Place the mixture in a 95°C water bath for 10 minutes (20 minutes for ≥100 nmol), then quickly place it in a 60°C water bath and cool, then freeze-dry to obtain double-stranded oligonucleotides and store at low temperature.

Complementary double-stranded oligonucleotides were prepared by combining equal molar single-stranded oligonucleotide solutions.

Examples 4 Biological Activity Analysis of Double-Stranded Oligonucleotides

Examples 4.1 Huh7 Cell transfection analysis

Dilute the cell suspension with 10% FBS DMEM to a final cell density of 2 × 10 ⁵ cells/mL. Add 20 μL/well of siRNA-RNAiMAX complex to a 96-well plate, and then add the cell suspension (100 μL/well). Incubate the cells at 37°C and 5% CO ₂ for 24 hours.

Intracellular RNA was isolated using the RNeasy kit (Qiagen-74182) according to the instruction manual. RNA was reverse transcribed using HiScript® III RT SuperMix for qPCR (+gDNA wiper) according to the instruction manual. The expression of the target gene was quantified using qPCR with gene-specific primers (e.g., with commercially available TaqMan® assays or primers as shown below). GAPDH was also measured as a housekeeping gene. The samples were processed in an Applied Biosystems Fast Real-Time PCR System for TaqMan cycling, cycling according to the following thermal profile: 95°C for 10 min, followed by 95°C for 15 sec, 60°C for 1 min, for 40 cycles.

Commercially available TaqMan® assays: ^TaqMan® Assays ID Suppliers VEGFA Thermo Hs00900055_m1 Thermo Fisher APOC3 Thermo Hs00163644_m1 Thermo Fisher PNPLA3 Thermo Hs00228747_m1 Thermo Fisher Antithrombin (Serpinc1) Thermo Hs00166654_m1 Thermo Fisher TMPRSS6 Thermo Hs00542191_m1 Thermo Fisher AGT Thermo Hs01586213_m1 Thermo Fisher PCSK9 Thermo Hs00545399_m1 Thermo Fisher ANGPTL3 Thermo Hs00205581_m1 Thermo Fisher C3 Thermo Hs00163811_m1 Thermo Fisher C5 Thermo Hs00156197_m1 Thermo Fisher TTR Thermo Hs00174914_m1 Thermo Fisher

Other introductions: Name Sequence ( 5' -3 ' ) INHBE-F (dT)(dC)(dA)(dC)(dA)(dG)(dA)(dC)(dT)(dC)(dC)(dA)(dC)(dT)(dT)(dC)(dA)(dG) (dC)(dC) INHBE-R (dA)(dA)(dA)(dG)(dA)(dG)(dT)(dG)(dC)(dC)(dA)(dG)(dG)(dA)(dA)(dG)(dG)(dG) (dT) INHBE- Probe (dC)(dA)(dG)(dC)(dC)(dA)(dC)(dA)(dG)(dG)(dC)(dG)(dG)(dG)(dC)(dA)(dT)(dG) (dG)(dT)(dA)(dC)(dA)(dG)(dG) KHK-F (dG)(dA)(dG)(dT)(dC)(dG)(dG)(dA)(dG)(dA)(dC)(dG)(dC)(dA)(dG)(dG)(dT)(dG) KHK-R (dT)(dT)(dT)(dT)(dA)(dA)(dA)(dG)(dC)(dG)(dC)(dG)(dG)(dC)(dT)(dA)(dA)(dG) (dC)(dG) KHK-Probe (dG)(dG)(dA)(dG)(dA)(dG)(dT)(dG)(dC)(dG)(dG)(dG)(dG)(dC)(dA)(dA)(dG)(dT) (dA)(dG)(dC)(dG) SOD1-F (dC)(dC)(dT)(dA)(dG)(dC)(dG)(dA)(dG)(dT)(dT)(dA)(dT)(dG)(dG)(dC)(dG)(dA) (dC)(dG) SOD1-R (dC)(dC)(dA)(dC)(dA)(dC)(dC)(dT)(dT)(dC)(dA)(dC)(dT)(dG)(dG)(dT)(dC)(dC) (dA)(dT) APP-F (dC)(dG)(dC)(dA)(dA)(dG)(dC)(dA)(dG)(dT)(dG)(dC)(dA)(dA)(dG)(dA)(dC)(dC) APP-R (dT)(dC)(dA)(dG)(dG)(dA)(dA)(dC)(dG)(dA)(dG)(dA)(dA)(dG)(dG)(dG)(dC)(dA) (dT)(dC) APP-Probe (dT)(dC)(dC)(dC)(dC)(dA)(dC)(dT)(dT)(dT)(dG)(dT)(dG)(dA)(dT)(dT)(dC)(dC) (dC)(dT)(dA)(dC)(dC)(dG) ANGPTL4-1F (dG)(dC)(dT)(dG)(dG)(dG)(dT)(dC)(dT)(dG)(dG)(dA)(dG)(dA)(dA)(dG)(dG)(dT) ANGPTL4-1R (dG)(dA)(dG)(dA)(dA)(dC)(dT)(dG)(dC)(dA)(dG)(dC)(dA)(dA)(dC)(dT)(dC)(dG) (dG)

surface 2 : Huh 7 Knockdown activity of test compounds in transfection assays Compound IC ₅₀ （ nM ） Target SS （ 5 '-3' ） AS ( 5' -3 ' ) ds1 ＜0.0024 APOC3 ACGGGACAGUAUUCUCAGUGA ACGAUCACUGAGAAUACUGUCCCGUUU ds2 0.060 INHBE ACCAGUCGUCCCAGAAUAACU CUGAAGUUAUUCUGGGACGACUGGUCU ds3 0.303 PNPLA3 UGAGCAAGAUUUGCAACUUGA ACGAUCAAGUUGCAAAUCUUGCUCAUG ds4 0.049 Antithrombin (Serpinc1) GGUUAACACCAUUUACUUCAA CCGAUUGAAGUAAAUGGUUAACCAG ds5 0.032 APP GGCUACGAAAAUCCAACCUAA UCGAUUAGGUUGGAUUUUCGUAGCCGU ds6 0.057 SOD1 CACUUUAAUCCUCUAUCCAGA AGGAUCUGGAUAGAGGAUUAAAGUGAG ds7 0.023 TMPRSS6 CCUUUGGAAUAAAGCUGCCUU CCGUAAGGCAGCUUUAUUCCAAAGGGC ds8 0.090 KHK GCAGGAAGCACUGAGAUUCGU CCGAACGAAUCUCAGUGCUUCCUGCAC ds9 ＜0.0024 PCSK9 CUAGACCUGUUUUGCUUUUGU UGGAACAAAAGCAAAACAGGUCUAGAA ds10 0.047 VEGF-A AUGCAGAUUAUGCGGAUCAAA ACGAUUUGAUCCGCAUAAUCUGCAUGG ds11 0.603 ANGPTL3 GCUCACAUAUUUGAUCAGUA CCGAUACUGAUCAAAUAUGUUGAGCUU ds12 0.003 C3 GAGCCGUUCUCUACAAUUACU GCGAAGUAAUUGUAGAGAACGGCUCGG ds13 0.432 C5 AAGCAAGAUAUUUUUAUAAUA ACGUUAUUAUAAAAAUAUCUUGCUUUU ds14 0.002 TTR UGGGAUUUCAUGUAACCAAGA CCGAUCUUGGUUACAUGAAAUCCCAUC

