Lipid oligonucleotide conjugate and use thereof

Abstract

A lipid oligonucleotide conjugate, an intermediate thereof, the use thereof and a pharmaceutical composition thereof. The lipid oligonucleotide conjugate can be used for the delivery of a variety of non-hepatic organs or tissue, including but not limited to muscles, fat, CNS, lungs, eyes, heart, etc., so as to achieve the treatment of diseases in the non-hepatic field.

Description

Lipid oligonucleotide conjugates and uses thereof Technical Field

[0001] The present invention relates to the delivery of oligonucleotides, in particular to the non-hepatic delivery of oligonucleotides. The present invention particularly relates to lipid-oligonucleotide conjugates, intermediates thereof, uses and pharmaceutical compositions. Background Art

[0002] Nucleic acid drugs are different from traditional small molecule drugs. Their advantages such as target specificity, wide therapeutic range and long-term effectiveness give them unique clinical applications. By utilizing the endocytosis mediated by the GalNAc ligand combined with the asialoprotein receptor (ASGPR), the efficient targeted delivery of nucleic acid drugs such as siRNA and ASO to the liver is perfectly achieved. At present, 6 GalNAc-nucleic acid drugs have been successfully launched on the market, and many drugs are in the clinical stage. However, the non-hepatic delivery of small nucleic acid drugs is still a huge challenge in this field, which also limits the further development of small nucleic acid drugs. Although there have been attempts at non-hepatic delivery of cholesterol nucleic acid conjugates, their low delivery efficiency and the toxicity caused by high-dose administration have limited the clinical application of this method.

[0003] Arrowhead Corporation published a lipid structure code-named LP300 in WO2023 / 245061, but the effect was limited.

[0004]

[0005] Therefore, designing and optimizing lipid structures to achieve non-hepatic delivery of lipid-nucleic acid conjugates through lipid-assisted membrane permeation to treat non-hepatic diseases has important clinical significance. Summary of the Invention

[0006] This invention aims to provide lipid ligands for nucleic acid drug delivery, enabling non-hepatic delivery of small nucleic acid drugs and expanding the scope of non-hepatic disease treatment with small nucleic acid. The lipid-oligonucleotide conjugates provided by this invention can be used for delivery to a variety of non-hepatic organs or tissues, including but not limited to muscle, fat, CNS, lungs, eyes, and heart, achieving treatment of non-hepatic diseases.

[0007] In one aspect, the present invention provides a lipid oligonucleotide conjugate comprising the structure of Formula Ia:

[0008]

[0009] in,

[0010] At least one of R1 and R2 is selected from Ra, -C(O)Ra, -C(O)NHRa, and C(O)ORa, wherein Ra is an optionally substituted C5-30 alkyl or alkenyl group, and

[0011] The other of R1 and R2 is selected from H, Rb, -C(O)Rb, -C(O)NHRb and C(O)ORb, wherein Rb is selected from optionally substituted C1-4 alkyl or alkenyl, optionally substituted C5-30 alkyl or alkenyl, optionally substituted C2-10 alkynyl, optionally substituted C1-10 alkoxy, optionally substituted C3-10 cycloalkyl, optionally substituted 6 to 14 membered aryl, optionally substituted 5 to 18 membered heteroaryl, optionally substituted 5 to 14 membered heterocyclyl;

[0012] The optional substitution is optionally substituted with Rc, wherein Rc is selected from C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 alkoxy, C3-10 cycloalkyl, 6 to 14 membered aryl, 5 to 18 membered heteroaryl, 5 to 14 membered heterocyclyl, halogen, hydroxyl, amino, guanidino, carboxyl, cyano, nitro, C1-3 alkylthio and thiol;

[0013] m1, m2 and m3 are independently an integer of 1 or 2;

[0014] p is 0, 1, or 2;

[0015] stands for oligonucleotide;

[0016] L stands for linker.

[0017] In another aspect, the present invention provides a lipid oligonucleotide conjugate represented by formula Ib,

[0018]

[0019] in,

[0020] At least one of R1 and R2 is selected from Ra, -C(O)Ra, -C(O)NHRa, and C(O)ORa, wherein Ra is an optionally substituted C5-30 alkyl or alkenyl group, and

[0021] The other of R1 and R2 is selected from H, Rb, -C(O)Rb, -C(O)NHRb and C(O)ORb, wherein Rb is selected from optionally substituted C1-4 alkyl or alkenyl, optionally substituted C5-30 alkyl or alkenyl, optionally substituted C2-10 alkynyl, optionally substituted C1-10 alkoxy, optionally substituted C3-10 cycloalkyl, optionally substituted 6 to 14 membered aryl, optionally substituted 5 to 18 membered heteroaryl, optionally substituted 5 to 14 membered heterocyclyl;

[0022] The optional substitution is optionally substituted with Rc, wherein Rc is selected from C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 alkoxy, C3-10 cycloalkyl, 6 to 14 membered aryl, 5 to 18 membered heteroaryl, 5 to 14 membered heterocyclyl, halogen, hydroxyl, amino, guanidino, carboxyl, cyano, nitro, C1-3 alkylthio and thiol;

[0023] L1 is The right side of the formula is connected to L2; L2 is wherein the left side of the formula is connected to L1; the wavy line represents the connection position; and wherein each n is independently an integer from 1 to 10, preferably an integer from 2 to 8, more preferably an integer from 4 to 6;

[0024] represents an oligonucleotide, preferably, the oligonucleotide is an antisense oligonucleotide or the sense strand or antisense strand of an RNAi agent; preferably, the 5' and / or 3' end of the oligonucleotide is connected to L2 via a phosphate group, a phosphorothioate group or a phosphonate group; preferably, the sense strand or antisense strand of the RNAi agent is paired with its antisense strand or sense strand to form a duplex; preferably, the RNAi agent is siRNA; preferably, the oligonucleotide is the sense strand of the siRNA; preferably, the antisense strand of the siRNA has a 5'-VP modification.

[0025] In another aspect, the present invention provides a lipid oligonucleotide conjugate represented by formula Ic,

[0026]

[0027] in,

[0028] R1 and R2 are independently optionally substituted C5-30 alkyl or alkenyl, preferably optionally substituted C12-24 alkyl or alkenyl; preferably optionally substituted C14-22 alkyl or alkenyl; more preferably optionally substituted C14, C15, C16, C18, C20 or C22 alkyl, monounsaturated alkenyl or polyunsaturated alkenyl; the optional substitution is optionally substituted by Rc, wherein Rc is selected from C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 alkoxy, C3-10 cycloalkyl, 6 to 14 membered aryl, 5 to 18 membered heteroaryl, 5 to 14 membered heterocyclyl, halogen, hydroxyl, amino, guanidino, carboxyl, cyano, nitro, C1-3 alkylthio and sulfhydryl;

[0029] L1 is The right side of the formula is connected to L2; L2 is wherein the left side of the formula is connected to L1; the wavy line represents the connection position; and wherein each n is independently an integer from 1 to 10, preferably an integer from 2 to 8, more preferably an integer from 4 to 6;

[0030] represents an oligonucleotide, preferably, the oligonucleotide is an antisense oligonucleotide or the sense strand or antisense strand of an RNAi agent; preferably, the 5' and / or 3' end of the oligonucleotide is connected to L2 via a phosphate group, a phosphorothioate group or a phosphonate group; preferably, the sense strand or antisense strand of the RNAi agent is paired with its antisense strand or sense strand to form a duplex; preferably, the RNAi agent is siRNA; preferably, the oligonucleotide is the sense strand of the siRNA; preferably, the antisense strand of the siRNA has a 5'-VP modification.

[0031] In another aspect, the present invention provides a ligand moiety of Formula II for forming a lipid oligonucleotide conjugate:

[0032]

[0033] in,

[0034] At least one of R1 and R2 is selected from Ra, -C(O)Ra, -C(O)NHRa, and C(O)ORa, wherein Ra is an optionally substituted C5-30 alkyl or alkenyl group, and

[0035] The other of R1 and R2 is selected from H, Rb, -C(O)Rb, -C(O)NHRb and C(O)ORb, wherein Rb is selected from optionally substituted C1-4 alkyl or alkenyl, optionally substituted C5-30 alkyl or alkenyl, optionally substituted C2-10 alkynyl, optionally substituted C1-10 alkoxy, optionally substituted C3-10 cycloalkyl, optionally substituted 6 to 14 membered aryl, optionally substituted 5 to 18 membered heteroaryl, optionally substituted 5 to 14 membered heterocyclyl;

[0036] The optional substitution is optionally substituted with Rc, wherein Rc is selected from C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 alkoxy, C3-10 cycloalkyl, 6 to 14 membered aryl, 5 to 18 membered heteroaryl, 5 to 14 membered heterocyclyl, halogen, hydroxyl, amino, guanidino, carboxyl, cyano, nitro, C1-3 alkylthio and thiol;

[0037] m1, m2 and m3 are independently an integer of 1 or 2;

[0038] p is 0, 1, or 2; and

[0039] represents a linker and the wavy line represents the location of attachment to the rest of the lipid-oligonucleotide conjugate.

[0040] In another aspect, the present invention provides the use of a ligand moiety represented by formula III in the preparation of a lipid oligonucleotide conjugate.

[0041]

[0042] in,

[0043] At least one of R1 and R2 is selected from Ra, -C(O)Ra, -C(O)NHRa, and C(O)ORa, wherein Ra is an optionally substituted C5-30 alkyl or alkenyl group, and

[0044] The other of R1 and R2 is selected from H, Rb, -C(O)Rb, -C(O)NHRb and C(O)ORb, wherein Rb is selected from optionally substituted C1-4 alkyl or alkenyl, optionally substituted C5-30 alkyl or alkenyl, optionally substituted C2-10 alkynyl, optionally substituted C1-10 alkoxy, optionally substituted C3-10 cycloalkyl, optionally substituted 6 to 14 membered aryl, optionally substituted 5 to 18 membered heteroaryl, optionally substituted 5 to 14 membered heterocyclyl;

[0045] The optional substitution is optionally substituted with Rc, wherein Rc is selected from C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 alkoxy, C3-10 cycloalkyl, 6 to 14 membered aryl, 5 to 18 membered heteroaryl, 5 to 14 membered heterocyclyl, halogen, hydroxyl, amino, guanidino, carboxyl, cyano, nitro, C1-3 alkylthio and thiol;

[0046] m1, m2 and m3 are independently an integer of 1 or 2;

[0047] p is 0, 1, or 2; and

[0048] The wavy line represents the location of attachment to the rest of the lipid-oligonucleotide conjugate.

[0049] Other aspects of the present invention will become apparent from the detailed description of the specification which follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] FIG1 . Inhibitory effects of lipid siRNA conjugates D1 and D2 synthesized according to an example of the present invention on SOD1 mRNA expression in quadriceps femoris.

[0051] FIG2 . Inhibitory effects of lipid siRNA conjugates D1 and D2 synthesized according to an example of the present invention on SOD1 mRNA expression in epididymal adipose tissue. DETAILED DESCRIPTION

[0052] definition

[0053] As used herein, "oligonucleotide" refers to a nucleotide sequence that forms a nucleotide chain via internucleotide connection, wherein each nucleoside and internucleotide connection can be modified or unmodified. Unless otherwise indicated, an oligonucleotide consists of 12-30 connected nucleotides. As used herein, an oligonucleotide can be an "antisense oligonucleotide" or "ASO," which refers to a single-stranded oligonucleotide that can specifically bind to a target RNA (such as mRNA) sequence and regulate protein expression. In addition, an oligonucleotide can also be one of the chains of an RNAi agent, such as the sense strand or antisense strand of an siRNA, in which case the oligonucleotide preferably forms a complete RNAi agent with the other parts of the RNAi agent. For example, when the oligonucleotide is the sense strand of an siRNA, it is preferred that the oligonucleotide is paired with the antisense strand of the siRNA to form a duplex (i.e., siRNA). Oligonucleotides include modified or unmodified oligonucleotides.

[0054] "Internucleotide linkage" means a covalent linkage between adjacent nucleotides in an oligonucleotide. As used herein, "modified internucleotide linkage" means any internucleotide linkage other than a phosphodiester internucleotide linkage. A "phosphorothioate internucleotide linkage" is a modified internucleotide linkage in which one of the non-bridging oxygen atoms of the phosphodiester internucleotide linkage is replaced by a sulfur atom.

[0055] "Deoxy region" means a region of 5-12 contiguous nucleotides in which at least 70% of the nucleosides are 2'-β-D-deoxynucleosides. In certain embodiments, each nucleoside is selected from 2'-β-D-deoxynucleosides, bicyclic nucleosides, and 2'-substituted nucleosides. In certain embodiments, the deoxy region supports RNase H activity. In certain embodiments, the deoxy region is a gap or internal region of a gapmer.

[0056] "Gap polymer" means an antisense oligonucleotide comprising an internal region, wherein the internal region is located between an external region having one or more nucleosides, has a plurality of nucleosides that support RNase H cleavage, wherein the nucleosides constituting the internal region are chemically different from the one or more nucleosides constituting the external region. The internal region can be referred to as a "gap", and the external region can be referred to as a "wing". The internal region is a deoxy region. The position of the internal region or gap refers to the order of the nucleosides in the internal region and is counted from the 5' end of the internal region. Unless otherwise indicated, "gap polymer" refers to a sugar motif. In certain embodiments, each nucleoside in the gap is a 2'-β-D-deoxynucleoside. In certain embodiments, the gap is included in a 2'-substituted nucleoside at position 1, 2, 3, 4 or 5 of the gap, and the remaining nucleosides in the gap are 2'-β-D-deoxynucleosides. As used herein, the term "MOE gap polymer" indicates a gap polymer having a gap comprising 2'-β-D-deoxynucleosides and a wing comprising 2'-MOE nucleosides. As used herein, the term "mixed-wing gapmer" refers to a gapmer having wings comprising modified nucleosides comprising at least two different sugar modifications. Unless otherwise indicated, a gapmer may comprise one or more modified internucleotide linkages and / or modified nucleobases, and such modifications do not necessarily follow the sugar-modified gapmer pattern.

[0057] As used herein, the term "RNAi agent" refers to an agent comprising an RNA molecule that can downregulate the expression of a target gene by an RNA interference mechanism when introduced into a cell. The term RNAi agent includes modified or unmodified RNAi agents. RNA interference refers to a process in which a nucleic acid molecule induces the cutting and degradation of a target RNA molecule (such as an mRNA molecule) in a sequence-specific manner, such as by an RNA-induced silencing complex (RISC) pathway. RNAi agents include siRNA, shRNA, and DNA / RNA hybrid molecules herein, sometimes also collectively referred to as double-stranded RNA (dsRNA), which include two antiparallel continuous nucleotide chains that are fully complementary to each other to hybridize to form a double-stranded region. "Hybridization" refers to the pairing of complementary polynucleotides, typically by hydrogen bonds (such as Watson-Crick hydrogen bonds, Wobble hydrogen bonds, Hoogsteen hydrogen bonds, or reverse Hoogsteen hydrogen bonds) between complementary bases in two polynucleotides. "Double-stranded region" refers to a region in two complementary or substantially complementary polynucleotides that form base pairs by hybridization, thereby forming a double chain between the two polynucleotide chains.

[0058] The term "antisense strand" refers to the strand of a dsRNA that contains a region that is substantially complementary to the target sequence. The term "positive strand" or "sense strand" refers to the strand of a dsRNA that contains a region that is substantially complementary to the antisense strand region as defined herein. The term "substantially complementary region" refers to a region that is fully complementary or incompletely complementary. When the complementary region is not fully complementary to the target sequence, mismatches may be located in the interior or terminal regions of the molecule. Typically, the most tolerable mismatches are located in the terminal regions, for example, 5, 4, 3, or 2 at the 5' and / or 3' ends of the dsRNA.

[0059] "siRNA" refers to a nucleic acid that forms double-stranded RNA that has the ability to reduce or inhibit the expression of a target gene when the siRNA and the target gene are present in the same cell. siRNAs are typically about 15 to about 30 base pairs in length, most typically about 19 to 25 base pairs in length, e.g., 19, 20, 21, 22, 23, 24, or 25 nucleotide pairs in length.

[0060] shRNA refers to a short hairpin RNA that includes two short inverted repeats and an intermediate stem-loop structure connecting the two. The stem-loop may contain at least one unpaired nucleotide, for example, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides. The stem-loop may be 10 or fewer nucleotides. The stem-loop may be 8 or fewer unpaired nucleotides. The stem-loop may be 4 to 10 unpaired nucleotides. The stem-loop may be 4 to 8 nucleotides.

[0061] The two substantially complementary chains of a dsRNA need not but may also be covalently linked. The maximum number of base pairs is the number of nucleotides in the shortest chain of the dsRNA minus any overhangs present in the duplex. In addition to the duplex structure, the dsRNA may also comprise one or more nucleotide overhangs. Overhanging nucleotides refer to one or more unpaired nucleotides that extend beyond the double-stranded region at the end of the chain. When the 3' end of a chain extends beyond the 5' end of the other chain, or when the 5' end of a line extends beyond the 3' end of the other line, nucleotide overhangs are usually produced. For example, at least one chain comprises a 3' overhang of at least 1 nucleotide, for example, 1 to 4 nucleotides overhang. For another example, at least one chain comprises a 5' overhang of at least 1 nucleotide, for example, 1 to 4 nucleotides overhang. In other embodiments, both the 3' end and the 5' end of a chain of the dsRNA comprise an overhang of at least 1 nucleotide.

[0062] As used herein, the term "blunt end" or "blunt end" with respect to dsRNA refers to the absence of unpaired nucleotides or nucleotide analogs at a given end of the dsRNA, i.e., the absence of nucleotide overhangs. One or both ends of a dsRNA may be blunt. If both ends of a dsRNA are blunt, the dsRNA is said to be blunt-ended. It should be noted that a "blunt-ended" dsRNA is a dsRNA with both ends blunt, i.e., there are no nucleotide overhangs at either end of the molecule. In most cases, such molecules are double-stranded throughout their entire length. As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the duplex structure of a dsRNA. For example, a nucleotide overhang is present when the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa. Nucleotide overhangs may comprise or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhangs may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, overhanging nucleotides may be present at the 5' end, the 3' end, or both ends of the antisense or sense strand of the dsRNA.

[0063] The dsRNA molecule may include chemical modifications to ribonucleotides, including modifications to the ribose, bases, or backbone components of the ribonucleic acid, as described herein or known in the art. Any such modifications, as used in double-stranded ribonucleic acid molecules (e.g., siRNA, shRNA, etc.), are encompassed by the term "dsRNA" for the purposes of this disclosure. "Modified" nucleotides refer to nucleotides that independently have modified sugar moieties, modified internucleotide linkages, and / or modified nucleobases. Thus, the term "modified nucleotides" includes substitutions, additions, or removals of, for example, functional groups or atoms of internucleoside linkages, sugar moieties, or nucleobases.

