Oligonucleotide delivery carrier and method of use thereof

Abstract

This application relates to nucleic acids, and more particularly to oligonucleotide agents comprising a target oligonucleotide and a non-target site covalently linked to the oligonucleotide, and to the pharmaceutical applications of the same.

Description

[Technical Field]

[0001] This application relates to the technical field of nucleic acids, and more particularly to oligonucleotide agents comprising double-stranded RNA (dsRNA, duplex) and a non-target site covalently conjugated to the dsRNA, and to the pharmaceutical applications of the same. (Cross-reference with related applications)

[0002] This application claims priority to the filing date of the provisional patent application "PCT / CN2023 / 085065," filed on March 30, 2023, and its disclosures are incorporated herein by reference in their entirety. (Array List)

[0003] This application includes a sequence listing submitted electronically in computer-readable format, which is incorporated herein by reference in its entirety. [Background technology]

[0004] Oligonucleotides are an innovative class of therapeutic agents that have attracted significant attention in the scientific community due to their flexibility in addressing a wide range of diseases through diverse mechanisms of action (MOAs). This category of therapeutic agents is broadly divided into single-stranded antisense oligonucleotides (ASOs) and double-stranded RNA molecules (dsRNAs), which, although used for different purposes, have similar chemical compositions.

[0005] dsRNA molecules, including small interfering RNAs (siRNAs) and small activating RNAs (saRNAs), are representative analogues within the dsRNA subclass. These molecules exhibit chemical similarities in their structural framework, length, strategies used for chemical modification, and terminal structure. Functionally, both siRNAs and saRNAs undergo a series of biological processes necessary for their mechanism of action. These include intracellular uptake, evasion from endosomal or lysosomal pathways, and interaction with the Argonaut (AGO) protein in the cytoplasm. This interaction causes the non-functional "passenger" strand to be cleaved and released, while the functional "guide" strand remains bound to AGO and exerts its mechanism of action. This common biological process suggests that siRNA and saRNA delivery technologies are mutually interchangeable, potentially providing a unified approach to the delivery of these therapeutic molecules.

[0006] Compared to ASOs, which can independently interact with intracellular uptake mechanisms via targeted chemical modifications, dsRNA molecules such as siRNA and saRNA require the support of a drug delivery system (DDS) to effectively reach target cells and exert therapeutic effects. Currently, various dsRNA DDS platforms, including polymer-based, lipid-based, and conjugate-based systems, are being developed for dsRNA delivery. However, the efficiency of these systems in reaching specific target organs, tissues, and cells remains limited, highlighting the need for innovative and advanced delivery solutions.

[0007] (overview) This application provides a novel oligonucleotide agent or oligonucleotide agent conjugate comprising a target oligonucleotide (e.g., double-stranded RNA) and a non-target site conjugated to the target oligonucleotide. The oligonucleotide agent constitutes a system having "self-delivery" properties. Surprisingly, the inventors have found that when the non-target site disclosed herein is conjugated to a target oligonucleotide (including double-stranded RNA, siRNA, or saRNA), favorable in vivo distribution and high in vivo activity are obtained in local administration to selected tissues and systemic delivery to multiple organs and tissues such as the liver, muscle, lungs, kidneys, bladder, brain, spinal cord, heart, eyes, and spleen.

[0008] In some embodiments, the following are provided: (a) a double-stranded oligonucleotide comprising a sense strand and an antisense strand, wherein the antisense strand is complementary to a target nucleic acid; and (b) a non-target site comprising one or more components covalently linked by at least one phosphorothioate bond, wherein the double-stranded oligonucleotide is conjugated to the non-target site to form an oligonucleotide agent.

[0009] In some embodiments, the double-stranded oligonucleotide is siRNA or saRNA.

[0010] The non-target sites disclosed herein may include one or more identical or different components (or "units"), which are covalently linked in series to form the framework of the non-target site. The non-target sites may have a linear or branched structure.In some embodiments, the constituent is a substituted or unsubstituted alkyl, aralkyl, alkoxy, aryloxy, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclic alkyl, heterocyclic alkenyl, heterocyclic alkynyl, aryl, heteroaryl, heterocyclic, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynyl heteroarylalkyl, alkynyl heteroarylalkyl, alkynyl heteroarylal Selected from alkennyl, alkynyl heteroaryl alkynyl, alkyl heterocyclic alkyl, alkyl heterocyclic alkenyl, alkyl heterocyclic alkynyl, alkenyl heterocyclic alkyl, alkenyl heterocyclic alkenyl, alkenyl heterocyclic alkynyl, alkynyl heterocyclic alkyl, alkynyl heterocyclic alkenyl, alkynyl heterocyclic alkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkyl heteroaryl, alkenyl heteroaryl, and alkynyl heteroaryl, and one or more of the methylene groups. However, these are interrupted or terminated by O, S, S(O), SO2, N(R')2, C(O), cleavable linker groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, and substituted or unsubstituted heterocyclic groups, furthermore, each R' is independently selected from hydrogen, substituted or unsubstituted alkyl, aryl, aralkyl, alkylaryl, alkoxy, aryloxy, acyl or aliphatic groups, which may be linear, branched, cyclic, polycyclic, unsaturated, hydroxylated, carbonylated, phosphorylated, and / or sulfurized.

[0011] In some embodiments, one or more of the components or units are nucleotides. In some embodiments, the components of the non-target site may be in their native form or chemically modified. In some embodiments, the nucleotides included in the non-target site are chemically modified and selected from the group consisting of 2′-fluoro-2′-deoxynucleoside (2′-F), 2′-O-methyl (2′-O-Me), 2′-O-(2-methoxyethyl) (2′-O-MOE), locked nucleic acid (LNA), bridged nucleic acid (BNA), peptide nucleic acid (PNA), 5′-(E)-vinylphosphonate, and 5-methylcytosine. In some embodiments, the non-target site does not contain any nucleotide components. Specifically, the non-target site may include: (a) a series of linked components, none of which are nucleotides; (b) one or more nucleotides interposed within the non-nucleotide components; (c) one or more non-nucleotide components interposed within a nucleotide; or (d) a sequence of a continuous nucleotide sequence and a sequence of continuously linked non-nucleotide components.

[0012] In some embodiments, one or more components or units of the non-target site are selected from the following: a) L1 or S18 (Spacer-18 Linker) (1,1-Bis(4-methoxyphenyl)-1-phenyl-2,5,8,11,14,17-Hexaoxanonadecan-19-yl(2-cyanoethyl)diisopropylphosphorumidite); b) L4 or C6 (spacer-C6 linker) (6-(bis(4-methoxyphenyl)(phenyl)methoxy)hexyl(2-cyanoethyl)diisopropylphosphorumidite); c) L6(1,1-bis(4-methoxyphenyl)-1-phenyl-2,5,8,11,14-pentaoxahexadecane-16-yl (2-cyanoethyl)diisopropylphosphorumidite); d) L9 or S9 (Spacer-9 Linker) (2- (2- (2- (Bis(4-methoxyphenyl)(phenyl)methoxy)ethoxy)ethoxy)ethyl(2-cyanoethyl)diisopropylphosphorumidite); e) L10 or C3 (spacer-C3 linker) (3-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl(2-cyanoethyl)diisopropylphosphorumidite); f) L12(d spacer)((2R,3S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofumapal-3-yl(2-cyanoethyl)diisopropylphosphorumidite); g) L13 or C12 (spacer-C12 linker) (12- (bis(4-methoxyphenyl)(phenyl)methoxy)dodecyl(2-cyanoethyl)diisopropylphosphorumidite); h) L14 (Spacer-L14 Linker) (((1r, 4r)-4- ((Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)cyclohexyl)methyl(d-cyanoethyl)diisopropylphosphorumidite); i) L15 (Spacer-L15 Linker) (4-(2-(Bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)phenethyl(2-cyanoethyl)diisopropylphosphorumidite); j) L16 (Spacer-L16 Linker) (2- (1- (2- (Bis(4-Methoxyphenyl)(Phenyl) Methoxy) Ethyl) Cyclohexyl) Ethyl (2-Cyanoethyl) Diisopropyl Phosphoramidite) k) C6x1((2S, 3S, 4S, 5S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-methoxy-4-(pento-4-in-1-yloxy)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphorumidite); i) C6x2((2S, 3S, 4S, 5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2-methoxy-4-(pento-4-in-1-yloxy)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphorumidite); m) C6x5(2- ((2- (bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(pento-4-in-1-yl)amino)ethyl(2-cyanoethyl)diisopropylphosphorumidite); n) C6x7((9H-Fluoren-9-yl)methyl(4-((2S,4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((bis(diisopropylamino)phosphanyl)oxy)pyrrolidine-1-yl)-4-oxobutyl)carbamate); o) L20 methyl 1-(5-(bis(4-methoxyphenyl)(phenyl)methoxy)pentyl)-2-(4-(((2-cyanoethoxy)(diisopropylamino)phosphanyl)oxy)butyl)-1H-benzo[d]imidazole-5-carboxylate; and

[0013] p) L42 6-((6-(bis(4-methoxyphenyl)(phenyl)methoxy)hexyl)disulfanyl)hexyl(2-cyanoethyl)diisopropylphosphorumidite.

[0014] In some embodiments, one or more components or units of a non-target site include at least one phosphorothioate modification in its backbone. These components or units of the non-target site may be identical or different. For example, a non-target site may include m such components (where m is, for example, an integer in the range of 1 to 50), all of which may be identical and covalently linked in series. In some embodiments, m-1 such components may be identical and the remaining one different. In some embodiments, m-2 such components may be identical and the remaining one different. While not intended to be bound by any theory, the location of a particular component in a non-target site is not limited, and there are no specific placement requirements as long as the component is conjugated to a target oligonucleotide and functions as a “scaffold” that assists its function.

[0015] The non-target site may be conjugated to the target oligonucleotide directly or via a linker. In some embodiments, the non-target site is conjugated to an RNA duplex (e.g., siRNA or saRNA), which consists of two complementary or partially complementary strands, one of which is covalently bound to the non-target site. The RNA duplex targets at least one nucleic acid sequence (e.g., mRNA or DNA) and is optionally chemically modified using oligonucleotide chemistry techniques (e.g., 2'-fluoro, 2'-O-methyl, phosphorothioate, mesylphosphoramide or boranophosphate backbone, LNA, etc.) to enhance in vivo activity, stability, and safety. Unlike the RNA duplex, the non-target site has neither the intention nor the ability to specifically target any nucleic acid sequence in the subject being administered the drug. Even if the non-target site unintentionally or inevitably induces “off-target” effects by interacting with nucleic acid sequences in the subject, the oligonucleotide agent still retains its “self-delivery” properties and constitutes one embodiment of the present invention disclosed herein. The non-target site may be chemically modified, for example, by containing a phosphorothioate bond, a mesylphosphoamidate bond, or a boranophosphate bond in its backbone, or by containing as such a component 2′-fluoro-2′-deoxynucleoside (2′-F), 2′-O-methyl (2′-O-Me), 2′-O-(2-methoxyethyl) (2′-O-MOE), locked nucleoic acid (LNA), bridged nucleoic acid (BNA), peptidonucleotide (PNA), 5'-(E)-vinylphosphonate, 5-methylcytosine, etc., thereby conferring advantageous physicochemical properties to improve the bioavailability and deliverability of the agent. As described herein, non-target sites offer specific advantages, such as unconventional chemical structures and modification patterns favorable for binding to cytoplasmic proteins and delivery, as well as in vivo distribution, bioavailability, stability, intracellular uptake, and other pharmacological properties, all without the concern of impairing double-strand activity.

[0016] In some embodiments, the non-target site is linked to the target oligonucleotide via a linker. The linker connecting the target oligonucleotide and the non-target site may be selected from natural or non-natural nucleotides, ethylene glycol, carbohydrates, alkyl chains, or any other linker available for covalently linking any two oligonucleotides. On the other hand, the linker may be considered part of the non-target site.

[0017] In some embodiments, the bond between the target oligonucleotide and the non-target site, or between adjacent components within the target oligonucleotide, is selected from ethylene glycol chains, alkyl chains, alkenyl chains, alkynyl chains, peptides, carbohydrates, thiol bonds, phosphodiesters, phosphorothioates, phosphoramidates, amides, carbamates, tetrazole bonds, or benzimidazole bonds.

[0018] Preferably, the non-target site contains at least one phosphorothioate (PS) bond. In some embodiments, the non-target site contains at least one phosphorothioate (PS) bond in the backbone. In some embodiments, the non-target site contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least thirty or more phosphorothioate (PS) bonds. In some embodiments, all adjacent components within the non-target site are linked to each other via PS bonds.

[0019] In addition to phosphorothioate (PS) bonds, non-target sites may include phosphodiester bonds, mesylphosphoramide bonds, and / or boranophosphate bonds. For example, some adjacent components may be linked to each other by phosphorothioate (PS) bonds, while other adjacent components may be linked to each other by phosphodiester bonds, mesylphosphoramide bonds, and / or boranephosphate bonds.

[0020] In some embodiments, one or more components of the non-target site are nucleotides, which may be RNA, DNA, BNA, LNA, or PNA. In some embodiments, the non-target site comprises m components, where m is an integer between 1 and 50. Furthermore, of the m components, n are nucleotides (natural or modified), where n is an integer between 0 and 49. The nucleotides constituting the non-target site may have a specific composition consisting of A, G, C, and / or U, and one or more nucleotides may have a 2'-O-methyl (2'Ome) modification.

[0021] In certain embodiments, the sense strand of the double-stranded oligonucleotide in the oligonucleotide preparation is at least 10 nucleotides long. In certain embodiments of this application, the sense strand has a nucleotide length in the range of 10 to 60 nucleotides.

[0022] In certain embodiments, the antisense strand of the double-stranded oligonucleotide in the oligonucleotide agent is at least 10 nucleotides long. In certain embodiments, the antisense strand has a nucleotide length in the range of 10 to 60 nucleotides.

[0023] In certain embodiments, the chemical modification in a double-stranded oligonucleotide is the addition of a 5'-phosphonate moiety at the 5' end of the nucleotide sequence. In certain embodiments, the chemical modification is the addition of a 5'-(E)-vinylphosphonate moiety. In certain embodiments, the chemical modification is the addition of a 5'-methylcytosine at the 5' end of the nucleotide sequence.

[0024] In some embodiments, the non-target site is conjugated at the 3' end of the double-stranded oligonucleotide, either directly or via a linker. In some embodiments, the non-target site is conjugated at the 5' end of the double-stranded oligonucleotide, either directly or via a linker. In some embodiments, the non-target site is conjugated within the double-stranded oligonucleotide, either directly or via a linker. In some embodiments, the double-stranded oligonucleotide is conjugated within the non-target site, either directly or via a linker.

[0025] In some embodiments, the non-target site is covalently conjugated to the sense strand, antisense strand, or both of the double-stranded oligonucleotide, either directly or via a linker. In some embodiments, the non-target site is covalently conjugated to the 3' end, 5' end, both ends, or inner nucleotide of the sense strand of the double-stranded oligonucleotide. In some embodiments, the non-target site is covalently conjugated to the 3' end, 5' end, both ends, or inner nucleotide of the antisense strand of the double-stranded oligonucleotide. In some embodiments, the inner nucleotide of the sense strand or antisense strand of the double-stranded oligonucleotide is substituted with a linker, and the non-target site is covalently conjugated to that linker.

[0026] In some embodiments, multiple non-target sites are covalently conjugated to the double-stranded oligonucleotide. In some embodiments, approximately 2 to 10 non-target sites are covalently conjugated to the double-stranded oligonucleotide.

[0027] In some embodiments, multiple double-stranded oligonucleotides are covalently conjugated to a non-target site. In some embodiments, approximately 2 to 10 double-stranded oligonucleotides are covalently conjugated to a non-target site.

[0028] In some embodiments, the terminal component of the non-target site is directly bonded to the double-stranded oligonucleotide via a phosphorothioate (PS) bond. In some other embodiments, the terminal component of the non-target site is linked to the double-stranded oligonucleotide via a linker, which is covalently bonded to the double-stranded oligonucleotide via a phosphorothioate (PS) bond.

[0029] In some embodiments, the bond between the linker and the double-stranded oligonucleotide includes a direct bond, an oxygen atom or a sulfur atom, or a unit selected from the following group: NR1, C(O), C(O)O, C(O)NR1, SO, SO2, and SO2NH, where R1 is hydrogen, acyl, aliphatic, or substituted aliphatic.

[0030] In some embodiments, the double-stranded RNA is designed to inhibit the expression of superoxide dismutase 1 (SOD1) in cells. In some other embodiments, the double-stranded RNA (dsRNA) is designed to activate the expression of survival motor neuron 2 (SMN2) protein in cells.

[0031] In some embodiments, the double-stranded RNA comprises a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence described in any one of SEQ ID NO: 1, 3, 56, or 61, and the antisense strand comprises a nucleotide sequence that is partially or completely complementary to the first strand.

[0032] In some embodiments, the antisense strand includes a nucleotide sequence that is partially complementary to any of SEQ ID NO: 1, 3, 56, or 61. In yet other embodiments, the antisense strand includes a nucleotide sequence that is at least 90% identical to any of the nucleotide sequences described in SEQ ID NO: 2, 4, 57, or 62.

[0033] In some embodiments, the sense strand of the double-stranded oligonucleotide has a nucleotide sequence that is at least 90% identical to a nucleotide sequence selected from RD-11810 (SEQ ID NO: 1), RD-12556 (SEQ ID NO: 3), RD-16988 (SEQ ID NO: 56), or RD-16990 (SEQ ID NO: 61).

[0034] In some embodiments, the antisense strand of the double-stranded oligonucleotide has a nucleotide sequence that is at least 90% identical to a nucleotide sequence selected from RD-11810 (SEQ ID NO: 2), RD-12556 (SEQ ID NO: 4), RD-16988 (SEQ ID NO: 57), or RD-16990 (SEQ ID NO: 62).

[0035] In some embodiments, a non-target site is further conjugated to one or more conjugation groups. In some embodiments, a double-stranded oligonucleotide is further conjugated to one or more conjugation groups. In some embodiments, the sense strand or antisense strand of the double-stranded oligonucleotide is further conjugated to one or more conjugation groups.

[0036] In some embodiments, the conjugation group is one or more selected from lipids, fatty acids, fluorescent dyes, ligands, sugars, peptides, and antibodies. In some embodiments, one or more conjugation groups are selected from cell-permeable peptides, polyethylene glycol, alkaloids, tryptamine, benzimidazole, quinolone, amino acids, cholesterol, glucose, and N-acetylgalactosamine.

[0037] In some embodiments, each of the sense strand and the antisense strand independently has a nucleotide length in the range of 15 to 35 nucleotides.

[0038] In some embodiments, the oligonucleotide agent comprises a small interfering RNA (siRNA), which forms a double-stranded structure comprising a sense strand and an antisense strand, and the oligonucleotide agent can inhibit the expression of a target gene in a cell. Specifically, the targeted gene or protein includes, but is not limited to, superoxide dismutase 1 (SOD1).

[0039] In some embodiments, the oligonucleotide agent comprises a small activated RNA (saRNA), which includes a sense strand and an antisense strand to form a double-stranded structure, and the oligonucleotide agent can activate the expression of a target gene in a cell. Specifically, the targeted gene or protein includes, but is not limited to, SMN2.

[0040] In some embodiments, the sense strand and antisense strand of the siRNA each independently have nucleotide sequences that have at least 85% homology to a nucleotide sequence pair selected from Table 5 or Table 14.

[0041] In some embodiments, non-target sites in oligonucleotide agents improve the stability, bioavailability, in vivo distribution, and / or intracellular uptake of double-stranded oligonucleotides compared to oligonucleotide agents without non-target sites.

[0042] In some embodiments, non-target sites in oligonucleotide agents increase the in vivo distribution of double-stranded oligonucleotides within one or more target tissues compared to oligonucleotide agents without non-target sites. In some embodiments, one or more target tissues are selected from the tissues of the brain, spinal cord, muscles, spleen, lungs, heart, liver, bladder, kidneys, and retina. In some embodiments, one or more target tissues are selected from the group consisting of: prefrontal cortex, cerebellum, cerebrum; cervical, thoracic, and lumbar vertebrae of the spinal cord; heart, biceps brachii, semitendinosus biceps femoris, platysma, and gluteus maximus.

[0043] Vectors and cells containing the oligonucleotide agents of this disclosure are also provided herein. In some embodiments, the cells are mammalian cells and optionally human cells. In some embodiments, the cells are host cells. In some embodiments, the cells are in vitro. In some embodiments, the cells are present in the body of a mammal.

[0044] Specific embodiments of this application relate to pharmaceutical compositions containing an oligonucleotide agent, including: (a) a double-stranded oligonucleotide comprising a sense strand and an antisense strand, wherein the antisense strand is complementary to a target nucleic acid; and (b) a non-target site comprising one or more components and covalently linked via at least one phosphorothioate (PS) bond, wherein the double-stranded oligonucleotide is conjugated to the non-target site to form an oligonucleotide agent. The target nucleic acid may be any target nucleic acid, including, but not limited to, the SOD1 gene, the SMN2 gene, and the like.

[0045] In certain embodiments, the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier selected from aqueous carriers, liposomes or LNPs, polymers, micelles, colloids, metal nanoparticles, nonmetal nanoparticles, bioconjugates, and polypeptides.

[0046] In certain embodiments, the pharmaceutical composition reduces or suppresses the transcription of the SOD1 gene or the SOD1 protein.

[0047] In certain embodiments, the pharmaceutical composition increases or activates the expression of the SMN2 gene or the SMN2 protein.

[0048] Kits comprising the oligonucleotide agents or pharmaceutical compositions described herein are also provided herein.

[0049] Specific embodiments relate to kits comprising the pharmaceutical compositions of the present disclosure.

[0050] Certain embodiments relate to a method for reducing or silencing the transcription of the SOD1 gene or protein, comprising administering a pharmaceutical composition of the present disclosure to a subject.

[0051] Certain embodiments relate to a method for treating or delaying the onset or progression of amyotrophic lateral sclerosis (ALS) in a subject, the method comprising: administering the subject to the pharmaceutical composition of the present disclosure. In certain embodiments, the subject has sporadic ALS (sALS). In certain embodiments, the subject has familial ALS (fALS).

[0052] A particular embodiment of this application relates to a method for treating or delaying the onset or progression of spinal muscular atrophy (SMA) in a subject, the method comprising: administering a pharmaceutical composition to the subject.

[0053] A particular embodiment of this application relates to a method for increasing or activating the expression of the SMN2 gene, comprising administering a pharmaceutical composition to a subject.

[0054] Certain embodiments of this application relate to a method for treating or delaying the onset or progression of spinal muscular atrophy (SMA) in a subject, the method comprising: administering the subject a pharmaceutical composition of the present disclosure.

[0055] In certain embodiments, the pharmaceutical composition reduces or suppresses the expression of the SOD1 gene or protein.

[0056] In certain embodiments, the non-target site of the oligonucleotide agent improves the stability, bioavailability, in vivo distribution, and / or intracellular uptake of the double-stranded oligonucleotide compared to oligonucleotide agents without the non-target site.

[0057] In certain embodiments, the non-target site of the oligonucleotide agent increases the in vivo distribution of one or more double-stranded oligonucleotides within target tissues compared to oligonucleotide agents without a non-target site.

[0058] In certain embodiments, the non-target site of the oligonucleotide agent improves the stability, in vivo distribution, bioavailability, and activity of double-stranded oligonucleotides in two or more target cell types within tissues compared to oligonucleotide agents without a non-target site. In certain embodiments, one or more target tissues are selected from tissues of the brain, spinal cord, muscle, spleen, lung, heart, liver, bladder, and kidney. In certain embodiments, one or more target tissues are selected from the group consisting of: prefrontal cortex, cerebellum, cerebrum; cervical, thoracic, and lumbar vertebrae of the spinal cord; heart, biceps brachii, semitendinosus biceps femoris, platysma, and gluteus maximus.

[0059] Certain embodiments of this application relate to the use of oligonucleotide agents of the present disclosure in the manufacture of a pharmaceutical product for treating or delaying the onset or progression of amyotrophic lateral sclerosis (ALS).

[0060] Specific embodiments of this application relate to the use of the pharmaceutical compositions of this disclosure in the manufacture of a medicament for treating or delaying the onset or progression of amyotrophic lateral sclerosis (ALS). In specific embodiments, ALS includes sporadic ALS (sALS) and / or familial ALS (fALS).

[0061] Certain embodiments of this application relate to oligonucleotide agents of the present disclosure for use in the treatment of or delaying the onset or progression of amyotrophic lateral sclerosis (ALS), wherein ALS optionally includes sporadic ALS (sALS) and / or familial ALS (fALS).

[0062] Certain embodiments of this application also relate to pharmaceutical compositions of the present disclosure for use in the treatment of or delaying the onset or progression of amyotrophic lateral sclerosis (ALS), wherein ALS optionally includes sporadic ALS (sALS) and / or familial ALS (fALS).