surface 3 : Huh 7 Knockdown activity of test compounds in transfection assays Target genes: AGT AS(5'-3'): (invAB)*c ZY(X2) *Gf*uac(Tgn)cucauugUfgGfaugac*g*a SS(5'-3')：g*u*caucCfaCfAfAfugagagUfa*c* (X1) Compound IC ₅₀ （ nM ） X1 X2 Y Z ds15 0.04 a u u u ds16 0.02 a u u a ds17 0.06 a u u c ds18 0.02 a u u g ds19 0.04 a u a u ds20 0.02 a u a a ds21 0.05 a u a c ds22 0.01 a u a g ds23 0.02 u a u u ds24 0.02 u a u a ds25 0.03 u a u c ds26 0.01 u a u g ds27 0.03 u a a u ds28 0.02 u a a a ds29 0.05 u a a c ds30 0.01 u a a g

surface 4 : Huh 7 Knockdown activity of test compounds in transfection assays Target genes: ANGPTL3 AS(5-3): (invAB)*c ZY(X2) *A*c*UfgAfuCfaAfaUfaUfgUfuGfaGfca*a SS(5-3): (invAB)*gcucaacaUfAfUfuugaucagu (X1) Compound IC ₅₀ （ nM ） X1 X2 Y Z ds47 0.27 a u u u ds48 0.15 a u u a ds49 0.26 a u u c ds50 0.10 a u u g ds51 0.26 a u a u ds52 0.19 a u a a ds53 0.30 a u a c ds54 0.14 a u a g ds55 0.18 u a u u ds56 0.16 u a u a ds57 0.29 u a u c ds58 0.10 u a u g ds59 0.23 u a a u ds60 0.13 u a a a ds61 0.20 u a a c ds62 0.08 u a a g

surface 5 : Huh 7 Knockdown activity of test compounds in transfection assays Target genes: AGT AS(5-3): (M06)* NZY(X2) *Gfuac(Tgn)cucauugugGfaugac*g*a SS(5-3)：g*u*caucCfaCfAfAfugagagua*c* (X1) Compound and ds67 of Relative IC ₅₀ X1 NZY(X2) ds64 1.14 u cgaa ds65 1.31 u cgua ds66 1.09 u cgga ds67 1.00 u cgca ds68 1.08 u ggaa ds69 2.59 u ugaa ds70 5.65 g cgac ds71 5.75 c cgag ds72 1.30 u ccgccgaa Relative _IC50 = (dsX) / ds67

surface 6 : Huh 7 Knockdown activity of test compounds in transfection assays Target genes: PCSK9 AS(5-3): NZY(X2) CfaAfAfAfgCfaAfaAfcAfgGfuCfuag*a*a SS(5-3): c*u*agacCfuGfu(dT)uugcuuuu*g* (X1) Compound and ds74 of Relative IC ₅₀ X1 NZY(X2) ds74 1.00 u (M06)*cgaa* ds75 2.04 u (M06)*cgua* ds76 1.09 u (M06)*cgga* ds77 1.74 u (M06)*cgca* ds78 1.64 u (M06)*agaa* ds79 2.79 u (M06)*ggua* ds80 5.43 g (M06)*cgac* ds81 5.83 c (M06)*cgag* ds82 1.23 u (M06)*gcuucgaa* Relative _IC50 = (dsX) / ds74

As shown in the above data, the dsRNA reagents disclosed herein show effective target gene expression inhibitory activity. For dsRNA reagents cross-targeting different genes, dsRNA reagents with purine (preferably guanine) at Z show better inhibitory activity than those with pyrimidine at Z. It can be seen that dsRNA reagents with adenine or uracil at X1/X2 show effective inhibitory activity, while dsRNA reagents with cytosine or guanine at X1/X2 show relatively moderate activity.

Surprisingly, dsRNA reagents with a blocking group (e.g., M03, M06, or invAB) or a ligand (e.g., VSDL-03A) at the 5' end of the antisense strand showed inhibitory activity against target gene expression, whereas other reported dsRNA reagents lost their activity significantly or completely when the 5' end of the antisense strand was linked to the blocking group or ligand. Although not wishing to be bound by theory, one of the plausible explanations may be that the blocking group or ligand is cleaved together with the cleavage region of the dsRNA disclosed herein, which then provides RNAi effects.

Examples 4.2 PHH Free photography KD analyze

Primary human hepatocytes (PHH) were mixed with appropriate medium, and the cell suspension was then adjusted to a final cell density of 6 × 10 ⁵ cells/mL. dsRNA was diluted and added to a collagen-I-coated 96-well plate at 10 μL/well, and the cell suspension was subsequently added (90 μL/well). Cells were cultured at 37°C and 5% CO ₂ for 48 hours.

Intracellular RNA was isolated using the RNeasy kit (Qiagen-74182) according to the instruction manual. RNA was reverse transcribed using HiScript® III RT SuperMix for qPCR (+gDNA wiper) according to the instruction manual. The expression of the target gene was quantified using qPCR with gene-specific primers (e.g., with commercially available TaqMan® assays or primers as shown below). GAPDH was also measured as a housekeeping gene. The samples were processed in an Applied Biosystems Fast Real-Time PCR System for TaqMan cycling, cycling according to the following thermal profile: 95°C for 10 min, followed by 95°C for 15 sec, 60°C for 1 min, for 40 cycles.