[0064] The term "alkyl" refers to a saturated straight or branched hydrocarbon chain. For alkyl groups with a specific number of carbon atoms, the term includes the corresponding n-alkyl group and its various isomeric forms (if any). For example, an alkyl group with 4 carbon atoms (C4 alkyl) includes n-butyl, isobutyl, sec-butyl and tert-butyl. Exemplary C1-10 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 2-ethylbutyl, 4,4-dimethylpentyl, 5,5-dimethylhexyl, 6,6-dimethylheptyl, etc.

[0065] The term "alkenyl" refers to a straight or branched hydrocarbon chain having one or more carbon-carbon double bonds. Exemplary C2-10 alkenyls include ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 3-methyl-2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 4-methyl-3-pentenyl, 1-hexenyl, 3-hexenyl, 5-hexenyl, etc.

[0066] The term "alkynyl" refers to a straight or branched hydrocarbon chain having one or more carbon-carbon triple bonds. Exemplary C2-10 alkynyl groups include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 4-methyl-2-pentynyl, etc.

[0067] The term "alkoxy" refers to an alkyl-O- group, wherein alkyl is as defined above. Examples of "C1-10 alkoxy" include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, hexyloxy, and the like. When halogenated, the alkoxy group may be substituted with 1 to 7, preferably 1 to 5, halogen atoms. Specific examples include difluoromethoxy, trifluoromethoxy, 2,2,2-trifluoroethoxy, and 4,4,4-trifluorobutoxy.

[0068] The term "cycloalkyl" refers to a cyclic saturated hydrocarbon group. Examples of C3-10 cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, bicyclo[3.2.1]octyl, and adamantyl.

[0069] The term "aryl" refers to aromatic monocyclic and polycyclic carbocyclic ring systems, wherein the individual carbocyclic rings in the polycyclic ring system are fused or connected to each other by single bonds. Suitable aryl groups include phenyl, naphthyl, 2,3-dihydro-1H-indenyl, and biphenyl. 6- to 14-membered aryl refers to aromatic ring systems that are 6-carbon monocyclic, 10-carbon bicyclic, 12-carbon biphenyl, or 14-carbon tricyclic.

[0070] The term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, the heteroatoms being selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S, respectively, if monocyclic, bicyclic, or tricyclic), wherein 0, 1, 2, 3, or 4 atoms of each ring are substituted with substituents. Examples of heteroaryl groups include pyridyl, furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thienyl, quinolinyl, indolyl, thiazolyl, and the like.

[0071] The term "heterocyclyl" refers to a non-aromatic monocyclic, bicyclic or tricyclic ring system. Thus, a 5- to 14-membered heterocyclyl refers to a 5-8-membered monocyclic, 8-12-membered bicyclic or 11-14-membered tricyclic ring system having 1-3 heteroatoms if a monocyclic ring, 1-6 heteroatoms if a bicyclic ring, or 1-9 heteroatoms if a tricyclic ring, the heteroatoms being selected from O, N or S (e.g., carbon atoms and 1-3, 1-6 or 1-9 O, N or S heteroatoms, respectively, if a monocyclic, bicyclic or tricyclic ring) or other suitable heteroatoms (such as P or Si), wherein 0, 1, 2 or 3 atoms of each ring are substituted by substituents. Examples of 5- to 14-membered heterocyclyls include piperazinyl, pyrrolidinyl, dioxane, morpholinyl, tetrahydrofuranyl, tetrahydropyrrolidinyl, etc.

[0072] The term "therapeutically effective amount" refers to an amount of a lipid oligonucleotide conjugate of the invention or a composition thereof effective to produce some desired therapeutic effect in at least a subpopulation of cells in an animal, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0073] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0074] The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle that participates in carrying or delivering the lipid oligonucleotide conjugate from one organ or part of the body to another organ or part of the body, such as a liquid or solid filler, diluent, excipient, manufacturing aid, or solvent encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to the patient.

[0075] The term "treatment" encompasses prevention, therapy, and cure. The patient receiving such treatment is generally any animal in need thereof, including primates (particularly humans) and other mammals such as horses, cattle, pigs, sheep, poultry, and pets.

[0076] Lipid oligonucleotide conjugates

[0077] A first aspect of the present invention provides lipid-oligonucleotide conjugates.

[0078] In some embodiments, the present invention provides a lipid oligonucleotide conjugate comprising the structure of Formula Ia:

[0079]

[0080] in,

[0081] At least one of R1 and R2 is selected from Ra, -C(O)Ra, -C(O)NHRa, and C(O)ORa, wherein Ra is an optionally substituted C5-30 alkyl or alkenyl group, and

[0082] The other of R1 and R2 is selected from H, Rb, -C(O)Rb, -C(O)NHRb and C(O)ORb, wherein Rb is selected from optionally substituted C1-4 alkyl or alkenyl, optionally substituted C5-30 alkyl or alkenyl, optionally substituted C2-10 alkynyl, optionally substituted C1-10 alkoxy, optionally substituted C3-10 cycloalkyl, optionally substituted 6 to 14 membered aryl, optionally substituted 5 to 18 membered heteroaryl, optionally substituted 5 to 14 membered heterocyclyl;

[0083] The optional substitution is optionally substituted with Rc, wherein Rc is selected from C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 alkoxy, C3-10 cycloalkyl, 6 to 14 membered aryl, 5 to 18 membered heteroaryl, 5 to 14 membered heterocyclyl, halogen, hydroxyl, amino, guanidino, carboxyl, cyano, nitro, C1-3 alkylthio and thiol;

[0084] m1, m2 and m3 are independently an integer of 1 or 2;

[0085] p is 0, 1, or 2;

[0086] stands for oligonucleotide;

[0087] L stands for linker.

[0088] In some embodiments, one of R1 and R2 is -C(O)Ra, wherein Ra is optionally substituted C5-30 alkyl or alkenyl, and the other of R1 and R2 is H or -C(O)C1-4 alkyl.

[0089] In some embodiments, R1 is -C(O)Ra, wherein Ra is optionally substituted C5-30 alkyl or alkenyl, and R2 is H or -C(O)C1-4 alkyl.

[0090] In some embodiments, R2 is -C(O)Ra, wherein Ra is optionally substituted C5-30 alkyl or alkenyl, and R1 is H or -C(O)C1-4 alkyl.

[0091] In a preferred embodiment, Ra is a C5-30 alkyl or alkenyl group. In a preferred embodiment, Ra is a C12-24 alkyl or alkenyl group. In a preferred embodiment, Ra is a C14-22 alkyl or alkenyl group. In a preferred embodiment, Ra is a C14, C15, C16, C18, C20 or C22 alkyl group, a monounsaturated alkenyl group or a polyunsaturated alkenyl group.

[0092] For example, Ra is selected from C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C10 alkyl, C11 alkyl, C12 alkyl, C13 alkyl, C14 alkyl, C15 alkyl, C16 alkyl, C17 alkyl, C18 alkyl, C19 alkyl, C20 alkyl, C21 alkyl, C22 alkyl, C23 alkyl, C24 alkyl, C25 alkyl, C26 alkyl, C27 alkyl, C28 alkyl, C29 alkyl, C30 alkyl, C5 monounsaturated alkenyl, C6 monounsaturated alkenyl, C7 monounsaturated alkenyl, C8 monounsaturated alkenyl, C9 monounsaturated alkenyl, C10 monounsaturated alkenyl, C11 monounsaturated alkenyl, C12 monounsaturated alkenyl, C13 monounsaturated alkenyl, C14 monounsaturated alkenyl, C15 monounsaturated alkenyl, C16 monounsaturated alkenyl, C17 monounsaturated alkenyl, C18 monounsaturated alkenyl, C19 monounsaturated alkenyl, C20 monounsaturated alkenyl, C21 monounsaturated alkenyl, C22 monounsaturated alkenyl, C C23 monounsaturated alkenyl, C24 monounsaturated alkenyl, C25 monounsaturated alkenyl, C26 monounsaturated alkenyl, C27 monounsaturated alkenyl, C28 monounsaturated alkenyl, C29 monounsaturated alkenyl, C30 monounsaturated alkenyl, C5 polyunsaturated alkenyl, C6 polyunsaturated alkenyl, C7 polyunsaturated alkenyl, C8 polyunsaturated alkenyl, C9 polyunsaturated alkenyl, C10 polyunsaturated alkenyl, C11 polyunsaturated alkenyl, C12 polyunsaturated alkenyl, C13 polyunsaturated alkenyl, C14 polyunsaturated alkenyl, C15 polyunsaturated alkenyl, C16 polyunsaturated alkenyl, C17 polyunsaturated alkenyl, C18 polyunsaturated alkenyl, C19 polyunsaturated alkenyl, C20 polyunsaturated alkenyl, C21 polyunsaturated alkenyl, C22 polyunsaturated alkenyl, C23 polyunsaturated alkenyl, C24 polyunsaturated alkenyl, C25 polyunsaturated alkenyl, C26 polyunsaturated alkenyl, C27 polyunsaturated alkenyl, C28 polyunsaturated alkenyl, C29 polyunsaturated alkenyl, and C30 polyunsaturated alkenyl.

[0093] In a preferred embodiment, both of R1 and R2 are and independently -C(O)Ra, and Ra is an optionally substituted C5-30 alkyl or alkenyl. In a preferred embodiment, both of R1 and R2 are -C(O)Ra, and Ra is an optionally substituted C5-30 alkyl or alkenyl. In a preferred embodiment, R1 and R2 are identical and Ra is a C5-30 alkyl or alkenyl. In a preferred embodiment, R1 and R2 are identical and Ra is a C12-24 alkyl or alkenyl. In a preferred embodiment, R1 and R2 are identical and Ra is a C14-22 alkyl or alkenyl. In a preferred embodiment, R1 and R2 are identical and Ra is a C14, C15, C16, C18, C20 or C22 alkyl, monounsaturated alkenyl or polyunsaturated alkenyl.

[0094] In a preferred embodiment, R1 and R2 are the same and Ra is selected from C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C10 alkyl, C11 alkyl, C12 alkyl, C13 alkyl, C14 alkyl, C15 alkyl, C16 alkyl, C17 alkyl, C18 alkyl, C19 alkyl, C20 alkyl, C21 alkyl, C22 alkyl, C23 alkyl, C24 alkyl, C25 alkyl, C26 alkyl, C27 alkyl, C28 alkyl, C29 alkyl, C30 alkyl, C31 alkyl, C32 alkyl, C33 alkyl, C34 alkyl, C35 alkyl, C36 alkyl, C37 alkyl, C38 alkyl, C39 alkyl, C40 alkyl, C41 alkyl, C42 alkyl, C43 alkyl, C44 alkyl, C45 alkyl, C46 alkyl, C47 alkyl, C48 alkyl, C49 alkyl, C50 alkyl, C51 alkyl, C52 alkyl, C53 alkyl, C54 alkyl, C55 alkyl, C56 alkyl, C57 alkyl, C58 alkyl, C59 alkyl, C60 alkyl, C61 alkyl, C62 alkyl, C63 alkyl, C64 alkyl, C65 alkyl, C66 alkyl, C67 alkyl, C68 alkyl, C69 alkyl, C70 alkyl, C71 alkyl, C72 alkyl, C73 alkyl, C74 alkyl, C75 alkyl, C76 alkyl, C77 alkyl, C78 alkyl, C79 alkyl, C80 alkyl, C81 alkyl, C82 Alkyl, C5 monounsaturated alkenyl, C6 monounsaturated alkenyl, C7 monounsaturated alkenyl, C8 monounsaturated alkenyl, C9 monounsaturated alkenyl, C10 monounsaturated alkenyl, C11 monounsaturated alkenyl, C12 monounsaturated alkenyl, C13 monounsaturated alkenyl, C14 monounsaturated alkenyl, C15 monounsaturated alkenyl, C16 monounsaturated alkenyl, C17 monounsaturated alkenyl, C18 monounsaturated alkenyl, C19 monounsaturated alkenyl, C20 monounsaturated alkenyl, C21 monounsaturated alkenyl, C22 monounsaturated alkenyl, C23 monounsaturated alkenyl, C24 monounsaturated alkenyl, C25 monounsaturated alkenyl, C26 monounsaturated alkenyl, C27 monounsaturated alkenyl, C28 monounsaturated alkenyl, C29 monounsaturated alkenyl, C30 monounsaturated alkenyl, C5 polyunsaturated alkenyl, C6 polyunsaturated alkenyl, C7 polyunsaturated alkenyl, C8 polyunsaturated alkenyl, C9 polyunsaturated alkenyl, C10 polyunsaturated alkenyl, C11 polyunsaturated alkenyl, C12 polyunsaturated alkenyl, C13 polyunsaturated alkenyl , C14 polyunsaturated alkenyl, C15 polyunsaturated alkenyl, C16 polyunsaturated alkenyl, C17 polyunsaturated alkenyl, C18 polyunsaturated alkenyl, C19 polyunsaturated alkenyl, C20 polyunsaturated alkenyl, C21 polyunsaturated alkenyl, C22 polyunsaturated alkenyl, C23 polyunsaturated alkenyl, C24 polyunsaturated alkenyl, C25 polyunsaturated alkenyl, C26 polyunsaturated alkenyl, C27 polyunsaturated alkenyl, C28 polyunsaturated alkenyl, C29 polyunsaturated alkenyl, and C30 polyunsaturated alkenyl.

[0095] In some embodiments, m1, m2, and m3 are all 1. In some embodiments, two of m1, m2, and m3 are 1, and the other is 2. In some embodiments, m1 and m2 are 1, and m3 is 2. In some embodiments, m1 and m3 are 1, and m2 is 2. In some embodiments, m2 and m3 are 1, and m1 is 2.

[0096] In some embodiments, p is 2. In some embodiments, p is 0. In a preferred embodiment, p is 1.

[0097] In some embodiments, the oligonucleotide is an antisense oligonucleotide or a sense strand or antisense strand of an RNAi agent. In some embodiments, the sense strand or antisense strand of the RNAi agent is paired with its antisense strand or sense strand to form a duplex. In a preferred embodiment, the RNAi agent is an siRNA.

[0098] In a preferred embodiment, the oligonucleotide is an antisense oligonucleotide (ASO). In a preferred embodiment, the oligonucleotide is the sense strand or antisense strand of siRNA. In a preferred embodiment, the oligonucleotide is the sense strand of siRNA. In a preferred embodiment, the sense strand or antisense strand of the siRNA is paired with its antisense strand or sense strand to form a duplex.

[0099] In some embodiments, oligonucleotides can be connected via 5' and / or 3' ends. In certain embodiments, oligonucleotides are connected via 5' ends, such as the 5' ends of the sense strand of ASOs or siRNAs. In certain embodiments, oligonucleotides are connected via 3' ends, such as the 3' ends of the sense strand of ASOs or siRNAs. In alternative embodiments, oligonucleotides are connected via 1, 2, 3 or 4 nucleotides before the 5' or 3' terminal nucleotides. In some embodiments, oligonucleotides are connected via the 2'-position of the sugar of the 3'-terminal nucleotides. In other embodiments, oligonucleotides are connected via the 2'-position of the sugar of the 5'-terminal nucleotides.

[0100] In a preferred embodiment, the 5' and / or 3' end of the oligonucleotide is linked to L via a phosphate group, a phosphorothioate group or a phosphonate group. In a preferred embodiment, the 5' and / or 3' end of the ASO or siRNA is linked to L via a phosphorothioate group.

[0101] In a preferred embodiment, the antisense strand of the siRNA has a 5'-VP modification.

[0102] "Linker" or L refers to the atom or a group of atoms that the lipid portion is covalently connected to the oligonucleotide portion. The length of the linker can be about 1 to about 30 atoms, about 2 to about 28 atoms, about 3 to about 26 atoms, about 4 to about 24 atoms, about 6 to about 20 atoms, about 7 to about 20 atoms, about 8 to about 20 atoms, about 8 to about 18 atoms, and about 12 to about 18 atoms. In some embodiments, the linker can include a bifunctional linking portion, which generally includes an alkyl portion with two functional groups. One of the functional groups is selected to be combined with the compound of interest (such as the sense or antisense strand of the RNAi agent chain), while the other functional group is selected to be combined with substantially any selected group, such as the lipid portion as described herein. In certain embodiments, the linker includes an oligomer of a chain structure or a repeating unit, such as ethylene glycol or an amino acid unit. The example of the functional group typically used in the bifunctional linking portion includes but is not limited to an electrophilic reagent for reacting with a nucleophilic group and a nucleophilic reagent for reacting with an electrophilic group. In some embodiments, the bifunctional linking moiety includes an amino group, a hydroxyl group, a carboxylic acid, a thiol group, an unsaturated bond (eg, a double bond or a triple bond), and the like.

[0103] Linkers that can be used to attach lipids to oligonucleotides include, but are not limited to, pyrrolidine, 8-amino-3,6-dioxooctanoic acid, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid, 6-aminohexanoic acid, substituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, or substituted or unsubstituted C2-C20 alkynyl. Preferred substituents for such linkers include, but are not limited to, hydroxyl, amino, alkoxy, carboxyl, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0104] In certain embodiments, joint is cleavable.Cleaving joint is a kind of enough stable outside cell but is cracked to release two parts that combine together by joint when entering target cell.In some embodiments, cleavable joint is in target cell or under the first reference condition (it can for example be selected as simulation or represent cell condition) than cracking in experimenter's blood fast at least 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least 100 times.

[0105] Cleavable linkers are susceptible to the effects of cleavage agents, such as pH, redox potential, or the presence of degrading molecules. Generally, cleavage agents are more prevalent in cells than in serum or blood, or are found at higher levels or activity in cells. Examples of such cleavage agents include: redox agents selected for specific substrates or having no substrate specificity, including, for example, oxidases or reductases present in cells; esterases; endosomes or reagents that can produce an acidic environment, such as those that result in a pH of 5 or less; enzymes that can hydrolyze or degrade acid-cleavable linkers as general acids, peptidases (which can be substrate-specific), and phosphatases.

[0106] Cleavable linker can comprise part that is sensitive to pH.The pH of human serum is 7.4, and average intracellular pH is slightly lower, and scope is about 7.1-7.3.Endosome has more acidic pH, and in the scope of 5.5-6.0, lysosome has more acidic pH (about 5.0).Some joints will have the cleavable group that is cracked under preferred pH, thereby RNA molecule is released from intracellular part, or is released in the expected organelle of cell.Joint can comprise the cleavable group that can be cracked by specific enzyme.The type of the cleavable group that is incorporated into the joint can depend on the cell that will target.

[0107] Other types of linkers suitable for attaching lipids to oligonucleotides are known in the art, such as those described in CN116133691A, WO2023245061A2, or WO2017053995A1, all of which are incorporated herein by reference in their entirety.