[0063] As a result of ongoing research, the inventors have surprisingly discovered that nucleotides are not required as essential components in single-stranded carriers for the delivery of double-stranded RNA (dsRNA) to target cells. This represents a significant advance in the field of nucleic acid-based therapeutics and offers many technical advantages over existing methodologies. The innovative aspects of this invention are characterized by the following beneficial effects:

[0064] 1. Expanding the delivery profile without compromising therapeutic efficacy: The technical solution of the present invention exhibits activity equivalent to or greater than that of prior art. The mechanism utilizing non-target sites, detailed in this application, is noteworthy as an example of an excellent approach in the development of oligonucleotide delivery technologies, including double-stranded RNA (dsRNA) such as siRNA and saRNA. This strategic design allows the target oligonucleotide to maintain its efficacy while possessing self-delivery properties conferred by the non-target site, thereby expanding the therapeutic applications of oligonucleotides, particularly in various organs, tissues, and cells, including the central nervous system.

[0065] 2. Low-Cost Synthesis: This invention utilizes readily available chemical linkers or spacers within its structure, rather than relying on expensive chemically modified nucleotide monomers. This strategic choice significantly reduces chemical synthesis costs, making the production of oligonucleotide agents more economically feasible. This cost advantage is crucial for the commercialization and widespread adoption of this invention.

[0066] 3. Improved therapeutic efficacy through enhanced target specificity and minimization of off-target effects: The present invention is designed to reduce or eliminate nucleotide content in non-target sites. This intentional modification is theoretically sound and practically effective in reducing the likelihood of off-target effects due to nonspecific base pairing. By reducing these unintended interactions, the present invention achieves high target specificity and therapeutic precision, minimizes potential side effects, and improves the overall efficacy of the treatment.

[0067] 4. Reduced Cytotoxicity: The inventors also found that oligonucleotide agents exhibited low cytotoxicity. This reduction was clearly observed and may be due to less chemical modification of the nucleotides compared to conventional delivery carriers. Further improvements to the cytotoxicity profile are possible through the refined design and configuration of non-target sites, as well as the improved conjugate morphology, which could minimize potential harm to healthy cells. This improvement is crucial, as it leads to an improved therapeutic index and a better safety profile for patients receiving treatment.

[0068] Novel features of the present invention are specifically described in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description illustrating exemplary embodiments in which the principles of the present invention are employed, and to the appended drawings (also referred to as "Figures" in this specification). [Brief explanation of the drawing]

[0069] [Figure 1]Figures 1A and 1B show screening data for the knockdown activity and cytotoxicity of 31 different linear oligonucleotide delivery carrier (cODV)-siRNA (cODV-siRNA) designs in primary mouse hepatocytes (PMH). PMH cells were transfected with the specified cODV-siRNAs (see Table 5) at 0.1 nM for 24 hours using RNAiMAX. A condition without oligonucleotides was used as a mock transfection. dsCon2 double-stranded siRNA was transfected as a non-targeted control. RD-12559 was used as a positive control with known knockdown activity. Figure 1A shows the mouse Sod1 mRNA levels after each cODV-siRNA treatment, quantified by two-step RT-qPCR using the gene-specific primer sets shown in Table 4. The geometric mean of Hmbs and Hprt1 mRNA levels was used as an internal reference. The values ​​on the vertical axis (y-axis) represent the relative Sod1 mRNA expression levels (mean ± standard error of transfection replication in 2 wells) compared to mock treatment, after normalization by Hmbs and HPrt1. Figure 1B shows the cytotoxicity levels of 31 different cODV-siRNAs (0.1 nM) in PMH cells, measured by propidium iodide (PI) staining. Optical density (OD) of PI staining at excitation wavelength 535 nm and fluorescence wavelength 615 nm was detected using a microplate reader system (Infinite M2000 Pro, Tecan). The values ​​on the vertical axis represent the relative PI staining values ​​for each cODV-siRNA compared to mock treatment, and show the mean ± standard error of transfection replication in 2 wells. [Figure 2]Figures 2A and 2B show the knockdown activity and cytotoxicity of cODV-siRNA designs administered to primary mouse hepatocytes (PMH) by free uptake. The specified cODV-siRNAs (see Table 5) were added to PMH cell culture medium at 1,000 nM for 3 days. RD-12559 was treated as a positive control with known knockdown activity. Figure 2A shows the relative Sod1 mRNA levels after each cODV-siRNA treatment, quantified by two-step RT-qPCR using the gene-specific primer sets shown in Table 4. The geometric mean of Hmbs and Hprt1 mRNA levels was used as an internal reference. The values ​​on the vertical axis represent the relative Sod1 mRNA expression levels (mean ± standard error in 2-well replication) to mock treatment, after normalization by Hmbs and Hprt1. Figure 2B shows the cytotoxicity levels of each cODV-siRNA (0.1 nM) in PMH cells, measured by propidium iodide (PI) staining. Using a microplate reader system, the optical density (OD) of PI staining at an excitation wavelength of 535 nm and an fluorescence wavelength of 615 nm was measured. The values ​​on the vertical axis represent the relative values ​​of PI staining with each cODV-siRNA compared to mock samples, showing the mean ± standard error in two wells of replication. [Figure 3] Figure 3 shows the body weight change in C57BL / 6J mice after intraventricular (ICV) injection of cODV-siRNA. Adult C57BL / 6J mice were administered 200 μg of specified cODV-siRNA (i.e., RD-13592, RD-13608, RD-13611, RD-13614, and RD-14794) via ICV injection. A baseline was established by injecting physiological saline as a vehicle control. Mice were euthanized 14 days after administration. The post-administration body weight change (g) of C57BL / 6J mice is shown as the mean (mean ± standard error) of three mice in each group. [Figure 4]Figure 4 shows the body weight change in C57BL / 6J mice after tail vein (IV) injection of cODV-siRNA. Adult C57BL / 6J mice were administered IV at a dose of 20 mg / kg using specified cODV-siRNAs (i.e., RD-13592, RD-13608, RD-13611, RD-13614, and RD-14794). A baseline was established by injection of physiological saline as a solvent control. Mice were euthanized 14 days after administration. The post-administration body weight change (g) of C57BL / 6J mice is shown as the mean (mean ± standard error) of three mice in each group. [Figure 5] Figures 5A and 5B show the in vivo knockdown activity of cODV-siRNA against rat Sod1 mRNA expression in adult SD rats administered by local intravitreous (IVT) injection. Adult SD rats were administered 30 μg of specified cODV-siRNA (i.e., RD-13592, RD-13596, RD-13600, RD-13604, RD-13608, RD-13611, RD-13615, RD-13619, RD-13625, RD-13184, and RD-13185) by IVT injection. Physiological saline was injected as a solvent control to establish baseline. RD-12556 was injected as a double-stranded control. SD rats were euthanized 14 days after administration. Figures 5A and 5B show the rat Sod1 mRNA levels in the retina quantified by two-step RT-qPCR using the rat gene-specific primer sets shown in Table 4. Gapdh was used as the internal reference for amplification. The values ​​on the vertical axis (y-axis) represent the relative levels of rat Sod1 mRNA expression in response to saline treatment, after normalization by Gapdh (mean ± standard error of 2-3 rats in each group). [Figure 6]Figures 6A–6D show the knockdown activity of cODV-siRNA against SOD1 mRNA expression levels in SK-N-AS and T98G cells. SK-N-AS and T98G cells were transfected with specified cODV-siRNAs (i.e., RD-16989, RD-16978, RD-16102, and RD-16979) at specified concentrations (i.e., 0.0001, 0.0002, 0.001, 0.004, 0.016, 0.063, 0.25, and 1) for 24 hours. Mock treatment without oligonucleotide addition was also performed (not shown). dsCon2 double-stranded cells were transfected as a non-target control (not shown). RD-16988 and RD-16990 were transfected as double-stranded controls. Residual human SOD1 mRNA levels, as shown in the figures, were quantified by two-step RT-qPCR using gene-specific primer sets. TBP was amplified as an internal reference. The values ​​on the vertical axis (y-axis) represent the remaining SOD1 mRNA after normalization by TBP, and are relative to the mock treatment (mean of four transfection wells ± SEM). [Figure 7] Figure 7 shows the knockdown activity of cODV-siRNA in human SOD1G93A (hSOD1G93A) mice to SOD1 mRNA expression. The specified cODV-siRNAs (RD-16145 and RD-16978) were administered intraventricularly (ICV) at a dose of 100 μg to hSOD1G93A mice. Artificial cerebrospinal fluid (aCSF) was administered as a solvent control to establish baseline expression. hSOD1G93A mice were euthanized 14 days after administration. The amount of residual SOD1 mRNA was quantified using a two-step RT-qPCR method with gene-specific primer sets, using samples extracted from brain (frontal cortex, cerebellum, cerebrum), spinal cord, and peripheral (liver) tissues. Mouse Rpl13a was amplified as an internal control. The residual SOD1 mRNA levels in each mouse tissue were normalized by Rpl13a and then expressed as relative values ​​to the aCSF group. The data are shown as the mean ± SEM values ​​obtained from 4 mice in each group. [Figure 8]Figure 8 shows the knockdown activity of cODV-siRNA in Sod1 mRNA expression in C57BL / 6J mice. The specified cODV-siRNAs (RD-16293, RD-16294, RD-16295, and RD-14794) were administered to C57BL / 6J mice at a dose of 200 μg via ICV. Artificial cerebrospinal fluid (aCSF) was administered as a solvent control to establish baseline expression. C57BL / 6J mice were euthanized 14 days after administration. Mouse Sod1 mRNA levels were quantified using two-step RT-qPCR with gene-specific primer sets, using samples extracted from brain (frontal cortex, cerebellum, cerebrum) and spinal cord (cervical, thoracic, lumbar) tissues. Mouse Rpl13a was amplified as an internal control. Sod1 mRNA levels in each mouse tissue were normalized by Rpl13a and then expressed as relative values ​​to the aCSF group. The data are shown as mean values ​​± SEM values ​​obtained from three mice in each group. [Figure 9] Figure 9 shows the knockdown activity of cODV-siRNA against Sod1 mRNA levels in Neuro-2a (N2a) cells. N2a cells were transfected with specified cODV-siRNAs (i.e., RD-18148, RD-18150, RD-18151, RD-18152, RD-18153, RD-18154, RD-18155, and RD-18156) at 0.1 nM for 24 hours. Transfection was performed under oligonucleotide-free conditions as a mock treatment. dsCon2M8 double-stranded siRNA was transfected as a non-targeted control. The residual Sod1 mRNA levels shown in the figure were quantified by two-step RT-qPCR using gene-specific primer sets. Mouse Rpl13a was amplified as an internal control. The values ​​on the vertical axis (y-axis) represent the amount of remaining Sod1 mRNA after normalization with Rpl13a, and are relative to the mock treatment (average of four transfection wells ± SEM). [Figure 10]Figure 10 shows the knockdown activity of cODV-siRNA against Sod1 mRNA levels in N2a cells. Specified cODV-siRNAs (i.e., RD-18151, RD-18317, RD-18318, RD-18319, RD-18320, RD-18321, RD-18322, RD-18323, RD-18153, RD-18325, RD-18326, RD-18327, RD-18329, RD-18150, and RD-18330) were transfected at 0.1 nM for 24 hours. Mock treatment without oligonucleotide addition was also performed. As a non-targeted control, dsCon2M8 double-stranded siRNA was transfected (not shown). The residual Sod1 mRNA levels shown in the figure were quantified by two-step RT-qPCR using a gene-specific primer set. Mouse Rpl13a was amplified as an internal control. The values ​​on the vertical axis (y-axis) represent the amount of Sod1 mRNA remaining after normalization with Rpl13a, and are relative to the mock treatment (mean of four transfection wells ± SEM). [Modes for carrying out the invention]

[0070] Detailed explanation While various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided only as examples. Numerous modifications, alterations, and substitutions can be made to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein can be adopted.

[0071] Before describing the present invention, it should be understood that the present invention is not limited to the specific embodiments described. Furthermore, since the scope of the present invention is limited only by the appended claims, it should be understood that the terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit them.

[0072] Where a range of values ​​is provided, unless the context explicitly indicates otherwise, each intermediate value between the upper and lower limits of that range, up to one-tenth of the unit of the lower limit, is also specifically disclosed. Each subrange between any stated value or intermediate value within a stated range and any other stated value or intermediate value within that stated range is included in the invention. The upper and lower limits of these subranges may be included in or excluded from the range independently, and each range that includes either one, both, or both upper limits in a subrange is also included in the invention, subject to any upper limits specifically excluded from the stated range. Where a stated range includes one or both limits, the range excluding one or both of the limits that they include is also included in the invention.

[0073] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the invention pertains. Any methods and materials similar or equivalent to those described herein may be used in carrying out or testing the invention, but only exemplary methods and materials are described herein. All publications referenced herein are incorporated herein by reference to disclose and describe methods and / or materials relating to which those publications are cited. It is understood that this disclosure takes precedence over the disclosures of incorporated publications insofar as there is a conflict.

[0074] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include the plural form unless the context makes it clear otherwise. Therefore, for example, a reference to "sample" includes multiple such samples, and a reference to "molecule" includes one or more molecules and their equivalents known to those skilled in the art.

[0075] The publications discussed herein are provided solely for disclosure prior to the filing date of this application. Nothing in this specification shall be construed as admitting that the present invention does not have prior rights to such publications by prior art. Furthermore, the publication dates provided may differ from the actual publication dates, which must be independently verified.

[0076] definition The terms “oligonucleotide agent” and “oligonucleotide conjugate” are synonymous and refer to a chimeric oligonucleotide molecule comprising a target oligonucleotide and a non-target site capable of facilitating its delivery. Target oligonucleotides include, but are not limited to, double-stranded nucleic acid molecules of DNA, RNA, or DNA / RNA hybrids, oligonucleotide chains in which deoxyribosyl and ribosyl sites are regularly or irregularly alternating, and modified and naturally occurring or unnaturally occurring skeletons of these oligonucleotides. Target oligonucleotides disclosed herein may be small nucleic acid molecules (siRNA), small activated nucleic acid molecules (saRNA), or antisense oligonucleotide molecules (ASO). Specifically, oligonucleotide agents that inhibit the mRNA transcription level of a target gene described herein are siRNA molecules conjugated to a non-target site, and oligonucleotide agents that activate the transcription of a target gene are saRNA molecules conjugated to a non-target site.

[0077] The terms "non-target site" or "chain oligonucleotide delivery vehicle (cODV)" refer to a portion of an oligonucleotide agent that is conjugated directly or via a linker to a target oligonucleotide, and are intended to facilitate the in vivo delivery of the target oligonucleotide, without having an intentional gene-targeting function. The non-target site comprises one or more covalently linked components, such as linkers, ligation groups, or nucleotides, and may be linear or branched. The components of the non-target site may be further chemically modified, for example, in the backbone or branched chain.

[0078] As used herein, the term “non-target” means that the site conjugated with the target oligonucleotide (e.g., siRNA, saRNA, etc.) does not specifically bind to the target sequence on which the target oligonucleotide acts, or ideally, to any other nucleotide sequence within an animal cell. The target oligonucleotides disclosed herein are nucleic acid sequences specifically complementary to the target sequence or the site. In some cases, “specifically complementary” may mean that the complementarity between the target oligonucleotide and the target sequence or region is at least about 95%. The non-target site is not intended to induce any biological activity by any known mechanism when the oligonucleotide is administered, nor is it intended to function against a complementary nucleic acid sequence (i.e., mRNA) as an ASO (e.g., “mixmer” or “gapmer”). The non-target site is intended to facilitate the in vivo distribution, entry into cells, and intracellular function of the target oligonucleotide (e.g., siRNA, saRNA, etc.) conjugated to that site when the oligonucleotide conjugate is administered.

[0079] As used herein, the term “backbone” means, with respect to a compound, the portion consisting of the longest carbon chain in the molecule (which may be modified to include other heteroatoms instead of carbon atoms) or the carbon chain containing a functional group. Bonds by functional groups in the backbone include, but are not limited to, -CH2-O-CH2-, CH2-OPO-, -OPO-CH2-, -CH2-NH-O-CH2-, -CH2-N(CH3)-O-CH2-, -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -ON(CH3)-CH2-CH2-. As shown herein, the backbone of a non-target site may be modified to include one or more phosphorothioate bonds.

[0080] As used herein, the terms “linker,” “chemical linker,” “spacer,” “component,” or “unit” are mutually interchangeable and refer to a molecule or chemical group that covalently connects two parts. This includes, but is not limited to, linkers commonly used to space two nucleotides (e.g., spacer-18 linker, spacer-C6 linker, L6, spacer-9 linker, spacer-C3 linker, L12(d-spacer), spacer-C12 linker, spacer-L14 linker, spacer-L15 linker, spacer-L16 linker, C6x1 linker, C6x2 linker, C6x5 linker, C6x7 linker, L20 linker, L42 linker, etc.). It also includes other chemical groups that can be used to provide a backbone, such as linear or branched aliphatic chains or substituted aliphatic chains, and disulfide bonds that connect two molecules. The term also includes nucleic acid or nucleic acid analog linkers, peptide linkers, etc. In this application, linkers within a non-target region belong to the components or units of the non-target region.

[0081] Where used herein, the terms “subject” and “individual” are used interchangeably herein to mean any living organism that can be treated with the agent of this application. The term “patient” refers to a human subject or individual, including infants, children and adults, who are subject to disclosure.

[0082] The “therapeutic effective amount” of a composition is an amount sufficient to achieve a desired therapeutic effect and therefore does not require a cure or complete remission. In embodiments of this application, the therapeutic effect is an improvement in any of the disease indicators, and the therapeutic effect amount is sufficient to cause an improvement in a clinically significant condition / symptom in the treated individual. The expressions “therapeutic effective amount” and “effective amount” are used herein to mean an amount sufficient to reduce a clinically significant deficit in the activity, function and response of the treated individual by at least about 15%, preferably at least 50%, more preferably at least 90%, or to increase, most preferably at least 50%, at least about 100%, at least about 200%, more preferably at least about 500%, and most preferably prevent.

[0083] The effective dose may vary depending on factors such as the subject's physique and weight, the type of disease, or the specific drug being administered. For example, the choice of drug can affect what constitutes the "effective dose." A person skilled in the art could study the factors included herein and make a determination regarding the effective dose of the drug in this application without conducting excessive experiments.

[0084] The administration method may affect what constitutes the effective dose. The agents of this application can be administered to a subject either before or after the diagnosis or manifestation of a disease. Furthermore, the doses can be divided into several doses and staggered doses, administered daily or continuously, or the doses can be administered by continuous infusion or as a bolus injection. In addition, the dose of one or more agents of this application may be increased or decreased proportionally, as indicated by the urgency of the therapeutic or preventive situation.

[0085] As used herein, the terms “treat,” “administer,” or “procedure” have the meanings commonly understood in medical technology and therefore include any beneficial or desired clinical outcome, without requiring a cure or complete remission. Non-limiting examples of such beneficial or desired clinical outcomes include an extension of survival compared to the expected survival without treatment, and relief of one or more symptoms including: weakness and atrophy of proximal skeletal muscles, inability to sit or walk independently, dysphagia, dyspnea, etc.

[0086] In this specification, “prevention” or “delay” of a disease means preventing the complete onset of the disease.

[0087] The term "biological sample" refers to any tissue, cell, fluid, or other substance derived from an organism (e.g., a human subject). In certain embodiments, the biological sample is serum or blood.

[0088] As used herein, the terms “sequence identity” or “sequence homology” refer to, for example, the similarity of one oligonucleotide strand (sense or antisense) of a saRNA or siRNA to a region on the coding or template strand of a promoter, or to the sequence of a target gene, by at least 80%. Unless otherwise specified, the terms “identity” and “homology” herein refer to the nucleotide itself, and modifications are not considered. For example, nucleotides A, mA(2'-O-methyl A), and fA(2'-fluoro A) are treated as the same nucleotide when calculating sequence identity or homology.

[0089] A "target sequence" refers to a sequence fragment in which the sense strand or antisense oligonucleotide of an siRNA or saRNA is homologous or complementary.

[0090] As used herein, the term “gapmer” refers to a short DNA antisense oligonucleotide (ASO) structure having modified RNA segments on both sides of a central DNA structure. In some embodiments, at least one of the modified RNA segments comprises one or more modified nucleotides selected from lock nucleic acids (LNAs) and 2'-OMe or 2'-F modified nucleotides to increase affinity to a target, increase nuclease resistance, decrease immunogenicity, and / or reduce toxicity. In some embodiments, the gapmer comprises at least one nucleotide modified with a phosphorothioate (PS) group. In some embodiments, the gapmer is designed to hybridize to a target portion of the RNA and repress a gene transcript via induction of RNase H cleavage.

[0091] As used herein, the term “mixer” refers to an antisense oligonucleotide (ASO) characterized as a mixed structure of DNA and a chemically modified nucleic acid analog. Optionally, a mixer consists of a fully modified nucleotide or nucleic acid analog. In some embodiments, a mixmer is designed to bind to and mask a complementary RNA sequence to sterically inhibit a protein, factor, or other RNA from interacting with a target RNA. In some embodiments, a mixmer is designed to alter the splicing of pre-mRNA by displacing a spliceosome. In some embodiments, a mixmer is designed to bind to and sequester a microRNA (miRNA), in which case it may be called an “antagomir” or “anti-miR.”

[0092] As used herein, the term “sense strand of double-stranded RNA (dsRNA) (e.g., siRNA, saRNA)” refers to a strand that has sequence homology or sequence identity with a fragment of the coding strand of the target gene sequence.

[0093] As used herein, the term “antisense strand of double-stranded RNA (dsRNA) (e.g., siRNA, saRNA)” refers to a strand having a sequence complementary to the sense strand. The antisense strand may interact with a target sequence to activate or upregulate gene expression, and the target sequence may be a fragment of the coding strand of a target gene.

[0094] As used herein, the term “first oligonucleotide strand” may be a sense strand or an antisense strand. For example, the sense strand of saRNA refers to an oligonucleotide strand homologous to the coding strand of the promoter DNA sequence of the saRNA's target gene. The sense strand of siRNA refers to an oligonucleotide strand homologous to the mRNA sequence of the siRNA's target gene. The antisense strand refers to an oligonucleotide strand complementary to the sense strand of dsRNA.

[0095] As used herein, the term “second oligonucleotide chain” may also refer to a sense chain or an antisense chain. If the first oligonucleotide chain is a sense chain, the second oligonucleotide chain is an antisense chain; if the first oligonucleotide chain is an antisense chain, the second oligonucleotide chain is a sense chain.

[0096] As used herein, the term “coding strand” refers to the DNA strand of a target gene that is not transcribed, whose nucleotide sequence is identical to that of the RNA produced by transcription (in RNA, the T in DNA is replaced by U). The coding strand of the double-stranded DNA sequence of the target gene promoter as described in this disclosure refers to the promoter sequence on the same DNA strand as the DNA coding strand of the target gene.

[0097] As used herein, the term “template strand” refers to another strand of double-stranded DNA of the target gene that is complementary to the coding strand and can be transcribed as a template to RNA complementary to the transcribed RNA bases (AU, GC). During transcription, RNA polymerase binds to the template strand and moves along the 3'→5' direction of the template strand, catalyzing RNA synthesis in the 5'→3' direction. The template strand of the double-stranded DNA sequence of the target gene promoter as described herein refers to the promoter sequence on the same DNA strand as the DNA template strand of the target gene.

[0098] As used herein, the term “overhang” refers to an oligonucleotide having a non-base-paired nucleotide (maybe multiple) at the oligonucleotide chain end (5' or 3') resulting from another chain extending beyond one of the chains in a double-stranded oligonucleotide. A single-stranded region extending beyond the 3' and / or 5' ends of a double helix is ​​called an overhang. In certain embodiments, an overhang is 0 to 6 nucleotides in length. An overhang of 0 nucleotides is understood to mean no overhang.

[0099] As used herein, the term “natural overhang” refers to an overhang consisting of one or more nucleotides identical or complementary to the corresponding position on the target sequence. A natural overhang on the sense strand consists of one or more nucleotides identical to the corresponding position on the mRNA or RNA target. A natural overhang on the antisense strand consists of one or more nucleotides complementary to the corresponding position on the mRNA or RNA target.

[0100] As used herein, the terms “gene activation,” “gene expression activation,” “gene upregulation,” and “gene expression upregulation” are interchangeable and refer to an increase or upregulation of the transcription, translation, expression, or activity of a particular nucleic acid sequence, determined by measuring the transcription level, mRNA level, protein level, enzyme activity, methylation status, chromatin status or arrangement, translation level, or activity or status in a cell or biological system. These activities or states can be determined directly or indirectly. Furthermore, “gene activation” or “gene expression activation” refers to an increase in activity related to a nucleic acid sequence, regardless of the mechanism of such activation. For example, gene activation occurs at the transcription level, increasing transcription to RNA, which is then translated into protein, thereby increasing protein expression.

[0101] As used herein, the terms “gene silencing,” “knockdown of gene expression,” “gene downregulation,” and “downregulation of gene expression” are interchangeable and refer to a reduction or downregulation of the transcription, translation, expression, or activity of a particular nucleic acid sequence, determined by measuring the transcription level, mRNA level, protein level, enzyme activity, methylation status, chromatin status or arrangement, translation level, or activity or status of the gene in a cellular or biological system. These activities or states can be determined directly or indirectly. Furthermore, “gene downregulation” or “downregulation of gene expression” refers to a decrease in activity associated with a nucleic acid sequence, regardless of the mechanism of such downregulation. For example, gene downregulation occurs at the transcription level, reducing or suppressing transcription to RNA, which is not translated into protein, thereby reducing or suppressing protein expression.