Commercially available ^TaqMan® assays: ^TaqMan® Assays ID Suppliers AGT Thermo Hs01586213_m1 Thermo Fisher ANGPTL3 Thermo Hs00205581_m1 Thermo Fisher Antithrombin (Serpinc1) Thermo Hs00166654_m1 Thermo Fisher PCSK9 Thermo Hs00545399_m1 Thermo Fisher

Other introductions: Name Sequence ( 5' -3' ) C3-F (dG)(dT)(dG)(dC)(dT)(dG)(dC)(dC)(dC)(dA)(dG)(dT)(dT)(dT)(dC)(dG)(dA)(dG) (dG)(dT)(dC)(dA)(dT)(dA) C3-R (dT)(dC)(dC)(dC)(dG)(dA)(dA)(dG)(dA)(dT)(dG)(dA)(dC)(dA)(dA)(dA)(dG)(dG) (dC)(dA)(dG)(dT)(dT) C3- Probe (dT)(dC)(dC)(dA)(dC)(dT)(dT)(dT)(dC)(dT)(dT)(dC)(dC)(dC)(dG)(dT)(dA)(dG)(dA)(dG)(dG)(dA)(dA)(dC)(dC)(dT) SOD1-F (dC)(dC)(dT)(dA)(dG)(dC)(dG)(dA)(dG)(dT)(dT)(dA)(dT)(dG)(dG)(dC)(dG)(dA) (dC)(dG) SOD1-R (dC)(dC)(dA)(dC)(dA)(dC)(dC)(dT)(dT)(dC)(dA)(dC)(dT)(dG)(dG)(dT)(dC)(dC) (dA)(dT) INHBE-F (dT)(dC)(dA)(dC)(dA)(dG)(dA)(dC)(dT)(dC)(dC)(dA)(dC)(dT)(dT)(dC)(dA)(dG) (dC)(dC) INHBE-R (dA)(dA)(dA)(dG)(dA)(dG)(dT)(dG)(dC)(dC)(dA)(dG)(dG)(dA)(dA)(dG)(dG)(dG) (dT) INHBE-Probe (dC)(dA)(dG)(dC)(dC)(dA)(dC)(dA)(dG)(dG)(dC)(dG)(dG)(dG)(dC)(dA)(dT)(dG) (dG)(dT)(dA)(dC)(dA)(dG)(dG) APP-F (dC)(dG)(dC)(dA)(dA)(dG)(dC)(dA)(dG)(dT)(dG)(dC)(dA)(dA)(dG)(dA)(dC)(dC) APP-R (dT)(dC)(dA)(dG)(dG)(dA)(dA)(dC)(dG)(dA)(dG)(dA)(dA)(dG)(dG)(dG)(dC)(dA) (dT)(dC) APP-Probe (dT)(dC)(dC)(dC)(dC)(dA)(dC)(dT)(dT)(dT)(dG)(dT)(dG)(dA)(dT)(dT)(dC)(dC)(dC)(dT)(dA)(dC)(dC)(dG)

surface 7 : Knockdown activity of test compounds in a free uptake assay in primary human hepatocytes Compound SS(5'-3') AS(5'-3') AGT _EC50 (pM) ds83 g*u*caucCfaCfAfAfugagagUfacu[L96] (invAB)*cgaa*Gfuac(Tgn)cucauugUfgGfaugac*g*a 564 ds84 g*u*caucCfaCfAfAfugagagUfacu[L96] (invAB)*CGAa*Gfuac(Tgn)cucauugUfgGfaugac*g*a 50.4 ds85 g*u*caucCfaCfAfAfugagagUfacu[L96] (invAB)*CfGfAfa*Gfuac(Tgn)cucauugUfgGfaugac*g*a 56.2 ds86 g*u*caucCfaCfAfAfugagagUfacu[L96] (invAB)*CfGfaa*Gfuac(Tgn)cucauugUfgGfaugac*g*a 52.3 ds87 g*u*caucCfaCfAfAfugagagUfacu[L96] (invAB)*Cfgaa*Gfuac(Tgn)cucauugUfgGfaugac*g*a 74.3 ds88 g*u*caucCfaCfAfAfugagagUfacu[L96] (invAB)*CfGfAa*Gfuac(Tgn)cucauugUfgGfaugac*g*a 76.1 ds89 g*u*caucCfaCfAfAfugagagUfacu[L96] (invAB)*CfGf(dA)a*Gfuac(Tgn)cucauugUfgGfaugac*g*a 70 ds90 g*u*caucCfaCfAfAfugagagUfacu[L96] (invAB)*cgAa*Gfuac(Tgn)cucauugUfgGfaugac*g*a 126.1 ds91 g*u*caucCfaCfAfAfugagagUfacu[L96] (invAB)*cg(dA)a*Gfuac(Tgn)cucauugUfgGfaugac*g*a 636.9 ds95 g*u*caucCfaCfAfAfugagagUfacu[L96] c*g*aaGfuac(Tgn)cucauugUfgGfaugac*g*a Inactive ds96 g*u*caucCfaCfAfAfugagagUfacu[L96] c*Gf*AfaGfuac(Tgn)cucauugUfgGfaugac*g*a 1400 ds97 g*u*caucCfaCfAfAfugagagUfacu[L96] cgaa*Gf*uac(Tgn)cucauugUfgGfaugac*g*a 130 ds99 g*u*caucCfaCfAfAfugagagUfacu[L96] (M06)*CfGfaa*Gfuac(Tgn)cucauugUfgGfaugac*g*a 10.5 ds100 g*u*caucCfaCfAfAfugagagUfacu[L96] (M03)*CfGfaa*Gfuac(Tgn)cucauugUfgGfaugac*g*a 21.3 ds101 g*u*caucCfaCfAfAfugagagUfaca[L96] u*Gf*uac(Tgn)cucauugUfgGfaugac*g*a 99.5

As shown in the table above, the cleavage zone with both 2'-F and 2'-OMe showed effective inhibitory activity. In a preferred embodiment, X2 and Y are modified with 2'-OMe, and Z and N1 are modified with 2'-F.