[0108] In some embodiments, L is -NH-(CH2)qO-, -NH-(CH2CH2O)q-, Wherein q and r are independently integers of 1 to 20, and the right side of the formula is connected to the oligonucleotide. For example, q and r can independently be an integer of 1 to 15, an integer of 1 to 10, an integer of 1 to 8, an integer of 1 to 6, an integer of 2 to 10, an integer of 3 to 10, an integer of 4 to 10, an integer of 5 to 10, an integer of 6 to 10, an integer of 10 to 15, or an integer of 10 to 20. For example, q and r can independently be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0109] In some embodiments, L is -NH-(CH2)3-O-, -NH-(CH2)4-O-, -NH-(CH2)5-O-, -NH-(CH2)6-O-, -NH-(CH2)7-O-, -NH-(CH2)8-O-, -NH-(CH2)9-O-, -NH-(CH2)10-O-, -NH-(CH2CH2O)-, -NH-(CH2CH2O)2-, -NH-(CH2CH2O)3-, -NH-(CH2CH2O)4-, -NH-(CH2CH2O)5-, or -NH-(CH2CH2O)6-.

[0110] In some embodiments, L comprises L1-L2, wherein L1 is The right side of the formula is connected to L2; L2 is wherein the left side of the formula is connected to L1; the wavy line represents the connection position; and wherein each n is independently an integer from 1 to 10, preferably an integer from 2 to 8, more preferably an integer from 4 to 6. For example, each n can independently be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0111] In some embodiments, L comprises or is:

[0112]

[0113]

[0114]

[0115]

[0116] wherein the wavy line represents the connection position and the right wavy line is connected to the oligonucleotide, preferably the phosphate group, phosphorothioate group or phosphonate group at the 5' and / or 3' end of the oligonucleotide, and each n in each formula is independently an integer from 1 to 10, preferably an integer from 2 to 8, more preferably an integer from 4 to 6.

[0117] In some embodiments, the present invention provides a lipid oligonucleotide conjugate represented by Formula Ib,

[0118]

[0119] in,

[0120] At least one of R1 and R2 is selected from Ra, -C(O)Ra, -C(O)NHRa, and C(O)ORa, wherein Ra is an optionally substituted C5-30 alkyl or alkenyl group, and

[0121] The other of R1 and R2 is selected from H, Rb, -C(O)Rb, -C(O)NHRb and C(O)ORb, wherein Rb is selected from optionally substituted C1-4 alkyl or alkenyl, optionally substituted C5-30 alkyl or alkenyl, optionally substituted C2-10 alkynyl, optionally substituted C1-10 alkoxy, optionally substituted C3-10 cycloalkyl, optionally substituted 6 to 14 membered aryl, optionally substituted 5 to 18 membered heteroaryl, optionally substituted 5 to 14 membered heterocyclyl;

[0122] The optional substitution is optionally substituted with Rc, wherein Rc is selected from C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 alkoxy, C3-10 cycloalkyl, 6 to 14 membered aryl, 5 to 18 membered heteroaryl, 5 to 14 membered heterocyclyl, halogen, hydroxyl, amino, guanidino, carboxyl, cyano, nitro, C1-3 alkylthio and thiol;

[0123] L1 is The right side of the formula is connected to L2; L2 is wherein the left side of the formula is connected to L1; the wavy line represents the connection position; and wherein each n is independently an integer from 1 to 10;

[0124] stands for oligonucleotide.

[0125] In some embodiments, the present invention provides a lipid oligonucleotide conjugate represented by Formula Ib,

[0126]

[0127] in,

[0128] Both of R1 and R2 are and are independently -C(O)Ra, Ra is an optionally substituted C5-30 alkyl or alkenyl group;

[0129] L1 is The right side of the formula is connected to L2; L2 is wherein the left side of the formula is connected to L1; the wavy line represents the connection position; and wherein each n is independently an integer from 1 to 10;

[0130] stands for oligonucleotide.

[0131] In some embodiments, the present invention provides a lipid oligonucleotide conjugate represented by Formula Ib,

[0132]

[0133] in,

[0134] Both of R1 and R2 are and are independently -C(O)Ra, and Ra is a C5-30 alkyl or alkenyl group, preferably a C12-24 alkyl or alkenyl group, more preferably a C14-22 alkyl or alkenyl group, still more preferably a C14, C15, C16, C18, C20 or C22 alkyl group, a monounsaturated alkenyl group or a polyunsaturated alkenyl group;

[0135] L1 is The right side of the formula is connected to L2; L2 is wherein the left side of the formula is connected to L1; the wavy line represents the connection position; and wherein each n is independently an integer from 1 to 10, preferably an integer from 2 to 8, more preferably an integer from 4 to 6;

[0136] represents an oligonucleotide, preferably, the oligonucleotide is an antisense oligonucleotide or the sense strand or antisense strand of an RNAi agent; preferably, the 5' and / or 3' end of the oligonucleotide is connected to L2 via a phosphate group, a phosphorothioate group or a phosphonate group; preferably, the sense strand or antisense strand of the RNAi agent is paired with its antisense strand or sense strand to form a duplex; preferably, the RNAi agent is siRNA; preferably, the oligonucleotide is the sense strand of the siRNA; preferably, the antisense strand of the siRNA has a 5'-VP modification.

[0137] In a preferred embodiment, the present invention provides a lipid oligonucleotide conjugate represented by formula Ic,

[0138]

[0139] in,

[0140] One of Ra1 and Ra2 is an optionally substituted C5-30 alkyl or alkenyl, preferably an optionally substituted C12-24 alkyl or alkenyl; preferably an optionally substituted C14-22 alkyl or alkenyl; more preferably an optionally substituted C14, C15, C16, C18, C20 or C22 alkyl, monounsaturated alkenyl or polyunsaturated alkenyl;

[0141] the other of Ra1 and Ra2 is optionally substituted C1-4 alkyl or alkenyl, optionally substituted C5-30 alkyl or alkenyl, optionally substituted C2-10 alkynyl, optionally substituted C1-10 alkoxy, optionally substituted C3-10 cycloalkyl, optionally substituted 6- to 14-membered aryl, optionally substituted 5- to 18-membered heteroaryl, or optionally substituted 5- to 14-membered heterocyclyl;

[0142] The optional substitution is optionally substituted with Rc, wherein Rc is selected from C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 alkoxy, C3-10 cycloalkyl, 6 to 14 membered aryl, 5 to 18 membered heteroaryl, 5 to 14 membered heterocyclyl, halogen, hydroxyl, amino, guanidino, carboxyl, cyano, nitro, C1-3 alkylthio and thiol;

[0143] L1 is The right side of the formula is connected to L2; L2 is wherein the left side of the formula is connected to L1; the wavy line represents the connection position; and wherein each n is independently an integer from 1 to 10;

[0144] stands for oligonucleotide.

[0145] In a preferred embodiment, the present invention provides a lipid oligonucleotide conjugate represented by formula Ic,

[0146]

[0147] in,

[0148] One of Ra1 and Ra2 is a C5-30 alkyl or alkenyl group, preferably a C12-24 alkyl or alkenyl group, more preferably a C14-22 alkyl or alkenyl group, still more preferably a C14, C15, C16, C18, C20 or C22 alkyl group, a monounsaturated alkenyl group or a polyunsaturated alkenyl group;

[0149] The other of Ra1 and Ra2 is C1-4 alkyl or alkenyl, C5-30 alkyl or alkenyl, C2-10 alkynyl, C1-10 alkoxy, C3-10 cycloalkyl, 6- to 14-membered aryl, 5- to 18-membered heteroaryl, or 5- to 14-membered heterocyclyl;

[0150] L1 is The right side of the formula is connected to L2; L2 is wherein the left side of the formula is connected to L1; the wavy line represents the connection position; and wherein each n is independently an integer from 1 to 10;

[0151] stands for oligonucleotide.

[0152] In a preferred embodiment, in Formula Ic, one of Ra1 and Ra2 is selected from C1-4 alkyl or alkenyl, and the other is selected from C5-30 alkyl or alkenyl. In these embodiments, one of Ra1 and Ra2 (e.g., Ra1) is selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, vinyl, propenyl, 1-butenyl, isobutylenyl, cis-2-butenyl, or trans-2-butenyl, and the other (e.g., Ra2) is selected from C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C10 alkyl, C11 alkyl, C12 alkyl, C13 alkyl, C14 alkyl, C15 alkyl, C16 alkyl, C17 alkyl, C18 alkyl, C19 alkyl, C20 alkyl, C21 alkyl, C22 alkyl, C23 alkyl, C24 alkyl, C25 alkyl, C26 alkyl, C27 alkyl, C28 alkyl, C29 alkyl, C30 alkyl, C31 alkyl, C32 alkyl, C33 alkyl, C34 alkyl, C35 alkyl, C36 alkyl, C37 alkyl, C38 alkyl, C39 alkyl, C40 alkyl, C41 alkyl, C42 alkyl, C43 alkyl, C44 alkyl, C45 alkyl, C46 alkyl, C47 alkyl, C48 alkyl, C49 ...4 alkyl, C45 alkyl, C46 alkyl, C47 alkyl, C20 alkyl, C21 alkyl, C22 alkyl, C23 alkyl, C24 alkyl, C25 alkyl, C26 alkyl, C27 alkyl, C28 alkyl, C29 alkyl, C30 alkyl, C5 monounsaturated alkenyl, C6 monounsaturated alkenyl, C7 monounsaturated alkenyl, C8 monounsaturated alkenyl, C9 monounsaturated alkenyl, C10 monounsaturated alkenyl, C11 monounsaturated alkenyl, C12 monounsaturated alkenyl, C13 monounsaturated alkenyl, C14 monounsaturated alkenyl, C15 monounsaturated alkenyl, C16 monounsaturated alkenyl, C17 monounsaturated alkenyl, C1 C8 monounsaturated alkenyl, C19 monounsaturated alkenyl, C20 monounsaturated alkenyl, C21 monounsaturated alkenyl, C22 monounsaturated alkenyl, C23 monounsaturated alkenyl, C24 monounsaturated alkenyl, C25 monounsaturated alkenyl, C26 monounsaturated alkenyl, C27 monounsaturated alkenyl, C28 monounsaturated alkenyl, C29 monounsaturated alkenyl, C30 monounsaturated alkenyl, C5 polyunsaturated alkenyl, C6 polyunsaturated alkenyl, C7 polyunsaturated alkenyl, C8 polyunsaturated alkenyl, C9 polyunsaturated alkenyl, C10 polyunsaturated alkenyl, C11 polyunsaturated alkenyl polyunsaturated alkenyl, C12 polyunsaturated alkenyl, C13 polyunsaturated alkenyl, C14 polyunsaturated alkenyl, C15 polyunsaturated alkenyl, C16 polyunsaturated alkenyl, C17 polyunsaturated alkenyl, C18 polyunsaturated alkenyl, C19 polyunsaturated alkenyl, C20 polyunsaturated alkenyl, C21 polyunsaturated alkenyl, C22 polyunsaturated alkenyl, C23 polyunsaturated alkenyl, C24 polyunsaturated alkenyl, C25 polyunsaturated alkenyl, C26 polyunsaturated alkenyl, C27 polyunsaturated alkenyl, C28 polyunsaturated alkenyl, C29 polyunsaturated alkenyl, and C30 polyunsaturated alkenyl.

[0153] In a preferred embodiment, in Formula Ic, Ra1 and Ra2 are independently selected from C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C10 alkyl, C11 alkyl, C12 alkyl, C13 alkyl, C14 alkyl, C15 alkyl, C16 alkyl, C17 alkyl, C18 alkyl, C19 alkyl, C20 alkyl, C21 alkyl, C22 alkyl, C23 alkyl, C24 alkyl, C25 alkyl, C26 alkyl, C27 alkyl, C28 alkyl, C29 alkyl , C30 alkyl, C5 monounsaturated alkenyl, C6 monounsaturated alkenyl, C7 monounsaturated alkenyl, C8 monounsaturated alkenyl, C9 monounsaturated alkenyl, C10 monounsaturated alkenyl, C11 monounsaturated alkenyl, C12 monounsaturated alkenyl, C13 monounsaturated alkenyl, C14 monounsaturated alkenyl, C15 monounsaturated alkenyl, C16 monounsaturated alkenyl, C17 monounsaturated alkenyl, C18 monounsaturated alkenyl, C19 monounsaturated alkenyl, C20 monounsaturated alkenyl, C21 monounsaturated alkenyl, C22 monounsaturated alkenyl, C23 monounsaturated alkenyl, C24 monounsaturated alkenyl, C25 monounsaturated alkenyl, C26 monounsaturated alkenyl, C27 monounsaturated alkenyl, C28 monounsaturated alkenyl, C29 monounsaturated alkenyl, C30 monounsaturated alkenyl, C5 polyunsaturated alkenyl, C6 polyunsaturated alkenyl, C7 polyunsaturated alkenyl, C8 polyunsaturated alkenyl, C9 polyunsaturated alkenyl, C10 polyunsaturated alkenyl, C11 polyunsaturated alkenyl, C12 polyunsaturated alkenyl, C13 polyunsaturated alkenyl polyunsaturated alkenyl, C14 polyunsaturated alkenyl, C15 polyunsaturated alkenyl, C16 polyunsaturated alkenyl, C17 polyunsaturated alkenyl, C18 polyunsaturated alkenyl, C19 polyunsaturated alkenyl, C20 polyunsaturated alkenyl, C21 polyunsaturated alkenyl, C22 polyunsaturated alkenyl, C23 polyunsaturated alkenyl, C24 polyunsaturated alkenyl, C25 polyunsaturated alkenyl, C26 polyunsaturated alkenyl, C27 polyunsaturated alkenyl, C28 polyunsaturated alkenyl, C29 polyunsaturated alkenyl, and C30 polyunsaturated alkenyl.

[0154] In a preferred embodiment, in Formula Ic, Ra1 and Ra2 are the same.

[0155] In a preferred embodiment, in Formula Ic, the oligonucleotide is an antisense oligonucleotide or the sense strand or antisense strand of an RNAi agent; preferably, the 5' and / or 3' end of the oligonucleotide is linked to L2 via a phosphate group, a phosphorothioate group, or a phosphonate group; preferably, the sense strand or antisense strand of the RNAi agent pairs with its antisense strand or sense strand to form a duplex; preferably, the RNAi agent is an siRNA; preferably, the oligonucleotide is the sense strand of the siRNA; preferably, the antisense strand of the siRNA has a 5'-VP modification.

[0156] In some embodiments, in Formula Ic, L1-L2 together form:

[0157]

[0158]

[0159]

[0160]

[0161] wherein the wavy line represents the connection position and the right wavy line is connected to the oligonucleotide, preferably the phosphate group, phosphorothioate group or phosphonate group at the 5' and / or 3' end of the oligonucleotide, and each n in each formula is independently an integer from 1 to 10, preferably an integer from 2 to 8, more preferably an integer from 4 to 6.

[0162] In some embodiments, the present invention provides a lipid oligonucleotide conjugate of the following structural formula:

[0163] in stands for oligonucleotide.

[0164] In a preferred embodiment, the oligonucleotide is an antisense oligonucleotide or the sense strand or antisense strand of an RNAi agent; preferably, the 5' and / or 3' end of the oligonucleotide is linked via a phosphate group, a phosphorothioate group or a phosphonate group; preferably, the sense strand or antisense strand of the RNAi agent pairs with its antisense strand or sense strand to form a duplex; preferably, the RNAi agent is an siRNA; preferably, the oligonucleotide is the sense strand of the siRNA; preferably, the antisense strand of the siRNA has a 5'-VP modification.

[0165] In a preferred embodiment, the lipid oligonucleotide conjugate is selected from any one of the following structures:

[0166] in, represents siRNA, X is S or O, and SS-5' / 3' represents the 5' or 3' end of the sense strand.

[0167] In some embodiments, the oligonucleotide is an antisense oligonucleotide. In some embodiments, the antisense oligonucleotide provided comprises modified nucleotides, such as modified sugar moieties and / or modified nucleobase moieties and / or modified internucleotide linkages. In preferred embodiments, the antisense oligonucleotide provided comprises modified nucleotides, including modified sugar moieties, modified nucleobase moieties, and modified internucleotide linkages.

[0168] In some embodiments, the antisense oligonucleotide comprises an oligonucleotide consisting of 12-30, 14-22, 14-20, 14-18, 14-20, 15-17, 15-25, or 16-20 linked nucleotides, wherein the antisense oligonucleotide comprises at least one modification selected from a modified sugar moiety, a modified nucleobase moiety, and a modified internucleotide linkage.

[0169] In some embodiments, the antisense oligonucleotide comprises an oligonucleotide in which each nucleotide is modified, including a modified sugar moiety and a modified internucleotide linkage.

[0170] In some embodiments, the antisense oligonucleotide comprises an oligonucleotide in which each nucleotide is modified, including a modified sugar moiety, a modified nucleobase moiety, and a modified internucleotide linkage.

[0171] In some embodiments, the modified sugar moiety can be at least one bicyclic sugar moiety, at least one non-bicyclic modified sugar moiety, and / or at least one sugar surrogate.

[0172] In some embodiments, the antisense oligonucleotide comprises at least one bicyclic sugar moiety. In some embodiments, the substituent bridges two atoms of the furanose ring to form a second ring, thereby producing a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety comprises a bridge between 4' and 2' furanose ring atoms. Examples of such 4' to 2' bridged sugar substituents include, but are not limited to: 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2' ("LNA"), 4'-CH2-S-2', 4'-(CH2)2-O-2' ("ENA"), 4'-CH(CH3)-O-2' (referred to as "constrained ethyl" or "cEt"), 4'-CH2-O-CH2-2', 4'-CH2-N(R)-2', 4'-CH(CH2OCH3)-O-2' ("constrained MOE" or "cMOE") and its analogs (see, e.g., Seth et al., US7,399,845; Bhat et al., US7,569,686; Swayze et al., US7,741,457; and Swayze et al., US8,022,193), 4'-C(CH3)(CH3)-O-2' and its analogs (see, e.g., Seth et al., US8,278,283), 4' -CH2-N(OCH3)-2' and its analogs (see, for example, Prakash et al., US8,278,425), 4'-CH2-ON(CH3)-2' (see, for example, Allerson et al., US7,696,345 and Allerson et al., US8,124,745), 4'-CH2-C(H)(CH3)-2' (see, for example, Zhou et al., J. Org. Chem., 2009, 74, 118-134), 4 '-CH2-C(=CH2)-2' and its analogs (see, for example, Seth et al., US8,278,426), 4'-C(RaRb)-N(R)-O-2', 4'-C(RaRb)-ON(R)-2', 4'-CH2-ON(R)-2' and 4'-CH2-N(R)-O-2', wherein each R, Ra and Rb are independently H, a protecting group or a C1-C12 alkyl group (see, for example, Imanishi et al., US7,427,672). In certain embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configurations. For example, LNA nucleosides (described herein) can be in an α-L configuration or in a β-D configuration. In a preferred embodiment, the bicyclic sugar moiety has a 4'-2' bridge, and the 4'-2' bridge is preferably cEt or LNA.