[0102] As used herein, the terms “inhibition of gene expression” or “inhibit gene expression” and “gene downregulation” or “downregulate gene expression” are interchangeable and mean a reduction in the transcription, translation, expression, or activity of a particular nucleic acid, determined by measuring the transcription level, mRNA level, protein level, enzyme activity, methylation status, chromatin status or arrangement, translation level, or the activity or status of the gene in a cell or biological system. These activities or states can be determined directly or indirectly. Furthermore, the terms “inhibition of gene expression,” “inhibit gene expression,” “gene downregulation,” or “downregulation of gene expression” refer to a reduction in activity related to the nucleotide sequence, regardless of the mechanism of the inhibition. For example, inhibition of gene expression occurs at the transcription level, reducing transcription to RNA, which is then translated into protein, thereby reducing protein expression.

[0103] As used herein, the terms “small interfering RNA,” “siRNA,” and “silencing RNA” are interchangeable and refer to ribonucleic acid molecules that can downregulate, knock down, or repress target gene expression. They may also be double-stranded nucleic acid molecules. They interfere with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription and blocking translation. siRNA primarily binds to target mRNA in the cytoplasm and downregulates gene expression at the post-transcriptional stage via RNA interference (RNAi) mechanisms. siRNA can be designed to target the mRNA sequences of genes such as SOD1, and by repressing their expression via RNAi, it may be possible to maximize treatment outcomes, for example, in ALS patients. siRNA is a molecule that has either native ribonucleotides or chemically modified nucleotides. This modification does not eliminate cellular activity, but rather leads to increased stability or increased cellular activity. Examples of chemical modifications include phosphorothioate groups, 2'-deoxyribonucleotides, 2'-OCH3-containing ribonucleotides, 2'-F-ribonucleotides, 2'-methoxyethylribonucleotides, and combinations thereof. siRNAs can have varying lengths (e.g., 10–200 bps) and structures (e.g., hairpin, single-stranded / double-stranded, bulge, nick / gap, mismatch) and are processed intracellularly to provide active gene silencing. Double-stranded siRNAs may have the same number of nucleotides on each strand (blunt ends) or asymmetric ends (overhangs). For example, a 1–2 nucleotide overhang may be present on the sense strand and / or antisense strand, and may also be present on the 5'- and / or 3'-ends of a given strand. The lengths of siRNA molecules are typically about 10–60, 10–50, 15–30, 17–29, 18–28, 19–27, 20–26, 21–25, and 22–24 base pairs, and are typically about 15, 16, 17, 18, 19, 20, 21, 23, 25, 30, 40, or 50 base pairs.Furthermore, the terms “small interfering RNA,” “silencing RNA,” and “siRNA” include nucleic acids other than ribonucleotides, including but not limited to modified nucleotides or analogues.

[0104] As used herein, the terms “small activated RNA,” “saRNA,” and “small activated ribonucleic acid” are interchangeable and refer to ribonucleic acid molecules that can upregulate the expression of a target gene. A saRNA may be a double-stranded nucleic acid molecule comprising a first nucleic acid strand containing a ribonucleotide sequence having sequence homology to the non-coding nucleic acid sequence (such as a promoter or enhancer) of the target gene, and a second nucleic acid strand containing a nucleotide sequence complementary to the first strand. A saRNA may also consist of a synthetic or vector-expressed single-stranded RNA molecule that readily forms a hairpin structure due to two complementary regions within the molecule, where the first region contains a ribonucleotide sequence having sequence homology to the target sequence of the gene's promoter, and the ribonucleotide sequence in the second region is complementary to the first region. The length of the double-stranded region of a saRNA molecule is typically about 10–60, 10–50, 10–40, 12–30, 14–28, 16–26, 18–24, 20–22 base pairs, and typically about 10, 13, 15, 17, 18, 19, 20, 21, 22, 25, 30, 40, 50, or 60 base pairs. Furthermore, the terms “small activated RNA,” “saRNA,” and “small activated ribonucleic acid” include non-ribonucleotide nucleic acids, including but not limited to modified nucleotides or analogues.

[0105] As used herein, the terms “isolated target site,” “target site,” and “isolated polynucleotide” are interchangeable and refer herein to nucleic acid target sites to which siRNA / saRNA is complementary or hybridizes. For example, an isolated nucleic acid sequence of a target site may include a nucleic acid sequence to which a region of siRNA / saRNA is complementary or hybridizes.

[0106] As used herein, the term “complementarity” refers to the ability to form base pairs between two oligonucleotide chains. Base pairs are generally formed by hydrogen bonds between nucleotides in antiparallel oligonucleotide chains. The bases of complementary oligonucleotide chains can be paired by the Watson-Crick method (AT, AU, CG, etc.) or by other methods that enable the formation of double helices (such as Hoogsteen or reverse Hoogsteen base pairing).

[0107] Complementarity can be categorized into complete and incomplete complementarity. "Complete complementarity" or "100% complementarity" means that each nucleotide in the first oligonucleotide chain can form a hydrogen bond with the corresponding nucleotide in the second oligonucleotide chain within the double-stranded region of the siRNA molecule, and there are no "misspairs" in the base pairs. "Incomplete complementarity," "partial complementarity," or "mismatch" means that not all nucleotide units in the two chains are bonded to each other by hydrogen bonds. For example, in the case of two oligonucleotide chains, each 20 nucleotides long in the double-stranded region, if only two base pairs can be formed by hydrogen bonds in this double-stranded region, the complementarity of the oligonucleotide chains is 10%. In the same example, if 18 base pairs in this double-stranded region can be formed by hydrogen bonds, the oligonucleotide chains have 90% complementarity. Substantial complementarity refers to a complementarity of at least approximately 75%, 79%, 80%, 85%, 90%, 95%, or 99%.

[0108] As used herein, the term “synthesis” refers to the method by which oligonucleotides are synthesized and includes any means by which RNA can be synthesized or chemically modified, such as chemical synthesis, in vitro transcription, and vector expression.

[0109] As used herein, the term "LNA" refers to a locating nucleic acid in which a 2'-oxygen atom and a 4'-carbon atom are linked by an extra bridge. As used herein, the term "BNA" refers to a 2'-O and 4'-aminoethylene crosslinked nucleic acid that may include a 5-membered or 6-membered crosslinked structure having an NO bond. As used herein, the term "PNA" refers to a nucleic acid mimetic having a pseudopeptide backbone consisting of an N-(2-aminoethyl)glycine unit having a nucleic acid base linked to a glycine nitrogen via a carbonylmethylene linker.

[0110] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by experts in the technical field to which this application pertains.

[0111] overview Aspects of this application include oligonucleotide agents that efficiently target one or more genes associated with a disease or symptom, and that include additional sites for improving delivery, chemical properties, in vivo distribution, bioavailability and other pharmacological properties without impairing the activity of the oligonucleotide.

[0112] This application is based on studies of compositions and methods that improve therapeutic effects against genetic diseases by activating or upregulating gene expression to increase the expression level of full-length genes or proteins, or by knocking out or repressing gene expression to decrease the expression level of full-length genes or proteins, through the combination of a target oligonucleotide (siRNA, saRNA, etc.) with a non-target site. As used herein, the term “chain oligonucleotide delivery vehicle (cODV)” refers to a portion of an oligonucleotide agent conjugated to a target oligonucleotide (directly or via a linker), intended to facilitate the in vivo delivery of the target oligonucleotide, and does not have the function of intentionally targeting genes. The non-target site comprises one or more covalently linked components, such as a linker, linking group, or nucleotide, and may be linear or branched. The components of the non-target site may be further chemically modified, for example, in the backbone or branched chain.

[0113] The inventors found that the non-target site does not inhibit siRNA-induced knockdown activity or saRNA-induced gene activation. Furthermore, the inventors found that the length, composition, modifications (e.g., 2'-Ome, 2'-MOE, 2'-F), binding components, and phosphorothioate (PS) backbone binding of the non-target site affect the in vivo activity of double-stranded RNA (dsRNA). Compared to dsRNA without a non-target site, cODV-dsRNA exhibits enhanced in vivo activity in the central nervous system (CNS) when locally administered to brain or spinal cord tissue.

[0114] Further aspects of this application include a method for treating amyotrophic lateral sclerosis (ALS) by administering an effective dose of an oligonucleotide agent containing SOD1-targeted siRNA. This siRNA inhibits the expression of the SOD1 gene via an RNAi silencing mechanism. The inventors have developed a potent inhibitory SOD1 siRNA for use in the treatment of ALS.

[0115] Oligonucleotide agents Aspects of this application relate to oligonucleotide agents comprising one or more components, each comprising a non-target site and a target double-stranded oligonucleotide, which are covalently conjugated, and the non-target site is covalently bonded by at least one phosphorothioate (PS) bond. The components are selected from chemical linkers, nucleotides, and other units that can function as a "scaffold" for the non-target site.

[0116] Aspects of this application relate to oligonucleotide agents comprising a non-target site and a target double-stranded oligonucleotide, which are covalently conjugated, and wherein at least two adjacent linkers, two adjacent nucleotides, or a spacer and an adjacent nucleotide are linked to each other by phosphorothioate (PS) bonds at the non-target site.

[0117] Aspects of this application relate to oligonucleotide agents comprising a non-target site and a target double-stranded oligonucleotide, which are covalently conjugated, wherein the non-target site is selected from the following: (a) a series of linked components, none of which contain nucleotides; (b) a series of non-nucleotide components with one or more nucleotides interposed within them; (c) a series of nucleotides with one or more non-nucleotide components interposed within them; (d) a series of continuous nucleotide sequences and sequences of non-nucleotide components linked together. Preferably, the non-nucleotide components are linkers such as those shown in Table 1.

[0118] Aspects of this application relate to oligonucleotide agents comprising a non-target site and a target double-stranded oligonucleotide, which are covalently conjugated, and in which the non-target site does not contain any nucleotides. In some embodiments, the non-target site contains one or more nucleotides, which may be natural, synthetic, or chemically modified.

[0119] Aspects of this application relate to an oligonucleotide agent comprising a non-target site and a target double-stranded oligonucleotide, which are covalently conjugated, and in which the non-target site is capable of promoting the delivery of the double-stranded oligonucleotide in the central nervous system (CNS).

[0120] In some embodiments, the oligonucleotide agent comprises a double-stranded oligonucleotide, which comprises a sense strand and an antisense strand, the antisense strand being complementary to the target nucleic acid. It also comprises a non-target site consisting of one or more linkers, one or more nucleotides, or a hybrid of both. The double-stranded oligonucleotide and the non-target site are conjugated covalently, either via the linker or directly, to form the oligonucleotide agent. In some embodiments, the sense strand of the double-stranded target oligonucleotide is covalently conjugated to a non-target site. In some embodiments, the antisense strand of the double-stranded target oligonucleotide is covalently conjugated to a non-target site. In some embodiments, the oligonucleotide agent has the following compound formula: [ka] And here: O refers to a double-stranded oligonucleotide, which includes a sense strand and an antisense strand, the antisense strand being complementary to the target nucleic acid (e.g., mammalian target nucleic acid). M refers to a non-target site containing one or more components linked by at least one phosphorothioate (PS) bond. L is an optional linker for covalently conjugating the double-stranded oligonucleotide and the non-target site. In some embodiments, the oligonucleotide agent has the following compound formula: [ka] Here, O is a double-stranded oligonucleotide comprising a sense strand and an antisense strand, the antisense strand being complementary to the target nucleic acid. M refers to a non-target site comprising one or more spacers, one or more nucleotides, or a hybrid of one or more spacers and nucleotides. L is a linker for covalently conjugating the double-stranded oligonucleotide to the non-target site. The compound also includes optional components Cx, Cy, and Cz, each independently selected from lipids, fatty acids, fluorescent dyes, ligands, sugars, peptides, antibodies, and other commonly used conjugation groups. In some embodiments, the compound of formula II contains one conjugation group. In some embodiments, the compound of formula II contains two conjugation groups. In some embodiments, the compound of formula II contains three conjugation groups.

[0121] In some embodiments, the double-stranded oligonucleotide is siRNA. In some embodiments, the double-stranded oligonucleotide is saRNA.

[0122] In some embodiments, if nucleotides at the 5' end, 3' end, or inside of the non-target site are present, they are conjugated to the linking component. In some embodiments, the inside nucleotides of the sense or antisense strand of the double-stranded oligonucleotide are substituted with the linking component, and the single-stranded oligonucleotide is covalently conjugated to the linking component. In some embodiments, the non-target site is covalently conjugated to the sense strand, antisense strand, or both of the double-stranded oligonucleotide via the linking component.

[0123] In some embodiments, the non-target site is covalently conjugated to the 3' end, 5' end, both 3' and 5' ends, or an internal nucleotide of the sense strand of the double-stranded oligonucleotide. In some embodiments, the non-target site is covalently conjugated to the 3' end, 5' end, both 3' and 5' ends, or an internal nucleotide of the antisense strand of the double-stranded oligonucleotide. Non-targeted site

[0124] Several previous studies have shown that siRNA capable of inhibiting SOD1 mRNA and reducing SOD1 protein expression can be used to treat SOD1 protein-related diseases, such as amyotrophic lateral sclerosis (ALS). However, the inventors have identified two unresolved problems: 1) insufficient potency of the SOD1 siRNA molecule, and 2) the lack of an efficient delivery method for delivering the siRNA molecule to cells in target organs or tissues.

[0125] Similarly, there are unresolved issues regarding saRNAs. These include 1) insufficient efficacy of saRNA molecules, and 2) the lack of efficient delivery methods for saRNA molecules to target organ or tissue cells.

[0126] Surprisingly, the present invention has found that when dsRNA agents, such as siRNA or saRNA, are conjugated to a non-targeting moiety disclosed herein, the bioavailability, in vivo distribution, and / or intracellular uptake of the dsRNA, as well as its in vivo efficacy, are significantly improved compared to oligonucleotide agents without a non-targeting moiety. In particular, in several in vivo examples in this application, it was confirmed that the non-targeting moiety of an oligonucleotide agent increases the in vivo distribution of dsRNA in one or more target tissues compared to oligonucleotide agents without a non-targeting moiety.

[0127] "Delivery into cells" refers to efficient uptake or absorption by cells, as understood by those skilled in the art, when referring to target double-stranded oligonucleotides, such as siRNA and double-stranded RNA agents (dsRNAs) like saRNA. The absorption or uptake of dsRNA can occur by unassisted diffusive or active cellular processes, or by auxiliaries or devices. The meaning of this term is not limited to in vitro cells; it is also possible to "introduce" dsRNA into cells, where the cells are part of a living organism. In such cases, introduction into cells also includes delivery to a living organism. For example, in vivo introduction can involve injecting dsRNA into a tissue site or administering it systemically. In vitro introduction into cells includes methods known in the art, such as electroporation, free uptake, and lipofection. Further approaches not known in the art are described below.

[0128] Generally, the non-targeting site of an oligonucleotide agent is a single-stranded region that works favorably for the oligonucleotide agent due to its delivery characteristics. Therefore, this non-targeting site does not target the nucleic acid sequence in the subject targeted by the dsRNA, nor "natural" nucleic acids derived from the subject, such as the target nucleic acid of the dsRNA. In some embodiments, the site does not target the nucleic acid in the subject targeted by the double-stranded RNA (dsRNA). In some embodiments, the site is not complementary to the nucleic acid targeted by the dsRNA. In some embodiments, the site is not complementary to the gene sequence or its mRNA transcript targeted by the dsRNA.

[0129] In some embodiments, one or more components of the non-target site have the following compound formula. [ka] Here, R1, R2, R3, and R4 are, independently of each other, hydrogen (H), substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclic alkyl, heterocyclic alkenyl, heterocyclic alkynyl, aryl, heteroaryl, heterocyclic, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroaryl Selected from: lukyl, alkyl heteroaryl alkenyl, alkyl heteroaryl alkinyl, alkenyl heteroaryl alkyl, alkenyl heteroaryl alkenyl, alkenyl heteroaryl alkinyl, alkinyl heteroaryl alkinyl, alkinyl heteroaryl alkyl, alkinyl heteroaryl alkenyl, alkinyl heteroaryl alkinyl, alkyl hetero ring alkyl, alkyl hetero ring alkenyl, alkyl hetero ring alkynyl, alkenyl hetero ring alkyl, alkenyl hetero ring alkenyl, alkenyl hetero ring alkynyl, alkinyl hetero ring alkyl, alkinyl hetero ring alkenyl, alkinyl hetero ring alkynyl, alkylaryl, alkenyl aryl, alkinyl aryl, alkyl heteroaryl, alkenyl heteroaryl, and alkinyl heteroaryl. Furthermore, one or more methylene groups in these groups may be interrupted or terminated by oxygen (O), sulfur (S), sulfoxide [S(O)], sulfone (SO2), N(R')2, carbonyl group [C(O)], cleavable linker, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycle. Each R' is independently selected from hydrogen, substituted or unsubstituted alkyl, aryl, aralkyl, alkylaryl, alkoxy, aryloxy, acyl, or aliphatic group.These groups may be linear, branched, cyclic, polycyclic, unsaturated, hydroxylated, carbonylated, phosphorylated, and / or sulfurized, preferably one or more components of the non-target site have phosphorothioate (PS) modifications in the skeleton. In some embodiments, R1, R2, R3, and R4 are independently selected from substituted or unsubstituted C1-C16 alkyl groups and aryl groups, respectively.

[0130] In some embodiments, the site comprises m components, of which n are nucleotides, where m is 1 to 50 and n is 0 to 20. In some embodiments, the non-nucleotide components in the non-target site are selected from spacer 18 linker, spacer C6 linker, L6, spacer 9 linker, spacer C3 linker, L12 (d spacer), spacer C12 linker, spacer L14 linker, spacer L15 linker, spacer L16 linker, C6x1 linker, C6x2 linker, C6x5 linker, C6x7 linker, L20 linker, L42 linker, and any other linker available for spacing between two nucleotides. For some purposes, the non-target site contains no nucleotides and contains at least 1, 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 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, and at least 30 or more linkers. The linkers may be the same or different, and can be selected from spacer-18 linker, spacer-C6 linker, L6, spacer-9 linker, spacer-C3 linker, L12 (d spacer), spacer-C12 linker, spacer-L14 linker, spacer-L15 linker, spacer-L16 linker, C6x1, C6x2, C6x5, C6x7, L20 linker, L42 linker, etc. The part in question may include a combination of one or more linkers selected from the following: Spacer-18 linker, Spacer-C6 linker, L6 linker, Spacer-9 linker, Spacer-C3 linker, L12(d spacer), Spacer-C12 linker, Spacer-L14 linker, Spacer-L15 linker, Spacer-L16 linker, C6x1, C6x2, C6x5, C6x7, L20 linker, and L42 linker.The same linker may be connected in series consecutively, or it may be interposed by different linkers.

[0131] In some embodiments, the portion includes zero, one or more S9 linkers and one or more linkers selected from the following: spacer-18 linker, spacer-C6 linker, L6, spacer-C3 linker, L12(d spacer), spacer-C12 linker, spacer-L14 linker, spacer-L15 linker, spacer-L16 linker, C6x1, C6x2, C6x5, C6x7, L20 linker, L42 linker. In some embodiments, the portion includes one or more spacer-18 linkers and one or more linkers selected from the following: S9 linker, spacer-C6 linker, L6 linker, spacer-C3 linker, L12(d spacer), spacer-C12 linker, spacer-L14 linker, spacer-L15 linker, spacer-L16 linker, C6x1, C6x2, C6x5, C6x7, L20 linker, L42 linker. In some embodiments, the portion includes one or more spacer-C6 linkers and one or more linkers selected from the following: S9 linker, spacer-18 linker, L6 linker, spacer-C3 linker, L12(d spacer), spacer-C12 linker, spacer-L14 linker, spacer-L15 linker, spacer-L16 linker, C6x1, C6x2, C6x5, C6x7, L20 linker, L42 linker. In some embodiments, the portion includes one or more L6 linkers and one or more linkers selected from the following: S9 linker, spacer-18 linker, spacer-C6 linker, spacer-C3 linker, L12(d spacer), spacer-C12 linker, spacer-L14 linker, spacer-L15 linker, spacer-L16 linker, C6x1, C6x2, C6x5, C6x7, L20 linker, L42 linker. In some embodiments, the portion includes one or more spacer-C3 linkers and one or more linkers selected from the following: S9 linker, spacer-18 linker, L6, spacer-C6 linker, L12(d spacer), spacer-C12 linker, spacer-L14 linker, spacer-L15 linker, spacer-L16 linker, C6x1, C6x2, C6x5, C6x7, L20 linker, L42 linker. In some embodiments, the part includes one or more L12(d spacers) and one or more linkers selected from the following: S9 linker, spacer-18 linker, L6, spacer-C3 linker, spacer-C6 linker, spacer-C12 linker, spacer-L14 linker, spacer-L15 linker, spacer-L16 linker, C6x1, C6x2, C6x5, C6x7, L20 linker, L42 linker. In some embodiments, the portion includes one or more spacer-C12 linkers and one or more linkers selected from the following: S9 linker, spacer-18 linker, L6, spacer-C3 linker, spacer-C6 linker, L12(d spacer), spacer-L14 linker, spacer-L15 linker, spacer-L16 linker, C6x1, C6x2, C6x5, C6x7, L20 linker, L42 linker. In some embodiments, the portion includes one or more spacer-L14 linkers and one or more linkers selected from the following: S9 linker, spacer-18 linker, L6, spacer-C3 linker, spacer-C6 linker, spacer-C12 linker, L12(d spacer), spacer-L15 linker, spacer-L16 linker, C6x1, C6x2, C6x5, C6x7, L20 linker, L42 linker. In some embodiments, the portion includes one or more spacer-L15 linkers and one or more linkers selected from the following: S9 linker, spacer-18 linker, L6, spacer-C3 linker, spacer-C6 linker, spacer-C12 linker, spacer-L14 linker, L12(d spacer), spacer-L16 linker, C6x1, C6x2, C6x5, C6x7, L20 linker, L42 linker.In some embodiments, the portion includes one or more spacer-L15 linkers and one or more linkers selected from the following: S9 linker, spacer-18 linker, L6, spacer-C3 linker, spacer-C6 linker, spacer-C12 linker, spacer-L14 linker, L12(d spacer), spacer-L16 linker, C6x1, C6x2, C6x5, C6x7, L20 linker, L42 linker. In some embodiments, the portion includes one or more spacer-L16 linkers and one or more linkers selected from the following: S9 linker, spacer-18 linker, L6, spacer-C3 linker, spacer-C6 linker, spacer-C12 linker, spacer-L14 linker, L12(d spacer), spacer-L15 linker, C6x1, C6x2, C6x5, C6x7, L20 linker, L42 linker. In some embodiments, the portion includes one or more C6x1 linkers and one or more linkers selected from the following: S9 linker, spacer-18 linker, L6, spacer-C3 linker, spacer-C6 linker, spacer-C12 linker, spacer-L14 linker, L12(d spacer), spacer-L15 linker, spacer-L16 linker, C6x2, C6x5, C6x7, L20 linker, L42 linker. In some embodiments, the portion includes one or more C6x2 linkers and one or more linkers selected from the following: S9 linker, spacer-18 linker, L6, spacer-C3 linker, spacer-C6 linker, spacer-C12 linker, spacer-L14 linker, L12(d spacer), spacer-L15 linker, spacer-L16 linker, C6x1, C6x5, C6x7, L20 linker, L42 linker.In some embodiments, the portion includes one or more C6x5 linkers and one or more linkers selected from the following: S9 linker, spacer-18 linker, L6, spacer-C3 linker, spacer-C6 linker, spacer-C12 linker, spacer-L14 linker, L12(d spacer), spacer-L15 linker, spacer-L16 linker, C6x2, C6x1, C6x7, L20 linker, L42 linker. In some embodiments, the portion includes one or more C6x7 linkers and one or more linkers selected from the following: S9 linker, spacer-18 linker, L6, spacer-C3 linker, spacer-C6 linker, spacer-C12 linker, spacer-L14 linker, L12(d spacer), spacer-L15 linker, spacer-L16 linker, C6x2, C6x5, C6x1, L20 linker, L42 linker. In some embodiments, the portion includes one or more L20 linkers and one or more linkers selected from the following: S9 linker, spacer-18 linker, L6, spacer-C3 linker, spacer-C6 linker, spacer-C12 linker, spacer-L14 linker, L12(d spacer), spacer-L15 linker, spacer-L16 linker, C6x2, C6x5, C6x1, C6x7 linker, L42 linker. In some embodiments, the portion includes one or more L42 linkers and one or more linkers selected from the following: S9 linker, spacer-18 linker, L6, spacer-C3 linker, spacer-C6 linker, spacer-C12 linker, spacer-L14 linker, L12(d spacer), spacer-L15 linker, spacer-L16 linker, C6x2, C6x5, C6x1, C6x7 linker, L20 linker.