It can also be seen that modifications of the internucleotide bonds between X2 and Y or Y and Z reduce the inhibitory activity of the dsRNA.

surface 8 ：Free uptake analysis of primary human hepatocytes Compound SS(5'-3') AS(5'-3') EC ₅₀ (nM) Target genes: INHBE ds102 a*c*cagucgUfCfCfcagaauaacu[L96] a*(dG)*uu(dA)u(dT)cugg(dG)aCfgacuggu*c*u 2.41 ds103 a*c*cagucgUfCfCfcagaauaacu[L96] (invAB)*CfGfaa*(dG)uu(dA)u(dT)cugg(dG)aCfgacuggu*c*u 0.19 Target genes: C5 ds104 a*a*GfcAfaGfaUfAfUfuUfuuAfuAfaua[L96] u*Af*UfuAfuaAfaAfauaUfcUfuGfcuu*u*u 0.44 ds105 a*a*GfcAfaGfaUfAfUfuUfuuAfuAfaua[L96] (invAB)*CfGfau*AfUfuAfuaAfaAfauaUfcUfuGfcuu*u*u 0.18 Target gene: antithrombin ( Serpinc1 ） ds106 Gf*g*UfuAfaCfaCfCfAfuUfuAfcUfuCfaAf[L96] u*Uf*gAfaGfuAfaAfuggUfgUfuAfaCfc*a*g 1.2 ds107 Gf*g*UfuAfaCfaCfCfAfuUfuAfcUfuCfaAf[L96] (invAB)*CfGfau*UfgAfaGfuAfaAfuggUfgUfuAfaCfc*a*g 0.33 Target genes: C3 ds108 g*a*gccgUfuCfUfCfuacaauuacu[L96] a*Gf*uaaUfuGfUfagagAfaCfggcuc*g*g 1.5 ds109 g*a*gccgUfuCfUfCfuacaauuacu[L96] (M06)*CfGfaa*GfuaaUfuGfUfagagAfaCfggcuc*g*g 0.68 Target genes: APP ds116 g*g*cuacga(dA)a(dA)uccaaccuaa[L96] u*Uf*aggu(Tgn)ggau(dT)uUfc(dG)uagcc*g*u 0.43 ds117 g*g*cuacga(dA)a(dA)uccaaccuaa[L96] (invAB)*CfGfau*Ufaggu(Tgn)ggau(dT)uUfc(dG)uagcc*g*u 0.61

As shown in the above table, the dsRNA reagents disclosed herein having a 5' extension on the antisense strand and having a cleavage region exhibited relatively more potent inhibitory activities against various genes and targets compared to the parental dsRNA reagent without a 5' extension.

Examples 4.3 In vivo mice HDI Model

On day 0, mice (BALB/C, 6-7 weeks, female) were given vehicle or test siRNA (5 mg/kg). On day 3, all mice were injected with 8% of the mouse body weight of plasmid DNA solution through the tail vein within 5 seconds (injection volume (mL) = mouse body weight (g) × 8%). The amount of plasmid injected into each mouse was 10 μg. All animals were sacrificed on day 4. Liver tissues from all groups were collected for target mRNA analysis by qPCR. RNA was reverse transcribed into cDNA using HiScript® III RT SuperMix (+gDNA wiper) (Vazyme-R323) for qPCR according to the manual. cDNA was quantified by qPCR. NEO (sequence information is shown in the table below) mRNA was also detected as an internal control. Name Primer sequence ( 5'-3' ) NEO-F (dG)(dA)(dT)(dG)(dG)(dA)(dT)(dT)(dG)(dC)(dA)(dC)(dG)(dC)(dA)(dG)(dG)(dT)(dT)(dC)(dT)(dC) NEO-R (dG)(dA)(dA)(dC)(dA)(dC)(dG)(dG)(dC)(dG)(dG)(dC)(dA)(dT)(dC)(dA)(dG)(dA)(dG)(dC) NEO-Probe (dC)(dG)(dC)(dT)(dT)(dG)(dG)(dG)(dT)(dG)(dG)(dA)(dG)(dA)(dG)(dG)(dC)(dT)(dA)(dT)(dT)(dC)(dG)(dG) (dC)(dT)(dA)(dT)(dG)(dA)

Test compound: Compound SS (5'-3') AS ( 5'-3' ) ds100 g*u*caucCfaCfAfAfugagagUfacu[L96] (M03)*CfGfaa*Gfuac(Tgn)cucauugUfgGfaugac*g*a ds101 g*u*caucCfaCfAfAfugagagUfaca[L96] u*Gf*uac(Tgn)cucauugUfgGfaugac*g*a

The results are shown in Figure 1.

As shown in FIG. 1 , the dsRNA reagent disclosed herein having a 5′ extension on the antisense strand and a cleavage region exhibited significantly more effective inhibition efficacy on the target gene compared to the parent dsRNA reagent without a 5′ extension.

Examples 4.4 In vivo rat CNS Targeted gene knockout model ( IT injection)

Test compounds were formulated up to 20 mg/mL in 10 mM PBS (pH 7.4) and administered as 50 μL IT injections (0.9 mg/dose) by lumbar puncture. Tail wiggles or tail tip twitches were considered a sign of successful procedure. Following dosing, anesthesia maintenance devices were removed and animals were returned to cages. Animals were sacrificed on day 14 and tissue samples were collected for qPCR analysis.

Measurement of SOD1 mRNA levels by qPCR

RNA from tissue samples was extracted by an automated nucleic acid extraction system. Samples were transferred to fresh RNase-free tubes for cDNA synthesis and qPCR. Introduction Sequence (5'-3') rSOD-1 F (dC)(dT)(dC)(dA)(dG)(dG)(dA)(dG)(dA)(dG)(dC)(dA)(dT)(dT)(dC)(dC)(dA)(dT)(dC)(dA)(dT)(dT)(dG) rSOD-1 R (dC)(dA)(dA)(dT)(dC)(dA)(dC)(dA)(dC)(dC)(dA)(dC)(dA)(dA)(dG)(dC)(dC)(dA)(dA)(dG)(dC)

Test compound: Target gene: Rat SOD-1 Compound SS ( 5'-3' ) AS ( 5'-3' ) ds110 c*a*uuu(C16U)AfaUfCfCfucacucua*a*a (VPu)*Uf*uagAfgUfGfaggaUfuAfaaaug*a*g ds111 c*a*uuuuAfaUfCfCfucacucua*a*u (VSDL-03A)*CfGfaa*UfuagAfgUfGfaggaUfuAfaaaug*a*g ds112 (VSDL-03A)*c*a*u*u*uuAfaUfCfCfucacucua*a*u (M06)*CfGfaa*UfuagAfgUfGfaggaUfuAfaaaug*a*g ds113 (VSDL-09)*c*a*u*u*uuAfaUfCfCfucacucua*a*u (M06)*CfGfaa*UfuagAfgUfGfaggaUfuAfaaaug*a*g ds114 c*a*uuu(C16U)AfaUfCfCfucacucua*a*a u*Uf*uagAfgUfGfaggaUfuAfaaaug*a*g ds115 c*a*uuu(C16U)AfaUfCfCfucacucua*a*u (invAB)*CfGfaa*Uf*uagAfgUfGfaggaUfuAfaaaug*a*g

The results are shown in Figures 2 and 3.