[0173] In some embodiments, the antisense oligonucleotide comprises at least one non-bicyclic modified sugar moiety. Such non-bridging substituents may be located at any position of the furanose moiety, including but not limited to substituents at the 2', 4', and / or 5' positions. In certain embodiments, one or more non-bridging substituents of the non-bicyclic modified sugar moiety are branched. Examples of suitable 2'-substituents for non-bicyclic modified sugar moieties include, but are not limited to, 2'-F, 2'-OCH3 ("OMe" or "O-methyl"), and 2'-O(CH2)2OCH3 ("MOE" or "O-methoxyethyl"). In certain embodiments, the 2'-substituent is selected from the group consisting of halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, O-C1-C10 alkoxy, substituted O-C1-C10 alkoxy, O-C1-C10 alkyl, substituted O-C1-C10 alkyl, S-alkyl, N(Rm)-alkyl, O-alkenyl, S-alkenyl, N(Rm)-alkenyl, O-alkynyl, S-alkynyl, N(Rm)-alkynyl, O-alkylene-O-alkyl, alkynyl, alkaryl, aryl alkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O(CH2)2ON(Rm)(Rn) or OCH2C(=O)-N(Rm)(Rn), wherein each Rm and Rn is independently H, an amino protecting group, or a substituted or unsubstituted C1-C10 alkyl, and the 2'-substituents described in Cook et al., US6,531,584; Cook et al., US5,859,221; and Cook et al., US6,005,087. Certain embodiments of these 2'-substituents may be further substituted with one or more substituents independently selected from the group consisting of hydroxy, amino, alkoxy, carboxyl, benzyl, phenyl, nitro(NO2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl, and alkynyl. Examples of suitable 4'-substituents for non-bicyclic modified sugar moieties include, but are not limited to, alkoxy (e.g., methoxy), alkyl, and those described in Manoharan et al., WO 2015 / 106128. Examples of suitable 5'-substituents for non-bicyclic modified sugar moieties include, but are not limited to, 5'-methyl (R or S), 5'-vinyl, and 5'-methoxy. In certain embodiments, non-bicyclic modified sugar moieties comprise more than one non-bridging sugar substituent, such as 2'-F-5'-methyl sugar moieties and modified sugar moieties and modified nucleosides described in Migawa et al., WO 2008 / 101157 and Rajeev et al., US 2013 / 0203836.

[0174] In certain embodiments, the 2'-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2'-substituent selected from the group consisting of F, NH2, N3, OCF3, OCH3, O(CH2)3NH2, CH2CH=CH2, OCH2CH=CH2, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(Rm)(Rn), O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamide (OCH2C(=O)—N(Rm)(Rn)), wherein each Rm and Rn is independently H, an amino protecting group, or a substituted or unsubstituted C1-C10 alkyl group.

[0175] In certain embodiments, the 2'-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2'-substituent selected from the group consisting of F, OCF3, OCH3, OC H2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(CH3)2, O(CH2)2O(CH2)2N(CH3)2, and OCH2C(=O)-N(H)CH3 ("NMA"). In certain embodiments, the 2'-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2'-substituent selected from the group consisting of F (2'-F), OCH3 (2'-OMe), and OCH2CH2OCH3 (2'-MOE).

[0176] In certain embodiments, the modified furanosyl sugar moiety and the nucleoside having such modified furanosyl sugar moiety are further defined by isomeric configurations. For example, the 2'-deoxyfuranosyl sugar moiety can be in seven isomeric configurations except the naturally occurring β-D-deoxyribosyl configuration. Such modified sugar moieties are described in, for example, WO 2019 / 157531, which is incorporated herein by reference. The 2'-modified sugar moiety has another stereocenter at the 2' position relative to the 2'-deoxyfuranosyl sugar moiety; therefore, such sugar moieties have a total of sixteen possible isomeric configurations. Unless otherwise specified, the 2'-modified sugar moieties described herein are in β-D-ribosyl isomeric configurations.

[0177] In some embodiments, the antisense oligonucleotide comprises at least one sugar surrogate. In some such embodiments, the oxygen atom of the sugar moiety is replaced by, for example, a sulfur, carbon, or nitrogen atom. In some such embodiments, such modified sugar moieties also include bridged and / or non-bridged substituents as described herein. For example, some sugar surrogate comprises a 4'-sulfur atom and a substitution at the 2' position (see, for example, Bhat et al., US7,875,733 and Bhat et al., US7,939,677) and / or the 5' position.

[0178] In certain embodiments, the sugar surrogate comprises a ring having other than 5 atoms. For example, in certain embodiments, the sugar surrogate comprises a 6-membered tetrahydropyran ("THP"). Such tetrahydropyrans may be further modified or substituted. Nucleosides comprising such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acids ("HNA"), anitol nucleic acids ("ANA"), mannitol nucleic acids ("MNA") (see, e.g., Leumann, CJ. Bioorg. & Med. Chem. 2002, 10, 841-854), and fluoro-HNA (F-HNA, also referred to as F-THP or 3'-fluorotetrahydropyran).

[0179] In certain embodiments, the sugar surrogate comprises a ring with more than 5 atoms and more than one heteroatom. For example, nucleosides comprising morpholinyl sugar moieties and their uses in oligonucleotides have been reported. In certain embodiments, the sugar surrogate comprises an acyclic portion. Examples of nucleosides and oligonucleotides comprising such acyclic sugar surrogate include, but are not limited to, peptide nucleic acids ("PNA"), acyclic butyl nucleic acids (see, for example, Kumar et al., Org. Biomol. Chem., 2013, 11, 5853-5865) and Manoharan et al., nucleosides and oligonucleotides described in WO2011 / 133876. By introducing a modified nucleobase with a cationic lipid or its equivalent covalently attached thereto, PNA can be made highly permeable to mammalian cell membranes (see, for example, WO2019022434 and WO2009113828). In a preferred embodiment, the sugar surrogate is selected from POM, PNA, THP and F-HNA.

[0180] In some embodiments, the antisense oligonucleotide comprises one or more nucleosides comprising unmodified nucleobases. In certain embodiments, the antisense oligonucleotide comprises one or more nucleosides comprising modified nucleobases. In certain embodiments, the antisense oligonucleotide comprises one or more nucleosides that do not comprise nucleobases, referred to as abasic nucleosides.

[0181] In certain embodiments, the modified nucleobase is selected from: a 5-substituted pyrimidine (eg, 5' methylcytosine, m5C), a 6-azapyrimidine, an alkyl or alkynyl substituted pyrimidine, an alkyl substituted purine, and N-2, N-6, and O-6 substituted purines. In certain embodiments, the modified nucleobase is selected from the group consisting of: 2-aminopropyladenine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (-C≡C-CH3) uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy, 8-aza and other 8-substituted Purines; 5-halo, particularly 5-bromo, 5-trifluoromethyl, 5-halouracil and 5-halocytosine; 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 5-methylcytosine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl4-N-benzoylcytosine, 5-methyl4-N-benzoyluracil, universal bases, hydrophobic bases, mixed bases, size-expanded bases and fluorinated bases. Other modified nucleobases include tricyclic pyrimidines, such as 1,3-diazaphenoxazine-2-one, 1,3-diazaphenthiazine-2-one, and 9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced by other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. In a preferred embodiment, the antisense oligonucleotide comprises one or more 5-methylcytosines (m5C).

[0182] In some embodiments, the modified nucleobase can be a nucleobase covalently attached to a cationic lipid. By introducing a modified nucleobase with a cationic lipid covalently attached thereto or its equivalent, antisense oligonucleotides can be made highly permeable to mammalian cell membranes. Suitable covalent attachment of cationic lipid modifications can be those described in WO2019022434A1, the entire contents of which are incorporated herein by reference.

[0183] In some embodiments, the antisense oligonucleotide comprises at least one modified internucleotide linkage. In preferred embodiments, each internucleotide linkage of the antisense oligonucleotide is a modified internucleotide linkage. In certain embodiments, any internucleotide linkage can be used to link the nucleosides of the antisense oligonucleotide together. Two main categories of internucleoside linking groups are defined based on the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleotide linkages include, but are not limited to, phosphodiester, which contains a phosphodiester bond ("P(O2)=O") (also known as an unmodified linkage or a naturally occurring linkage); phosphotriester; methylphosphonate; phosphoramidate (PN); phosphorothioate ("P(O2)=S", PS) and phosphorodithioate ("HS-P=S"). Phosphoramidates (PN) may have the formula -N(R)P(=X)(OH)O-, -OP(=X)(OH)N(R)-, -OP(NR)(=X)O-, -N(S02R)P(=X)(OH)O-, -OP(=X)(OH)N(S02R)-, or -OP(NS02R)(=X)O- ("MsPA"), wherein X is O or S, R may be an optionally substituted alkyl, aryl, heteroaryl, or heterocycloalkyl group; or NR may be an optionally substituted cycloguanidinyl moiety, an optionally substituted triazolyl, or a Tmg group. More PN structures can be found in WO2023220744, WO 2019 / 032612, and WO2021 / 030778, which are incorporated herein by reference in their entireties.

[0184] Representative non-phosphorus-containing internucleoside linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiesters, thionocarbamates (-OC(=O)(NH)-S-); siloxanes (-O-SiH2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Modified internucleotide linkages can be used to alter, and generally increase, the nuclease resistance of oligonucleotides compared to naturally occurring phosphodiester internucleotide linkages. In certain embodiments, internucleotide linkages with chiral atoms can be prepared as racemic mixtures or as separated enantiomers. Methods for preparing phosphorus-containing and non-phosphorus-containing internucleotide linkages are well known to those skilled in the art.

[0185] Representative internucleotide connections with chiral centers include, but are not limited to, alkylphosphonates and phosphorothioates. Antisense oligonucleotides comprising internucleotide connections with chiral centers can be prepared into a population of antisense oligonucleotides comprising stereo-random internucleotide connections, or into a population of antisense oligonucleotides comprising internucleotide connections of phosphorothioates in a specific stereochemical configuration. In certain embodiments, the population of antisense oligonucleotides comprises internucleotide connections of phosphorothioates, wherein all of the internucleotide connections of phosphorothioates are stereo-random. Such antisense oligonucleotides can be generated using a synthetic method that randomly selects the stereochemical configuration of each internucleotide connection of phosphorothioates. Nevertheless, as is well understood by those skilled in the art, each individual phosphorothioate of each individual oligonucleotide molecule has a determined stereo configuration. In certain embodiments, the population of antisense oligonucleotides is enriched with antisense oligonucleotides comprising one or more internucleotide connections of specific phosphorothioates in a specific independently selected stereochemical configuration. In certain embodiments, a specific internucleotide connection of phosphorothioates of a specific configuration is present in at least 65% of the molecules in the population. In certain embodiments, there is a specific phosphorothioate internucleotide connection of a specific configuration in at least 70% of the molecules in the population. In certain embodiments, there is a specific phosphorothioate internucleotide connection of a specific configuration in at least 80% of the molecules in the population. In certain embodiments, there is a specific phosphorothioate internucleotide connection of a specific configuration in at least 90% of the molecules in the population. In certain embodiments, there is a specific phosphorothioate internucleotide connection of a specific configuration in at least 99% of the molecules in the population. Such chiral enriched populations of antisense oligonucleotides can be generated using synthetic methods known in the art, such as the methods described in the following documents: Oka et al., JACS 125, 8307 (2003); Wan et al., Nuc. Acid. Res. 42, 13456 (2014); and WO 2017 / 015555. In certain embodiments, the population of antisense oligonucleotides is enriched with antisense oligonucleotides having at least one indicated phosphorothioate in (Sp) configuration. In certain embodiments, the population of antisense oligonucleotides is enriched for antisense oligonucleotides having at least one phosphorothioate in the (Rp) configuration.

[0186] In some embodiments, the antisense oligonucleotide includes a gap polymer (Gapmer). The gap polymer is defined by two external regions or " wings" and a central or internal region or " gap ". The three regions (5' wing, gap and 3' wing) of the gap polymer motif form a contiguous sequence of nucleosides, wherein at least some sugar moieties of the nucleosides of each wing are different from at least some sugar moieties of the nucleosides of the gap. Specifically, at least the sugar moiety of the nucleosides closest to the gap in each wing (the most 3' end nucleosides of the 5' wing and the most 5' end nucleosides of the 3' wing) is different from the sugar moiety of the adjacent gap nucleosides, thereby defining the boundary between the wing and the gap (that is, the wing / gap boundary). In certain embodiments, the sugar moieties in the gap are identical to each other. In certain embodiments, the gap includes one or more nucleosides, and the sugar moieties possessed by the nucleosides are different from the sugar moieties of one or more other nucleosides of the gap. In certain embodiments, the sugar motifs of two wings are identical to each other (symmetrical gap polymers). In certain embodiments, the sugar motifs of 5' wing are different from the sugar motifs of 3' wing (asymmetric gap polymers).

[0187] In some embodiments, the gapmer comprises: (a) a 5' region consisting of 1-6 linked 5' region nucleotides; (b) a central region consisting of 6-10 linked central region nucleotides; and (c) a 3' region consisting of 1-6 linked 3' region nucleotides; wherein each of the nucleotides in the 5' region and the 3' region comprises a sugar moiety modified from 2'-MOE, LNA and cEt, and at least 6 of the nucleotides in the central region comprise a 2'-deoxy sugar moiety.

[0188] In some embodiments, the gapmer has a sugar motif (5' to 3') selected from the following: eekddddddddddkke, ekkddddddddddkke, kkkdyddddddddkkk, kkkddydddddddkkk, kkkdddyddddddkkk, kkkddddddddddddkkk, or eeeeeddddddddddeeeee; wherein e represents a 2'-MOE sugar moiety, k represents a cEt sugar moiety, d represents a 2'-deoxy sugar moiety, and y represents a 2'-OMe sugar moiety.

[0189] In a preferred embodiment, the gapmer has a eeeeeddddddddddeeeee sugar motif (5' to 3'), wherein e represents a 2'-MOE sugar moiety and d represents a 2'-deoxy sugar moiety.

[0190] In some embodiments, the oligonucleotide is the sense strand or antisense strand of the RNAi agent. In such embodiments, the lipid-oligonucleotide conjugate preferably also comprises the antisense strand or sense strand of the RNAi agent, such that the sense strand and the antisense strand pair to form a double-stranded region.

[0191] In some embodiments, the length of the double-stranded region is about 17 to about 23 base pairs. For example, a double-stranded region of suitable length is about 17 to about 22 base pairs, about 17 to about 21 base pairs, about 17 to about 20 base pairs, about 17 to about 19 base pairs, about 17 to about 18 base pairs, about 18 to about 23 base pairs, about 18 to about 22 base pairs, about 18 to about 21 base pairs, about 18 to about 20 base pairs, about 19 to 23 base pairs, about 19 to 22 base pairs, about 19 to 21 base pairs, about 20 to 23 base pairs, about 20 to 22 base pairs, or about 21 to 23 base pairs. In certain embodiments, the length of the double-stranded region is about 18 to about 21 base pairs. In other embodiments, the length of the double-stranded region is about 19 base pairs.

[0192] In some embodiments, the sense and antisense strands in the RNAi agents of the invention are each independently about 17 to about 23 nucleotides in length, e.g., about 18 to about 23 nucleotides, about 19 to about 23 nucleotides, about 20 to about 23 nucleotides, about 21 to about 23 nucleotides, about 17 to about 22 nucleotides, about 17 to about 21 nucleotides, about 17 to about 20 nucleotides, about 17 to about 19 nucleotides, about 18 to about 22 nucleotides, about 18 to about 21 nucleotides, about 18 to about 20 nucleotides, about 19 to about 22 nucleotides, about 19 to about 21 nucleotides, or about 20 to about 22 nucleotides. In certain embodiments, the sense and antisense strands are each independently about 17, about 18, about 19, about 20, about 21, about 22, or about 23 nucleotides in length.

[0193] In some embodiments, the sense strand and the antisense strand have the same length, but form a double-stranded region that is shorter than the strand, so that the RNAi agent for inhibiting the expression of the target gene has two nucleotide protrusions. For example, in one embodiment, the RNAi agent for inhibiting the expression of the target gene includes (i) a sense strand and an antisense strand that are 21 nucleotides in length, (ii) a double-stranded region that is 19 base pairs in length, and (iii) a nucleotide protrusion with one unpaired nucleotide at the 3' end of the sense strand and the 3' end of the antisense strand. In another embodiment, the RNAi agent for inhibiting the expression of the target gene includes (i) a sense strand and an antisense strand that are 23 nucleotides in length, (ii) a double-stranded region that is 21 base pairs in length, and (iii) a nucleotide protrusion with one unpaired nucleotide at the 3' end of the sense strand and the 3' end of the antisense strand.

[0194] In other embodiments, the sense strand and the antisense strand have the same length and form a double-stranded region over their entire length so that no nucleotides protrude from either end of the double-stranded molecule. In one such embodiment, the RNAi agent for suppressing the expression of a target gene is flat-ended and includes (i) a sense strand and an antisense strand each having a length of 21 nucleotides, and (ii) a double-stranded region having a length of 21 base pairs. In another such embodiment, the RNAi agent for suppressing the expression of a target gene is flat-ended and includes (i) a sense strand and an antisense strand each having a length of 23 nucleotides, and (ii) a double-stranded region having a length of 23 base pairs. In another such embodiment, the RNAi agent for suppressing the expression of a target gene is flat-ended and includes (i) a sense strand and an antisense strand each having a length of 19 nucleotides, and (ii) a double-stranded region having a length of 19 base pairs.

[0195] In other embodiments, the sense strand or antisense strand is longer than the other strand, and the two strands form a double-stranded region with a length equal to the length of the short strand, so that the RNAi agent for inhibiting target gene expression includes at least one nucleotide overhang. For example, in some embodiments, the sense strand is 1 to 4 nucleotides longer than the antisense strand, and the double-stranded region formed by the two strands is equal to the length of the antisense strand, so that the sense strand forms an overhang with 1 to 4 unpaired nucleotides. In other embodiments, the antisense strand is 1 to 4 nucleotides longer than the sense strand, and the double-stranded region formed by the two strands is equal to the length of the sense strand, so that the antisense strand forms an overhang with 1 to 4 unpaired nucleotides. In some embodiments, the length of the nucleotide overhang is 1, 2, 3 or 4 nucleotides. In a specific embodiment, the overhang includes 2 nucleotides. In certain embodiments, the overhang includes a single nucleotide.