[0132] In some specific embodiments, the site includes 2 L10 linkers, 4 L10 linkers, 6 L10 linkers, 8 L10 linkers, 10 L10 linkers, 12 L10 linkers, 14 L10 linkers, 16 L10 linkers, 18 L10 linkers, 20 L10 linkers, 22 L10 linkers, 24 L10 linkers, 26 L10 linkers, 28 L10 linkers, 30 or more L10 linkers. Further, the site includes an S9 linker, and the S9 linker may be located inside or at the end of the site.

[0133] In some specific embodiments, in addition to one linker that binds to a target oligonucleotide, the site includes 2 L12 linkers, 4 L12 linkers, 6 L12 linkers, 8 L12 linkers, 10 L12 linkers, 12 L12 linkers, 14 L12 linkers, 16 L12 linkers, 18 L12 linkers, 20 L12 linkers, 22 L12 linkers, 24 L12 linkers, 26 L12 linkers, 28 L12 linkers, 30 or more L12 linkers.

[0134] In specific embodiments, the non-target site is S9-(L10)2, S9-(L10)3, S9-(L10)4, S9-(L10)5, S9-(L10)6, S9-(L10)7, S9-(L10)8, S9-(L10)9, S9-(L10) 10 、S9-(L10) 12 、S9-(L10) 14 、S9-(L10) 16 、S9-(L10) 18 、S9-(L10) 20 、S9-(L10) 21 、S9-(L10) 22 、S9-(L10) 23 、S9-(L10) 24 、S9-(L10) 25 、S9-(L10) 26 、S9-(L10) 27 、S9-(L10) 28 、S9-(L10) 29 、S9-(L10) 30This includes, for example. The bonds between S9 and L10 and / or between two L10s may be replaced with PS bonds.

[0135] In certain embodiments, the non-target regions are S9-L12, S9-(L12)2, S9-(L12)3, S9-(L12)4, S9-(L12)5, S9-(L12)6, S9-(L12)7, S9-(L12)8, S9-(L12)9, S9-(L12) 10 S9-(L12) 11 S9-(L12) 12 S9-(L12) 13 S9-(L12) 14 S9-(L12) 15 S9-(L12) 16 S9-(L12) 17 S9-(L12) 18 S9-(L12) 19 S9-(L12) 20 S9-(L12) 21 S9-(L12) 22 S9-(L12) 23 S9-(L12) 24 S9-(L12) 25 S9-(L12) 26 S9-(L12) 27 S9-(L12) 28 S9-(L12) 29 S9-(L12) 30 This includes, for example. The bonds between S9 and L12 and / or between two L12s may be replaced with PS bonds.

[0136] In certain embodiments, the non-target areas are L20-L12, L20-(L12)2, L20-(L12)3, L20-(L12)4, L20-(L12)5, L20-(L12)6, L20-(L12)7, L20-(L12)8, L20-(L12)9, L20-(L12) 10 L20-(L12) 11 L20-(L12) 12 L20-(L12) 13 L20-(L12) 14 L20-(L12) 15 L20-(L12)16 L20-(L12) 17 L20-(L12) 18 L20-(L12) 19 L20-(L12) 20 L20-(L12) 21 L20-(L12) 22 L20-(L12) 23 L20-(L12) 24 L20-(L12) 25 L20-(L12) 26 L20-(L12) 27 L20-(L12) 28 L20-(L12) 29 L20-(L12) 30 This includes, for example. The bonds between L20 and L12 and / or between two L12s may be replaced with PS bonds.

[0137] In certain embodiments, the non-target regions are L42-L12, L42-(L12)2, L42-(L12)3, L42-(L12)4, L42-(L12)5, L42-(L12)6, L42-(L12)7, L42-(L12)8, L42-(L12)9, L42-(L12) 10 L42-(L12) 11 L42-(L12) 12 L42-(L12) 13 L42-(L12) 14 L42-(L12) 15 L42-(L12) 16 L42-(L12) 17 L42-(L12) 18 L42-(L12) 19 L42-(L12) 20 L42-(L12) 21 L42-(L12) 22 L42-(L12) 23 L42-(L12) 24 L42-(L12) 25 L42-(L12) 26 L42-(L12) 27 L42-(L12) 28 L42-(L12) 29 L42-(L12)30 including etc. The bond between L42 and L12 and / or between two L12s may be replaced by a PS bond.

[0138] In certain embodiments, the non-target site is S9-(L10)2, S9-(L10)4, S9-(L10)6, S9-(L10)8, S9-(L10) 10 , S9-(L10) 12 , S9-(L10) 14 , S9-(L10) 16 , S9-(L10) 18 , S9-(L10) 20 , S9-(L10) 22 , S9-(L10)<​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​12 , or L20-(L12) 18 The linker comprises a phosphodiester bond, and at least one phosphodiester bond between two adjacent linkers may be substituted with a phosphorothioate (PS) bond, a mesylphosphoramidate bond, or a boranophosphate bond.

[0141] In certain embodiments, the non-target site is L42-(L12)6 or L42-(L12) 12 The linker comprises a phosphodiester bond, and at least one phosphodiester bond between two adjacent linkers may be substituted with a phosphorothioate (PS) bond, a mesylphosphoramidate bond, or a boranophosphate bond.

[0142] Optionally, the site may be conjugated to a target oligonucleotide via a linker or linking component. The bond between the linker and the terminal nucleotide in the target oligonucleotide may be replaced by a phosphorothioate (PS) bond. In some embodiments, the site includes one or more nucleotides in addition to one or more linkers. The one or more nucleotides may be positioned between the linkers or linked consecutively to form a nucleotide sequence. For example, the linker connecting the target oligonucleotide and the non-target site is a spacer 9 linker. The linker between the target oligonucleotide and the site may be treated as belonging to the non-target site.

[0143] In some embodiments, the site can modulate the in vivo distribution, bioavailability, and / or intracellular uptake of the oligonucleotide agent within target tissue or target cells. In some embodiments, oligonucleotide agents having the site exhibit improved efficacy, activity, pharmacokinetics, and / or pharmacodynamics throughout the central nervous system compared to oligonucleotide agents without the site. In some embodiments, the efficacy, activity, pharmacokinetics, and / or pharmacodynamics of the oligonucleotide agent are improved in specific regions of the brain and spinal cord. In some embodiments, endosomal and / or lysosomal escape of the oligonucleotide agent is improved.

[0144] The site may include a series of linkers and / or nucleotides modified to further enhance its ability to deliver target oligonucleotides. In some embodiments, the site may include one or more chemically modified nucleotides, or within the site, at least one phosphodiester bond between two adjacent linkers, between two adjacent nucleotides, or between a linker and an adjacent nucleotide may be replaced with a phosphorothioate bond or a boranophosphate bond. Chemical modifications of the site include, but are not limited to, modification of the 2'-OH of the ribose in the nucleotide, modification or deletion of a base in the nucleotide, locking or bridging of nucleic acids, nucleotides that are peptide nucleic acids, nucleotides that are deoxyribonucleotides (DNA), nucleotides having a 5'-phosphate moiety, nucleotides having a 5'-(E)-vinylphosphonate moiety, nucleotides having a 5-methylcytosine moiety, and the like. Examples of chemical modifications at the site are described in further detail below.

[0145] At this site, at least one phosphodiester bond on the backbone of the nucleotide sequence may be replaced with a phosphorothioate (PS) bond. In some embodiments, the site includes multiple phosphorothioate (PS) backbone modifications, for example having at least 2, 3, 4, 5, 6, or more than 6 PS backbone modifications. In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the phosphodiester bonds on the backbone may be replaced with phosphorothioate (PS) bonds. As a non-limiting example, the site containing 14 linkers may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 PS backbone modifications. In some embodiments, the site containing m linkers and n nucleotides may include m + n - 1 PS modifications.

[0146] The site may have a specific composition of nucleotides and non-nucleotide chemical groups. Thus, one aspect of the present application relates to an oligonucleotide agent capable of inhibiting the expression of superoxide dismutase 1 (SOD1), and the oligonucleotide agent includes small interfering RNA (siRNA) and the site.

[0147] The site may have a specific composition of nucleotides and non-nucleotide chemical groups. Thus, one aspect of the present application relates to an oligonucleotide agent capable of activating the expression of SMN2, and the oligonucleotide agent includes small activating RNA (saRNA) and the site.

[0148] In some embodiments, the oligonucleotide agent includes one or more conjugate sites, thereby enhancing the in vivo distribution and intracellular uptake of the oligonucleotide agent in specific tissues, and further increasing the permeability through membranes such as the blood-brain barrier.

[0149] In some embodiments, the double-stranded RNA (dsRNA) and the site are covalently linked with or without one or more linking components to form an oligonucleotide agent.

[0150] In another aspect of the present application, an oligonucleotide agent comprising siRNA and a non-target site is provided. In some embodiments, the oligonucleotide agent comprises a non-target site linked to a double-stranded RNA (dsRNA). In some embodiments, the dsRNA is a natural nucleic acid. In some embodiments, the natural nucleic acid is a target nucleic acid. In certain embodiments, the natural nucleic acid is an intracellular nucleic acid.

[0151] In another aspect of the present application, an oligonucleotide agent comprising saRNA and a non-target site is provided. In some embodiments, the oligonucleotide agent comprises a non-target site linked to a double-stranded RNA (dsRNA). In some embodiments, the dsRNA is a natural nucleic acid. In some embodiments, the natural nucleic acid is a target nucleic acid. In certain embodiments, the natural nucleic acid is an intracellular nucleic acid.

[0152] In some embodiments, the site comprises one or more nucleotides. The nucleotides may be randomly selected. The nucleotides may be RNA, DNA, BNA, LNA, PNA, or a combination thereof.

[0153] ]>In some embodiments, the site interacts with one or more of a protein within the cell membrane, a plasma protein, a peptide, a ligand, a lipid, a fatty acid, a saccharide, a proteoglycan, and zwitterionic phosphocholine. Such interaction of the site increases the in vivo distribution and enrichment of the target double-stranded oligonucleotide of the oligonucleotide agent, enabling local delivery to various target tissues and cells of interest. Furthermore, such interaction can reduce or eliminate the cytotoxicity of the oligonucleotide agent and ensure strong "on-target" activity without having an obvious effect on cell viability.

[0154] In certain embodiments, one or more proteins that interact with the site are selected from the following group: serum albumin, IgG, apolipoprotein AI, apolipoprotein A-II, complement factor C3, transferrin, α-1 antitrypsin, haptoglobin, hemopexin, fibrinogen, α-2-macroglobulin, prealbumin / TTR, antithrombin III, α-1-antichymotrypsin, β-2-glycoprotein, ceruloplasmin, α-1 acid glycoprotein, complement component C1q, complement factor C4, histidine-rich glycoprotein, plasminogen, fibronectin, ApoB100, factor H, apolipoprotein E, and factor V.

[0155] In yet another embodiment, the protein that interacts with the site is selected from ASGPR, EGFR, LDLR, M6PR, TLR, Stabilin, SRB, Nucleolin, AP2M1, EEA1, Rab5C, Rab7a, STX5, P115, COPII, M6PR, GCC2, ANXA2, TCP1, ALIX, TSG101, VPS28, GLP-1, and HSP-90.

[0156] In some embodiments, the interaction of the site is mediated by direct binding or by one or more conjugate ligands, the ligands being covalently linked to the site or a double-stranded oligonucleotide, or both. In certain embodiments, the one or more conjugate ligands include lipids, fatty acids, fluorescent dyes, sugars, peptides, antibodies, and other commonly used conjugation ligands.

[0157] In certain embodiments, the conjugate ligand is selected from one or more of the following: cell-permeable peptides, polyethylene glycol, alkaloids, tryptamine, benzimidazole, quinolone, amino acids, cholesterol, glucose, and N-acetylgalactosamine. In certain embodiments, one or more conjugate ligands are fatty acids.

[0158] In some embodiments, oligonucleotide agents containing one or more conjugate ligands improve the in vivo distribution of the oligonucleotide agent in specific tissues, reduce or eliminate the cytotoxicity of the oligonucleotide agent, and increase membrane permeability and transmissibility, such as across the blood-brain barrier.

[0159] In certain embodiments, the site includes a chemically modified nucleotide sequence.

[0160] In some embodiments, the oligonucleotide agents of this application include, for example, two, three, four, five, six, seven, nine, or ten non-target sites, which are covalently conjugated to double-stranded RNA, with or without the use of one or more linkers. The number of such sites may vary as needed to 2-10, 2-100, 2-1,000, or 2-10,000, and these may be conjugated to double-stranded RNA via multivalent linkers, such as branched or linear polymer linkers. In some embodiments, multiple non-target sites are covalently linked to two or more double-stranded RNAs, for example, two, three, four, five, six, seven, nine, or ten or more double-stranded RNAs (including saRNA and / or siRNA), in a single agent.

[0161] In some embodiments, the oligonucleotide agent of this application comprises one non-target site and a plurality of double-stranded RNAs, for example, 2, 3, 4, 5, 6, 7, 9, or 10 double-stranded RNAs, which are linked to the non-target site and the double-stranded RNAs, with or without linkers. The number of double-stranded RNAs, including saRNA and / or siRNA, may vary as needed to 2-10, 2-100, 2-1,000, or 2-10,000, which may be bound to the site via polyvalent linkers, such as branched or linear polymer linkers. Target oligonucleotides

[0162] In some embodiments, the target oligonucleotide comprises a double-stranded oligonucleotide. In some embodiments, the double-stranded oligonucleotide is a double-stranded RNA (dsRNA). The dsRNA may be any dsRNA considered useful, but examples of dsRNAs found to be useful in this disclosure include, but are not limited to, siRNA and saRNA.

[0163] In some embodiments, the double-stranded oligonucleotide comprises a sense strand and an antisense strand, the antisense strand having complementarity with the target nucleic acid. In some embodiments, the antisense strand having complementarity with the target nucleic acid is located in the promoter sequence. In some embodiments, the antisense strand having complementarity with the target nucleic acid is located in the coding sequence or template sequence of the gene. In some embodiments, either the sense strand or the antisense strand has complementarity with the target nucleic acid, which is a gene transcript, e.g., mRNA or premRNA.

[0164] In some embodiments, the dsRNA includes a sense strand consisting of at least 17 consecutive nucleotides. In some embodiments, the dsRNA includes a sense strand consisting of at least 18 consecutive nucleotides. In some embodiments, the dsRNA includes a sense strand consisting of up to 60 consecutive nucleotides.

[0165] In some embodiments, the sense strand and antisense strand each independently have lengths of about 10 nucleotides or more, about 15 nucleotides or more, about 20 nucleotides or more, about 25 nucleotides or more, about 30 nucleotides or more, about 35 nucleotides or more, about 40 nucleotides or more, about 45 nucleotides or more, about 50 nucleotides or more, about 55 nucleotides or more, or about 60 nucleotides or more. In some embodiments, the sense strand and antisense strand each independently have lengths of 10 to 100 nucleotides, for example, 10 to 20 nucleotides, 10 to 50 nucleotides, 10 to 90 nucleotides, 20 to 95 nucleotides, 30 to 70 nucleotides, 40 to 80 nucleotides, 50 to 100 nucleotides, 10 to 40 nucleotides, or 10 to 30 nucleotides. In some embodiments, the sense strand is 10 to 60 nucleotides long (for example, 10 to 20 nucleotides, 10 to 50 nucleotides, 10 to 40 nucleotides, or 10 to 30 nucleotides). In some embodiments, the sense strand has a nucleotide length in the range of 27 to 41 nucleotides. In some embodiments, the antisense strand is 19 to 30 nucleotides long. In some embodiments, the antisense strand is 18 to 26 nucleotides long.

[0166] Double-stranded oligonucleotides may include modified sequences to further increase their ability to regulate stability and / or gene expression. In some embodiments, the sequences of double-stranded oligonucleotides include one or more chemically modified nucleotides, or at least one phosphodiester bond between two adjacent nucleotides in the oligonucleotide sequence being replaced with a phosphorothioate bond or a boranophosphate bond. Chemical modifications of double-stranded oligonucleotides include, but are not limited to, modifications of the 2'-OH of the ribose in the nucleotide, modifications or absence of bases in the nucleotide, lock nucleic acids or crosslinking nuclei, nucleotides that are peptide nucleic acids, nucleotides that are deoxyribonucleotides (DNA), nucleotides having a 5'-phosphate moiety, nucleotides having a 5'-(E)-vinylphosphonate moiety, nucleotides having a 5'-methylcytosine moiety, and the like.

[0167] Exemplary short interfering RNA (siRNA) Embodiments of this application are based on the disclosure that oligonucleotide agents (e.g., siRNA, also referred herein as “SOD1 gene siRNA,” “SOD1 siRNA,” or “siSOD1”) can inhibit or downregulate the expression of the SOD1 gene in cells. A reduction in functional SOD1 gene transcript after administration of the oligonucleotide agents of this application can achieve a significant reduction or downregulation of SOD1 mRNA and SOD1 protein levels in mammalian cells or the central nervous system.

[0168] In particular, the inventors have found a functional oligonucleotide agent capable of inhibiting the expression of superoxide dismutase 1 (SOD1) consisting of siRNA. Here, the siRNA includes a sense strand and an antisense strand that form a double strand, and the antisense strand has at least 85% nucleotide sequence complementarity or homology to a part of the nucleotide sequence of SOD1 mRNA, and consists of at least 10 consecutive nucleotides having 0, 1, 2 or 3 mismatches.

[0169] In some embodiments, the difference or mismatch is located in the middle or 3' end of the oligonucleotide sequence of the siRNA. The methods and principles of siRNA molecular design are well known to those skilled in the art. For example, Place et. al., Molecular Therapy-Nucleic Acids (2012) 1, e15; and Li et.al., PNAS, 2006, vol. 103, no. 46, 17337-17342, are hereby incorporated by reference in their entirety.

[0170] As a beneficial result, target sequences (e.g., isolated nucleic acid sequences containing the target sequence), when interacting with siRNA, can inhibit / downregulate SOD1 mRNA transcripts by at least 10% compared to baseline levels of SOD1 mRNA. Based at least in part on these findings, this application features siRNA, compositions, and pharmaceutical compositions for inhibiting / downregulating SOD1 mRNA transcripts by at least 10% compared to baseline levels of SOD1 mRNA. In some embodiments, the siRNA inhibits or downregulates SOD1 mRNA by 10% or more. For example, siRNA inhibits or downregulates SOD 1 mRNA by at least approximately 15%, at least approximately 20%, at least approximately 25%, at least approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 100%, or more than 100% compared to baseline levels of SOD 1 mRNA.

[0171] Furthermore, this specification also provides a method for preventing or treating a disease or condition induced in an individual by overexpression of the SOD1 protein, mutation in the SOD1 gene, and / or high or abnormal SOD1 levels, the method comprising administering any of the siRNAs, compositions, and / or pharmaceutical compositions described herein to the individual.

[0172] In some embodiments, the antisense strands disclosed herein are capable of interacting with the target nucleic acid sequence of the SOD1 gene mRNA in a sequence-specific manner, meaning that the antisense strands are capable of hybridizing with the target nucleic acid via hydrogen bonding. In some embodiments, the antisense strand has a nucleotide sequence that, when described in the 5' to 3' direction, constitutes the reverse complement of the target portion of the target nucleic acid to which it is targeted. In certain such embodiments, the antisense strand has a nucleotide sequence that, when described in the 5' to 3' direction, constitutes the reverse complement of the target portion in a fragment of the SOD1 gene transcript.

[0173] Examples of small activated RNAs (saRNAs) Embodiments of this application are based on the disclosure that oligonucleotide agents (e.g., saRNA, also referred herein as “SMN2 gene saRNA,” “SMN2 saRNA,” or “saSMN2”) can activate or upregulate the expression of the SMN2 gene within cells. A decrease in functional SMN2 gene transcripts following administration of the oligonucleotide agents of this application may achieve a significant increase or upregulation of SMN2 mRNA and SMN2 protein levels in mammalian cells or the central nervous system.

[0174] In particular, the present inventors disclose a functional oligonucleotide agent capable of activating SMN2 expression, the oligonucleotide agent comprising saRNA, the saRNA comprising a sense strand and an antisense strand forming a double helix, the antisense strand comprising a nucleotide sequence containing at least 10 consecutive nucleotides, having 0, 1, 2, or 3 mismatches, and having at least 85% nucleotide sequence complementarity or homology with a portion of the nucleotide sequence of SMN2 mRNA.

[0175] In some embodiments, the difference or mismatch is located in the middle or at the 3' end of the oligonucleotide sequence of the saRNA. Methods and principles of saRNA molecular design are well known to those skilled in the art, for example, Place et. al., Molecular Therapy-Nucleic Acids (2012) 1, e15; and Li et. al., PNAS, 2006, vol. 103, no. 46, 17337-17342, which are incorporated herein by reference in their entirety.

[0176] chemical modification All nucleotides contained in the oligonucleotides or non-target sites (if any) described herein may be natural, i.e., unmodified nucleotides, or at least one nucleotide may be a chemically modified nucleotide. Non-limiting examples of chemical modifications include one or more combinations of: a) modification of phosphodiester bonds between two nucleotides, between two linkers, or between nucleotides and linkers in an oligonucleotide; b) modification of the 2'-OH of ribose in a nucleotide; c) modification of a base in a nucleotide; d) at least one nucleotide in the oligonucleotide sequence is a locked nucleotide; e) at least one nucleotide in the oligonucleotide sequence is a deoxyribonucleotide (DNA).

[0177] In some embodiments, the nucleotides or oligonucleotides of this application are chemically modified to enhance stability or other beneficial properties. Nucleic acids characterized in this application include those from conventional methods, such as "Current protocols in nucleic acid chemistry," Beaucage, SL et al. (Edrs.), John Wiley & Sons, Inc., which is incorporated herein by reference. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, Modifications include, for example, (a) terminal modifications (e.g., 5' terminal modifications [phosphorylation, conjugation, inversion bonding, etc.], 3' terminal modifications [conjugation, DNA nucleotides, inversion bonding, etc.]), (b) base modifications (e.g., stabilization bases, destabilization bases, or substitutions to bases that form base pairs with extended complementary partners, base removal [abasic nucleotides], or conjugate bases), (c) sugar modifications (e.g., modifications at the 2' or 4' position) or sugar substitutions, and (d) skeletal modifications (including modifications or substitutions of phosphodiester bonds). Specific examples of siRNA molecules that can be used in this application include, but are not limited to, RNA with a modified skeleton or RNA without natural nucleoside-to-nucleoside bonds. In some embodiments, RNA with a modified skeleton includes, among other things, RNA without phosphorus atoms in the skeleton. In some embodiments, modified RNA without a phosphorus atom in its internucleoside backbone may also be an oligonucleoside. In some embodiments, the modified oligonucleotide will have a phosphorus atom in its internucleoside backbone.

[0178] The chemical modifications of nucleotides or linkers in this disclosure are well known to those skilled in the art, and modifications of phosphodiester bonds refer to modifications of oxygen in the phosphodiester bond, including phosphorothioate modifications and boronated phosphate modifications. The modifications disclosed herein stabilize the oligonucleotide structure and maintain high specificity and high affinity for base pairing. The modifications disclosed herein also stabilize the structure of non-target sites and maintain delivery support properties, including bioavailability, in vivo distribution and / or intracellular uptake, of oligonucleotide agents in various tissues (prefrontal cortex, cerebellum, spinal cord [e.g., cervical, thoracic, and lumbar vertebrae], muscle, liver, and kidney).

[0179] In some embodiments, the oligonucleotide agent disclosed herein is a chemical modification in which a phosphodiester bond is replaced with a phosphorothioate (PS) bond in the backbone. In some embodiments, the oligonucleotide agent disclosed herein includes at least one PS backbone modification. In some embodiments, the non-target site includes at least one PS backbone modification. In some embodiments, the oligonucleotide agent includes at least two PS, at least three PS, at least four PS, at least five PS, at least six PS, or more than six PS backbone modifications. In some embodiments, approximately 90% to 95% of the phosphodiester backbone bonds in the non-target site are replaced with phosphorothioate (PS) bonds. In some embodiments, the oligonucleotide agent includes at least one PS backbone modification at the 5' end, 3' end, or internal site of the sense strand of dsRNA. In some embodiments, the oligonucleotide agent includes at least one PS backbone modification on the 5' end, 3' end, or internal site of the antisense strand of dsRNA. In some embodiments, the oligonucleotide agent includes at least one PS backbone modification at the 5' end, 3' end, or internal site of the non-target site.

[0180] In some embodiments, the nucleotide or oligonucleotide in this application comprises at least one chemically modified nucleotide modified with a 2'-OH group of the pentose of the nucleotide, for example, 2'-fluoro modification, 2'-oxymethyl modification, 2'-oxyethylidene methoxy modification, 2,4'-dinitrophenol modification, loc nucleic acid (LNA), 2'-amino modification, or 2'-deoxy modification, for example, 2'-deoxy-2'-fluoro modified nucleotide, 2'-deoxy modified nucleotide.

[0181] In some embodiments, the nucleotides or oligonucleotides in this application include at least one chemically modified nucleotide modified with a base of the nucleotide, for example, 5'-bromouracil modification, 5'-iodouracil modification, N-methyluracil modification, or 2,6-diaminopurine modification.

[0182] In some embodiments, the chemical modification of a nucleotide or oligonucleotide in this application is the addition of an (E)-vinylphosphonate moiety at the 5' end of a sense or antisense sequence. In some embodiments, the chemical modification of at least one chemically modified nucleotide is the addition of a 5'-methylcytosine moiety at the 5' end of a sense or antisense sequence.