As shown in Figures 2 and 3, the dsRNA reagent disclosed herein having a 5' extension and a cleavage region and a VSDL ligand on the antisense strand showed comparable inhibitory efficacy on the target gene compared to the parental dsRNA reagent having a 5'VPu modification and C16U. However, compared to the dsRNA reagent having a 5'VPu modification and C16U, the dsRNA reagent disclosed herein showed more tissue-specific target gene knockdown.

Examples 4.5 In vivo mice IVT Model

Topical antibiotics (tobramycin) were applied twice to both eyes, one day before and the next day after IVT injections into mice. On day 0, 1× PBS or siRNA formulated in 1× PBS was administered to both eyes by IVT injection at a dose of 3 μg per dose. All animals were sacrificed on day 7, and whole eyes were sampled for qPCR analysis.

Test compound: Target gene: mTTR Compound SS ( 5'-3' ) AS ( 5'-3' ) ds118 a*a*cag(C16U)GfuUfCfUfugcucuau*a*a (VPu)*Uf*auaGfagcaagaAfcAfcuguu*u*u ds119 a*a*caguGfuUfCfUfugcu*c*u*a*u*a*a (VSDL-03A)*CfGfau*UfauaGfagcaagaAfcAfcuguu*u*u

The results are shown in Figure 4.

As shown in FIG. 4 , the dsRNA reagent disclosed herein having a 5′ extension as well as a cleavage region and a VSDL ligand on the antisense strand exhibited a stronger inhibitory effect on the target gene compared to the parental dsRNA reagent having a 5′VPu modification and C16U.

Examples 4.6 Rat liver tissue homogenization reaction

Exactly 1.00 mg of the test compound was dissolved in 0.961 mL of water to obtain a working solution with a calibration concentration of 1,000,000 ng/mL. The final sample concentration of 10,000 ng/mL was obtained by homogenizing the working solution with 20% rat liver tissue and then incubating at 37°C for 48 hours. 50.0 μL of IS working solution was added to the incubated sample for extraction (phenol/chloroform/isoamyl alcohol = 25/24/1, v/v/v) to perform liquid-liquid extraction. After centrifugation, 300 μL of the supernatant was transferred to solid phase extraction. Then, the sample was transferred to the equilibrated SPE plate, and the SPE plate was washed and eluted. The collected eluate was evaporated. (LC-HRMA: liquid chromatography-high resolution mass spectrometer; LC: Shimadzu, LC-30AD; HRMA: Q Exactive Plus or Q Exactive Focus (Thermo San Jose, CA))

Test compound: Compound SS ( 5'-3' ) AS ( 5'-3' ) ds99 g*u*caucCfaCfAfAfugagagUfacu[L96] (M06)*CfGfaa*Gfuac(Tgn)cucauugUfgGfaugac*g*a

The results are shown in Figure 5. After 48 hours of culture, the observed products included unconsumed test compound (31%), test compound with one nucleotide cleaved at the 3' end (15%), test compound with two nucleotides cleaved at the 3' end and M06 cleavage (18%), test compound with three nucleotides cleaved at the 5' end (15%+5%+10%) and other minor metabolites (5%).

In summary, (1) the only observed 5' cleavage product was the test compound with three nucleotides cleaved at the 5' end; (2) the observed 3' cleavage products included the test compounds with one or two nucleotides cleaved at the 3' end; and (3) the cleavage product of the test compound with three nucleotides cleaved at the 5' end was the major product among all cleavage products.

Examples 4.7 NHP Long-term plasma target gene knockout model

The in vivo target gene knockdown effect of modified siRNA was evaluated in cynomolgus monkeys. Animals (N=2 per group) received a single 2 mg/kg subcutaneous dose of dsRNA test sample on day 1. Blood was obtained on days -1, 2, 7, 14, 28, 42, 56, and 70 after dosing. Circulating AGT levels were quantified using an ELISA (Sino Biological KIT10994) specific for human proangiotensin (and cross-reactive with cynomolgus monkeys) according to the manufacturer's protocol. Data are expressed as a percentage of baseline values and are presented as the mean plus/minus the standard error of the mean.

Test compound: Compound SS ( 5'-3' ) AS ( 5'-3' ) ds86 g*u*caucCfaCfAfAfugagagUfacu[L96] (invAB)*CfGfaa*Gfuac(Tgn)cucauugUfgGfaugac*g*a ds101 g*u*caucCfaCfAfAfugagagUfaca[L96] u*Gf*uac(Tgn)cucauugUfgGfaugac*g*a

The results are shown in Figure 6.

Examples 4.8 Repeated-dose tolerance study in rats

Repeated dose tolerance study in rats. Test compounds (ds86, ds99) were administered subcutaneously once a week at doses higher than 250 mg/kg for 3 weeks (3 doses in total). (300 mg/kg*1, 250 mg/kg*2). Compound SS ( 5'-3' ) AS ( 5'-3' ) ds99 g*u*caucCfaCfAfAfugagagUfacu[L96] (M06)*CfGfaa*Gfuac(Tgn)cucauugUfgGfaugac*g*a ds86 g*u*caucCfaCfAfAfugagagUfacu[L96] (invAB)*CfGfaa*Gfuac(Tgn)cucauugUfgGfaugac*g*a

Results: Clinical observation and pathology plus gross anatomy revealed no abnormalities.

Histopathology:

Kidney: Mild basophilic granules and tubular cell vacuolation were present in both groups.

Liver: Mildly to moderately vacuolated and pigmented hepatocytes and Kupffer cells were present in both groups.

Injection site: Mild to moderate mixed cell inflammation.

Conclusions: The results are consistent with uptake and clearance of GalNAc siRNA administration and are not considered adverse.

without

The drawings cited herein form a part of this specification. The features shown in the drawings only show some embodiments of the present application, rather than all embodiments of the present application, unless the specific description explicitly indicates otherwise, and the reader of this specification should not make the opposite inference.