[0196] The nucleotides that protrude can be ribonucleotides or modified nucleotides as described herein. In some embodiments, the nucleotides that protrude are 2'-modified nucleotides (e.g., 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides) or combinations thereof. For example, in one embodiment, the nucleotides that protrude are deoxyribonucleotides, such as deoxythymidine. In another embodiment, the nucleotides that protrude are 2'-O-methyl-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-methoxyethyl-modified nucleotides, abasic nucleotides, inverted abasic nucleotides, inverted nucleotides or combinations thereof. In other embodiments, the protrusion comprises a 5'-uridine-uridine-3' (5'-UU-3') dinucleotide. In such embodiments, the UU dinucleotide can include a ribonucleotide or modified nucleotide, such as a 2'-modified nucleotide. In other embodiments, the protrusion comprises a 5'-deoxythymidine-deoxythymidine-3' (5'-dTdT-3') dinucleotide. When there is a nucleotide overhang in the antisense strand, the nucleotides in the overhang may be complementary to the target gene sequence, form a mismatch with the target gene sequence, or contain some other sequence (such as UU, TT, AA, GG, etc.).

[0197] The nucleotide overhang may be at the 5' end or the 3' end of one or both strands. For example, in one embodiment, the RNAi agent used to inhibit the expression of a target gene comprises nucleotide overhangs at the 5' end and the 3' end of the antisense strand. In another embodiment, the RNAi agent used to inhibit the expression of a target gene comprises nucleotide overhangs at the 5' end and the 3' end of the sense strand. In some embodiments, the RNAi agent used to inhibit the expression of a target gene includes nucleotide overhangs at the 5' end of the sense strand and the 5' end of the antisense strand. In other embodiments, the RNAi agent used to inhibit the expression of a target gene includes nucleotide overhangs at the 3' end of the sense strand and the 3' end of the antisense strand. In some embodiments, the RNAi agent used to inhibit the expression of a target gene includes only a nucleotide overhang at the 5' end of the sense strand. In some embodiments, the RNAi agent used to inhibit the expression of a target gene includes only a nucleotide overhang at the 3' end of the sense strand. In some embodiments, the RNAi agent used to inhibit the expression of a target gene includes only a nucleotide overhang at the 3' end of the antisense strand. In some embodiments, the RNAi agent used to inhibit the expression of a target gene includes only a nucleotide overhang at the 5' end of the antisense strand. In some embodiments, a RNAi agent used to inhibit target gene expression includes only nucleotide overhangs at the 5' end of the sense strand.

[0198] The RNAi agent for inhibiting the expression of the target gene can include a nucleotide overhang at one end of the double-stranded RNA molecule and a flat end at the other end. "Flat end" means that the sense strand and the antisense strand are completely base-paired at the ends of the molecule, and no unpaired nucleotides extend beyond the double-stranded region. In some embodiments, the RNAi agent for inhibiting the expression of the target gene includes a nucleotide overhang at the 3' end of the sense strand and a flat end at the 5' end of the sense strand and the 3' end of the antisense strand. In other embodiments, the RNAi agent for inhibiting the expression of the target gene includes a nucleotide overhang at the 3' end of the antisense strand and a flat end at the 5' end of the antisense strand and the 3' end of the sense strand.

[0199] Specifically, for example, in one embodiment, the RNAi agent for inhibiting the expression of the target gene comprises (i) a sense strand of 19 nucleotides in length, (ii) an antisense strand of 21 nucleotides in length, and the two strands form a double-stranded region whose length is equal to the chain length of the sense strand. In another embodiment, the RNAi agent for inhibiting the expression of the target gene comprises (i) a sense strand of 21 nucleotides in length, (ii) an antisense strand of 23 nucleotides in length, and the two strands form a double-stranded region whose length is equal to the chain length of the sense strand.

[0200] In some embodiments, the RNAi agent that can form the lipid oligonucleotide conjugate of the present invention comprises a sense strand and an antisense strand that form a double-stranded region, the antisense strand is no longer than 23 nucleotides, and the RNAi agent includes an overhang and a blunt end, and the overhang preferably has 2 unpaired nucleotides.

[0201] In some embodiments, the RNAi agent that can form the lipid oligonucleotide conjugate of the present invention comprises a sense strand and an antisense strand that form a double-stranded region, the antisense strand is no longer than 23 nucleotides, and the RNAi agent includes an overhang and a blunt end, the overhang preferably having 2 unpaired nucleotides, wherein the overhang is formed at the 3' end of the antisense strand, and the blunt end is formed at the 3' end of the sense strand and the 5' end of the antisense strand.

[0202] In some embodiments, the RNAi agent that can form the lipid oligonucleotide conjugate of the present invention comprises a sense strand and an antisense strand that form a double-stranded region, wherein the double-stranded region is 19 base pairs in length, the antisense strand is no longer than 23 nucleotides in length, and the RNAi agent includes an overhang and a blunt end, wherein the overhang preferably has 2 unpaired nucleotides, wherein the overhang is formed at the 3' end of the antisense strand, and the blunt end is formed at the 3' end of the sense strand and the 5' end of the antisense strand.

[0203] In some embodiments, the RNAi agent that can form the lipid oligonucleotide conjugate of the present invention comprises a sense strand and an antisense strand that form a double-stranded region, wherein the double-stranded region is 19 base pairs in length, the antisense strand is no longer than 23 nucleotides in length, and the sense strand is 19 to 21 nucleotides in length, and the RNAi agent includes an overhang and a blunt end, wherein the overhang preferably has 2 unpaired nucleotides, wherein the overhang is formed at the 3' end of the antisense strand, and the blunt end is formed at the 3' end of the sense strand and the 5' end of the antisense strand.

[0204] In some embodiments, the RNAi agent that can form the lipid oligonucleotide conjugate of the present invention comprises a sense strand and an antisense strand that form a double-stranded region, wherein the double-stranded region is 19 base pairs in length, the antisense strand is 21 nucleotides in length, and the sense strand is 19 nucleotides in length, and the RNAi agent includes an overhang and a blunt end, wherein the overhang preferably has 2 unpaired nucleotides, wherein the overhang is formed at the 3' end of the antisense strand, and the blunt end is formed at the 3' end of the sense strand and the 5' end of the antisense strand.

[0205] In some embodiments, the RNAi agent of the present invention that can form the lipid oligonucleotide conjugate of the present invention comprises a sense strand and an antisense strand that form a double-stranded region, wherein the double-stranded region is 19 base pairs in length, the sense strand is 19 nucleotides in length, and the RNAi agent includes an overhang and a blunt end, wherein the overhang preferably has 2 unpaired nucleotides, wherein the overhang is formed at the 3' end of the antisense strand, and the blunt end is formed at the 3' end of the sense strand and the 5' end of the antisense strand.

[0206] In the present invention, the sense strand and / or antisense strand of the RNAi agent may comprise at least one modified nucleotide. In some embodiments, the sense strand and antisense strand of the RNAi agent respectively comprise at least one modified nucleotide. In some embodiments, each nucleotide of the sense strand and antisense strand of the RNAi agent is modified.

[0207] In any of the above embodiments, the modified nucleotides are independently selected from 2'-deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides (2'-OMe), 2'-fluorine modified nucleotides (2'-F), 2'-deoxy modified nucleotides, locked nucleic acids (LNA), open ring nucleic acids (UNA), bridge nucleic acids (BNA), glycol nucleic acids (GNA), athreose nucleic acids (TNA), conformationally restricted nucleotides, restricted ethyl nucleotides (cEt), 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-O -methoxyethyl modified nucleotides (2'-MOE), abasic nucleotides, inverted abasic nucleotides, inverted nucleotides, morpholino nucleotides (MOP), phosphoramidates (PN), tetrahydropyran modified nucleotides (THP), 1,5-anhydrohexitol modified (HNA) nucleotides, cyclohexenyl modified nucleotides, nucleotides containing phosphorothioate groups (PS), nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphate, nucleotides containing 5'-phosphate mimetics, nucleotides covalently linked to cationic lipids, nucleotides containing 5'-vinylphosphonate (5'-VP), and combinations thereof.

[0208] In a preferred embodiment, the modified nucleotides are independently selected from 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, nucleotides comprising thiophosphate internucleotide linkages, and combinations thereof. In a preferred embodiment, each nucleotide of the sense strand and / or antisense strand of the RNAi agent is modified. In a preferred embodiment, each nucleotide of the sense strand and / or antisense strand of the RNAi agent is modified, and the modified nucleotides are independently selected from 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, nucleotides comprising thiophosphate internucleotide linkages, and combinations thereof.

[0209] In a preferred embodiment, each nucleotide of the sense and antisense strands of the RNAi agent is modified, and the modification is selected from one of STC (Alnylam), ESC (Alnylam), Advanced ESC (Alnylam), ESC+ (Alnylam), AD1-3 (Arrowhead), AD5 (Arrowhead) and GalXC (Dicerna) (see, for example, Hu B, Zhong L, Weng Y, et al. Therapeutic siRNA: state of the art. Signal Transduct Target Ther. 2020; 5(1): 101).

[0210] In a preferred embodiment, the 5'-terminal nucleotide of the antisense strand of the RNAi agent contains a phosphate or phosphate analog modification, preferably 5'-VP (see Parmar R, Willoughby JL, Liu J, et al. 5'-(E)-Vinylphosphonate: A Stable Phosphate Mimic Can Improve the RNAi Activity of siRNA-GalNAcConjugates. Chembiochem. 2016; 17(11): 985-989).

[0211] In some embodiments, each nucleotide of the antisense strand of the RNAi agent is modified, and in the 5' to 3' direction, the nucleotides at positions 2, 5, 7, and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense strand is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides.

[0212] In some embodiments, each nucleotide of the antisense strand of the RNAi agent is modified, and in the 5' to 3' direction, the nucleotides at positions 2, 5, 7, 12, and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides.

[0213] In some embodiments, each nucleotide of the antisense strand of the RNAi agent is modified, and in the 5' to 3' direction, the nucleotides at positions 2, 5, 7, 14, and 16 of the antisense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the antisense strand are 2'-methoxy-modified nucleotides.

[0214] In some embodiments, each nucleotide of the antisense strand of the RNAi agent is modified, and in the 5' to 3' direction, the nucleotides at positions 2, 5, 7, and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense strand is a 2'-fluoro-modified nucleotide, the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides, and the antisense strand has at least one phosphorothioate internucleotide linkage. In a preferred embodiment, the phosphorothioate internucleotide linkage is present in one or more of the following: between the first and second nucleotides at the 5' end of the antisense strand; between the second and third nucleotides at the 5' end of the antisense strand; between the first and second nucleotides at the 3' end of the antisense strand; and between the second and third nucleotides at the 3' end of the antisense strand. In a preferred embodiment, the phosphorothioate internucleotide linkage exists between the first and second nucleotides at the 5' end of the antisense strand; between the second and third nucleotides at the 5' end of the antisense strand; between the first and second nucleotides at the 3' end of the antisense strand; and between the second and third nucleotides at the 3' end of the antisense strand.

[0215] In some embodiments, each nucleotide of the sense strand and antisense strand of the RNAi agent is modified, and in the 5' to 3' direction, the nucleotides at positions 2, 5, 7, and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense strand is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the nucleotides at positions 7 and 9 of the sense strand are 2'-fluoro-modified nucleotides, one or two of the nucleotides at positions 5, 8, and 11 of the sense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense strand are all 2'-methoxy-modified nucleotides.

[0216] For example, each nucleotide of the sense strand and antisense strand of the RNAi agent is modified, and in the 5' to 3' direction, the nucleotides at positions 2, 5, 7 and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense strand is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the nucleotides at positions 5, 7, 8 and 9 of the sense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense strand are all 2'-methoxy-modified nucleotides.

[0217] For example, each nucleotide of the sense strand and antisense strand of the RNAi agent is modified, and in the 5' to 3' direction, the nucleotides at positions 2, 5, 7, and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense strand is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the nucleotides at positions 7, 8, and 9 of the sense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense strand are all 2'-methoxy-modified nucleotides.

[0218] For example, each nucleotide of the sense strand and antisense strand of the RNAi agent is modified, and in the 5' to 3' direction, the nucleotides at positions 2, 5, 7 and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense strand is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the nucleotides at positions 5, 7 and 9 of the sense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense strand are all 2'-methoxy-modified nucleotides.

[0219] For example, each nucleotide of the sense strand and the antisense strand is modified, and in the 5' to 3' direction, the nucleotides at positions 2, 5, 7 and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense strand is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the nucleotides at positions 7, 9 and 11 of the sense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense strand are all 2'-methoxy-modified nucleotides.

[0220] In a preferred embodiment, the sense strand has at least one phosphorothioate internucleotide linkage. In a preferred embodiment, the phosphorothioate internucleotide linkage is present at one or more of the following positions: (i) between the first and second nucleotides at the 5' end of the sense strand; (ii) between the second and third nucleotides at the 5' end of the sense strand; (iii) between the first and second nucleotides at the 3' end of the sense strand; and (iv) between the second and third nucleotides at the 3' end of the sense strand. In a more preferred embodiment, the phosphorothioate internucleotide linkage is present between the first and second nucleotides at the 5' end of the sense strand; and between the second and third nucleotides at the 5' end of the sense strand. In a more preferred embodiment, the phosphorothioate internucleotide linkage exists between the first and second nucleotides at the 5' end of the sense strand; between the second and third nucleotides at the 5' end of the sense strand; between the first and second nucleotides at the 3' end of the sense strand; and between the second and third nucleotides at the 3' end of the sense strand.

[0221] In a preferred embodiment, the antisense strand has at least one phosphorothioate internucleotide linkage. Preferably, the phosphorothioate internucleotide linkage is present in one or more of the following: between the first and second nucleotides at the 5' end of the antisense strand; between the second and third nucleotides at the 5' end of the antisense strand; between the first and second nucleotides at the 3' end of the antisense strand; and between the second and third nucleotides at the 3' end of the antisense strand.

[0222] Thus, in some embodiments, each nucleotide of the sense and antisense strands of the RNAi agent is modified, and in the 5' to 3' direction, the nucleotides at positions 2, 5, 7, and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense strand is a 2'-fluoro-modified nucleotide, the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides, and the antisense strand has at least one phosphorothioate internucleotide linkage, the phosphorothioate internucleotide linkage being present in one or more of the following: between the first and second nucleotides at the 5' end of the antisense strand; between the second and third nucleotides at the 5' end of the antisense strand; between the first and second nucleotides at the 3' end of the antisense strand; and between the second and third nucleotides at the 3' end of the antisense strand; in the 5' to 3' direction, the 7th and 9th nucleotides of the sense strand are 2'-fluoro-modified nucleotides, one or two of the 5th, 8th and 11th nucleotides of the sense strand are 2'-fluoro-modified nucleotides, the nucleotides at the remaining positions of the sense strand are all 2'-methoxy-modified nucleotides, and the sense strand has at least one phosphorothioate bond internucleotide connection, and the phosphorothioate bond internucleotide connection exists between the first and second nucleotides at the 5' end of the sense strand and / or between the second and third nucleotides at the 5' end of the sense strand.

[0223] In some embodiments, each nucleotide of the sense strand and the antisense strand of the RNAi agent is modified, and in the 5' to 3' direction, the nucleotides at positions 2, 5, 7, and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense strand is a 2'-fluoro-modified nucleotide, the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides, and the antisense strand has at least one phosphorothioate internucleotide linkage, the phosphorothioate internucleotide linkage being present between: the first nucleotide and the second nucleotide at the 5' end of the antisense strand, between the second nucleotide and the third nucleotide at the 5' end of the antisense strand, and between the first nucleotide and the second nucleotide at the 5' end of the antisense strand. between the first and second nucleotides at the 3' end, and between the second and third nucleotides at the 3' end of the antisense chain; in the direction from the 5' end to the 3' end, the nucleotides at positions 7 and 9 of the sense chain are 2'-fluoro-modified nucleotides, one or two of the nucleotides at positions 5, 8 and 11 of the sense chain are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense chain are all 2'-methoxy-modified nucleotides, and the sense chain has at least one phosphorothioate bond between nucleotides, and the phosphorothioate bond between nucleotides exists between the first and second nucleotides at the 5' end of the sense chain and between the second and third nucleotides at the 5' end of the sense chain.

[0224] Intermediate compounds, ligand moieties and uses

[0225] Another aspect of the present invention provides an intermediate compound selected from the group consisting of compounds of the following structural formulas:

[0226] The present invention also provides a ligand moiety of formula II for forming a lipid oligonucleotide conjugate:

[0227]

[0228] in,

[0229] At least one of R1 and R2 is selected from Ra, -C(O)Ra, -C(O)NHRa, and C(O)ORa, wherein Ra is an optionally substituted C5-30 alkyl or alkenyl group, and

[0230] The other of R1 and R2 is selected from H, Rb, -C(O)Rb, -C(O)NHRb and C(O)ORb, wherein Rb is selected from optionally substituted C1-4 alkyl or alkenyl, optionally substituted C5-30 alkyl or alkenyl, optionally substituted C2-10 alkynyl, optionally substituted C1-10 alkoxy, optionally substituted C3-10 cycloalkyl, optionally substituted 6 to 14 membered aryl, optionally substituted 5 to 18 membered heteroaryl, optionally substituted 5 to 14 membered heterocyclyl;

[0231] The optional substitution is optionally substituted with Rc, wherein Rc is selected from C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 alkoxy, C3-10 cycloalkyl, 6 to 14 membered aryl, 5 to 18 membered heteroaryl, 5 to 14 membered heterocyclyl, halogen, hydroxyl, amino, guanidino, carboxyl, cyano, nitro, C1-3 alkylthio and thiol;

[0232] m1, m2 and m3 are independently an integer of 1 or 2;

[0233] p is 0, 1, or 2; and

[0234] represents a linker and the wavy line represents the location of attachment to the rest of the lipid-oligonucleotide conjugate.

[0235] In some embodiments, one of R1 and R2 is -C(O)Ra, wherein Ra is optionally substituted C5-30 alkyl or alkenyl, and the other of R1 and R2 is H or -C(O)C1-4 alkyl or alkenyl.

[0236] In some embodiments, R1 is -C(O)Ra, wherein Ra is optionally substituted C5-30 alkyl or alkenyl, and R2 is H or -C(O)C1-4 alkyl or alkenyl.

[0237] In some embodiments, R2 is -C(O)Ra, wherein Ra is optionally substituted C5-30 alkyl or alkenyl, and R1 is H or -C(O)C1-4 alkyl or alkenyl.

[0238] In a preferred embodiment, Ra is a C5-30 alkyl or alkenyl group. In a preferred embodiment, Ra is a C12-24 alkyl or alkenyl group. In a preferred embodiment, Ra is a C14-22 alkyl or alkenyl group. In a preferred embodiment, Ra is a C14, C15, C16, C18, C20 or C22 alkyl group, a monounsaturated alkenyl group or a polyunsaturated alkenyl group.