[0183] In some embodiments, the nucleotides or oligonucleotides in this application are modified with a base, for example, 5'-bromouracil modification, 5'-iodouracil modification, N-methyluracil modification, or 2,6-diaminopurine modification. In some embodiments, at least one oligonucleotide in the oligonucleotide preparation includes at least one modified nucleotide, for example, a 2'-O-methyl modified nucleotide, a nucleotide containing a 5'-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a basic nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, and a non-natural base consisting of a nucleotide. In some embodiments, the first and second dsRNAs include "endo-light" modifications with a 2'-O-methyl modified nucleotide and a nucleotide containing a 5'-phosphorothioate group.

[0184] Modified skeletons include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramides and aminoalkyl phosphoramidates, thionophosphoramides, thioalkyl phosphonates, thioalkyl phosphotriesters, and boranophosphates having the usual 3'-5' bond, their 2'-5' bonded analogues, and those with reverse polarity where adjacent pairs of nucleoside units are bonded from 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0185] Non-limiting examples of the preparation of phosphorus-containing conjugates include, but are not limited to, the following: US 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590, 6,534,639, 6,608,035, 6,683,167, 6,858,715, 6,867,294, 6,878,805, 7,015,315, 7,041,816, 7,273,933, 7,321,029, and U.S. Patent RE39464, which are incorporated in their entirety by reference, respectively, in particular herein.

[0186] In some embodiments, the nucleotide or oligonucleotide comprises one or more RNA, DNA, BNA, LNA, or peptide nucleic acid (PNA).

[0187] In other RNA mimics suitable for or intended for use with siRNA, both the sugar and nucleoside bonds of the nucleotide unit, i.e., the backbone, are replaced with novel groups. The base unit is maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, an RNA mimic that has been shown to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobase is retained and is directly or indirectly bound to the aza nitrogen atom of the amide moiety, or to the S or O atom in the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patents 5,539,082; 5,714,331; and 5,719,262, which are incorporated herein by reference, respectively. Further teachings on PNA compounds can be found, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0188] In some embodiments, if nucleotides at non-target sites are present, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% are chemically modified nucleotides.

[0189] In some embodiments, the sense and antisense strands of the oligonucleotide agent independently contain at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of nucleotides that are chemically modified nucleotides.

[0190] These modifications can increase the bioavailability of oligonucleotides, their affinity for target sequences, and enhance their resistance to intracellular nuclease hydrolysis.

[0191] Furthermore, to facilitate the entry of oligonucleotides into cells, based on the above modifications, lipophilic groups such as cholesterol can be introduced to the ends of the sense or antisense strands of the oligonucleotides to facilitate their action through the cell membrane, which consists of lipid bilayers, the nuclear membrane, and gene promoter regions within the nucleus.

[0192] In some embodiments, the oligonucleotide agents disclosed herein have the effect of inactivating or downregulating the expression of one or more genes within a cell upon contact with the cell, preferably in an amount of at least 10% (e.g., at least 15%, at least 20%, at least 25%, 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%, at least 90%, or at least 95%).

[0193] In some embodiments, the oligonucleotide agents disclosed herein have the effect of activating or upregulating the expression of one or more genes within a cell upon contact with the cell, preferably in an amount of at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 500%, at least 800%, at least 1000%, at least 2000%, or at least 5000%).

[0194] One aspect of this application provides cells comprising the oligonucleotide agent of this application or nucleic acids encoding the oligonucleotide agent of this application. In one embodiment, the cells are mammalian cells, preferably human cells. Such cells may be extra vivo cells such as cell lines or cells present in the body of a mammal such as a human, including an infant, child or adult.

[0195] In some embodiments, at least one chemically modified oligonucleotide is present at the non-target site. In some embodiments, at least one chemically modified oligonucleotide is present at the target double-stranded oligonucleotide. In certain embodiments, at least one chemically modified oligonucleotide is present at both the non-target site and the target double-stranded oligonucleotide.

[0196] Covalent linking or conjugation One aspect disclosed herein relates to an oligonucleotide agent comprising a covalently linked target double-stranded oligonucleotide and a non-target site.

[0197] In some embodiments, the target double-stranded oligonucleotide and the non-target site are covalently linked by a linking component.

[0198] In some embodiments, a double-stranded oligonucleotide and a non-target site are linked by a covalent linker. In some embodiments, the linker is a disulfide linker. Various combinations of strands can be linked. For example, the dsRNA sense strand and the non-target site may be covalently linked, or the dsRNA antisense strand and the non-target site may be covalently linked.

[0199] In some embodiments, the sense strand of the double-stranded target oligonucleotide is covalently conjugated to a non-target site. In some embodiments, the antisense strand of the double-stranded target oligonucleotide is covalently conjugated to a non-target site.

[0200] In some embodiments, either the oligonucleotide or the linker in the oligonucleotide agent of this application includes a linking component.

[0201] Linkers are typically formed by direct bonds, or by atoms such as oxygen or sulfur, NR 1 , C(O), C(O)O, C(O)NR 1Units such as SO, SO2, SO2NH, or substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylal Nyl, alkenyl heteroarylalkynyl, alkynyl heteroarylalkyl, alkynyl heteroarylalkenyl, alkynyl heteroarylalkynyl, alkyl heterocyclylalkyl, alkyl heterocyclylalkenyl, alkyl heterocyclylalkynyl, alkenyl heterocyclylalkyl, alkenyl heterocyclylalkenyl, alkenyl heterocyclylalkynyl, alkynyl heterocyclylalkyl, alkynyl heterocyclylalkenyl, alkynyl heterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkyl heteroaryl, alkenyl heteroaryl, alkynyl heteroaryl, where one or more methylene groups are O, S, S(O), SO2, N(R')2, C(O), cleavable linking groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, substituted or unsubstituted heterocycles; where R1 is hydrogen, acyl, aliphatic or substituted aliphatic. Each R' is independently selected from hydrogen, substituted or unsubstituted alkyl, aryl, aralkyl, alkylaryl, alkoxy, allyloxy, acyl, or aliphatic group. These groups may be linear, branched, cyclic, polycyclic, unsaturated, hydroxylated, carbonylated, phosphorylated, and / or sulfurized.

[0202] The subject's combination may include, but is not limited to, various types of linker functionality, including, severable and non-severable linkers, as well as reversible and irreversible linkers.

[0203] In some embodiments, the linker is a cleavable linker. A cleavable linker is one that, depending on processes within the target cell, releases two bound parts, such as a non-target site and the dsRNA, such as reduction in the cytoplasm, exposure to acidic conditions in lysosomes or endosomes, or cleavage by specific intracellular enzymes (e.g., proteases). Thus, a cleavable linker allows the dsRNA to be released in its original form after the conjugate has been internalized and processed within the target cell. Cleavable linkers include, but are not limited to, those whose binding is enzymatically cleavable (e.g., peptide linkers), those whose binding is cleavable under reducing conditions (e.g., disulfide linkers), and those whose binding is cleavable under acidic conditions (e.g., hydrazones and carbonates).

[0204] In some embodiments, the linking component is selected from one or more of the following: ethylene glycol chains, alkyl chains, peptides, nucleic acids, carbohydrates, thiol linkages, phosphodiesters, phosphorothioates, phosphoramidates, amides, and carbamates. In some embodiments, the linking component includes, but is not limited to, the following: a) L1 or S18 (Spacer-18 Linker) (1,1-Bis(4-methoxyphenyl)-1-phenyl-2,5,8,11,14,17-Hexaoxanonadecan-19-yl(2-cyanoethyl)diisopropylphosphorumidite); b) L4 or C6 (spacer-C6 linker) (6-(bis(4-methoxyphenyl)(phenyl)methoxy)hexyl(2-cyanoethyl)diisopropylphosphorumidite); c) L6(1,1-bis(4-methoxyphenyl)-1-phenyl-2,5,8,11,14-pentaoxahexadecane-16-yl (2-cyanoethyl)diisopropylphosphorumidite); d) L9 or S9 (Spacer-9 Linker) (2- (2- (2- (Bis(4-methoxyphenyl)(phenyl)methoxy)ethoxy)ethoxy)ethyl(2-cyanoethyl)diisopropylphosphorumidite); e) L10 or C3 (spacer-C3 linker) (3-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl(2-cyanoethyl)diisopropylphosphorumidite); f) L12(d spacer) ((2R, 3S)-2- ((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphorumidite); g) L13 or C12 (spacer-C12 linker) (12- (bis(4-methoxyphenyl)(phenyl)methoxy)dodecyl(2-cyanoethyl)diisopropylphosphorumidite); h) L14 (Spacer-L14 Linker) (((1r, 4r)-4- ((Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)cyclohexyl)methyl(d-cyanoethyl)diisopropylphosphorumidite); i) L15 (Spacer-L15 Linker) (4-(2-(Bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)phenethyl(2-cyanoethyl)diisopropylphosphorumidite); j) L16 (Spacer-L16 Linker) (2- (1- (2- (Bis(4-Methoxyphenyl)(Phenyl) Methoxy) Ethyl) Cyclohexyl) Ethyl (2-Cyanoethyl) Diisopropyl Phosphoramidite) k) C6x1((2S, 3S, 4S, 5S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-methoxy-4-(pento-4-in-1-yloxy)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphorumidite); i) C6x2((2S, 3S, 4S, 5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2-methoxy-4-(pento-4-in-1-yloxy)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphorumidite); m) C6x5(2- ((2- (bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(pento-4-in-1-yl)amino)ethyl(2-cyanoethyl)diisopropylphosphorumidite); and n) C6x7((9H-Fluoren-9-yl)methyl(4-((2S,4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((bis(diisopropylamino)phosphanyl)oxy)pyrrolidine-1-yl)-4-oxobutyl)carbamate). o) L20 methyl 1-(5-(bis(4-methoxyphenyl)(phenyl)methoxy)pentyl)-2-(4-(((2-cyanoethoxy)(diisopropylamino)phosphanyl)oxy)butyl)-1H-benzo[d]imidazole-5-carboxylate; and p) L42 6-((6-(bis(4-methoxyphenyl)(phenyl)methoxy)hexyl)disulfanyl)hexyl(2-cyanoethyl)diisopropylphosphorumidite.

[0205] In certain embodiments, the linking component comprises the compound structure shown in Table 1. In certain embodiments, one or more linking components are linked in series to constitute a non-target site. In certain embodiments, the linking component is conjugated to a nucleotide in a non-target site or a double-stranded oligonucleotide. In certain embodiments, the linking component is bound to a nucleoside position selected from 5'-phosphate, 3'-base, and 2'-H / OH of a nucleotide in a single-stranded or double-stranded oligonucleotide. In certain embodiments, the linking component is spacer phosphoramidite 18 (phosphoramidic acid, N,N-bis(1-methylethyl)-,19,19-bis(4-methoxyphenyl)-19-phenyl-3,6,9,12,15,18-hexaoxanonadecyl-1-yl 2-cyanoethyl ester). [Table 1] JPEG2026514403000005.jpg248170JPEG2026514403000006.jpg248170JPEG2026514403000007.jpg248170

[0206] In some embodiments, the double-stranded target oligonucleotide and the non-target site are covalently linked by a phosphodiester bond. In some embodiments, the double-stranded target oligonucleotide and the non-target site are covalently linked by a phosphorothioate (PS) bond.

[0207] In some embodiments, the double-stranded target oligonucleotide includes a sense strand covalently bonded to a non-target site. In some embodiments, the double-stranded target oligonucleotide includes an antisense strand covalently bonded to a non-target site.

[0208] In some embodiments, the double-stranded target oligonucleotide and the non-target site are covalently linked via one or more nucleotides.

[0209] In some embodiments, the double-stranded target oligonucleotide and the non-target site are covalently linked via one or more linkers.

[0210] Non-limiting examples of covalent linkers are described in U.S. Patent Publication No. 20200332292, which is incorporated herein by reference in its entirety. Covalent linkers can link double-stranded target oligonucleotides to non-target sites. In some embodiments, covalent linkers can link two sense strands, two antisense strands, one sense strand and one antisense strand, two sense strands and one antisense strand, two antisense strands and one sense strand, two sense strands and two antisense strands, an antisense strand and a single-stranded oligonucleotide, a sense strand and an inactivated oligonucleotide, and the like.

[0211] In certain embodiments, the covalent linker comprises nucleic acids (e.g., RNA and / or DNA) and / or peptides. The linker may be single-stranded, double-stranded, partially single-stranded, or partially double-stranded. In some embodiments, the linker comprises disulfide bonds. The linker may be cleavable or non-cleavable.

[0212] In certain embodiments, the covalent linker includes: dTsdTuu=(5′-2′deoxythymidyl-3′-thiophosphate-5′-2′deoxythymidyl-3′-phosphate-5′-uridyl-3′-phosphate-5′-uridyl-3′-phosphate);rUsrU(thiophosphate linker:5′-uridyl-3′-thiophosphate-5′-uridyl-3′-phosphate);rUrU linker;dTsdTaa(aadTsdT, 5′-2′deoxythymidyl-3′-thiophosphate-5′-2′deoxythymidyl-3′-phosphate-5′-adenyl-3′-phosphate-5′-adenyl-3′-phosphate);dTsdT(5′-2′deoxythymidyl-3′-thiophosphate-5′-2′deoxythymidyl-3′-phosphate);or dTsdTuu=uudTsdT=5′-2′deoxythymidyl-3′-thiophosphate-5′-2′deoxythymidyl-3′-phosphate-5′-uridyl-3′-phosphate-5′-uridyl-3′-phosphate.

[0213] If the covalent linker is RNA, the RNA linker can consist of any combination of nucleotides. The combination of nucleotides may be adenine, uracil, guanosine, cytosine, or any combination thereof. The RNA linker may be of any length. In some embodiments, the RNA linker is 2 to 50 nucleotides long. If the RNA linker is 2 to 50 nucleotides long, the RNA linker may have any intervening length, such as 5 to 10, 10 to 15, or 15 to 20 nucleotides long. In some embodiments, the covalent linker is polyRNA such as poly(5′-adenyl-3′-phosphate-AAAAAAA) or poly(5′-cytidyl-3′-phosphate-5′-uridyl-3′-phosphate-CUCUCU)), e.g., X nIt includes a single-stranded polyRNA linker, where n is an integer from 2 to 50, preferably an integer from 4 to 15, and most preferably an integer from 7 to 8. Modified nucleotides or mixtures of nucleotides may also be present in the polyRNA linker. If the covalent linker is DNA, the DNA linker may consist of any combination of nucleotides. The combination of nucleotides may be adenine, thymine, guanosine, cytosine, or any combination thereof. The DNA linker may be of any length. In some embodiments, the DNA linker is 1 to 50 nucleotides long. If the DNA linker is 1 to 50 nucleotides long, the DNA linker may have any intervening length, such as 5 to 10, 10 to 15, or 15 to 20 nucleotides long. The covalent linker may be polyDNA, e.g., poly(5′-2′deoxythymidyl-3′-phosphate-TTTTTTTT), where n is an integer from 2 to 50, preferably an integer from 4 to 15, and most preferably an integer from 7 to 8. Modified nucleotides or mixtures of nucleotides may also be present in the polyDNA linker. A single-stranded polyDNA linker where n is an integer from 2 to 50, preferably an integer from 4 to 15, and most preferably an integer from 7 to 8. Modified nucleotides or mixtures of nucleotides may also be present in the polyDNA linker.

[0214] In some embodiments, the covalent linker comprises a disulfide bond, optionally a bis-hexyl-disulfide linker. In one embodiment, the disulfide linker is [ka]

[0215] In some embodiments, the covalent linker includes a peptide bond, for example, an amino acid. In one embodiment, the covalent linker is a 1-10 amino acid long linker, preferably consisting of 4-5 amino acids, and optionally X-Gly-Phe-Gly-Y, where X and Y represent any amino acids.

[0216] In some embodiments, the covalent linker includes a hexaethylene glycol linker, HEG.

[0217] Orientation and position of covalent bonds One aspect of this application relates to forming an oligonucleotide agent by covalently linking a double-stranded target oligonucleotide with a non-target site. In some embodiments, the orientation of the linkage and the positional relationship between the double-stranded target oligonucleotide and the non-target site can improve stability, oligonucleotide activity, or other beneficial properties (e.g., maximization of target gene expression, or increase or decrease in mRNA or protein expression levels in one or more target genes).

[0218] In some embodiments, the non-target site is covalently linked to one of the following: a) the 3′ end of the sense or antisense strand of the double-stranded target oligonucleotide; b) the 5′ end of the sense or antisense strand of the double-stranded target oligonucleotide; or c) an internal nucleotide between the 5′ and 3′ ends of the sense or antisense strand of the double-stranded target oligonucleotide. In some embodiments, the internal nucleotide of the sense or antisense strand of the double-stranded target oligonucleotide is located at the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, or 13th nucleotide position from the 5′ end of the sense or antisense strand; or it is located at the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, or 13th nucleotide position from the 3′ end of the sense or antisense strand.

[0219] In some embodiments, the internal nucleotides of the sense or antisense strand of the double-stranded target oligonucleotide are substituted with one or more conjugating components or spacers, which are covalently bonded to a non-target moiety at their 5' or 3' end (i.e., internally conjugated). In some embodiments, internally conjugated cODVs have enhanced potency compared to 3' or 5' end conjugated cODVs (i.e., cODVs in which the non-target site is conjugated to the 3' or 5' end of the sense or antisense strand of the double-stranded target oligonucleotide).

[0220] In certain embodiments, the 5' end of the single-stranded oligonucleotide is bound to the linking component. In some embodiments, the 3' end of the single-stranded oligonucleotide is bound to the linking component.

[0221] In certain embodiments, the linking component or spacer comprises the compounds shown in Table 1. Agents that reduce / upregulate the expression of target genes or proteins.

[0222] Double-stranded oligonucleotides can be designed for target genes associated with various diseases and disorders. In some embodiments, oligonucleotide agents reduce the expression of the SOD1 gene or SOD1 protein. Administration of oligonucleotide agents to patients can treat or delay the onset of ALS, such as familial ALS, sporadic ALS, or Lieutenant Lou Gehrig's disease.

[0223] Double-stranded oligonucleotides can be designed for target genes associated with various diseases and disorders. In some embodiments, oligonucleotide agents increase the expression of the SMN2 gene or SMN2 protein. Administering oligonucleotide agents to patients can treat or delay the progression or severity of spinal muscular atrophy (SMA).

[0224] Administration may be carried out via any route of administration deemed useful. In some embodiments, the route of administration is local to a site in the central nervous system. In some embodiments, the route of administration is systemic.

[0225] In certain embodiments, the double-stranded target oligonucleotide of an oligonucleotide agent that reduces the expression of a target gene or protein is an siRNA. The siRNA inactivates or downregulates the expression of a target gene, its mRNA transcript, or protein in cells where the target gene, its mRNA transcript, or protein is abnormally or excessively expressed.

[0226] In certain embodiments, the double-stranded target oligonucleotide in an oligonucleotide agent that increases the expression of a target gene or protein is a saRNA. The saRNA activates or increases the expression of the target gene, its mRNA transcript, or protein in cells where the gene is abnormally or underexpressed.

[0227] In specific embodiments of this application, the siRNA comprises a sense nucleic acid strand and an antisense nucleic acid strand, wherein the sense nucleic acid strand comprises at least one region complementary to at least one region on the antisense nucleic acid strand, forming a double-stranded nucleic acid structure capable of inactivating protein expression in a cell.

[0228] In specific embodiments of this application, the saRNA comprises a sense nucleic acid strand and an antisense nucleic acid strand, wherein the sense nucleic acid strand comprises at least one region complementary to at least one region on the antisense nucleic acid strand, forming a double-stranded nucleic acid structure capable of activating protein expression in a cell.

[0229] In a particular embodiment of this application, the sense nucleic acid strand and the antisense nucleic acid strand are located on two different nucleic acid strands. In a particular embodiment of this application, the sense nucleic acid fragment and the antisense nucleic acid fragment are located on the same nucleic acid strand, forming a hairpin-type single-stranded nucleic acid molecule, and the complementary regions of the sense nucleic acid fragment and the antisense nucleic acid fragment form a double-stranded nucleic acid structure.

[0230] In some embodiments, the oligonucleotide agent comprises a nucleotide sequence of an antisense strand having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identity with SEQ ID NO: 4, which is complementary to a sense strand fragment having a cODV structure of any of SEQ ID NO: 6 to 39.

[0231] In some embodiments, the oligonucleotide agent comprises an antisense strand nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identity with SEQ ID NO: 57 that is complementary to a sense strand fragment having any of the cODV structures of SEQ ID NO: 60.

[0232] In some embodiments, the oligonucleotide agent comprises an antisense strand nucleotide sequence having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identity with SEQ ID NO: 62, which is complementary to a sense strand fragment having any of the cODV structures of SEQ ID NO: 64.

[0233] In some embodiments, the oligonucleotide agent comprises a nucleotide sequence of an antisense strand having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identity with SEQ ID NO: 4, which is complementary to a sense strand fragment having a cODV structure of SEQ ID NO: 39, 65, 66, and 67.

[0234] In some embodiments, the oligonucleotide agent comprises a nucleotide sequence of an antisense strand having at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identity with SEQ ID NO: 69, which is complementary to a sense strand fragment having a cODV structure of any of SEQ ID NO: 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, and 87.

[0235] In some embodiments, the siRNA includes a sense strand nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to a nucleotide sequence selected from SEQ ID NO: 1, 3, 56, and 61. In some embodiments, the siRNA includes an antisense strand nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to a nucleotide sequence selected from SEQ ID NO: 2, 4, 57, and 62.

[0236] Furthermore, in order to facilitate the introduction of siRNA into cells, chemical conjugation groups other than the non-target sites disclosed herein may be introduced to the terminus of the sense or antisense strand of the siRNA based on the above modifications, thereby promoting its action via the mRNA region in the cell membrane, nuclear membrane, and nucleus, which are composed of lipid bilayers.

[0237] Furthermore, in order to facilitate the introduction of saRNA into cells, chemical conjugation groups other than the non-target sites disclosed herein may be introduced to the ends of the sense or antisense strands of saRNA based on the above modifications, thereby promoting action via mRNA regions in the cell membrane, nuclear membrane, and nucleus, which are composed of lipid bilayers.

[0238] In some embodiments, the siRNAs disclosed herein are covalently bound to one or more conjugation groups. In some embodiments, the conjugation groups modify one or more properties of the conjugated oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, intracellular uptake, charge, and clearance. In some embodiments, the conjugation groups impart novel properties to the conjugated oligonucleotide, such as a fluorescent dye or reporter group that enables detection of the oligonucleotide.Specific conjugation groups and conjugate moieties include, for example, the cholesterol moiety (Letsinger et al., ProcNatl.Acad.Sci.USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg.Med.Chem.Lett., 1994, 4, 1053-1060), thioethers, for example, hexyl-S-tritylthiol (Manoharan et al., Ann.NYAcad.Sci., 1992, 660, 306-309; Manoharan et al., Bioorg.Med.Chem.Lett., 1993, 3, 2765-2770), and thiocholesterol (Oberhauser et al., Nucl.Acids Res., 1992, 20, 533-538), aliphatic chains, e.g., dodecane-diol or undecyl residues (Saison-Behmoaras et al., EMBO 1, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), adamantane acetate palmityl moiety (Mishra et al., Biochim.Biophys.Acta, 1995, 1264, 229-237), octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., 1996, 277, 923-937), tocopherol group (Nishina et al., Molecular Therapy Nucleic Acids, 2015, 4, e220; and Nishina et al., Molecular Therapy, 2008, 16, 734-740), or GalNAc cluster (e.g., WO2014 / 179620).

[0239] In some embodiments, the siRNA / saRNA of this application relates to a sense or antisense strand of siRNA conjugated to one or more conjugation groups selected from: intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates, vitamin moieties, polyethylene glycol, thioethers, polyethers, cholesterol, thiocholesterol, cholic acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, fluorescent dyes, and dyes.

[0240] In some embodiments, the conjugation group is, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansyl sarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, benzothiazide, chlorothiazide, diazepine, indomethicine, barbiturates, cephalosporins, sulfonamides, antidiabetic drugs, antibacterial agents or antibiotics.

[0241] In some embodiments, the siRNA / saRNA of this application is conjugated to one or more conjugation groups selected from lipids, fatty acids, fluorescent dyes, ligands, sugars, peptides, and antibodies.

[0242] In some embodiments, the siRNA / saRNA of this application relates to a sense or antisense strand of siRNA / saRNA conjugated to one or more conjugation groups selected from cell-permeable peptides, polyethylene glycol, alkaloids, tryptamine, benzimidazole, quinolone, amino acid, cholesterol, glucose, and N-acetylgalactosamine.

[0243] In some embodiments, siRNA / saRNA conjugated to one or more conjugation groups disclosed in the embodiments is directly contacted, transported, delivered, or administered to cells or subjects. Methods for regulating gene expression

[0244] In some embodiments, the oligonucleotide agents of this application are useful in therapeutic approaches for treating diseases such as spinal muscular atrophy (SMA) or ALS.