Figure 1 shows the relative expression of AGT mRNA in liver of the test compounds (ds101 and ds100).

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

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

Figure 4 shows the relative expression of mTTR of the tested compounds (ds118 and ds119).

Figure 5 shows the results of liver tissue homogenate treatment responses to test compounds.

Figure 6 shows the relative plasma AGT content of the test compounds (ds101 and ds86) in the NHP model.

TW202519663A_113127147_SEQL.xml

Claims (56)

A double-stranded oligonucleotide reagent comprising a sense strand and an antisense strand, wherein: The sense strand and the antisense strand form a double-stranded portion having a length of 15 to 27 base pairs and a 5' extension in the antisense strand; The 5' extension is at least three nucleotides in length and can be cleaved from the 3'-most nucleotide in the 5' extension, and the cleaved double-stranded oligonucleotide reagent can silence the target RNA or inhibit the expression of the target gene by RNA interference. The double-stranded oligonucleotide reagent of claim 1, wherein the double-stranded oligonucleotide reagent further comprises a 3' extension in the antisense strand. A double-stranded oligonucleotide reagent as in any of the preceding claims, wherein the double-stranded portion is formed by base pairing between a first segment in the antisense strand and a second segment in the sense strand, wherein the first segment and the second segment have equal lengths. A double-stranded oligonucleotide reagent as claimed in any of the preceding claims, wherein the length of the double-stranded portion is: a) 15 to 25 nucleotide pairs, 15 to 24 nucleotide pairs, 15 to 23 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, 19 to 21 nucleotide pairs; or b) 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. The double-stranded oligonucleotide reagent of any preceding claim, wherein the sense strand has a length of 15 to 35, 16 to 35, 16 to 30, 16 to 27, 16 to 26, 16 to 25, 16 to 21, 17 to 35, 17 to 30, 17 to 25, 17 to 21, 18 to 35, 18 to 30, 18 to 25, 18 to 23, 18 to 21, 19 to 35, 19 to 30, 19 to 25, 19 to 21, 20 to 35, 20 to 30, 20 to 25, 20 to 23, 21 to 35, 21 to 30, 21 to 25, 21 to 23, e.g., 25, 24, 23, 22 or 21 nucleotides (nt). A double-stranded oligonucleotide reagent as in any of the preceding claims, wherein the length of the 5' extension is at least 3, 4, 5, 6, 7 or more nucleotides, and/or the length of the 3' extension is at least 1, 2 or more nucleotides. A double-stranded oligonucleotide reagent as in any of the preceding claims, wherein the first segment comprises a targeting region that is substantially complementary to a portion of the target RNA or target mRNA encoding the target gene. A double-stranded oligonucleotide reagent as claimed in any preceding claim, wherein the 5' extension is capable of enzymatic cleavage from the 3'-most nucleotide in the 5' extension, optionally the 5' extension is capable of specific cleavage by an endonuclease or optionally by a ribonuclease (RNase). A double-stranded oligonucleotide reagent as in any of the preceding claims, wherein the antisense strand comprises a cleavage region, the cleavage region comprising a nucleotide sequence as shown in Formula A: (3'-5') X2-Y-Z, which is capable of cleavage between X2 and Y, wherein X2 is the 5'-most nucleotide of the first fragment, and Y-Z are the two 3'-most nucleotides of the 5' extension. A double-stranded oligonucleotide reagent as in any of the preceding claims, wherein the cleavage region further comprises a fourth nucleotide (N1), the fourth nucleotide being the 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, which is capable of cleaving between X2 and Y. A double-stranded oligonucleotide reagent as in any one of claims 1 to 9, wherein the cleavage region further comprises a third fragment (N), the third fragment comprises at least one nucleotide, wherein N1 is the 3'-most nucleotide of the third fragment, and the cleavage region comprises a nucleotide sequence shown in formula B': (3'-5') X2-Y-Z-N, which is capable of cleaving between X2 and Y, the length of N is 1 to 10 nucleotides, preferably the length of N is 1 to 5 nucleotides, and further preferably the length of N is 1 nucleotide. A double-stranded oligonucleotide reagent as claimed in any one of claims 9 to 11, wherein Z is selected from G or A or a natural or non-natural analogue thereof. A double-stranded oligonucleotide reagent as in any one of claims 9 to 12, wherein Z is selected from G or a natural or non-natural analog thereof. A double-stranded oligonucleotide reagent as claimed in any one of claims 9 to 13, wherein X2 is selected from A or U or a natural or non-natural analogue thereof. A double-stranded oligonucleotide reagent as in any one of claims 9 to 11, wherein the formula A has a nucleotide sequence from 3' to 5' selected from the group consisting of: UUG, UAG, AUG, AAG, UUA, UAA, AUA, AAA, UCG, UGG, ACG, AGG, UCA, UGA, ACA and AGA, or a natural or non-natural analogue thereof. A double-stranded oligonucleotide reagent as claimed in any one of claims 9 to 15, wherein the nucleotide Y is selected from A or U or a natural or non-natural analogue thereof. A double-stranded oligonucleotide reagent as in any one of claims 9 to 16, wherein the formula A has a nucleotide sequence from 3' to 5' selected from the group consisting of: UUG, UAG, AUG, AAG, UUA, UAA, AUA and AAA. A double-stranded oligonucleotide reagent as claimed in any of the preceding claims, wherein at least one nucleotide in the cleavage region is a modified nucleotide, preferably all nucleotides in the cleavage region are modified nucleotides. A double-stranded oligonucleotide reagent as claimed in claim 18, wherein the modified nucleotide has a modified base, a modified sugar or a modified internucleotide bond. A double-stranded oligonucleotide reagent as claimed in claim 18 or 19, wherein: a) the modified base includes 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 sugar includes 2'-modified sugar, 3'-modified sugar and 5'-modified sugar, such as 2'-O-methyl (2'-OMe) modification, 2'-deoxy-2'-fluoro (2'-F) modification, 2'-O-methoxyethyl (2'-O-MOE) modification, 2'-deoxy (2'-d) modification, 5'-morpholine (5'-Mo) modification, unlocking nucleic acid (UNA) modification, glycol nucleic acid (GNA) modification, locking nucleic acid (LNA) modification, tricyclic-DNA (tcDNA) modification, (S)-constrained ethyl bridged nucleic acid ((S)-cEt-BNA) modification, 5'-(E)-vinylphosphonate modification, (VP) modification, 2'-O-C16 modification, conjugation with inverted abasic nucleotide at the 5' or 3' end (invAB), replacement with inverted abasic nucleotide (invAb), replacement with 2,4-difluoromethylphenyl ribonucleotide (rF), replacement with (S)-glycerol nucleic acid, replacement