[0239] For example, Ra is selected from C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C10 alkyl, C11 alkyl, C12 alkyl, C13 alkyl, C14 alkyl, C15 alkyl, C16 alkyl, C17 alkyl, C18 alkyl, C19 alkyl, C20 alkyl, C21 alkyl, C22 alkyl, C23 alkyl, C24 alkyl, C25 alkyl, C26 alkyl, C27 alkyl, C28 alkyl, C29 alkyl, C30 alkyl, C5 monounsaturated alkenyl, C6 monounsaturated alkenyl, C7 monounsaturated alkenyl, C8 monounsaturated alkenyl, C9 monounsaturated alkenyl, C10 monounsaturated alkenyl, C11 monounsaturated alkenyl, C12 monounsaturated alkenyl, C13 monounsaturated alkenyl, C14 monounsaturated alkenyl, C15 monounsaturated alkenyl, C16 monounsaturated alkenyl, C17 monounsaturated alkenyl, C18 monounsaturated alkenyl, C19 monounsaturated alkenyl, C20 monounsaturated alkenyl, C21 monounsaturated alkenyl, C22 monounsaturated alkenyl, C C23 monounsaturated alkenyl, C24 monounsaturated alkenyl, C25 monounsaturated alkenyl, C26 monounsaturated alkenyl, C27 monounsaturated alkenyl, C28 monounsaturated alkenyl, C29 monounsaturated alkenyl, C30 monounsaturated alkenyl, C5 polyunsaturated alkenyl, C6 polyunsaturated alkenyl, C7 polyunsaturated alkenyl, C8 polyunsaturated alkenyl, C9 polyunsaturated alkenyl, C10 polyunsaturated alkenyl, C11 polyunsaturated alkenyl, C12 polyunsaturated alkenyl, C13 polyunsaturated alkenyl, C14 polyunsaturated alkenyl, C15 polyunsaturated alkenyl, C16 polyunsaturated alkenyl, C17 polyunsaturated alkenyl, C18 polyunsaturated alkenyl, C19 polyunsaturated alkenyl, C20 polyunsaturated alkenyl, C21 polyunsaturated alkenyl, C22 polyunsaturated alkenyl, C23 polyunsaturated alkenyl, C24 polyunsaturated alkenyl, C25 polyunsaturated alkenyl, C26 polyunsaturated alkenyl, C27 polyunsaturated alkenyl, C28 polyunsaturated alkenyl, C29 polyunsaturated alkenyl, and C30 polyunsaturated alkenyl.

[0240] In a preferred embodiment, both of R1 and R2 are and independently -C(O)Ra, and Ra is an optionally substituted C5-30 alkyl or alkenyl. In a preferred embodiment, both of R1 and R2 are -C(O)Ra, and Ra is an optionally substituted C5-30 alkyl or alkenyl. In a preferred embodiment, R1 and R2 are identical and Ra is a C5-30 alkyl or alkenyl. In a preferred embodiment, R1 and R2 are identical and Ra is a C12-24 alkyl or alkenyl. In a preferred embodiment, R1 and R2 are identical and Ra is a C14-22 alkyl or alkenyl. In a preferred embodiment, R1 and R2 are identical and Ra is a C14, C15, C16, C18, C20 or C22 alkyl, monounsaturated alkenyl or polyunsaturated alkenyl.

[0241] In a preferred embodiment, R1 and R2 are the same and Ra is selected from C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C10 alkyl, C11 alkyl, C12 alkyl, C13 alkyl, C14 alkyl, C15 alkyl, C16 alkyl, C17 alkyl, C18 alkyl, C19 alkyl, C20 alkyl, C21 alkyl, C22 alkyl, C23 alkyl, C24 alkyl, C25 alkyl, C26 alkyl, C27 alkyl, C28 alkyl, C29 alkyl, C30 alkyl, C31 alkyl, C32 alkyl, C33 alkyl, C34 alkyl, C35 alkyl, C36 alkyl, C37 alkyl, C38 alkyl, C39 alkyl, C40 alkyl, C41 alkyl, C42 alkyl, C43 alkyl, C44 alkyl, C45 alkyl, C46 alkyl, C47 alkyl, C48 alkyl, C49 alkyl, C50 alkyl, C51 alkyl, C52 alkyl, C53 alkyl, C54 alkyl, C55 alkyl, C56 alkyl, C57 alkyl, C58 alkyl, C59 alkyl, C60 alkyl, C61 alkyl, C62 alkyl, C63 alkyl, C64 alkyl, C65 alkyl, C66 alkyl, C67 alkyl, C68 alkyl, C69 alkyl, C70 alkyl, C71 alkyl, C72 alkyl, C73 alkyl, C74 alkyl, C75 alkyl, C76 alkyl, C77 alkyl, C78 alkyl, C79 alkyl, C80 alkyl, C81 alkyl, C82 Alkyl, C5 monounsaturated alkenyl, C6 monounsaturated alkenyl, C7 monounsaturated alkenyl, C8 monounsaturated alkenyl, C9 monounsaturated alkenyl, C10 monounsaturated alkenyl, C11 monounsaturated alkenyl, C12 monounsaturated alkenyl, C13 monounsaturated alkenyl, C14 monounsaturated alkenyl, C15 monounsaturated alkenyl, C16 monounsaturated alkenyl, C17 monounsaturated alkenyl, C18 monounsaturated alkenyl, C19 monounsaturated alkenyl, C20 monounsaturated alkenyl, C21 monounsaturated alkenyl, C22 monounsaturated alkenyl, C23 monounsaturated alkenyl, C24 monounsaturated alkenyl, C25 monounsaturated alkenyl, C26 monounsaturated alkenyl, C27 monounsaturated alkenyl, C28 monounsaturated alkenyl, C29 monounsaturated alkenyl, C30 monounsaturated alkenyl, C5 polyunsaturated alkenyl, C6 polyunsaturated alkenyl, C7 polyunsaturated alkenyl, C8 polyunsaturated alkenyl, C9 polyunsaturated alkenyl, C10 polyunsaturated alkenyl, C11 polyunsaturated alkenyl, C12 polyunsaturated alkenyl, C13 polyunsaturated alkenyl , C14 polyunsaturated alkenyl, C15 polyunsaturated alkenyl, C16 polyunsaturated alkenyl, C17 polyunsaturated alkenyl, C18 polyunsaturated alkenyl, C19 polyunsaturated alkenyl, C20 polyunsaturated alkenyl, C21 polyunsaturated alkenyl, C22 polyunsaturated alkenyl, C23 polyunsaturated alkenyl, C24 polyunsaturated alkenyl, C25 polyunsaturated alkenyl, C26 polyunsaturated alkenyl, C27 polyunsaturated alkenyl, C28 polyunsaturated alkenyl, C29 polyunsaturated alkenyl, and C30 polyunsaturated alkenyl.

[0242] In some embodiments, m1, m2, and m3 are all 1. In some embodiments, two of m1, m2, and m3 are 1, and the other is 2. In some embodiments, m1 and m2 are 1, and m3 is 2. In some embodiments, m1 and m3 are 1, and m2 is 2. In some embodiments, m2 and m3 are 1, and m1 is 2.

[0243] In some embodiments, p is 2. In some embodiments, p is 0. In a preferred embodiment, p is 1.

[0244] In some embodiments, the oligonucleotide is an antisense oligonucleotide or a sense strand or antisense strand of an RNAi agent. In some embodiments, the sense strand or antisense strand of the RNAi agent is paired with its antisense strand or sense strand to form a duplex. In a preferred embodiment, the RNAi agent is an siRNA.

[0245] In a preferred embodiment, the oligonucleotide is an antisense oligonucleotide (ASO). In a preferred embodiment, the oligonucleotide is the sense strand or antisense strand of siRNA. In a preferred embodiment, the oligonucleotide is the sense strand of siRNA. In a preferred embodiment, the sense strand or antisense strand of the siRNA is paired with its antisense strand or sense strand to form a duplex.

[0246] In some embodiments, wherein L1 is wherein each n is independently an integer from 1 to 10, preferably an integer from 2 to 8, more preferably an integer from 4 to 6; and the right side of the formula is connected to the rest of the lipid oligonucleotide conjugate.

[0247] In a specific embodiment, the structure of the ligand is:

[0248]

[0249] Another aspect of the present invention provides the use of a ligand moiety represented by formula III in the preparation of a lipid oligonucleotide conjugate.

[0250]

[0251] in,

[0252] At least one of R1 and R2 is selected from Ra, -C(O)Ra, -C(O)NHRa, and C(O)ORa, wherein Ra is an optionally substituted C5-30 alkyl or alkenyl group, and

[0253] The other of R1 and R2 is selected from H, Rb, -C(O)Rb, -C(O)NHRb and C(O)ORb, wherein Rb is selected from optionally substituted C1-4 alkyl or alkenyl, optionally substituted C5-30 alkyl or alkenyl, optionally substituted C2-10 alkynyl, optionally substituted C1-10 alkoxy, optionally substituted C3-10 cycloalkyl, optionally substituted 6 to 14 membered aryl, optionally substituted 5 to 18 membered heteroaryl, optionally substituted 5 to 14 membered heterocyclyl;

[0254] The optional substitution is optionally substituted with Rc, wherein Rc is selected from C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 alkoxy, C3-10 cycloalkyl, 6 to 14 membered aryl, 5 to 18 membered heteroaryl, 5 to 14 membered heterocyclyl, halogen, hydroxyl, amino, guanidino, carboxyl, cyano, nitro, C1-3 alkylthio and thiol;

[0255] m1, m2 and m3 are independently an integer of 1 or 2;

[0256] p is 0, 1, or 2; and

[0257] The wavy line represents the location of attachment to the rest of the lipid-oligonucleotide conjugate.

[0258] In some embodiments, one of R1 and R2 is -C(O)Ra, wherein Ra is optionally substituted C5-30 alkyl or alkenyl, and the other of R1 and R2 is H or -C(O)C1-4 alkyl or alkenyl.

[0259] In some embodiments, R1 is -C(O)Ra, wherein Ra is optionally substituted C5-30 alkyl or alkenyl, and R2 is H or -C(O)C1-4 alkyl or alkenyl.

[0260] In some embodiments, R2 is -C(O)Ra, wherein Ra is optionally substituted C5-30 alkyl or alkenyl, and R1 is H or -C(O)C1-4 alkyl or alkenyl.

[0261] In a preferred embodiment, Ra is a C5-30 alkyl or alkenyl group. In a preferred embodiment, Ra is a C12-24 alkyl or alkenyl group. In a preferred embodiment, Ra is a C14-22 alkyl or alkenyl group. In a preferred embodiment, Ra is a C14, C15, C16, C18, C20 or C22 alkyl group, a monounsaturated alkenyl group or a polyunsaturated alkenyl group.

[0262] For example, Ra is selected from C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C10 alkyl, C11 alkyl, C12 alkyl, C13 alkyl, C14 alkyl, C15 alkyl, C16 alkyl, C17 alkyl, C18 alkyl, C19 alkyl, C20 alkyl, C21 alkyl, C22 alkyl, C23 alkyl, C24 alkyl, C25 alkyl, C26 alkyl, C27 alkyl, C28 alkyl, C29 alkyl, C30 alkyl, C5 monounsaturated alkenyl, C6 monounsaturated alkenyl, C7 monounsaturated alkenyl, C8 monounsaturated alkenyl, C9 monounsaturated alkenyl, C10 monounsaturated alkenyl, C11 monounsaturated alkenyl, C12 monounsaturated alkenyl, C13 monounsaturated alkenyl, C14 monounsaturated alkenyl, C15 monounsaturated alkenyl, C16 monounsaturated alkenyl, C17 monounsaturated alkenyl, C18 monounsaturated alkenyl, C19 monounsaturated alkenyl, C20 monounsaturated alkenyl, C21 monounsaturated alkenyl, C22 monounsaturated alkenyl, C C23 monounsaturated alkenyl, C24 monounsaturated alkenyl, C25 monounsaturated alkenyl, C26 monounsaturated alkenyl, C27 monounsaturated alkenyl, C28 monounsaturated alkenyl, C29 monounsaturated alkenyl, C30 monounsaturated alkenyl, C5 polyunsaturated alkenyl, C6 polyunsaturated alkenyl, C7 polyunsaturated alkenyl, C8 polyunsaturated alkenyl, C9 polyunsaturated alkenyl, C10 polyunsaturated alkenyl, C11 polyunsaturated alkenyl, C12 polyunsaturated alkenyl, C13 polyunsaturated alkenyl, C14 polyunsaturated alkenyl, C15 polyunsaturated alkenyl, C16 polyunsaturated alkenyl, C17 polyunsaturated alkenyl, C18 polyunsaturated alkenyl, C19 polyunsaturated alkenyl, C20 polyunsaturated alkenyl, C21 polyunsaturated alkenyl, C22 polyunsaturated alkenyl, C23 polyunsaturated alkenyl, C24 polyunsaturated alkenyl, C25 polyunsaturated alkenyl, C26 polyunsaturated alkenyl, C27 polyunsaturated alkenyl, C28 polyunsaturated alkenyl, C29 polyunsaturated alkenyl, and C30 polyunsaturated alkenyl.

[0263] In a preferred embodiment, both of R1 and R2 are and independently -C(O)Ra, and Ra is an optionally substituted C5-30 alkyl or alkenyl. In a preferred embodiment, both of R1 and R2 are -C(O)Ra, and Ra is an optionally substituted C5-30 alkyl or alkenyl. In a preferred embodiment, R1 and R2 are identical and Ra is a C5-30 alkyl or alkenyl. In a preferred embodiment, R1 and R2 are identical and Ra is a C12-24 alkyl or alkenyl. In a preferred embodiment, R1 and R2 are identical and Ra is a C14-22 alkyl or alkenyl. In a preferred embodiment, R1 and R2 are identical and Ra is a C14, C15, C16, C18, C20 or C22 alkyl, monounsaturated alkenyl or polyunsaturated alkenyl.

[0264] In a preferred embodiment, R1 and R2 are the same and Ra is selected from C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C10 alkyl, C11 alkyl, C12 alkyl, C13 alkyl, C14 alkyl, C15 alkyl, C16 alkyl, C17 alkyl, C18 alkyl, C19 alkyl, C20 alkyl, C21 alkyl, C22 alkyl, C23 alkyl, C24 alkyl, C25 alkyl, C26 alkyl, C27 alkyl, C28 alkyl, C29 alkyl, C30 alkyl, C31 alkyl, C32 alkyl, C33 alkyl, C34 alkyl, C35 alkyl, C36 alkyl, C37 alkyl, C38 alkyl, C39 alkyl, C40 alkyl, C41 alkyl, C42 alkyl, C43 alkyl, C44 alkyl, C45 alkyl, C46 alkyl, C47 alkyl, C48 alkyl, C49 alkyl, C50 alkyl, C51 alkyl, C52 alkyl, C53 alkyl, C54 alkyl, C55 alkyl, C56 alkyl, C57 alkyl, C58 alkyl, C59 alkyl, C60 alkyl, C61 alkyl, C62 alkyl, C63 alkyl, C64 alkyl, C65 alkyl, C66 alkyl, C67 alkyl, C68 alkyl, C69 alkyl, C70 alkyl, C71 alkyl, C72 alkyl, C73 alkyl, C74 alkyl, C75 alkyl, C76 alkyl, C77 alkyl, C78 alkyl, C79 alkyl, C80 alkyl, C81 alkyl, C82 Alkyl, C5 monounsaturated alkenyl, C6 monounsaturated alkenyl, C7 monounsaturated alkenyl, C8 monounsaturated alkenyl, C9 monounsaturated alkenyl, C10 monounsaturated alkenyl, C11 monounsaturated alkenyl, C12 monounsaturated alkenyl, C13 monounsaturated alkenyl, C14 monounsaturated alkenyl, C15 monounsaturated alkenyl, C16 monounsaturated alkenyl, C17 monounsaturated alkenyl, C18 monounsaturated alkenyl, C19 monounsaturated alkenyl, C20 monounsaturated alkenyl, C21 monounsaturated alkenyl, C22 monounsaturated alkenyl, C23 monounsaturated alkenyl, C24 monounsaturated alkenyl, C25 monounsaturated alkenyl, C26 monounsaturated alkenyl, C27 monounsaturated alkenyl, C28 monounsaturated alkenyl, C29 monounsaturated alkenyl, C30 monounsaturated alkenyl, C5 polyunsaturated alkenyl, C6 polyunsaturated alkenyl, C7 polyunsaturated alkenyl, C8 polyunsaturated alkenyl, C9 polyunsaturated alkenyl, C10 polyunsaturated alkenyl, C11 polyunsaturated alkenyl, C12 polyunsaturated alkenyl, C13 polyunsaturated alkenyl , C14 polyunsaturated alkenyl, C15 polyunsaturated alkenyl, C16 polyunsaturated alkenyl, C17 polyunsaturated alkenyl, C18 polyunsaturated alkenyl, C19 polyunsaturated alkenyl, C20 polyunsaturated alkenyl, C21 polyunsaturated alkenyl, C22 polyunsaturated alkenyl, C23 polyunsaturated alkenyl, C24 polyunsaturated alkenyl, C25 polyunsaturated alkenyl, C26 polyunsaturated alkenyl, C27 polyunsaturated alkenyl, C28 polyunsaturated alkenyl, C29 polyunsaturated alkenyl, and C30 polyunsaturated alkenyl.

[0265] In some embodiments, m1, m2, and m3 are all 1. In some embodiments, two of m1, m2, and m3 are 1, and the other is 2. In some embodiments, m1 and m2 are 1, and m3 is 2. In some embodiments, m1 and m3 are 1, and m2 is 2. In some embodiments, m2 and m3 are 1, and m1 is 2.

[0266] In some embodiments, p is 2. In some embodiments, p is 0. In a preferred embodiment, p is 1.

[0267] In some embodiments, the oligonucleotide is an antisense oligonucleotide or a sense strand or antisense strand of an RNAi agent. In some embodiments, the sense strand or antisense strand of the RNAi agent is paired with its antisense strand or sense strand to form a duplex. In a preferred embodiment, the RNAi agent is an siRNA.

[0268] In a preferred embodiment, the oligonucleotide is an antisense oligonucleotide (ASO). In a preferred embodiment, the oligonucleotide is the sense strand or antisense strand of siRNA. In a preferred embodiment, the oligonucleotide is the sense strand of siRNA. In a preferred embodiment, the sense strand or antisense strand of the siRNA is paired with its antisense strand or sense strand to form a duplex.