[0245] In non-limiting embodiments, this application provides a method for reducing or silencing levels of mRNA transcripts of the SOD1 gene or SOD1 protein in cells or organisms, comprising administering a pharmaceutical composition disclosed herein to a subject.

[0246] In some embodiments, non-target sites on oligonucleotide agents improve the stability, bioavailability, in vivo distribution, and / or intracellular uptake of siRNA / saRNA compared to oligonucleotide agents without non-target sites. In some embodiments, non-target sites on oligonucleotide agents increase the in vivo distribution of siRNA / saRNA in one or more target tissues compared to oligonucleotide agents without non-target sites. In some embodiments, non-target sites on oligonucleotide agents increase the in vivo distribution of siRNA / saRNA in two or more target tissues compared to oligonucleotide agents without non-target sites.

[0247] In some embodiments, one or more target tissues are selected from the tissues of the brain, spinal cord, muscles, spleen, lungs, heart, liver, bladder, and kidneys. In some embodiments, one or more target tissues are selected from the prefrontal cortex, cerebellum, cerebrum; cervical, thoracic, and lumbar vertebrae of the spinal cord; heart, biceps brachii, semitendinosus biceps femoris, platysma, and gluteus maximus.

[0248] In some embodiments, the oligonucleotide agents of this application achieve a reduction in full-length protein of less than that achieved by administering the same amount of double-stranded oligonucleotides, such as siRNA substances that do not have an ODV structure, used individually, with higher potency, reduced toxicity, or undesirable side effects. In some embodiments, the oligonucleotide agents of this application achieve a reduction in full-length protein of less than the additive effect of treatment with the same amount of double-stranded target oligonucleotides used individually.

[0249] In some embodiments, the oligonucleotide agents of this application achieve a greater increase in full-length protein, higher activity, lower toxicity, and reduced side effects compared to administering the same amount of double-stranded oligonucleotides that do not have a cODV structure (e.g., saRNA substances used alone).

[0250] In any of the embodiments provided herein, such cells may be in vitro, such as in a cell line, or they may be present in the body of a mammal, such as a human. In some embodiments, the human is a subject or individual suffering from SOD1 protein-related disease or ALS.

[0251] Another aspect of this application relates to the use of the oligonucleotide agent of this application, nucleic acids encoding two or more oligonucleotides of the oligonucleotide agent of this application, or compositions comprising the oligonucleotide agent of this application, or nucleic acids encoding two or more oligonucleotides of the oligonucleotide agent of this application, for the preparation of therapeutic agents or for delaying the onset of SMN deficiency-related conditions or ALS. The subjects may be mammals such as humans. The subjects may be infants, children or adults.

[0252] In certain embodiments, the oligonucleotide agents of the present invention achieve a reduction in full-length SOD1 protein of a smaller amount than that achieved by administering the same amount of double-stranded oligonucleotide substance used individually, while reducing toxicity or undesirable side effects. In some embodiments, the oligonucleotide agents of the present application achieve a reduction in full-length SOD1 protein of a smaller amount than the additive effect of treatment with the same amount of double-stranded targeting oligonucleotide used individually.

[0253] In certain embodiments, the effect of the oligonucleotide agent of the present invention achieves a greater clinical improvement compared to the effect of any one of the substances used individually in equal amounts. In certain embodiments, the effect of the oligonucleotide agent achieves an additive or greater clinical improvement compared to the effect of any one of the double-stranded oligonucleotides used individually in equal amounts.

[0254] In any embodiment provided herein, such oligonucleotide agents, nucleic acids encoding the oligonucleotide agents of the present invention, or compositions comprising such oligonucleotide agents or nucleic acids encoding the oligonucleotide agents of the present invention may be introduced directly into cells or produced intracellularly upon introduction into cells, preferably mammalian cells, more preferably human cells, containing a nucleotide sequence encoding the oligonucleotide agent. Such cells may be ex vivo, such as cell lines, or may be present in the body of a mammal, such as a human. In some embodiments, the human is a patient or individual suffering from SMN deficiency-related disease or ALS. In certain embodiments, a composition comprising a sufficient amount of each of the aforementioned oligonucleotide agents or nucleic acids encoding the oligonucleotide agents of the present invention to effectively treat ALS.

[0255] In certain embodiments, baseline measurements are obtained from a biological sample as defined herein, obtained from an individual prior to administration of the therapy described herein. In certain embodiments, the biological sample is peripheral blood mononuclear cells, plasma, serum, skin tissue, or cerebrospinal fluid (CSF). Cells containing siRNA

[0256] The oligonucleotide agents disclosed herein can effectively inhibit or downregulate the expression of target genes within cells after contact with them. For example, they can reduce expression levels by at least 10% (e.g., compared to baseline transcription levels).

[0257] In some embodiments, this application relates to cells containing oligonucleotide agents disclosed herein. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells, for example, human cells from various tissues of organs including the brain, spinal cord, muscles, spleen, lungs, heart, liver, bladder, and kidneys. In some embodiments, the cells in the target tissue include a group selected from: the prefrontal cortex, cerebellum, and cerebrum; the cervical, thoracic, and lumbar vertebrae of the spinal cord; the heart, biceps brachii, semitendinosus, platysma, and gluteus maximus.

[0258] The cells disclosed herein may be in vitro or ex vivo, such as cell lines, and may be present in the body of a mammal, such as a human body.

[0259] In some embodiments, the cells are derived from the CNS tissue of subjects suffering from ALS. Oligonucleotide composition

[0260] Another aspect of this application provides a pharmaceutical composition comprising a double-stranded targeted oligonucleotide and a non-targeted single-stranded oligonucleotide described in this application.

[0261] This application provides a composition or pharmaceutical composition that can downregulate the level of SOD1 mRNA transcripts by an RNA interference mechanism (MoA), comprising an oligonucleotide agent disclosed herein, for the treatment or prevention of the onset of SOD1-related diseases (particularly ALS).

[0262] In some embodiments, this application relates to a composition or pharmaceutical composition comprising siRNA / saRNA as described herein. In some embodiments, this application relates to a composition or pharmaceutical composition comprising siRNA / saRNA and a non-targeting site by a linker component described herein. In some embodiments, this application relates to a composition or pharmaceutical composition comprising covalently linked siRNA / saRNA and a non-targeting site by a linking component described herein.

[0263] In some embodiments, the pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier. In one embodiment, the pharmaceutically acceptable carrier comprises one or more of the following: aqueous carriers, liposomes or LNPs, polymers, micelles, colloids, metal nanoparticles, nonmetal nanoparticles, bioconjugates (e.g., GalNAc), and polypeptides.

[0264] In some embodiments, the composition comprises 1 to 150 nM of the oligonucleotide agent of the present invention.

[0265] Another aspect of this application relates to uses of the oligonucleotide agents described herein, nucleic acids encoding the oligonucleotide agents, or compositions comprising the oligonucleotide agents or nucleic acids, wherein a double-stranded targeted oligonucleotide and a single-stranded oligonucleotide are covalently linked and are used in the preparation of one or more compositions for regulating (enhancing or suppressing) the expression of one or more genes or proteins expressed by cells.

[0266] Another embodiment provides a pharmaceutical composition or medicinal product comprising the agent of the present application and a therapeutically inert carrier, diluent, or pharmaceutically acceptable excipient, as well as a method of using the agent of the present application to prepare such a composition and medicinal product.

[0267] The oligonucleotide composition of this application may be delivered optionally by parenteral infusion, including intrathecal, intramuscular, intravenous, intra-arterial, intraperitoneal, intravesical, intraventricular, intravitreous, or subcutaneous administration; or by oral, intranasal, inhalation, vaginal, or rectal administration.

[0268] Typical formulations are prepared by mixing the drug of this application with a carrier or excipient. Suitable carriers and excipients are well known to those skilled in the art and are described in detail, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems by Ansel HC et al. (2004, Lippincott, Williams & Wilkins, Philadelphia), Remington: The Science and Practice of Pharmacy by Gennaro AR et al. (2000, Lippincott, Williams & Wilkins, Philadelphia), and Handbook of Pharmaceutical Excipients by Rowe RC (2005, Pharmaceutical Press, Chicago).

[0269] The compositions of this application are formulated, administered, and given in a manner consistent with good medical practice. Factors to be considered in this context include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the administration schedule, and other factors known to healthcare professionals.

[0270] In another embodiment, this application provides the use of oligonucleotide agents or compositions according to any one of the embodiments described herein in the manufacture of pharmaceuticals for the treatment of gene or protein-related conditions in an individual. In use according to a particular embodiment, the condition may include SMN deficiency-related conditions, including ALS. In use according to a particular embodiment, the condition may include SMN deficiency-related conditions constituting hereditary neuromuscular diseases, preferably spinal muscular atrophy. In other embodiments, the condition may include immune-related conditions, such as cancer. Use according to a particular embodiment is also provided, where the individual is a mammal, preferably a human. Administration regimen and route of administration

[0271] Aspects of this application relate to pharmaceutical compositions comprising the oligonucleotide agent of this application. In some embodiments, the application relates to pharmaceutical compositions comprising the oligonucleotide agent of this application and a pharmaceutically acceptable carrier, a therapeutically inactive carrier, a diluent, or a pharmaceutically acceptable excipient. The pharmaceutical compositions disclosed herein are developed as pharmaceuticals for the prevention or treatment of SOD1 protein-related diseases or ALS.

[0272] kit In another embodiment, any of the compositions described herein may be provided in one or more kits, which optionally include instructions for use of the composition. That is, the kit may include instructions for use of the oligonucleotide agent or composition in any of the methods described herein. As used herein, “kit” typically defines a package, assembly, or container (such as an insulated container) that includes one or more components or embodiments of this application and / or other components related to this application, such as those described above. Any reagent or component of the kit may be provided in liquid form (e.g., in solution) or solid form (e.g., dry powder, frozen product).

[0273] In summary, the present invention provides a technologically superior approach to nucleic acid-based therapeutics that achieves improved self-delivery properties, enhanced efficacy, reduced cytotoxicity, lower synthesis costs, and minimized off-target effects. These advantages make the present invention a promising candidate for future development and commercialization, with the ultimate goal of improving patient outcomes through safer and more effective treatments.

[0274] (Embodiment) This application provides the following specific embodiments. Embodiment 1: An oligonucleotide agent conjugated with a non-target site that facilitates delivery to a target oligonucleotide, wherein the non-target site comprises one or more structural units covalently bonded in series to form its backbone, and at least two adjacent structural units are linked by phosphorothioate (PS) bonds, and each structural unit of the non-target site is selected from a chemical linker and a nucleotide. Here, non-target sites include: Cases in which one or more chemical linkers are interposed within a nucleotide; cases in which one or more nucleotides are interposed within a chemical linker; a sequence of consecutive nucleotides and a sequence of chemical linkers continuously bound thereto; or a sequence of continuously bound chemical linkers that does not contain any nucleotides. Here, at least one phosphodiester bond between two adjacent nucleotides, between two adjacent linkers, or between a nucleotide and an adjacent linker is replaced by a phosphorothioate (PS) bond, a mesylphosphoramide bond, or a boranophosphate bond.

[0275] Embodiment 2: An oligonucleotide agent according to Embodiment 1, wherein the chemical linker is selected from the following: a) L1 or S18 (Spacer-18 Linker) (1,1-Bis(4-methoxyphenyl)-1-phenyl-2,5,8,11,14,17-Hexaoxanonadecan-19-yl(2-cyanoethyl)diisopropylphosphorumidite); b) L4 or C6 (spacer-C6 linker) (6-(bis(4-methoxyphenyl)(phenyl)methoxy)hexyl(2-cyanoethyl)diisopropylphosphorumidite); c) L6(1,1-bis(4-methoxyphenyl)-1-phenyl-2,5,8,11,14-pentaoxahexadecane-16-yl (2-cyanoethyl)diisopropylphosphorumidite); d) L9 or S9 (Spacer-9 Linker) (2- (2- (2- (Bis(4-methoxyphenyl)(phenyl)methoxy)ethoxy)ethoxy)ethyl(2-cyanoethyl)diisopropylphosphorumidite); e) L10 or C3 (spacer-C3 linker) (3-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl(2-cyanoethyl)diisopropylphosphorumidite); f) L12(d spacer) ((2R, 3S)-2- ((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphorumidite); g) L13 or C12 (spacer-C12 linker) (12- (bis(4-methoxyphenyl)(phenyl)methoxy)dodecyl(2-cyanoethyl)diisopropylphosphorumidite); h) L14 (Spacer-L14 Linker) (((1r, 4r)-4- ((Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)cyclohexyl)methyl(d-cyanoethyl)diisopropylphosphorumidite); i) L15 (Spacer-L15 Linker) (4-(2-(Bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)phenethyl(2-cyanoethyl)diisopropylphosphorumidite); j) L16 (Spacer-L16 Linker) (2- (1- (2- (Bis(4-Methoxyphenyl)(Phenyl) Methoxy) Ethyl) Cyclohexyl) Ethyl (2-Cyanoethyl) Diisopropyl Phosphoramidite) k) C6x1((2S, 3S, 4S, 5S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-methoxy-4-(pento-4-in-1-yloxy)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphorumidite); l) C6x2((2S, 3S, 4S, 5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2-methoxy-4-(pento-4-in-1-yloxy)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphorumidite); m) C6x5(2- ((2- (bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(pento-4-in-1-yl)amino)ethyl(2-cyanoethyl)diisopropylphosphorumidite); n) C6x7((9H-Fluoren-9-yl)methyl(4-((2S,4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((bis(diisopropylamino)phosphanyl)oxy)pyrrolidine-1-yl)-4-oxobutyl)carbamate). o) L20 methyl 1-(5-(bis(4-methoxyphenyl)(phenyl)methoxy)pentyl)-2-(4-(((2-cyanoethoxy)(diisopropylamino)phosphanyl)oxy)butyl)-1H-benzo[d]imidazole-5-carboxylate; and p) L42 6-((6-(bis(4-methoxyphenyl)(phenyl)methoxy)hexyl)disulfanyl)hexyl(2-cyanoethyl)diisopropylphosphorumidite.

[0276] Embodiment 3: An oligonucleotide agent according to Embodiment 1, wherein the chemical linker is a chemical group selected from the following: substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclic alkyl, heterocyclic alkenyl, heterocyclic alkynyl, aryl, heteroaryl, heterocyclic, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylaryl These are alkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkenylheteroarylalkynyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclicalkyl, alkylheterocyclicalkenyl, alkylheterocyclicalkenyl, alkylheterocyclicalkenyl, alkenylheterocyclicalkenyl, alkenylheterocyclicalkenyl, alkenylheterocyclicalkenyl, alkenylheterocyclicalkenyl, alkenylheterocyclicalkenyl, alkenylheterocyclicalkenyl, alkenylheterocyclicalkenyl, alkylaryl, alkenylaryl, alkenylaryl, alkenylheteroaryl, and alkynylheteroaryl.Here, one or more methylene groups may be interrupted or terminated by O, S, S(O), SO2, N(R')2, C(O), a cleavable linking group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, or a substituted or unsubstituted heterocyclic group, and each R' is independently selected from hydrogen, a substituted or unsubstituted alkyl group, an aryl group, an aralkyl group, an alkylaryl group, an alkoxy group, an aryloxy group, an acyl group, or an aliphatic group (linear, branched, or cyclic).

[0277] Embodiment 4: An oligonucleotide agent according to any one of Embodiments 1 to 3, comprising m chemical linkers with identical or different non-target sites and n nucleotides with identical or different non-target sites, where m is an integer from 1 to 40 and n is an integer from 0 to 30.

[0278] Embodiment 5: An oligonucleotide agent according to any one of Embodiments 1 to 4, wherein the non-target site includes a sequence of continuously linked chemical linkers represented by the formula (linker1)x - (linker2)y, where linker1 is a first chemical linker, linker2 is a second chemical linker different from the first chemical linker, and x and y are integers satisfying 0 < x + y < 50. For example, non-target sites include any of the following: S9, (S9)2, (S9)3, (S9)4, (S9)5, (S9)6, (S9)7, (S9)8, (S9)9, (S9) 10 (S9) 11 (S9) 12 (S9) 13 (S9) 14 (S9) 15 (S9) 16 (S9) 17 (S9) 18 (S9) 19 (S9) 20 (S9) 21 (S9) 22 , (S9)23 , (S9) 24 , (S9) 25 , (S9) 26 , (S9) 27 , (S9) 28 , (S9) 29 , or (S9) 30 , L10, (L10)2, (L10)3, (L10)4, (L10)5, (L10)6, (L10)7, (L10)8, (L10)9, (L10) 10 , (L10) 11 , (L10) 12 , (L10) 13 , (L10) 14 , (L10) 15 , (L10) 16 , (L10) 17 , (L10) 18 , (L10) 19 , (L10) 20 , (L10) 21 , (L10) 22 , (L10) 23 , (L10) 24 , (L10) 25 , (L10) 26 , (L10) 27 , (L10) 28 , (L10) 29 , or (L10) 30 , L12, (L12)2, (L12)3, (L12)4, (L12)5, (L12)6, (L12)7, (L12)8, (L12)9, (L12)10, (L12)11, (L12)12, (L12)13, (L12)14, (L12)15, (L12)16, (L12)17, (L12)18, (L12)19, (L12)20, (L12)21, (L12)22, (L12)23, (L12)24, (L12)25, (L12)26, (L12)27, (L12)28, (L12)29, or (L12)30, S9-L10, S9-(L10)2, S9-(L10)3, S9-(L10)4, S9-(L10)5, S9-(L10)6, S9-(L10)7, S9-(L10)8, S9-(L10)9, S9-(L10) 10 , S9-(L10) 11 , S9-(L10) 12 , S9-(L10) 13 , S9-(L10) 14 , S9-(L10) 15 , S9-(L10) 16 , S9-(L10) 17 , S9-(L10) 18 , S9-(L10) 19 , S9-(L10) 20 , S9-(L10) 21 , S9-(L10) 22 , S9-(L10) 23 , S9-(L10) 24 , S9-(L10) 25 , S9-(L10) 26 , S9-(L10) 27 , S9-(L10) 28 , S9-(L10) 29 , or S9-(L10) 30 , S9-L12, S9-(L12)2, S9-(L12)3, S9-(L12)4, S9-(L12)5, S9-(L12)6, S9-(L12)7, S9-(L12)8, S9-(L12)9, S9-(L12) 10 , S9-(L12) 11 , S9-(L12) 12 , S9-(L12) 13 , S9-(L12) 14 , S9-(L12) 15 , S9-(L12) 16 , S9-(L12) 17 , S9-(L12) 18 , S9-(L12) 19 , S9-(L12) 20 , S9-(L12) 21 , S9-(L12) 22 , S9-(L12)23 , S9-(L12) 24 , S9-(L12) 25 , S9-(L12) 26 , S9-(L12) 27 , S9-(L12) 28 , S9-(L12) 29 , or S9-(L12) 30 , L20, (L20)2, (L20)3, (L20)4, (L20)5, (L20)6, (L20)7, (L20)8, (L20)9, (L20) 10 , (L20) 11 , (L20) 12 , (L20) 13 , (L20) 14 , (L20) 15 , (L20) 16 , (L20) 17 , (L20) 18 , (L20) 19 , (L20) 20 , (L20) 21 , (L20) 22 , (L20) 23 , (L20) 24 , (L20) 25 , (L20) 26 , (L20) 27 , (L20) 28 , (L20) 29 , or (L20) 30 , L42, (L42)2, (L42)3, (L42)4, (L42)5, (L42)6, (L42)7, (L42)8, (L42)9, (L42) 10 , (L42) 11 , (L42) 12 , (L42) 13 , (L42) 14 , (L42) 15 , (L42) 16 , (L42) 17 , (L42) 18 , (L42) 19 , (L42) 20 , (L42) 21, (L42) 22 , (L42) 23 , (L42) 24 , (L42) 25 , (L42) 26 , (L42) 27 , (L42) 28 , (L42) 29 , or (L42) 30 , L20-L12, L20-(L12)2, L20-(L12)3, L20-(L12)4, L20-(L12)5, L20-(L12)6, L20-(L12)7, L20-(L12)8, L20-(L12)9, L20-(L12) 10 , L20-(L12) 11 , L20-(L12) 12 , L20-(L12) 13 , L20-(L12) 14 , L20-(L12) 15 , L20-(L12) 16 , L20-(L12) 17 , L20-(L12) 18 , L20-(L12) 19 , L20-(L12) 20 , L20-(L12) 21 , L20-(L12) 22 , L20-(L12) 23 , L20-(L12) 24 , L20-(L12) 25 , L20-(L12) 26 , L20-(L12) 27 , L20-(L12) 28 , L20-(L12) 29 , or L20-(L12) 30 , or L42-L12, L42-(L12)2, L42-(L12)3, L42-(L12)4, L42-(L12)5, L42-(L12)6, L42-(L12)7, L42-(L12)8, L42-(L12)9, L42-(L12) 10 , L42-(L12) 11 , L42-(L12) 12, L42-(L12) 13 , L42-(L12) 14 , L42-(L12) 15 , L42-(L12) 16 , L42-(L12) 17 , L42-(L12) 18 , L42-(L12) 19 , L42-(L12) 20 , L42-(L12) 21 , L42-(L12) 22 , L42-(L12) 23 , L42-(L12) 24 , L42-(L12) 25 , L42-(L12) 26 , L42-(L12) 27 , L42-(L12) 28 , L42-(L12) 29 , or L42-(L12) 30 , Here, at least one phosphodiester bond between two adjacent linkers is replaced by a phosphorothioate (PS) bond, a mesylphosphoramidate bond, or a boranophosphate bond.

[0279] Embodiment 6: An oligonucleotide agent according to any one of Embodiments 1 to 5, wherein the non-target site contains 1 to about 50, about 2 to about 48, about 3 to about 46, about 4 to about 44, about 5 to about 42, about 6 to about 40, about 7 to about 38, about 8 to about 36, about 9 to about 34, about 10 to about 32, about 11 to about 30, about 12 to about 28, about 13 to about 26, about 14 to about 24, about 15 to about 22, about 16 to about 20, or about 17 to about 18 phosphorothioate (PS) bonds in its skeleton.

[0280] Embodiment 7: An oligonucleotide agent according to any one of Embodiments 1 to 5, wherein the non-target site contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 or more phosphorothioate (PS) bonds in its skeleton.

[0281] Embodiment 8: An oligonucleotide agent according to any one of Embodiments 1 to 7, wherein the target oligonucleotide is an antisense oligonucleotide, or a double-stranded oligonucleotide (e.g., siRNA or saRNA) comprising a sense strand and an antisense strand.

[0282] Embodiment 9: An oligonucleotide agent according to Embodiment 8, wherein the non-target site is conjugated to the sense strand or antisense strand of a double-stranded oligonucleotide.

[0283] Embodiment 10: An oligonucleotide agent according to any one of Embodiments 1 to 9, wherein, if present, the nucleotides at the non-target site are unchemically modified nucleotides, or at least one nucleotide is a chemically modified nucleotide.

[0284] Embodiment 11: An oligonucleotide agent according to any one of Embodiments 1 to 10, wherein all nucleotides of the target oligonucleotide are unchemically modified nucleotides, or at least one nucleotide is chemically modified nucleotide, or at least one phosphodiester bond between two adjacent nucleotides in the target oligonucleotide is replaced with a phosphorothioate (PS) bond, a mesylphosphoramidate bond, or a boranophosphate bond. Embodiment 12: An oligonucleotide agent according to any one of Embodiments 10 to 11, wherein the chemically modified nucleotide comprises one or more of the following modifications: a) Modification of the 2'-OH group of ribose in a nucleotide; b) Modification or non-modification of the base portion on the nucleoside ring in a nucleotide; c) that the nucleotide is a locked nucleic acid or a cross-linked nucleic acid, and d) The nucleotide is a deoxyribonucleotide (DNA).

[0285] Embodiment 13: The oligonucleotide agent according to Embodiment 12, wherein the chemically modified nucleotide has a 2'-OH ribose modification selected from 2'-fluoro-2'-deoxynucleoside (2'-F) modification, 2'-O-methyl (2'-O-Me) modification, and 2'-O-(2-methoxyethyl) (2'-O-MOE) modification.

[0286] Embodiment 14: An oligonucleotide agent according to any one of Embodiments 1 to 13, wherein, if present, the nucleotide in the non-target site is selected from RNA, DNA, cross-linked nucleic acid (BNA), locked nucleic acid (LNA), or peptide nucleic acid (PNA).

[0287] Embodiment 15: An oligonucleotide agent according to any one of Embodiments 10 to 11, wherein at least one chemically modified nucleotide is a nucleotide to which a 5'-phosphate group, 5-methylcytosine, or 5'-(E)-vinylphosphonate has been added.

[0288] Embodiment 16: An oligonucleotide agent according to any one of Embodiments 1 to 15, wherein the target oligonucleotide and the non-target site are directly conjugated, for example, via a phosphorothioate (PS) bond.

[0289] Embodiment 17: An oligonucleotide agent according to Embodiment 16, wherein the terminal unit or internal unit of the non-target site is conjugated to the target oligonucleotide.

[0290] Embodiment 18: An oligonucleotide agent according to any one of Embodiments 1 to 17, wherein the non-target site is conjugated to the 3' end, 5' end, both the 3' and 5' ends, or an internal nucleotide of the sense strand or antisense strand of a double-stranded oligonucleotide.