with inosine (I), conjugation with M03 at the 5' or 3' end, conjugation with M06 at the 5' or 3' end, and conjugation with a ligand, such as a GalNAc ligand, a lipophilic ligand, or other ligands targeting a receptor capable of promoting endocytosis of the siRNA conjugate, such as a TfR targeting ligand, an LDL-R targeting ligand, or an integrin targeting ligand, and/or c) The modified internucleotide bonds include methylphosphonate (MP), methoxypropylmethylphosphonate (MOP), phosphorothioate (PS), phosphorodithioate (PS2), phosphoroselenate, phosphorodiselenoate, aniline phosphorothioate, aniline phosphate, phosphoamidate and PNA. A double-stranded oligonucleotide reagent as claimed in any one of claims 4 to 20, wherein: a) at least one of the internucleotide bonds between nucleotides in 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; a) none of the internucleotide bonds between nucleotides in the cleavage region are phosphorothioate bonds; d) at least one of the internucleotide bonds between nucleotides in the cleavage region is a phosphodiester bond; e) the internucleotide bond between X2 and Y is a phosphodiester bond; f) the internucleotide bond between Y and Z is a phosphodiester bond; and/or g) each of the internucleotide bonds between nucleotides in the cleavage region is a phosphodiester bond. A double-stranded oligonucleotide reagent as claimed in any one of claims 4 to 21, wherein: b) at least one nucleotide in the cleavage region is modified with 2'-OMe or 2'-F; c) each nucleotide in the cleavage region is modified with 2'-OMe or 2'-F; d) at least one nucleotide in the cleavage region is modified with 2'-F; e) no more than two nucleotides in the cleavage region are modified with 2'-F; f) Z in the formula A or the formula B is modified with 2'-F, and optionally X2 in the formula A or the formula B is modified with 2'-F; g) N1 in the formula A or the formula B is modified with 2'-F; h) X2 and Y in the formula A or the formula B are both modified with 2'-OMe, and Z and N1 in the formula A or the formula B are both modified with 2'-F; and/or i) X2, Y and Z in the formula A or the formula B are all modified with 2'-OMe, and N1 in the formula A or the formula B is modified with 2'-F. A double-stranded oligonucleotide reagent as in any of the preceding claims, wherein the double-stranded oligonucleotide reagent further comprises at least one phosphorothioate or methylphosphonate internucleotide bond. A double-stranded oligonucleotide reagent as claimed in any of the preceding claims, wherein the double-stranded oligonucleotide reagent further comprises: a) at least one phosphorothioate or methylphosphonate internucleotide bond at position 1 and/or 2 (counted from its 5' end) of the first fragment; and/or b) at least one phosphorothioate or methylphosphonate internucleotide bond at position 1 and/or 2 (counted from its 3' end) of the first fragment. A double-stranded oligonucleotide reagent as in any of the preceding claims, wherein the double-stranded oligonucleotide reagent further comprises: a) 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 its 5' end) of the second fragment; and/or b) 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 its 3' end) of the second fragment. A double-stranded oligonucleotide reagent as in any of the preceding claims, wherein the antisense strand further comprises a blocking group at the 5' end, optionally the blocking group is linked to the 5' end of the 5' extension. A double-stranded oligonucleotide reagent as claimed in claim 26, wherein the blocking group comprises an abamatic residue, a reverse abamatic residue, M03 or M06. A double-stranded oligonucleotide reagent as claimed in claim 26 or 27, wherein the blocking group is linked to the 5' end of the 5' extension via an internucleotide bond, and the internucleotide bond is optionally modified or unmodified. A double-stranded oligonucleotide reagent as claimed in claim 28, wherein the blocking group is linked to the 5' end of the 5' extension via a phosphorothioate bond. A double-stranded oligonucleotide reagent as claimed in any preceding claim, wherein the sense strand is complementary to the antisense strand over its entire length. A double-stranded oligonucleotide reagent as in any one of claims 9 to 30, wherein cleavage at the cleavage region produces a blunt end at the 5' end of the first fragment. A double-stranded oligonucleotide reagent as claimed in any of the preceding claims, wherein: a) each nucleotide of the first segment of the antisense strand is modified; b) each nucleotide of the antisense strand is modified; c) each nucleotide of the second segment of the sense strand is modified; and/or d) each nucleotide of the sense strand is modified. A double-stranded oligonucleotide reagent as claimed in any of the preceding claims, wherein each nucleotide of the antisense strand is modified with 2'-F or with 2'-OMe; and/or each nucleotide of the sense strand is modified with 2'-F or with 2'-OMe. A double-stranded oligonucleotide reagent as claimed in any of the preceding claims, wherein the sense strand further comprises: a) a motif of three consecutive 2'-F modified nucleotides, optionally the motif occurs at positions 9, 10 and 11 counting from the 5' terminal nucleotide of the sense strand; and/or b) a 2'-F modified nucleotide at position 7 and/or 18 counting from the 5' terminal nucleotide of the sense strand; and/or c) an alternating motif at positions 9 to 13 counting from the 5' end, optionally the 2'-F modification and the 2'-OMe modification occur at alternating nucleotides in the motif. A double-stranded oligonucleotide reagent as in any of the preceding claims, wherein the first segment of the antisense strand and/or the second segment of the sense strand further comprises an alternating motif, wherein modifications occur at alternating nucleotides in the motif, optionally 2'-F modifications occur at alternating nucleotides in the motif and/or 2'-OMe modifications occur at alternating nucleotides in the motif. A double-stranded oligonucleotide reagent as in any of the preceding claims, wherein the first segment of the antisense strand and/or the second segment of the sense strand comprises a modification selected from the group consisting of: 2'-O-methyl (2'-OMe) modification, 2'-deoxy-2'-fluoro (2'-F) modification, 2'-O-methoxyethyl (2'-O-MOE) modification, 2'-deoxy (2'-d) modification, 5'-morpholine (5'-Mo) modification, unblocked nucleic acid (UNA) modification, glycol nucleic acid (GNA) modification, Locked nucleic acid (LNA) modification, tricyclic-DNA (tcDNA) modification, (S)-constrained ethyl bridged nucleic acid ((S)-cEt-BNA) modification, 5'-(E)-vinylphosphonate (VP) modification, 2'-O-C16 modification, ligation to inverted abasic nucleotide at the 5' or 3' end (invAB), replacement with inverted abasic nucleotide (invAb), replacement with 