[0269] Pharmaceutical compositions, treatment methods, and uses

[0270] The present invention also includes pharmaceutical compositions or preparations comprising lipid oligonucleotide conjugates as described herein and pharmaceutically acceptable carriers, excipients or diluents. Such compositions and preparations can be used to reduce the expression of target genes in patients in need. In the case of considering clinical applications, pharmaceutical compositions and preparations will be prepared in a form suitable for the intended application. Typically, this will require preparation of a composition that is substantially free of pyrogens and other impurities that may be harmful to humans or animals.

[0271] The composition and method for preparing the pharmaceutical composition depend on many standards, including but not limited to route of administration, the type and degree of the disease to be treated or the condition or the dosage to be administered. In some embodiments, the pharmaceutical composition is prepared based on the expected route of delivery. For example, in certain embodiments, the pharmaceutical composition is formulated for parenteral delivery. Parenteral administration forms include intravenous, intraarterial, subcutaneous, intrathecal, intraperitoneal or intramuscular injection or infusion. In one embodiment, the pharmaceutical composition is formulated for intravenous delivery. In such an embodiment, the pharmaceutical composition can include a lipid-based delivery vehicle. In another embodiment, the pharmaceutical composition is formulated for subcutaneous delivery.

[0272] In an embodiment, the lipid-oligonucleotide conjugate is preferably delivered to a specific tissue in the body after administration. In an embodiment, the lipid-oligonucleotide conjugate is, for example, delivered to cardiac tissue after administration. In an embodiment, the lipid-oligonucleotide conjugate is, for example, delivered to spleen tissue after administration. In an embodiment, the lipid-oligonucleotide conjugate is, for example, delivered to kidney tissue after administration. In an embodiment, the lipid-oligonucleotide conjugate is, for example, delivered to skin tissue after administration. In an embodiment, the lipid-oligonucleotide conjugate is, for example, delivered to muscle tissue after administration. In an embodiment, the lipid-oligonucleotide conjugate is, for example, delivered to lung tissue after administration. In an embodiment, the lipid-oligonucleotide conjugate is, for example, delivered to adipose tissue after administration. In an embodiment, the lipid-oligonucleotide conjugate is, for example, delivered to brain tissue after administration.

[0273] In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount or a preventive effective amount of a lipid oligonucleotide conjugate as described herein." effective amount" refers to an amount sufficient to produce a useful or desired clinical outcome. In some embodiments, an effective amount is an amount sufficient to reduce target gene expression in a patient's specific tissue or cell type (e.g., liver or hepatocyte). The effective amount of the lipid oligonucleotide conjugate of the present invention can be from about 0.01 mg / kg body weight to about 100 mg / kg body weight, and can be administered every day, every week, every month, or at longer intervals. Accurately determine specific effective dosage and administration frequency may be based on several factors, including the patient's size, age, and general condition, the type of disease to be treated (e.g., myocardial infarction, coronary artery disease, peripheral arterial disease, stroke), the specific lipid oligonucleotide conjugate used, and route of administration.

[0274] The administration of the pharmaceutical composition of the present invention can be carried out by any common route, as long as the target tissue can be obtained by the route. These routes include, but are not limited to, parenteral (e.g., subcutaneous, intramuscular, intraperitoneal or intravenous), oral, nasal, oral, intradermal, transdermal and sublingual routes. In some embodiments, the pharmaceutical composition is administered parenterally. For example, in certain embodiments, the pharmaceutical composition is administered intravenously. In other embodiments, the pharmaceutical composition is administered subcutaneously. In other embodiments, the pharmaceutical composition is administered pulmonary or intranasally.

[0275] Pharmaceutical compositions suitable for injection include, for example, sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In general, these preparations are sterile and, to a certain extent, fluid and easy to inject. The preparation should remain stable under production and storage conditions and should be preserved to prevent contamination by microorganisms such as bacteria and fungi. Suitable solvents or dispersion media can include, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. For example, suitable fluidity can be maintained by using a coating such as lecithin, by maintaining the desired particle size in the case of dispersion, and by using a surfactant. The effects of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferred to include isotonic agents, such as sugar or sodium chloride. Prolonged absorption of injectable compositions can be achieved by using agents that delay absorption in the composition, such as aluminum monostearate and gelatin.

[0276] Sterile injectable solutions can be prepared by adding an appropriate amount of the active compound to a solvent along with any other ingredients (e.g., those listed above) and then sterilizing by filtration. Typically, dispersions are prepared by adding the various sterilized active ingredients to a dispersion medium containing an alkaline dispersion medium and the desired other ingredients, e.g., as described above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods include vacuum drying and freeze drying techniques, which produce a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution thereof.

[0277] The compositions of the present invention can generally be formulated in neutral form or salt form. Pharmaceutically acceptable salts include, for example, acid addition salts (formed by free amino groups) derived from inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed with free carboxyl groups can also be derived from inorganic bases (such as sodium, potassium, ammonium, calcium or iron oxide) or organic bases (such as isopropylamine, trimethylamine, histidine, procaine, etc.). In some embodiments, the lipid oligonucleotide conjugates of the present invention are formulated as sodium salts.

[0278] For example, for parenteral administration in the form of an aqueous solution, the solution is generally appropriately buffered, and the liquid diluent is first made isotonic with, for example, enough saline or glucose. Such an aqueous solution can be used for, for example, intravenous, intramuscular, subcutaneous, and intraperitoneal administration. Preferably, a sterile aqueous medium is used. For example, a single dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion liquid, or injected at the infusion site of the suggestion. For human administration, the preparation should meet the sterility, pyrogenicity, general safety, and purity standards required by the local Food and Drug Administration. In certain embodiments, the pharmaceutical composition of the present invention comprises sterile saline solution and lipid oligonucleotide conjugate as described herein or consists of the two. In other embodiments, the pharmaceutical composition of the present invention comprises lipid oligonucleotide conjugate as described herein and sterile water (e.g., water for injection, WFI) or consists of the two. In other embodiments, the pharmaceutical composition of the present invention comprises lipid oligonucleotide conjugate as described herein and phosphate buffered saline (PBS) or consists of it.

[0279] In some embodiments, the pharmaceutical compositions of the present invention are packaged with or stored within a drug delivery device. Devices for injecting formulations include, but are not limited to, injection ports, prefilled syringes, autoinjectors, syringe pumps, intracorporeal syringes, and injection pens. Devices for aerosolizing or powdered formulations include, but are not limited to, inhalers, insufflators, aspirators, and the like. Thus, the present invention includes a drug delivery device containing a pharmaceutical composition of the present invention for use in treating or preventing one or more diseases or conditions described herein.

[0280] In some embodiments of the present invention, the target gene that the oligonucleotide in the lipid-oligonucleotide conjugate can target can be selected from the following group, which consists of the following items: SOD1, MALAT1, apolipoprotein B, apolipoprotein C III, endothelial lipase, p53, clusterin, signal transducer and activator of transcription 3 (STAT3), cell signaling molecule (Mothers against decapentaplegic homolog) 7 (SMAD7), intercellular adhesion molecule 1 (CD54), dystrophin (e.g., myotonic dystrophy protein kinase (DMPK), thyroxine transducer (TTR), huntingtin protein (HTT), microRNA-122 (miR-122).

[0281] In certain embodiments, the expression of the target gene in the cell is reduced by at least 40%, at least 45% or at least 50% by the lipid oligonucleotide conjugates of the present invention. In some embodiments, the expression of the target gene in the cell is reduced by at least 60%, at least 65%, at least 70%, at least 75%, at least 80% or at least 85% by the lipid oligonucleotide conjugates of the present invention. In other embodiments, the expression of the target gene in the cell is reduced by about 90% or more, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more by the lipid oligonucleotide conjugates of the present invention. The percentage reduction of target gene expression can be measured by any method described herein and other methods known in the art.

[0282] In certain embodiments, the present invention provides a method for reducing target gene expression in patients in need, comprising administering any lipid oligonucleotide conjugate described herein to the patient. Preferably, the target gene expression level in the patient's cells is reduced compared to the target gene expression level in patients who have not received the lipid oligonucleotide conjugate, or compared to the target gene expression level in the patient before administering the lipid oligonucleotide conjugate. In some embodiments, after administering the lipid oligonucleotide conjugate of the present invention, the expression of the target gene in the patient is reduced by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90%, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. The percentage reduction of target gene expression can be measured by any method described herein and other methods known in the art. In certain embodiments, the percent reduction in target gene expression is determined by assessing target gene protein levels in the patient's serum or plasma according to the methods described herein.

[0283] In certain other embodiments, the patient in need of reducing target gene expression is a patient with elevated levels of circulating target gene. Therefore, in some embodiments, the present invention provides a method for reducing the target gene protein serum or plasma level in a patient in need by administering any lipid oligonucleotide conjugate described herein to the patient. In some embodiments, the present invention includes the use of any lipid oligonucleotide conjugate described herein in the preparation of a medicament for reducing the target gene protein serum or plasma level in a patient in need. In other embodiments, the present invention provides a target gene targeted lipid oligonucleotide conjugate for use in a method for reducing the target gene protein serum or plasma level in a patient in need.

[0284] The present invention also provides the use of the lipid-oligonucleotide conjugates, intermediate compounds, or ligand moieties described herein in the preparation of lipid-oligonucleotide conjugates.

[0285] Example

[0286] Example 1. Synthesis of LL1p

[0287] Synthesis of compound 1-2

[0288] 1,4,7-Triazolidine (50.00 g, 386.99 mmol) was dissolved in dichloromethane (500 mL) at 25°C, and triethylamine (97.90 g, 967.47 mmol) was added. Di-tert-butyl dicarbonate (160.47 g, 735.28 mmol) was slowly added dropwise to the mixture at 0°C, followed by stirring at 25°C for 12 hours. After completion of the reaction, the mixture was diluted with water (1000 mL) and extracted with dichloromethane (300 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and dried. The residue was purified by flash silica gel chromatography (eluent: 0-25% ethyl acetate / petroleum ether) to afford compound 1-2 (50.00 g, 39.22% yield) as a white solid. 1 H NMR (400MHz, CDCl3) δ3.54-3.40 (m, 4H), 3.35-3.15 (m, 4H), 2.93 (d, J = 4.9Hz, 4H), 1.48 (s, 17H).

[0289] Synthesis of compounds 1-3

[0290] At 25°C, compound 1-2 (50.00 g, 151.78 mmol) and benzyl bromoacetate (38.24 g, 166.95 mmol) were dissolved in acetonitrile (500 mL), potassium carbonate (41.95 g, 303.55 mmol) was added, and then stirred at 70°C for 16 hours. After the reaction was completed, water (300 mL) was added to dilute the mixture and extracted with dichloromethane (300 mL*3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and dried, and the residue was purified by flash silica gel chromatography (eluent: 0-25% ethyl acetate / petroleum ether) to obtain compound 1-3 (70.00 g, 96.57% yield) as a white solid. MS m / z (ESI): 478.3 [M+H] + ; 1H NMR (400MHz, CDCl3) δ7.44-7.31(m,5H),5.24-5.10(m,2H),3.56-3.40(m,6 H), 3.35-3.17 (m, 4H), 2.87 (dd, J = 5.5, 10.8Hz, 4H), 1.48 (d, J = 4.5Hz, 18H).

[0291] Synthesis of compounds 1-4

[0292] At 25°C, compound 1-3 (70.00 g, 146.57 mmol) was dissolved in dioxane (100 mL), and hydrochloric acid / dioxane (2 M, 500 mL) was added, followed by stirring at 25°C for 16 hours. After the reaction was complete, the organic phase was dried to obtain compound 1-4 (40.00 g, crude) as a white solid. MS m / z (ESI): 278.2 [M+H] + .

[0293] Synthesis of compounds 1-5

[0294] Compound 1-4 (40.00 g, 144.22 mmol) was dissolved in dichloromethane (500 mL) at 25°C, triethylamine (87.56 g, 865.30 mmol) was added, and palmitoyl chloride (79.28 g, 288.43 mmol) was slowly added dropwise to the mixture at -60°C, followed by stirring at -60°C for 3 hours. After the reaction was complete, water (500 mL) was added to dilute the mixture and extracted with dichloromethane (300 mL*3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and dried. The residue was purified by flash silica gel chromatography (eluent: 0-10% ethyl acetate / petroleum ether) to obtain compound 1-5 (85.00 g, 78.15% yield) as a white solid. MS m / z (ESI): 754.7 [M+H] + ; 1 H NMR (400MHz, CDCl3) δ7.34-7.24(m,5H),5.09-5.05(m,2H),3.69-3.51(m,4H),3.38(s,2H),3.3 1-3.16(m,4H),2.95-2.71(m,4H),2.24-2.10(m,4H),1.26-1.14(m,52H),0.81(t,J=6.8Hz,6H).

[0295] Synthesis of compounds 1-6

[0296] At 25°C, compound 1-5 (100.00 g, 144.22 mmol) was dissolved in tetrahydrofuran (1000 mL) in a hydrogenation bottle, then purged with nitrogen three times. Wet palladium hydroxide (9.31 g, 20% purity) was added and the atmosphere in the hydrogenation bottle was purged with nitrogen three times and hydrogen three times to 15 psi, and then stirred at 25°C for 16 hours. After the reaction was completed, the reaction solution was filtered and dried. The residue was purified by methanol slurrying to obtain white solid compound 1-6 (65.00 g, 73.82% yield). MS m / z (ESI): 664.6 [M+H] + ; 1 H NMR (400MHz, CDCl3) δ3.81-3.62(m,4H),3.52-3.28(m,6H),3.03-2.78(m,4H),2.38-2.21(m,4H),1.63(s,4H),1.27(m,48H),0.89(t,J=6.7Hz,6H).

[0297] Synthesis of compounds 1-7

[0298] At 25 ° C, compound 1-6 (7.00 g, 10.54 mmol) and 1-hydroxybenzotriazole (2.85 g, 21.08 mmol) were dissolved in tetrahydrofuran (70 mL), tert-butyl 9-aminononyl ester (2.90 g, 12.65 mmol), N, N-diisopropylethylamine (4.09 g, 31.62 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.04 g, 21.08 mmol) were added, and then stirred at 25 ° C for 3.5 hours. After the reaction was completed, water (30 mL) was added to dilute and extracted with dichloromethane (10 mL * 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and spun down to dryness. The residue was purified by reverse-phase HPLC (column: C18 150×40 mm; mobile phase: [water (ammonia v / v)-(acetonitrile-tetrahydrofuran 2 / 1)]; gradient: 90% to 100% B, time: 9 min) to obtain compound 1-7 (9.00 g, 97.53% yield) as a yellow oil. MS m / z (ESI): 875.8 [M+H] + ; 1H NMR (400MHz, CDCl3) δ6.85-6.71(m,1H),3.75-3.58(m,4H),3.50-3.33(m,4H),3.29-3.17(m,4H),2.78(d,J=4.5Hz,4H ),2.35-2.27(m,4H),2.21(t,J=7.5Hz,2H),1.69-1.55(m,6H),1.46(s,9H),1.38-1.22(m,58H),0.90(t,J=6.8Hz,6H).

[0299] Synthesis of compounds 1-8

[0300] At 25°C, compound 1-7 (6.50 g, 7.43 mmol) was dissolved in dichloromethane (50 mL), trifluoroacetic acid (25 mL) was added, and then stirred at 25°C for 3 hours. After the reaction was completed, water (50 mL) was added to dilute and extracted with dichloromethane (50 mL*3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and dried, and the residue was purified by flash silica gel chromatography (eluent: 0-25% tetrahydrofuran / petroleum ether) to obtain white solid compound 1-8 (5.50 g, 90.41% yield). MS m / z (ESI): 819.8 [M+H] + ; 1 H NMR (400MHz, CDCl3) δ7.44-7.31(m,5H),5.24-5.10(m,2H),3.56-3.40(m,6 H), 3.35-3.17 (m, 4H), 2.87 (dd, J = 5.5, 10.8Hz, 4H), 1.48 (d, J = 4.5Hz, 18H).

[0301] Synthesis of compound LL1p

[0302] Compound 1-8 (2.00 g, 2.44 mmol) was dissolved in dichloromethane (300 mL) at 25°C, and 2,3,4,5,6-pentafluorophenol (539.19 mg, 2.93 mmol) was added, followed by stirring at 25°C for 3 hours. After the reaction was complete, the organic phase was dried, and the residue was purified by flash silica gel chromatography (eluent: 0-20% tetrahydrofuran / petroleum ether) to obtain compound LL1p (1.20 g, 49.89% yield) as a colorless amorphous solid. MS m / z (ESI): 985.7 [M+H] + ; 1H NMR (400MHz, CDCl3) δ6.78-6.62(m,1H),3.67-3.50(m,4H),3.42-3.24(m,4H),3.21-3.09(m,4H),2.76-2.64(m, 4H), 2.59 (t, J = 7.4Hz, 2H), 2.22 (q, J = 8.2Hz, 4H), 1.77-1.32 (m, 12H), 1.27-1.13 (m, 52H), 0.81 (t, J = 6.8Hz, 6H).

[0303] Example 2. Synthesis of LL2p

[0304] Synthesis of compound 2-1

[0305] At 25 ° C, compound 1-6 (7.00 g, 10.54 mmol) and 1-hydroxybenzotriazole (2.85 g, 21.08 mmol) were dissolved in tetrahydrofuran (70 mL), and tert-butyl 15-amino-4,7,10,13-tetraoxapentadecanoate (3.73 g, 11.60 mmol), N,N-diisopropylethylamine (4.09 g, 31.62 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.04 g, 21.08 mmol) were added, and then stirred at 25 ° C for 3.5 hours. After the reaction was completed, water (30 mL) was added to dilute and extracted with dichloromethane (10 mL * 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and spun down to dryness. The residue was purified by reverse-phase HPLC (column: C18 150×40 mm; mobile phase: [water (ammonia v / v)-(acetonitrile-tetrahydrofuran 2 / 1)]; gradient: 85% to 100% B, time: 9 min) to obtain compound 2-1 (10.00 g, 98.06% yield) as a yellow oil. MS m / z (ESI): 967.8 [M+H] + ; 1 H NMR (400MHz, CDCl3) δ7.25-7.06(m,1H),3.71(d,J=7.8Hz,4H),3.65-3.62(m,10H),3.57(t,J=5.0Hz,2H),3.47(d,J=5.0Hz,6H),3.26-3.18(m,2H), 2.88-2.73(m,4H),2.52(t,J=6.5Hz,2H),2.38-2.23(m,4H),1.78(s,4H), 1.70-1.58(m,4H),1.46(s,9H),1.34-1.24(m,48H),0.90(t,J=6.7Hz,6H).