[0291] Embodiment 19: An oligonucleotide agent according to any one of Embodiments 1 to 18, wherein the non-target site is selected from the following: 1) A single chemical linker that does not contain nucleotides, wherein the chemical linker is conjugated to the terminal of a target oligonucleotide via a phosphorothioate (PS) bond; 2) A sequence of chemical linkers that do not contain nucleotides, wherein the sequence of chemical linkers is conjugated to one end of a target oligonucleotide (optionally, for example, via 1 to 20 deoxyribonucleotides (DNA)); 3) Two consecutive chemical linker sequences that do not contain nucleotides, each of which is conjugated to both ends of a target oligonucleotide (optionally, for example, via 1 to 20 deoxyribonucleotides (DNA)); 4) A sequence comprising one or more consecutive nucleotide sequences and a sequence of chemical linkers, wherein the nucleotides are interposed within the chemical linkers (preferably, each nucleotide is interposed between two chemical linkers, or one or more consecutive nucleotide sequences are interposed within the chemical linkers). Here, at least one phosphodiester bond between two adjacent nucleotides, between two adjacent linkers, or between a nucleotide and an adjacent linker is replaced with a phosphorothioate (PS) bond.

[0292] Embodiment 20: An oligonucleotide agent according to any one of Embodiments 1 to 19, wherein the internal nucleotides of the sense strand or antisense strand of a double-stranded oligonucleotide are substituted with a linking component, and a single-stranded oligonucleotide is covalently bonded to the linking component.

[0293] Embodiment 21: An oligonucleotide agent according to any one of Embodiments 1 to 20, wherein 2 to 10 non-target sites are conjugated to a double-stranded oligonucleotide, or 2 to 10 double-stranded oligonucleotides are conjugated to a non-target site.

[0294] Embodiment 22: The oligonucleotide agent according to Embodiment 20, wherein the linking component is selected from one or more of the following: ethylene glycol chain, alkyl chain, alkenyl chain, alkynyl chain, peptide, carbohydrate, thiol bond, phosphodiester, phosphorothioate, phosphoramidate, amide, carbamate, tetrazole bond, and benzimidazole bond.

[0295] Embodiment 23: An oligonucleotide agent according to any one of Embodiments 1 to 22, wherein a non-target site and / or a double-stranded oligonucleotide is bound to one or more conjugation groups.

[0296] Embodiment 24: An oligonucleotide agent according to Embodiment 23, wherein one or more conjugation groups are selected from lipids, fatty acids, fluorescent dyes, ligands, sugars, peptides, and antibodies, and optionally, the conjugation groups are further selected from cell-permeable peptides, polyethylene glycol, alkaloids, tryptamine, benzimidazole, quinolone, amino acids, cholesterol, glucose, and N-acetylgalactosamine.

[0297] Embodiment 25: An oligonucleotide agent according to any one of Embodiments 1 to 24, wherein the nucleotide sequence of the sense strand has at least 90% identity with any of the nucleotide sequences described in SEQ ID NO: 1, 3, 56, and 61.

[0298] Embodiment 26: An oligonucleotide agent according to any one of Embodiments 1 to 25, wherein the nucleotide sequence of the antisense chain has at least 90% identity with any of the nucleotide sequences described in SEQ ID NO: 2, 4, 57, 62, and 69.

[0299] Embodiment 27: An oligonucleotide agent according to any one of Embodiments 1 to 24, comprising any sense strand sequence described in SEQ ID NO: 6 to 39, 60, 64, 65, 66, 67, 70 to 87.

[0300] Embodiment 28: An oligonucleotide agent according to any one of Embodiments 1 to 27, wherein the non-target site of the oligonucleotide agent improves the stability, bioavailability, in vivo distribution, and / or intracellular uptake of the target oligonucleotide compared to an oligonucleotide agent without a non-target site.

[0301] Embodiment 29: An oligonucleotide agent according to any one of Embodiments 1 to 28, wherein the non-target site of the oligonucleotide agent increases the in vivo distribution of target oligonucleotides in one or more target tissues, compared to an oligonucleotide agent that does not have a non-target site.

[0302] Embodiment 30: An oligonucleotide agent according to Embodiment 29, wherein one or more target tissues are selected from the brain, spinal cord, muscle, spleen, lung, heart, liver, bladder, and kidney.

[0303] Embodiment 31: An oligonucleotide agent according to Embodiment 29, wherein one or more target tissues are selected from the prefrontal cortex, cerebellum, cerebrum, cervical spine, thoracic spine, lumbar spine, heart, biceps brachii muscle, semitendinosus muscle, platysma muscle, and gluteus maximus muscle.

[0304] Embodiment 32: A vector comprising an oligonucleotide agent according to any one of Embodiments 1 to 31.

[0305] Embodiment 33: A cell comprising an oligonucleotide agent according to any one of Embodiments 1 to 31.

[0306] Embodiment 34: The cells described in Embodiment 33, wherein the cells are mammalian cells and optionally human cells.

[0307] Embodiment 35: A cell according to any one of Embodiments 33 to 34, wherein the cell is a host cell.

[0308] Embodiment 36: A cell according to any one of Embodiments 33 to 35, wherein the cell exists in vitro (in a test tube) or in a mammalian body.

[0309] Embodiment 37: A pharmaceutical composition comprising an oligonucleotide agent according to any one of Embodiments 1 to 30 and / or cells according to any one of Embodiments 33 to 36.

[0310] Embodiment 38: A pharmaceutical composition according to Embodiment 37, wherein the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier selected from aqueous carriers, liposomes or LNPs, polymers, micelles, colloids, metal nanoparticles, nonmetal nanoparticles, bioconjugates, and polypeptides.

[0311] Embodiment 39: A pharmaceutical composition according to any one of Embodiments 37 to 38, wherein the pharmaceutical composition inhibits SOD1 gene expression or reduces SOD1 protein.

[0312] Embodiment 40: A pharmaceutical composition according to any one of Embodiments 37 to 38, wherein the pharmaceutical composition activates SMN2 gene expression or increases SMN2 protein.

[0313] Embodiment 41: A kit comprising an oligonucleotide agent according to any one of Embodiments 1 to 31 or a pharmaceutical composition according to any one of Embodiments 37 to 40.

[0314] Embodiment 42: A method for inhibiting SOD1 gene expression or reducing SOD1 protein, comprising administering a pharmaceutical composition according to any one of Embodiments 37 to 39 to a subject.

[0315] Embodiment 43: A method for treating or delaying the onset or progression of amyotrophic lateral sclerosis (ALS), comprising administering a pharmaceutical composition according to any one of Embodiments 37 to 39 to a subject.

[0316] Embodiment 44: The method according to Embodiment 43, wherein the subject is sporadic ALS (sALS) or familial ALS (fALS).

[0317] Embodiment 45: A method for activating SMN2 gene expression or increasing SMN2 protein, comprising administering a pharmaceutical composition described in any one of Embodiments 37-38 and 40 to a subject.

[0318] Embodiment 46: A method for treating or delaying the onset or progression of spinal muscular atrophy (SMA), comprising administering a pharmaceutical composition according to any one of Embodiments 37 to 40 to a subject.

[0319] Embodiment 47: A method according to any one of Embodiments 42 to 46, wherein the non-target site of the oligonucleotide agent improves the stability, bioavailability, in vivo distribution and / or intracellular uptake of the double-stranded oligonucleotide compared to an oligonucleotide agent without a non-target site.

[0320] Embodiment 48: A method according to any one of Embodiments 42 to 47, wherein the non-target sites of the oligonucleotide agent increase the in vivo distribution of double-stranded oligonucleotides in one or more target tissues, compared to an oligonucleotide agent without a non-target site.

[0321] Embodiment 49: A method according to any one of Embodiments 42 to 48, wherein the non-target site of the oligonucleotide agent increases the in vivo distribution of double-stranded oligonucleotides in two or more target cell types within the tissue, compared to an oligonucleotide agent without a non-target site.

[0322] Embodiment 50: Use of an oligonucleotide agent according to any one of Embodiments 1 to 31 or a pharmaceutical composition according to any one of Embodiments 37 to 40 in the manufacture of a pharmaceutical product for treating or delaying the onset or progression of amyotrophic lateral sclerosis (ALS).

[0323] Embodiment 51: An oligonucleotide agent according to any one of Embodiments 1 to 31 or a pharmaceutical composition according to any one of Embodiments 37 to 40, which is used to treat or delay the onset or progression of amyotrophic lateral sclerosis (ALS).

[0324] Embodiment 52: A kit comprising a container containing an oligonucleotide agent according to any one of Embodiments 1 to 31.

[0325] This application further provides the following specific embodiments. Embodiment 1a: An oligonucleotide agent in which a non-target site is conjugated to a target oligonucleotide to facilitate delivery, wherein the non-target site comprises one or more structural units covalently bonded in series to form its backbone, and at least two adjacent structural units are linked by phosphorothioate (PS) bonds.

[0326] Embodiment 2a: An oligonucleotide agent according to Embodiment 1a, wherein each unit in the non-target site is selected from a chemical group, a chemical linker, and a nucleotide.

[0327] Embodiment 3a: An oligonucleotide agent according to Embodiment 2a, wherein the chemical group is selected from the following: substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclic alkyl, heterocyclic alkenyl, heterocyclic alkynyl, aryl, heteroaryl, heterocyclic, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylal These are nyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclicalkyl, alkylheterocyclicalkenyl, alkylheterocyclicalkenyl, alkylheterocyclicalkenyl, alkenylheterocyclicalkenyl, alkenylheterocyclicalkenyl, alkenylheterocyclicalkenyl, alkynylheterocyclicalkenyl, alkynylheterocyclicalkenyl, alkynylheterocyclicalkenyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, and alkynylheteroaryl. Here, one or more methylene groups may be interrupted or terminated by O, S, S(O), SO2, N(R')2, C(O), a cleavable linking group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, or a substituted or unsubstituted heterocyclic group, and each R' is independently selected from hydrogen, a substituted or unsubstituted alkyl group, an aryl group, an aralkyl group, an alkylaryl group, an alkoxy group, an aryloxy group, an acyl group, or an aliphatic group (linear, branched, or cyclic).

[0328] Embodiment 4a: An oligonucleotide agent according to Embodiment 2a or 3a, wherein the chemical linker is selected from the following: a) L1 or S18 (Spacer-18 Linker) (1,1-Bis(4-methoxyphenyl)-1-phenyl-2,5,8,11,14,17-Hexaoxanonadecan-19-yl(2-cyanoethyl)diisopropylphosphorumidite); b) L4 or C6 (spacer-C6 linker) (6-(bis(4-methoxyphenyl)(phenyl)methoxy)hexyl(2-cyanoethyl)diisopropylphosphorumidite); c) L6(1,1-bis(4-methoxyphenyl)-1-phenyl-2,5,8,11,14-pentaoxahexadecane-16-yl (2-cyanoethyl)diisopropylphosphorumidite); d) L9 or S9 (Spacer-9 Linker) (2- (2- (2- (Bis(4-methoxyphenyl)(phenyl)methoxy)ethoxy)ethoxy)ethyl(2-cyanoethyl)diisopropylphosphorumidite); e) L10 or C3 (spacer-C3 linker) (3-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl(2-cyanoethyl)diisopropylphosphorumidite); f) L12(d spacer) ((2R, 3S)-2- ((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphorumidite); g) L13 or C12 (spacer-C12 linker) (12- (bis(4-methoxyphenyl)(phenyl)methoxy)dodecyl(2-cyanoethyl)diisopropylphosphorumidite); h) L14 (Spacer-L14 Linker) (((1r, 4r)-4- ((Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)cyclohexyl)methyl(d-cyanoethyl)diisopropylphosphorumidite); i) L15 (Spacer-L15 Linker) (4-(2-(Bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)phenethyl(2-cyanoethyl)diisopropylphosphorumidite); j) L16 (Spacer-L16 Linker) (2- (1- (2- (Bis(4-Methoxyphenyl)(Phenyl) Methoxy) Ethyl) Cyclohexyl) Ethyl (2-Cyanoethyl) Diisopropyl Phosphoramidite) k) C6x1((2S, 3S, 4S, 5S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-methoxy-4-(pento-4-in-1-yloxy)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphorumidite); l) C6x2((2S, 3S, 4S, 5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2-methoxy-4-(pento-4-in-1-yloxy)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphorumidite); m) C6x5(2- ((2- (bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(pento-4-in-1-yl)amino)ethyl(2-cyanoethyl)diisopropylphosphorumidite); and n) C6x7((9H-Fluoren-9-yl)methyl(4-((2S,4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((bis(diisopropylamino)phosphanyl)oxy)pyrrolidine-1-yl)-4-oxobutyl)carbamate).

[0329] Embodiment 5a: An oligonucleotide agent according to any one of Embodiments 1a to 4a, wherein the non-target site includes the following: a) One or more chemical linkers interposed within a nucleotide b) One or more nucleotides interposed within the chemical linker c) Consecutive nucleotide sequences and sequentially linked chemical linker sequences d) A sequence of chemical linkers that do not contain nucleotides. Here, at least one phosphodiester bond between two adjacent nucleotides, between two adjacent linkers, or between a nucleotide and an adjacent linker is replaced by a phosphorothioate (PS) bond, a mesylphosphoramide bond, or a boranophosphate bond.

[0330] Embodiment 6a: An oligonucleotide agent according to Embodiment 5a or the like, comprising m chemical linkers with identical or different non-target sites and n nucleotides with identical or different non-target sites, where m is an integer from 1 to 40 and n is an integer from 0 to 30.

[0331] Embodiment 7a: An oligonucleotide agent according to Embodiment 6a, wherein the non-target site is (linker1) x -(linker2) y A sequence of continuously linked chemical linkers represented by the formula , where linker1 is the first chemical linker, linker2 is a second chemical linker distinct from the first, and x and y are integers satisfying 0 < x + y < 50. For example, non-target sites include: S9, L10, S9-L10, S9-(L10)2, S9-(L10)3, S9-(L10)4, S9-(L10)5, S9-(L10)6, S9-(L10)7, S9-(L10)8, S9-(L10)9, S9-(L10) 10 S9-(L10) 11 S9-(L10) 12 S9-(L10) 13 S9-(L10)14 、S9-(L10) 15 、S9-(L10) 16 、S9-(L10) 17 、S9-(L10) 18 、S9-(L10) 19 、S9-(L10) 20 、S9-(L10) 21 、S9-(L10) 22 、S9-(L10) 23 、S9-(L10) 24 、S9-(L10) 25 、S9-(L10) 26 、S9-(L10) 27 、S9-(L10) 28 、S9-(L10) 29 、S9-(L10) 30 、S9-L12、S9-(L12)2、S9-(L12)3、S9-(L12)4、S9-(L12)5、S9-(L12)6、S9-(L12)7、S9-(L12)8、S9-(L12)9、S9-(L12) 10 、S9-(L12) 11 、S9-(L12) 12 、S9-(L12) 13 、S9-(L12) 14 、S9-(L12) 15 、S9-(L12) 16 、S9-(L12) 17 、S9-(L12) 18 、S9-(L12) 19 、S9-(L12) 20 、S9-(L12) 21 、S9-(L12) 22 、S9-(L12) 23 、S9-(L12) 24 、S9-(L12) 25 、S9-(L12) 26 、S9-(L12) 27 、S9-(L12) 28 、S9-(L12) 29 、S9-(L12) 30 。 Here, at least one phosphodiester bond between two adjacent linkers is replaced by a phosphorothioate (PS) bond, a mesylphosphoramidate bond, or a boranophosphate bond.

[0332] Embodiment 8a: An oligonucleotide agent according to any one of Embodiments 1a to 7a, wherein the non-target site contains 1 to about 50, about 2 to about 48, about 3 to about 46, about 4 to about 44, about 5 to about 42, about 6 to about 40, about 7 to about 38, about 8 to about 36, about 9 to about 34, about 10 to about 32, about 11 to about 30, about 12 to about 28, about 13 to about 26, about 14 to about 24, about 15 to about 22, about 16 to about 20, or about 17 to about 18 phosphorothioate (PS) bonds in its skeleton.

[0333] Embodiment 9a: An oligonucleotide agent according to any one of Embodiments 1a to 7a, wherein the non-target site contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 113, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more phosphorothioate (PS) bonds in its skeleton.

[0334] Embodiment 10a: An oligonucleotide agent according to any one of Embodiments 1a to 9a, wherein the target oligonucleotide is an antisense oligonucleotide, or a double-stranded oligonucleotide (e.g., siRNA or saRNA) containing a sense strand and an antisense strand.

[0335] Embodiment 11a: An oligonucleotide agent according to Embodiment 10a, wherein the non-target site is conjugated to the sense strand or antisense strand of a double-stranded oligonucleotide.

[0336] Embodiment 12a: An oligonucleotide agent according to any one of Embodiments 2a to 11a, wherein, if present, the nucleotides in the non-target site are unchemically modified nucleotides, or at least one nucleotide is a chemically modified nucleotide.

[0337] Embodiment 13a: An oligonucleotide agent according to any one of Embodiments 1a to 12a, wherein all nucleotides of the target oligonucleotide are unchemically modified nucleotides, or at least one nucleotide is chemically modified nucleotide, or at least one phosphodiester bond between two adjacent nucleotides in the target oligonucleotide is replaced with a phosphorothioate (PS) bond, a mesylphosphoramidate bond, or a boranophosphate bond.

[0338] Embodiment 14a: An oligonucleotide agent according to any one of Embodiments 12a to 13a, wherein the chemically modified nucleotide comprises one or more of the following modifications: a) Modification of the 2'-OH group of ribose in a nucleotide; b) Modification or non-modification of the base portion on the nucleoside ring in a nucleotide; c) that the nucleotide is a locked nucleic acid or a cross-linked nucleic acid, and d) The nucleotide is a deoxyribonucleotide (DNA).

[0339] Embodiment 15a: The oligonucleotide agent according to Embodiment 14a, wherein the chemically modified nucleotide has a 2'-OH ribose modification selected from 2'-fluoro-2'-deoxynucleoside (2'-F) modification, 2'-O-methyl (2'-O-Me) modification, and 2'-O-(2-methoxyethyl) (2'-O-MOE) modification.

[0340] Embodiment 16a: An oligonucleotide agent according to any one of Embodiments 2a to 15a, wherein, if present, the nucleotide in the non-target site is selected from RNA, DNA, cross-linked nucleic acid (BNA), locked nucleic acid (LNA), or peptide nucleic acid (PNA).

[0341] Embodiment 17a: An oligonucleotide agent according to any one of Embodiments 12a to 13a, wherein at least one chemically modified nucleotide is a nucleotide to which a 5'-phosphate group, 5-methylcytosine, or 5'-(E)-vinylphosphonate has been added.

[0342] Embodiment 18a: An oligonucleotide agent according to any one of Embodiments 1a to 17a, wherein the target oligonucleotide and the non-target site are directly conjugated, for example, via a phosphorothioate (PS) bond.

[0343] Embodiment 19a: An oligonucleotide agent according to Embodiment 18a, wherein the terminal unit or internal unit of the non-target site is conjugated to the target oligonucleotide.

[0344] Embodiment 20a: An oligonucleotide agent according to any one of Embodiments 10a to 19a, wherein the non-target site is conjugated to the 3' end, 5' end, both the 3' and 5' ends, or an internal nucleotide of the sense strand or antisense strand of a double-stranded oligonucleotide.

[0345] Embodiment 21a: An oligonucleotide agent according to any one of Embodiments 1a to 20a, wherein the internal nucleotides of the sense strand or antisense strand of a double-stranded oligonucleotide are substituted with a linking component, and a single-stranded oligonucleotide is covalently bonded to the linking component.

[0346] Embodiment 22a: An oligonucleotide agent according to any one of Embodiments 10a to 21a, wherein 2 to 10 non-target sites are conjugated to a double-stranded oligonucleotide, or 2 to 10 double-stranded oligonucleotides are conjugated to a non-target site.

[0347] Embodiment 23a: The oligonucleotide agent according to Embodiment 21a, wherein the linking component is selected from one or more of the following: ethylene glycol chain, alkyl chain, alkenyl chain, alkynyl chain, peptide, carbohydrate, thiol bond, phosphodiester, phosphorothioate, phosphoramidate, amide, carbamate, tetrazole bond, and benzimidazole bond.

[0348] Embodiment 24a: An oligonucleotide agent according to any one of Embodiments 1a to 23a, wherein a non-target site and / or a double-stranded oligonucleotide is conjugated to one or more conjugation groups.

[0349] Embodiment 25a: An oligonucleotide agent according to Embodiment 24a, wherein one or more conjugation groups are selected from lipids, fatty acids, fluorescent dyes, ligands, sugars, peptides, and antibodies, and optionally, the conjugation groups are further selected from cell-permeable peptides, polyethylene glycol, alkaloids, tryptamine, benzimidazole, quinolone, amino acids, cholesterol, glucose, and N-acetylgalactosamine.

[0350] Embodiment 26a: An oligonucleotide agent according to any one of Embodiments 1a to 25a, wherein the nucleotide sequence has at least 90% identity with any of the nucleotide sequences described in SEQ ID NO: 1, 3 and 56 to 66.

[0351] Embodiment 27a: An oligonucleotide agent according to any one of Embodiments 1 to 26a, wherein the nucleotide sequence has at least 90% identity with any of the nucleotide sequences described in SEQ ID NO: 2, 4, 57 and 67 to 77.

[0352] Embodiment 28a: An oligonucleotide agent according to any one of Embodiments 1a to 25a, comprising the sequence according to any one of SEQ ID NO: 5 to 40.

[0353] Embodiment 29a: An oligonucleotide agent according to any one of Embodiments 1a to 28a, wherein the non-target site of the oligonucleotide agent improves the stability, bioavailability, in vivo distribution, and / or intracellular uptake of the target oligonucleotide compared to an oligonucleotide agent without a non-target site.

[0354] Embodiment 30a: An oligonucleotide agent according to any one of Embodiments 1a to 29a, wherein the non-target sites of the oligonucleotide agent increase the in vivo distribution of target oligonucleotides in one or more target tissues, compared to an oligonucleotide agent that does not have non-target sites.

[0355] Embodiment 31a: An oligonucleotide agent according to Embodiment 30a, wherein one or more target tissues are selected from the brain, spinal cord, muscle, spleen, lung, heart, liver, bladder, and kidney.

[0356] Embodiment 32a: An oligonucleotide agent according to Embodiment 30a, wherein one or more target tissues are selected from the prefrontal cortex, cerebellum, cerebrum, cervical spine, thoracic spine, lumbar spine, heart, biceps brachii muscle, semitendinosus muscle, platysma muscle, and gluteus maximus muscle.

[0357] Embodiment 33a: A compound to be conjugated to a target oligonucleotide, the compound comprising one or more structural units covalently bonded in series to form its skeleton, wherein at least two adjacent structural units are linked by phosphorothioate (PS) bonds, and each structural unit is selected from a chemical group, a chemical linker, and a nucleotide.

[0358] Embodiment 34a: A compound according to Embodiment 33a, wherein the chemical linker of the compound is selected from spacer 18 linker, spacer C6 linker, L6, spacer 9 linker, spacer C3 linker, L12(d spacer), spacer C12 linker, spacer L14 linker, spacer L15 linker, spacer L16 linker, C6x1 linker, C6x2 linker, C6x5 linker, C6x7 linker, and any other linker available for spacing between two nucleotides.

[0359] Embodiment 35a: A vector comprising an oligonucleotide agent according to any one of Embodiments 1a to 32a.

[0360] Embodiment 36a: A cell comprising an oligonucleotide agent according to any one of Embodiments 1a to 32a.

[0361] Embodiment 37a: The cells described in Embodiment 36a, wherein the cells are mammalian cells and optionally human cells.

[0362] Embodiment 38a: A cell according to any one of Embodiments 36a to 37a, wherein the cell is a host cell.

[0363] Embodiment 39a: A cell according to any one of Embodiments 36a to 38a, wherein the cell exists in vitro (in a test tube) or in a mammalian body.

[0364] Embodiment 40a: A pharmaceutical composition comprising an oligonucleotide agent according to any one of Embodiments 1a to 32a and / or cells according to any one of Embodiments 36a to 39a.

[0365] Embodiment 41a: A pharmaceutical composition according to Embodiment 40a, wherein the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier selected from aqueous carriers, liposomes or LNPs, polymers, micelles, colloids, metal nanoparticles, nonmetal nanoparticles, bioconjugates, and polypeptides.

[0366] Embodiment 42a: A pharmaceutical composition according to any one of Embodiments 40a to 41a, wherein the pharmaceutical composition inhibits SOD1 gene expression or reduces SOD1 protein.

[0367] Embodiment 43a: A pharmaceutical composition according to any one of Embodiments 40a to 41a, wherein the pharmaceutical composition activates SMN2 gene expression or increases SMN2 protein.

[0368] Embodiment 44a: A kit comprising an oligonucleotide agent according to any one of Embodiments 1a to 32a or a pharmaceutical composition according to any one of Embodiments 40a to 43a.

[0369] Embodiment 45a: A method for inhibiting SOD1 gene expression or reducing SOD1 protein, comprising administering a pharmaceutical composition described in any one of Embodiments 40a to 42a to a subject.