2,4-difluorotolyl ribonucleotide (rF), replacement with (S)-glycerol nucleic acid, and replacement with inosine (I). A double-stranded oligonucleotide reagent comprising a structure represented by formula (C): Right shares (5'→3'): Second segment Antisense strand (3'→5'): First Clip 5' extension Formula (C), wherein the first segment in the antisense strand and the second segment in the sense strand form a double-stranded portion by base pairing, wherein the first segment and the second segment have equal lengths; wherein the length of the 5' extension comprises at least three nucleotides; wherein the antisense strand comprises a cleavage region, the cleavage region comprises the 5'-most nucleotide (X2) of the first segment and the 3'-most two nucleotides (YZ) of the 5' extension, and the cleavage region comprises a nucleotide sequence shown in Formula A: (3'-5') X2-YZ, which is capable of cleavage between X2 and Y, and the formula A is as defined in any one of claims 9 to 36; wherein the sense strand is 15 to 35, 15 to 23, 15 to 22, or 15 to 21 nucleotides in length; and wherein the antisense strand is 25 to 35, 26 to 35, 26 to 30, 25 to 27, or 26 to 27 nucleotides in length, and optionally, the antisense strand further comprises a capping group at the 5' end, optionally the capping group is linked to the 5' end of the 5' extension. A double-stranded oligonucleotide reagent as claimed in claim 37, wherein the length of the sense strand is 17 to 23, 17 to 22, 21 to 23 or 17 to 21 nucleotides; and the length of the antisense strand is 25 to 30, 25 to 27 or 26 to 27 nucleotides. The double-stranded oligonucleotide reagent of claim 37, wherein the sense strand and the antisense strand have the following lengths: 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. A double-stranded oligonucleotide reagent comprising a structure represented by formula (D): Right shares (5'→3'): Second segment Antisense strand (3'→5'): 3' extension First Clip 5' extension Formula (D), wherein the first segment in the antisense strand forms a double-stranded portion with the second segment in the sense strand by base pairing, wherein the first segment and the second segment have equal lengths; the length of the 5' extension comprises at least three nucleotides; the antisense strand comprises a cleavage region, the cleavage region comprises the 5'-most nucleotide (X2) of the first segment and the 3'-most two nucleotides (YZ) of the 5' extension, and the cleavage region comprises a nucleotide sequence shown in Formula A: (3'-5') X2-YZ, which can be enzymatically cleaved between X2 and Y, and upon cleavage, the 5' extension is removed from its 3'-most nucleotide (Y), and the formula A is as defined in any one of claims 9 to 36, wherein the sense strand is 15 to 35, 15 to 23, 15 to 22, 15 to 21, 16 to 25, 17 to 23, 18 to 23, 19 to 23, 19 to 21, 20 to 23, 20 to 21, 21 to 23, e.g., 17, 18, 19, 20, 21, 22, or 23 nucleotides in length; and wherein the antisense strand is 25 to 35, 25 to 30, 26 to 35, 26 to 30, 26 to 27, e.g., 25, 26, 27, 28, 29, or 30 nucleotides in length, and optionally the antisense strand further comprises a capping group at the 5' end, optionally the capping group is linked to the 5' end of the 5' extension. A double-stranded oligonucleotide reagent as claimed in 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. The double-stranded oligonucleotide reagent of claim 40, wherein the sense strand and the antisense strand have the following lengths: a) 19 nt and 25 nt, respectively; b) 20 nt and 25 nt, respectively; c) 21 nt and 25 nt, respectively; d) 19 nt and 26 nt, respectively; e) 20 nt and 26 nt, respectively; f) 21 nt and 26 nt, respectively; or g) 21 nt and 27 nt, respectively. The double-stranded oligonucleotide reagent of any of the preceding claims, wherein the double-stranded oligonucleotide reagent further comprises a ligand operably linked to the sense strand or the antisense strand. The double-stranded oligonucleotide reagent of claim 43, wherein the ligand is operably linked to the 5' end of the sense strand or the 5' end of the antisense strand. A double-stranded oligonucleotide reagent as claimed in claim 43 or 44, wherein the ligand is linked to the double-stranded oligonucleotide reagent via a phosphorothioate. A double-stranded oligonucleotide reagent as in any one of claims 43 to 45, wherein the ligand is a GalNAc ligand, a lipophilic ligand or other ligand targeting a receptor capable of promoting endocytosis of siRNA complexes, such as a TfR targeting ligand, an LDL-R targeting ligand or an integrin targeting ligand. A double-stranded oligonucleotide reagent as in any of claims 44 to 46, wherein 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. The double-stranded oligonucleotide reagent of any of the preceding 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, mArlC1, TSHR, CB1 and LPA. A double-stranded oligonucleotide reagent comprises a double-stranded oligonucleotide operably linked to a blocking group, wherein the blocking group comprises M03 or M06. A double-stranded oligonucleotide reagent 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. A double-stranded oligonucleotide reagent as claimed in claim 49 or 50, wherein: 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 a target RNA or mRNA encoding a target gene, and the sense strand is sufficiently complementary to a targeted region of the antisense strand; c) the capping group is bound to the 5' end of the antisense strand; and/or d) the ligand is bound to the 5' end of the antisense strand, the 5' end of the sense strand, or the 3' end of the sense strand; and/or e) the double-stranded oligonucleotide is operably linked to the capping group at one end and to the ligand at the other end, and/or f) This double-stranded oligonucleotide reagent can silence target RNA or inhibit the expression of target genes through RNA interference. A pharmaceutical composition comprising the double-stranded oligonucleotide reagent of any of the preceding claims and a pharmaceutically acceptable carrier. A method for inhibiting the expression of a target gene in an individual in need thereof, the method comprising administering to the individual a pharmaceutically effective amount of a double-stranded oligonucleotide reagent as described in any one of claims 1 to 51 or a pharmaceutical composition as described in claim 52. The method of 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, mArlC1, TSHR, CB1 and LPA. A method for treating a disease or condition in an individual in need thereof, the method comprising administering to the individual a pharmaceutically effective amount of a double-stranded oligonucleotide reagent as described in any one of claims 1 to 51 or a pharmaceutical composition as described in claim 52. The method of claim 55, wherein the disease or condition is associated with the target gene.