[0306] Synthesis of compound 2-2

[0307] At 25°C, compound 2-1 (7.00 g, 7.24 mmol) was dissolved in dichloromethane (50 mL), trifluoroacetic acid (25 mL) was added, and then stirred at 25°C for 3 hours. After the reaction was completed, water (50 mL) was added to dilute and extracted with dichloromethane (50 mL*3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and dried, and the residue was purified by flash silica gel chromatography (eluent: 0-25% tetrahydrofuran / petroleum ether) to obtain white solid compound 2-2 (6.50 g, 98.57% yield). MS m / z (ESI): 911.8 [M+H] + ; 1 H NMR (400MHz, CDCl3) δ7.69-7.33(m,1H),4.40-3.21(m,20H),2.49-2.26(m ,6H),1.52(s,2H),1.64(s,5H),1.40-1.22(m,56H),0.90(t,J=6.7Hz,6H).

[0308] Synthesis of compound LL2p

[0309] Compound 2-2 (2.00 g, 2.44 mmol) was dissolved in dichloromethane (30 mL) at 25°C, and 2,3,4,5,6-pentafluorophenol (539.19 mg, 2.93 mmol) was added, followed by stirring at 25°C for 3 hours. After the reaction was complete, the organic phase was dried, and the residue was purified by flash silica gel chromatography (eluent: 0-20% tetrahydrofuran / petroleum ether) to obtain compound LL2p (1.20 g, 50.75% yield) as a colorless amorphous solid. MS m / z (ESI): 1077.7 [M+H] + ; 1 H NMR (400MHz, CDCl3) δ7.24-6.99(m,1H),3.89(t,J=6.2Hz,2H),3.74-3.61(m,16H),3.60-3.55(m,2H),3.51-3.32(m,6H),3.23 (s,2H),2.96(t,J=6.2Hz,2H),2.87-2.73(m,4H),2.36-2.24(m,4H),1.71-1.57(m,4H),1.35-1.24(m,48H),0.97-0.82(m,6H).

[0310] Example 3. Synthesis of lipid oligonucleic acid conjugates

[0311] Oligonucleotide synthesis

[0312] The oligonucleotide synthesis method is similar to conventional phosphoramidite solid-phase synthesis (all synthesized by Suzhou Beixin Biotechnology Co., Ltd.), consisting of four steps: deprotection, coupling, capping, and oxidation or sulfurization. Starting with a solid support, the nucleoside monomers are sequentially linked from the 3' to the 5' end. Nucleoside phosphoramidite monomer raw materials, such as 2'-F RNA and 2'-OMe RNA, were purchased from Shanghai Zhaowei Technology Development Co., Ltd. After solid-phase synthesis, the solid support was transferred to a centrifuge tube and soaked in a 3:1 solution of 28% ammonia and ethanol at 50°C for 16 hours to cleave the oligonucleotide from the solid support into solution. The supernatant was centrifuged and transferred to another centrifuge tube, concentrated and evaporated to dryness, then redissolved in deionized water and purified using a C18 reverse-phase chromatography column with a mobile phase of 0.1 M TEAA and acetonitrile. The target oligonucleotide was collected, lyophilized, identified as the target product by LC-MS, and quantified by UV spectroscopy (260 nm). The obtained sense strand and antisense strand are annealed according to the molar ratio to obtain complementary paired double-stranded siRNA, and the concentration is adjusted to the required value.

[0313] Synthesis of lipid-oligonucleic acid conjugates

[0314] The synthesized -NH2-containing oligonucleotide (1.0 equiv.) was dissolved in DMSO, and LL1p and LL2p compounds (15 equiv.) dissolved in DMSO were added. Triethylamine was then added and the mixture was vortexed at 25°C for 2 h. After completion of the reaction, HPLC reverse-phase purification was performed to obtain the desired oligonucleotide. The target oligonucleotide was collected, lyophilized, and identified as the target product by LC-MS. The target product was then quantified by UV light (260 nm). The resulting sense and antisense strands were annealed at a fixed molar ratio to yield complementary double-stranded siRNAs, which were then adjusted to the desired concentration. Example lipid-oligonucleotide conjugates synthesized are shown in Table 1.

[0315] Table 1 Lipid-siRNA conjugates targeting superoxide dismutase 1 (SOD1)

[0316] Where m represents 2'-O-methyl modification, f represents 2'-fluoro modification, * represents phosphorothioate modification, SS represents sense strand, and AS represents antisense strand.

[0317] NH2C6:

[0318] LL1:

[0319]

[0320] LL2:

[0321]

[0322] VPmU*:

[0323]

[0324] Example 4. Evaluation of the efficacy of lipid oligonucleic acid conjugates in mice

[0325] Male C57BL / 6 mice, 6-8 weeks old (N=6 per group), were intravenously injected with a single dose of 2 mg / kg or 10 mg / kg of lipid-siRNA conjugates D1 and D2.

[0326] On day 14 after administration, quadriceps and epididymal adipose tissues were collected, immediately snap-frozen in liquid nitrogen, and stored at -80°C. RNA was extracted and purified from tissue samples using a silica-based adsorption column method. The resulting RNA was used for cDNA synthesis using a reverse transcription kit (TAKARA, RR047A). SOD1 mRNA levels were quantified by RT-qPCR using a Bio-Rad CFX96. The relative changes in SOD1 mRNA expression were determined using the ΔΔCt method, with double normalization to the mean values ​​of the GAPDH and control groups. The results are shown in Figures 1 and 2, respectively, demonstrating that lipid siRNA conjugates D1 and D2 both exhibited dose-dependent inhibition of SOD1 mRNA expression in quadriceps (Figure 1) and epididymal adipose tissue (Figure 2).

Claims

1. A lipid oligonucleotide conjugate comprising the structure of Formula Ia: in, At least one of R1 and R2 is selected from Ra, -C(O)Ra, -C(O)NHRa, and C(O)ORa, wherein Ra is an optionally substituted C5-30 alkyl or alkenyl group, and The other of R1 and R2 is selected from H, Rb, -C(O)Rb, -C(O)NHRb and C(O)ORb, wherein Rb is selected from optionally substituted C1-4 alkyl or alkenyl, optionally substituted C5-30 alkyl or alkenyl, optionally substituted C2-10 alkynyl, optionally substituted C1-10 alkoxy, optionally substituted C3-10 cycloalkyl, optionally substituted 6 to 14 membered aryl, optionally substituted 5 to 18 membered heteroaryl, optionally substituted 5 to 14 membered heterocyclyl; The optional substitution is optionally substituted with Rc, wherein Rc is selected from C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 alkoxy, C3-10 cycloalkyl, 6 to 14 membered aryl, 5 to 18 membered heteroaryl, 5 to 14 membered heterocyclyl, halogen, hydroxyl, amino, guanidino, carboxyl, cyano, nitro, C1-3 alkylthio and thiol; m1, m2 and m3 are independently an integer of 1 or 2; p is 0, 1, or 2; stands for oligonucleotide; L stands for linker.

2. The lipid oligonucleotide conjugate according to claim 1, wherein at least one or both of R1 and R2 are independently -C(O)Ra, wherein Ra is an optionally substituted C5-30 alkyl or alkenyl group; Preferably, Ra is an optionally substituted C12-24 alkyl or alkenyl; Preferably, Ra is an optionally substituted C14-22 alkyl or alkenyl; More preferably, Ra is optionally substituted C14, C15, C16, C18, C20 or C22 alkyl, monounsaturated alkenyl or polyunsaturated alkenyl.

3. The lipid oligonucleotide conjugate according to claim 1 or 2, wherein m1, m2 and m3 are all 1, or two of m1, m2 and m3 are 1 and the other is 2.

4. The lipid oligonucleotide conjugate according to any one of claims 1 to 3, wherein p is 1.

5. The lipid-oligonucleotide conjugate according to any one of claims 1 to 4, wherein the oligonucleotide is an antisense oligonucleotide or a sense strand or antisense strand of an RNAi agent; Preferably, the 5' and / or 3' end of the oligonucleotide is linked to L via a phosphate group, a phosphorothioate group or a phosphonate group; Preferably, the sense strand or antisense strand of the RNAi agent pairs with its antisense strand or sense strand to form a duplex; Preferably, the RNAi agent is siRNA; Preferably, the oligonucleotide is the sense strand of siRNA; Preferably, the antisense strand of the siRNA has a 5'-VP modification.

6. The lipid oligonucleotide conjugate according to any one of claims 1 to 5, wherein: L is -NH-(CH2)qO-, -NH-(CH2CH2O)q-, wherein q and r are independently integers from 1 to 20, and the right side of the formula is connected to an oligonucleotide; or L contains L1-L2, where L1 is The right side of the formula is connected to L2; L2 is wherein the left side of the formula is connected to L1; the wavy line represents the connection position; and wherein each n is independently an integer from 1 to 10, preferably an integer from 2 to 8, more preferably an integer from 4 to 6.

7. The lipid oligonucleotide conjugate of formula Ib, in, At least one of R1 and R2 is selected from Ra, -C(O)Ra, -C(O)NHRa, and C(O)ORa, wherein Ra is an optionally substituted C5-30 alkyl or alkenyl group, and The other of R1 and R2 is selected from H, Rb, -C(O)Rb, -C(O)NHRb and C(O)ORb, wherein Rb is selected from optionally substituted C1-4 alkyl or alkenyl, optionally substituted C5-30 alkyl or alkenyl, optionally substituted C2-10 alkynyl, optionally substituted C1-10 alkoxy, optionally substituted C3-10 cycloalkyl, optionally substituted 6 to 14 membered aryl, optionally substituted 5 to 18 membered heteroaryl, optionally substituted 5 to 14 membered heterocyclyl; The optional substitution is optionally substituted with Rc, wherein Rc is selected from C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 alkoxy, C3-10 cycloalkyl, 6 to 14 membered aryl, 5 to 18 membered heteroaryl, 5 to 14 membered heterocyclyl, halogen, hydroxyl, amino, guanidino, carboxyl, cyano, nitro, C1-3 alkylthio and thiol; L1 is The right side of the formula is connected to L2; L2 is wherein the left side of the formula is connected to L1; the wavy line represents the connection position; and wherein each n is independently an integer from 1 to 10, preferably an integer from 2 to 8, more preferably an integer from 4 to 6; represents an oligonucleotide, preferably, the oligonucleotide is an antisense oligonucleotide or the sense strand or antisense strand of an RNAi agent; preferably, the 5' and / or 3' end of the oligonucleotide is connected to L2 via a phosphate group, a phosphorothioate group or a phosphonate group; preferably, the sense strand or antisense strand of the RNAi agent is paired with its antisense strand or sense strand to form a duplex; preferably, the RNAi agent is siRNA; preferably, the oligonucleotide is the sense strand of the siRNA; preferably, the antisense strand of the siRNA has a 5'-VP modification.

8. The lipid oligonucleotide conjugate of formula Ic, in, At least one of Ra1 and Ra2 is an optionally substituted C5-30 alkyl or alkenyl, preferably an optionally substituted C12-24 alkyl or alkenyl; preferably an optionally substituted C14-22 alkyl or alkenyl; more preferably an optionally substituted C14, C15, C16, C18, C20 or C22 alkyl, monounsaturated alkenyl or polyunsaturated alkenyl; the other of Ra1 and Ra2 is optionally substituted C1-4 alkyl or alkenyl, optionally substituted C5-30 alkyl or alkenyl, optionally substituted C2-10 alkynyl, optionally substituted C1-10 alkoxy, optionally substituted C3-10 cycloalkyl, optionally substituted 6- to 14-membered aryl, optionally substituted 5- to 18-membered heteroaryl, or optionally substituted 5- to 14-membered heterocyclyl; The optional substitution is optionally substituted with Rc, wherein Rc is selected from C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 alkoxy, C3-10 cycloalkyl, 6 to 14 membered aryl, 5 to 18 membered heteroaryl, 5 to 14 membered heterocyclyl, halogen, hydroxyl, amino, guanidino, carboxyl, cyano, nitro, C1-3 alkylthio and thiol; L1 is The right side of the formula is connected to L2; L2 is wherein the left side of the formula is connected to L1; the wavy line represents the connection position; and wherein each n is independently an integer from 1 to 10, preferably an integer from 2 to 8, more preferably an integer from 4 to 6; represents an oligonucleotide, preferably, the oligonucleotide is an antisense oligonucleotide or the sense strand or antisense strand of an RNAi agent; preferably, the 5' and / or 3' end of the oligonucleotide is connected to L2 via a phosphate group, a phosphorothioate group or a phosphonate group; preferably, the sense strand or antisense strand of the RNAi agent is paired with its antisense strand or sense strand to form a duplex; preferably, the RNAi agent is siRNA; preferably, the oligonucleotide is the sense strand of the siRNA; preferably, the antisense strand of the siRNA has a 5'-VP modification.

9. A lipid oligonucleotide conjugate selected from the following structural formulas: in Representing an oligonucleotide, preferably, the oligonucleotide is an antisense oligonucleotide or the sense strand or antisense strand of an RNAi agent; preferably, the 5' and / or 3' end of the oligonucleotide is connected via a phosphate group, a phosphorothioate group or a phosphonate group; preferably, the sense strand or antisense strand of the RNAi agent is paired with its antisense strand or sense strand to form a duplex; preferably, the RNAi agent is siRNA; preferably, the oligonucleotide is the sense strand of the siRNA; preferably, the antisense strand of the siRNA has a 5'-VP modification.

10. The lipid oligonucleotide conjugate according to claim 9 is selected from any one of the following structures: in, represents siRNA, X is S or O, and SS-5' / 3' represents the 5' or 3' end of the sense strand.

11. A compound selected from the following structural formula:

12. The ligand moiety of formula II for forming a lipid-oligonucleotide conjugate: in, At least one of R1 and R2 is selected from Ra, -C(O)Ra, -C(O)NHRa, and C(O)ORa, wherein Ra is an optionally substituted C5-30 alkyl or alkenyl group, and The other of R1 and R2 is selected from H, Rb, -C(O)Rb, -C(O)NHRb and C(O)ORb, wherein Rb is selected from optionally substituted C1-4 alkyl or alkenyl, optionally substituted C5-30 alkyl or alkenyl, optionally substituted C2-10 alkynyl, optionally substituted C1-10 alkoxy, optionally substituted C3-10 cycloalkyl, optionally substituted 6 to 14 membered aryl, optionally substituted 5 to 18 membered heteroaryl, optionally substituted 5 to 14 membered heterocyclyl; The optional substitution is optionally substituted with Rc, wherein Rc is selected from C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 alkoxy, C3-10 cycloalkyl, 6 to 14 membered aryl, 5 to 18 membered heteroaryl, 5 to 14 membered heterocyclyl, halogen, hydroxyl, amino, guanidino, carboxyl, cyano, nitro, C1-3 alkylthio and thiol; m1, m2 and m3 are independently an integer of 1 or 2; p is 0, 1, or 2; and represents a linker and the wavy line represents the location of attachment to the rest of the lipid-oligonucleotide conjugate.

13. The ligand moiety of claim 12, wherein for wherein each n is independently an integer from 1 to 10, preferably an integer from 2 to 8, more preferably an integer from 4 to 6; and the right side of the formula is connected to the rest of the lipid oligonucleotide conjugate.

14. The ligand portion of claim 12 or 13, having one or more of the following characteristics: (a) wherein at least one or both of R1 and R2 are independently -C(O)Ra, wherein Ra is an optionally substituted C5-30 alkyl or alkenyl; preferably, Ra is an optionally substituted C12-24 alkyl or alkenyl; preferably, Ra is an optionally substituted C14-22 alkyl or alkenyl; more preferably, Ra is an optionally substituted C14, C15, C16, C18, C20 or C22 alkyl, monounsaturated alkenyl or polyunsaturated alkenyl; (b) m1, m2, and m3 are all 1, or two of m1, m2, and m3 are 1 and the other is 2; (c) p is 1; (d) The oligonucleotide is an antisense oligonucleotide or the sense strand or antisense strand of an RNAi agent; preferably, the sense strand or antisense strand of the RNAi agent pairs with its antisense strand or sense strand to form a duplex; preferably, the RNAi agent is an siRNA; preferably, the oligonucleotide is the sense strand of the siRNA; preferably, the antisense strand of the siRNA has a 5'-VP modification.

15. The ligand portion according to claim 12, which is 16. Use of the ligand moiety represented by formula III in the preparation of lipid oligonucleotide conjugates, in, At least one of R1 and R2 is selected from Ra, -C(O)Ra, -C(O)NHRa, and C(O)ORa, wherein Ra is an optionally substituted C5-30 alkyl or alkenyl group, and The other of R1 and R2 is selected from H, Rb, -C(O)Rb, -C(O)NHRb and C(O)ORb, wherein Rb is selected from optionally substituted C1-4 alkyl or alkenyl, optionally substituted C5-30 alkyl or alkenyl, optionally substituted C2-10 alkynyl, optionally substituted C1-10 alkoxy, optionally substituted C3-10 cycloalkyl, optionally substituted 6 to 14 membered aryl, optionally substituted 5 to 18 membered heteroaryl, optionally substituted 5 to 14 membered heterocyclyl; The optional substitution is optionally substituted with Rc, wherein Rc is selected from C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 alkoxy, C3-10 cycloalkyl, 6 to 14 membered aryl, 5 to 18 membered heteroaryl, 5 to 14 membered heterocyclyl, halogen, hydroxyl, amino, guanidino, carboxyl, cyano, nitro, C1-3 alkylthio and thiol; m1, m2 and m3 are independently an integer of 1 or 2; p is 0, 1, or 2; and The wavy line represents the location of attachment to the rest of the lipid-oligonucleotide conjugate.

17. The use according to claim 16, having one or more of the following characteristics: (a) wherein at least one or both of R1 and R2 are independently -C(O)Ra, wherein Ra is an optionally substituted C5-30 alkyl or alkenyl; preferably, Ra is an optionally substituted C12-24 alkyl or alkenyl; preferably, Ra is an optionally substituted C14-22 alkyl or alkenyl; more preferably, Ra is an optionally substituted C14, C15, C16, C18, C20 or C22 alkyl, monounsaturated alkenyl or polyunsaturated alkenyl; (b) m1, m2, and m3 are all 1, or two of m1, m2, and m3 are 1 and the other is 2; (c) p is 1; (d) The oligonucleotide is an antisense oligonucleotide or the sense strand or antisense strand of an RNAi agent; preferably, the sense strand or antisense strand of the RNAi agent pairs with its antisense strand or sense strand to form a duplex; preferably, the RNAi agent is an siRNA; preferably, the oligonucleotide is the sense strand of the siRNA; preferably, the antisense strand of the siRNA has a 5'-VP modification.

18. A pharmaceutical composition comprising the lipid oligonucleotide conjugate according to any one of claims 1 to 10; and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition is formulated as an intravenous or subcutaneous injection.

19. Use of the lipid-oligonucleotide conjugate of any one of claims 1 to 10, the compound of claim 11, or the ligand moiety of any one of claims 12 to 15 in the preparation of a lipid-oligonucleotide conjugate.

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