[0370] Embodiment 46a: A method for treating or delaying the onset or progression of amyotrophic lateral sclerosis (ALS), comprising administering a pharmaceutical composition described in any one of Embodiments 40a to 42a to a subject.

[0371] Embodiment 47a: The method according to Embodiment 46a, wherein the subject is sporadic ALS (sALS) or familial ALS (fALS).

[0372] Embodiment 48a: A method for activating SMN2 gene expression or increasing SMN2 protein, comprising administering a pharmaceutical composition described in any one of Embodiments 40a to 41a and 43a to a subject.

[0373] Embodiment 49a: A method for treating or delaying the onset or progression of spinal muscular atrophy (SMA), comprising administering a pharmaceutical composition according to any one of Embodiments 40a to 41a and 43a to a subject.

[0374] Embodiment 50a: A method according to any one of Embodiments 45a to 49a, wherein the non-target site of the oligonucleotide agent improves the stability, bioavailability, in vivo distribution and / or intracellular uptake of the double-stranded oligonucleotide compared to an oligonucleotide agent without a non-target site.

[0375] Embodiment 51a: A method according to any one of Embodiments 45a to 50a, wherein the non-target sites of the oligonucleotide agent increase the in vivo distribution of double-stranded oligonucleotides in one or more target tissues, compared to an oligonucleotide agent without a non-target site.

[0376] Embodiment 52a: A method according to any one of Embodiments 45a to 51a, wherein the non-target site of the oligonucleotide agent increases the in vivo distribution of double-stranded oligonucleotides in two or more target cell types within the tissue, compared to an oligonucleotide agent without a non-target site.

[0377] Embodiment 53a: Use of an oligonucleotide agent according to any one of Embodiments 1a to 32a or a pharmaceutical composition according to any one of Embodiments 40a to 43a in the manufacture of a pharmaceutical product that treats or delays the onset or progression of amyotrophic lateral sclerosis (ALS).

[0378] Embodiment 54a: An oligonucleotide agent according to any one of Embodiments 1a to 32a or a pharmaceutical composition according to any one of Embodiments 40a to 43a, which is used to treat or delay the onset or progression of amyotrophic lateral sclerosis (ALS).

[0379] Embodiment 55a: A kit comprising a container containing an oligonucleotide agent according to any one of Embodiments 1a to 32a. [Examples]

[0380] The following examples are provided to give a complete disclosure and explanation of the manufacturing method and use of the present invention to those skilled in the art, and are not intended to limit the scope of what the inventors consider to be the present invention, nor are they intended to represent all or only experiments. Efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., quantity, temperature, etc.), but some experimental errors and deviations should be explained. Unless otherwise stated, parts are parts by weight, molecular weight is weight-average molecular weight, temperature is Celsius, and pressure is atmospheric pressure or near atmospheric pressure. Standard abbreviations may be used. For example, bp is base pair, kb is kilobase, pl is picoliters, s or sec is seconds, min is minutes, h or hr is hours, aa is amino acid, nt is nucleotide, im is intramuscular, ip is intraperitoneal; sc is subcutaneous; icv or icv or ICV is intracerebral, etc.

[0381] Materials and methods General method Starting materials, reagents, and solvents for organic synthesis were purchased commercially and used as is unless otherwise specified. Reaction products were purified by column chromatography using silica gel (200-300 mesh) and eluted with hexane / ethyl acetate or DCM / MeOH. Thin-layer chromatography (TLC) was performed using pre-coated silica gel GF plates and visualized using KMnO4 staining. 1H-NMR spectra were recorded at 400 MHz or 500 MHz (Varian) using CDCl3 with TMS. Mass spectra (MS) were recorded by ESI or matrix-assisted laser desorption / ionization (MALDI) using LC / MS (Agilent Technologies 1260 Infinity II / 6120 Quadrupole) and time-of-flight mass spectrometer.

[0382] oligonucleotide synthesis (1) Single-strand synthesis The oligonucleotides used were synthesized by solid-phase synthesis using a K&A DNA synthesizer (K&A Laboratories GbR, Schaafheim, Germany). Briefly, during solid-phase synthesis, phosphoramidite monomers (0.1 M in acetonitrile or dichloromethane) containing various linkers and conjugations were sequentially added to a solid support to generate the desired full-length oligonucleotides. Each base addition cycle consisted of four chemical reactions: detritylation, coupling, oxidation / thiolation, and capping.

[0383] Detritylation was performed using 3% dichloroacetic acid (DCA) in DCM for 45 seconds, and capping was performed using 16% N-methylimidazole in THF (CAP A) and THF:acetic anhydride:2,6-lutidine (80:10:10, v / v / v) (CAP B) for 20 seconds. Sulfidation was performed using a 0.1 M solution of xanthan hydride in pyridine / ACN (50:50, v / v) for 3 minutes. Oxidation was performed using 0.02 M iodine in THF:pyridine:water (70:20:10, v / v / v) for 60 seconds. The coupling time for all phosphoramidites was 360 seconds.

[0384] Deprotection Step I (Base Deprotection): After the synthesis was complete, the solid support was transferred to a screw-capped microcentrifuge tube. At a 1 μM synthesis scale, a mixture of 33% methylamine in ethanol and 1 ml of ammonium hydroxide was added. The tube containing the solid support was then heated in an oven at 60°C to 65°C for 15 minutes and cooled to room temperature. The cleavage solution was collected and evaporated to dryness using Speedvac.

[0385] Deprotection Step II (Removal of 2'-TBDMS Group): If 2'-TBDMS groups remained in the crude RNA oligonucleotide, it was dissolved in 0.1 ml of DMSO. After adding 1 ml of triethylamine 3HF, the tube was capped and the mixture was shaken vigorously to completely dissolve. The bottle was heated in an oven at 60°C to 65°C for 3 to 3.5 hours. The tube was removed from the oven and allowed to cool to room temperature. The solution containing the completely desilylated oligonucleotide was cooled on dry ice. 2 ml of ice-cold n-butanol (-20°C) was carefully added in 0.5 ml increments to precipitate the oligonucleotide. The precipitate was filtered, washed with 1 ml of ice-cold n-butanol, and then dissolved in 2 M TEAA (triethylammonium acetate). Next, the crude oligonucleotide was purified by exchange (IEX) HPLC using a source 15Q column. The purity of the fraction was determined by Column DNA Pac electrophoresis mobility shift assay (EMSA). Analysis was performed by ion exchange (IEX) HPLC using PA100. After desalting the purified single-chain solution, two complementary single-chain oligonucleotides were annealed to create double-chain oligonucleotides, which were then freeze-dried to obtain a powder.

[0386] (2) Single-strand purification Oligonucleotide purification was performed using an AKTA explorer 10 equipped with a Source 15Q 4.6 / 100 PE column under the following conditions: Buffer A: (10 mM Tris-HCl, 1 mM EDTA, pH 7.5), Buffer B: (10 mM Tris-HCl, 1 mM EDTA, 2M NaCl, pH 7.5), Gradient: The concentration of Buffer B was changed from 10% to 60% over 25 minutes at a flow rate of 1 ml / min. Pure oligonucleotides were recovered and desalted using a HiPrep 26 / 10 desalting column.

[0387] (3) Annealing for double helix formation In the case of double-chain proteins, a desalted and purified single-chain solution was prepared, and then equal volumes of sense and antisense chains were mixed in a tube at equimolar concentrations. The tube was placed in a 95°C heat block for 5 minutes, cooled to room temperature, and then freeze-dried to obtain a powder.

[0388] These linker components referred to in this application can also be used as amidites according to the oligonucleotide synthesis protocol.

[0389] RP-HPLC and ESI-MS To confirm the purity of the oligonucleotides, they were analyzed using reverse-phase chromatography (i.e., RP-HPLC) with acetonitrile application and a detection wavelength of 260 nm (Waters XBridge oligonucleotide BEH C18 130A). Electrospray ionization mass spectrometry (ESI-MS) was performed in negative ion mode on desalted oligonucleotides suspended in water / acetonitrile (50:50) containing 1% (vol / vol) triethylamine.

[0390] Electrophoretic mobility shift assay (EMSA) To evaluate the in vitro protein-binding activity of cODV oligonucleotides, oligonucleotides were diluted to 1 μM using 1×PBS. 10 μL of the diluted oligonucleotide was then mixed with an equal volume of plasma from C57BL / 6J mice (Code ID: 201, Beijing Vital River Laboratory Animal Technology Co., Ltd.) to a final concentration of 0.5 μM (total volume 20 μL). Each sample was centrifuged at 1000 g for 30 seconds and incubated at 37°C for 1 hour. Subsequently, 2 μL of 10× loading buffer (Code No. 9157, TaKaRa, Japan) was added to each mixed sample to prepare a 22 μL stock solution. 10 μL of the stock solution was loaded into wells of a 4% agarose gel and separated by electrophoresis at 120 V for 60 minutes. The same volume of oligonucleotide (unmixed with plasma) diluted in PBS was loaded into adjacent wells and used as an input control and size reference. Mouse plasma (unmixed with oligonucleotide) was used as a negative control. After electrophoresis, the gel was imaged using the ChemiDoc MP system, and the intensity of each oligonucleotide band was quantified. Oligonucleotide bands in the plasma mixed sample that showed the same electrophoretic velocity as the input were defined as the unbound fraction. For each sample, the unbound percentage (%) was calculated using the following formula IV: Unbound fraction (%) = Oligonucleotide band intensity (plasma mixed sample) / Oligonucleotide band intensity (input) × 100 (Formula IV)

[0391] C57BL / 6J mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were anesthetized with isoflurane, and their livers were sequentially perfused with initial perfusion reagents and digestion reagents. The livers were transferred to a 10 cm dish and finely digested in culture medium using forceps. The resulting cell suspension was filtered through a 70-75 μm membrane and collected in a 50 mL conical tube. The cells were then centrifuged at 100 × g for 2 minutes at 4°C using a swing-rotor centrifuge. After removing the supernatant, the cells were washed with 20 mL of cold PBS (this procedure was repeated twice). Only cells with a viability of 80% or higher were used in the next assay. The cells were seeded on cell culture plates pre-coated with collagen I, and the assay was started when 90-95% confluence was reached after 4-12 hours.

[0392] Cell culture and processing PMH cells were cultured in modified Willian's Medium E (WME) medium (A12176-01, Gibco, Thermo Fisher Scientific, Carlsbad, CA) and supplemented with 1% insulin (S6955, Selleck, US) and 1% penicillin / streptomycin (Gibco). SK-N-AS cells (Procell, Wuhan, China, Cat# CL-0621) were maintained in DMEM medium (Gibco, Thermo Fisher Scientific, Carlsbad, CA) and supplemented with 10% calf serum (Sigma-Aldrich) and 1% penicillin / streptomycin. T98G cells (Cobioer, Cat# CBP60301) were cultured in modified MEM medium (Gibco, Thermo Fisher Scientific, Carlsbad, CA) and supplemented with 10% calf serum and 1% penicillin / streptomycin. Neuro-2a (N-2a) cells (BNCC338529, Beijing, China) were cultured in EMEM medium (Gibco, Thermo Fisher Scientific, Carlsbad, CA) supplemented with 10% calf serum and 1% penicillin / streptomycin. All cell lines were cultured in a humid atmosphere at 5% CO2 and 37°C. Transfection was performed in growth medium using Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. A mock treatment was performed without the addition of oligonucleotides, and dsCon2 and dsCon2M8 double-stranded controls were used as non-target controls for transfection. In PMH cells, oligonucleotides were added directly to the medium at a final concentration of 1000 nM without the use of additional transfection reagents, and free uptake was performed for 3 days.

[0393] RNA extraction and two-step reverse transcription quantitative PCR (RT-qPCR) (1) RNA extraction and two-step RT-qPCR Total cellular RNA was extracted from the treated cells using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. RNA from animal tissues was separated using the MagPure Total RNA Micro LQ Kit (Magen, R6621, Guangzhou, Guangdong, China) in combination with the auto-pure96 automated extraction system (ALLSHENG, Hangzhou, Zhejiang, China). The obtained RNA (approximately 1 μg) was subjected to a PrimeScript electrophoresis mobility shift assay (EMSA). cDNA was prepared by reverse transcription using an RT reagent kit (with gDNA Eraser, Takara Bio Inc., RR047A, Shiga, Japan). The obtained cDNA was then subjected to a TB Green(R) Premix Ex Taq electrophoresis mobility shift assay (EMSA) on a Roche LightCycler 480 Multiwell Plate 384 (Roche Inc., 4729749001, USA). Amplification was performed using reagent II (Takara Bio Inc., RR820A, Shiga, Japan) and specific primers for target gene amplification. To confirm the specificity of the primers, a melting curve was performed after amplification.

[0394] The reaction conditions were as follows: Reverse transcription reaction (stage 1): 5 minutes at 42°C, 10 seconds at 95°C. PCR reaction (stage 2): 5 seconds at 95°C, 30 seconds at 60°C, 10 seconds at 72°C, repeated for 40 cycles. Furthermore, melting curve analysis (stage 3) was performed. The PCR reaction conditions are shown in Tables 2 and 3. The primer sequences are shown in Table 4. [Table 2]

[0395] [Table 3]

[0396] [Table 4]

[0397] 1) Use of a single reference gene in two-step RT-qPCR To calculate the relative expression level (Erel) of target gene mRNA in dsRNA transfected samples compared with control treatments (Mock, aCSF, or physiological saline), the Ct values ​​of the target gene and internal reference gene were substituted into the following formula V.

number

[0398] Here, CtTm is the Ct value of the target gene in the control treatment sample; CtTs is the Ct value of the target gene in the dsRNA treatment sample; CtRm is the Ct value of the internal reference gene in the control treatment sample; and CtRs is the Ct value of the internal reference gene in the dsRNA treatment sample.

[0399] 2) Use of dual reference genes in two-step RT-qPCR Relative expression level of target gene mRNA in dsRNA transfected samples (E rel To calculate the result by comparing it with a control treatment (Mock, aCSF, or physiological saline), the Ct values ​​of the target gene and two types of internal reference genes were substituted into the following formula VI.

number

[0400] Here, CtT m This is the Ct value of the target gene in the mock-treated sample, CtT s This refers to the Ct value of the target gene and CtR1 in the dsRNA-treated sample. m This is the Ct value of internal reference gene 1 in the control treatment sample, CtR1 s This is the Ct value of internal reference gene 1 in the dsRNA-treated sample, CtR2 mThis is the Ct value of internal reference gene 2 in the control treatment sample, CtR2 s This is the Ct value of internal reference gene 2 in the dsRNA-treated sample.

[0401] Propidium iodide (PI) staining PMH cells were cultured in 96-well plates for 24 hours or 3 days after siRNA treatment. Cells were washed with cold PBS and lysed with 40 μL / well of Cell Lysis Buffer (0.25% Igeal CA-630, 140 mM NaCl, 2 mM DTT, 10 mM Tris, pH 7.4) containing 1.5 M PI. Plates were incubated on ice for 5 minutes before measuring optical density (OD) at excitation wavelength 535 nm and emission wavelength 615 nm using a microplate reader system (Infinite M2000 Pro, TECAN).

[0402] Animal experimentation treatment All animal experimentation procedures were performed by accredited laboratory staff approved by the Institutional Animal Care and Use Committee, using protocols that complied with local and state regulations. C57BL / 6 mice (4-5 weeks old) were purchased from JOINN Biologics (Suzhou, Jiangsu Province, China). Sprague-Dawley (SD) rats (6 weeks old) were sourced from the Nantong University Laboratory Animal Center (SCXK2019-0001, Nantong, Jiangsu Province, China) and were SPF (Specific Pathogen Free) rats. SD rats received intravitreous (IVT) administration to the left eye. hSOD1G93A transgenic parent mice (Strain ID #004435) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and imported into China via Nantong University (Jiangsu Province, China). The mice were brought to the animal facility at 6 weeks old and subsequently bred at Nantong University to supply the animals used in this study. For in vivo studies, stock solutions were prepared by dissolving lyophilized oligonucleotides in physiological saline or artificial cerebrospinal fluid (aCSF) immediately before use to dilute them to the specified dosage concentration.

[0403] Intraventricular (ICV) injection Aveltin (1.2%) was prepared fresh and sterilized through a 0.2 micron filter. Mice were injected intraperitoneally (IP) at a dose of 0.30–0.35 ml per 10 g of body weight using a stereotactic device to rapidly induce anesthesia for up to 30 minutes. An incision of approximately 11.5 mm was made in the scalp of the animals, and a 25-gauge needle attached to a Hamilton syringe containing the appropriate siRNA or saRNA preparation was placed at the bregma level. The needle was moved to the appropriate anterior-posterior and medial / lateral coordinates (0.2 mm anterior-posterior, 1 mm medial / lateral). A total of 10 μL was injected into the lateral ventricle at a rate of approximately 1 μl / second. After treatment, the needle was slowly withdrawn and the wound was sutured. Tail vein (IV) administration

[0404] Mice were exposed to an infrared lamp for 2-3 minutes to dilate their veins, then restrained in a restrainer with their tails straightened. The tails were wiped with 75% ethanol, and the needle was inserted 2-4 mm parallel to the tail vein, with the tip of the needle held upwards. The prepared administration solution was slowly injected, and if administered correctly, it should inject without resistance. The recommended dose of the test substance was 200 μg, and the injection rate should not exceed 5 mL / min. After administration, the injection site was firmly compressed with a cotton swab or finger to prevent backflow of the administration solution or blood.

[0405] Intravitreous (IVT) administration SD rats were housed for at least 3 days at the Ractigen animal facility (Nantong, Jiangsu Province, China) before intravitreous (IVT) administration. First, the rats were anesthetized in an induction chamber using 5% isoflurane (100% medical oxygen, 2 L / min) and confirmed to be unresponsive to toe-pinching stimuli. They were then transferred to an experimental operating table, fitted with a custom-made face mask, and the IVT administration procedure was performed while continuously infusing **2% isoflurane (100% medical oxygen, 1.5 L / min)**. Before IVT administration, 0.5% alcaine was instilled into the injection eye (left eye) to provide local anesthesia. Next, anterior chamber paracentesis was performed using a 30G needle, and approximately 5 μL of aqueous humor was drained. The compound corresponding to each group was dissolved in 4 μL of physiological saline and packed into a 30G needle. IVT administration involved inserting the needle into the vitreous cavity at a 45° angle through the sclera, injecting it into the posterior ventricle, and holding it for 5 seconds to prevent leakage. After administration, antibiotic eye drops were administered to prevent post-IVT infection.

[0406] statistical analysis Differences in continuous variables between groups were evaluated using Dunnett's multiple comparison test with one-way ANOVA. A p-value less than 0.05 was considered statistically significant. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0407] Example 1: Design of various chain-type oligonucleotide delivery vehicle (cODV) structures Multiple cODV structures were designed and conjugated to double-stranded siRNA (siSOD1, RD-12556) via different linker designs. As a result, 35 cODV-siRNA variants were generated (RD-12559, RD-13592, RD-13593, RD-13594, RD-13595, RD-13596, RD-13597, RD-13598, RD-13599, RD-13600, RD-13601, RD-13602, RD-13603, RD-13604, RD-13605, RD-13606). (RD-13607, RD-13608, RD-13609, RD-13610, RD-13611, RD-13612, RD-13613, RD-13614, RD-13615, RD-13616, RD-13617, RD-13618, RD-13619, RD-13623, RD-13624, RD-13625, RD-14794, RD-13184, and RD-13185). A list of these designs and cODV-siRNA variants is shown in Table 5. "No linker" indicates that the siRNA is not conjugated with a non-target site. All single-stranded oligonucleotide sequences with chemical modifications including nucleate analogs, backbone substitutions, linkers, and non-target sites were synthesized as single molecules on a solid support. Next, cODV double-stranded oligonucleotides were synthesized by annealing complementary single-stranded oligonucleotides. These compounds were chemically synthesized using the methods described in the "Materials and Methods" section. [Table 5] JPEG2026514403000015.jpg249170JPEG2026514403000016.jpg249170JPEG20265144030 00017.jpg249170JPEG2026514403000018.jpg249170JPEG2026514403000019.jpg249170

[0408] Example 2: Evaluation of the protein-binding ability of cODV double-stranded bodies by EMSA The self-delivery capability of cODV-siRNA was hypothesized to be due to increased protein binding ability conferred by the linker and / or the number of linker components contained within the structure. To test this hypothesis, each cODV-siRNA was mixed with C57BL / 6J mouse plasma (primarily containing albumin) and prepared to a final concentration of 0.5 μM, then incubated at 37°C for 1 hour (see the "Materials and Methods" section for details). Separation was then performed by 4% agarose gel electrophoresis, and changes in mobility shift were quantified. Gel separation was also performed on the same compounds unmixed with plasma and used as an input control. RD-11810 was used as a double-strand control for double-stranded siRNA without cODV binding. For each compound, the oligonucleotide band intensity was quantified using the ChemiDoc MP system, and the proportion of the unbound protein fraction of cODV-siRNA was calculated as the ratio of the band intensity of the free oligonucleotide band in the plasma-mixed sample to the band intensity in the input sample. A lower unbound percentage indicates higher protein binding ability. The calculated unbound fraction percentages are summarized in Table 6, using the value for RD-11810 (100%), a cODV-unbound control, as the baseline. As shown in Table 6, all cODV-siRNA structures showed a reduced unbound fraction compared to the siRNA double-stranded body without a cODV structure (RD-11810), indicating that conjugation to non-target sites improved the protein binding ability of the cODV-siRNA structure. The reduction in the unbound fraction ranged from 92% for RD-13592 to the lowest at 22% for RD-13619.

[0409] [Table 6]

[0410] Example 3: In vitro delivery activity and cytotoxicity of cODV-siRNA in PMH cells To evaluate the in vitro delivery activity of cODV-siRNA, PMH cells were transfected with specified cODV-siRNAs (see Table 5) at 0.1 nM for 24 hours using RNAiMAX. dsCon2 and RD-12559 were used as a non-targeted double-strand control and a positive control with known knockdown activity, respectively. Sod1 mRNA levels were quantified by two-step RT-qPCR using gene-specific primer sets. As shown in Figure 1A, cODV-siRNA strongly knocked down Sod1 mRNA expression after 24 hours of treatment compared to the positive control RD-12559 (dotted line). Table 7 summarizes the Sod1 mRNA knockdown levels after cODV-siRNA treatment in PMH cells.

[0411] [Table 7]

[0412] The cytotoxicity of 31 cODV compounds was evaluated by PI staining 24 hours after treatment. As shown in Figure 1B, all siRNAs showed minimal changes in cell viability at 0.1 nM, with the exception of seven cODV-siRNAs (RD-13616, RD-13617, RD-13618, RD-13619, RD-13623, RD-13624, and RD-13625) which showed a slight decrease in cell viability, indicating that these cODV designs exhibit minimal in vitro cytotoxicity.

[0413] To further evaluate the in vitro delivery activity of the newly designed cODV-siRNA, a free uptake assay was performed in which cODV-siRNA (see Table 5) was directly added to freshly isolated PMH cells at 1000 nM for 3 days without the use of transfection reagents. RD-12559 was used as a positive control with known knockdown activity. Sod1 mRNA levels were quantified by two-step RT-qPCR using a gene-specific primer set. As shown in Figure 2A, cODV-siRNA showed a moderate knockdown of Sod1 mRNA expression after a 3-day treatment period compared to the positive control RD-12559 (dotted line). Table 8 summarizes the Sod1 mRNA knockdown levels after cODV-siRNA treatment in PMH cells.

[0414] [Table 8]

[0415] The undesirable cytotoxicity of the specified cODV-siRNAs was assessed by PI staining 72 hours after treatment. As shown in Figure 2B, all cODV-siRNAs showed minimal changes in cell viability, suggesting that these cODV designs exhibit minimal cytotoxicity.

[0416] Example 4: In vivo knockdown activity 7 days after single administration of cODV-siRNA by ICV or IV to C57BL / 6J mice. To test cODV-siRNA activity in the CNS and peripheral tissues, adult C57BL / 6J mice were injected with 200 μg of cODV-siRNA (i.e., RD-13184 and RD-13185) and a double-stranded control siRNA (i.e., RD-12556) via ICV. Physiological saline was used as a solvent control to establish baseline Sod1 mRNA expression levels. Mice were sacrificed 7 days post-administration, and Sod1 mRNA knockdown was quantified in the brain (cerebellum, etc.), spinal cord (cervical, thoracic, lumbar vertebrae, etc.), and peripheral tissues (liver, etc.) by two-step RT-qPCR. Tbp was amplified and used as an internal reference. The results of Sod1 mRNA knockdown after cODV-siRNA treatment in the central nervous system and peripheral tissues are summarized in Table 9.

[0417] To test cODV-siRNA activity in peripheral and skeletal muscle tissues, adult C57BL / 6J mice were intravenously injected with the designated cODV-siRNAs (i.e., RD-13184 and RD-13185) and a double-stranded siRNA control (i.e., RD-12556) at a dose of 20 mg / kg. Physiological saline was used as a solvent control to establish baseline Sod1 mRNA expression levels. Mice were sacrificed 7 days post-administration, and Sod1 mRNA knockdown in peripheral tissues (i.e., liver, spleen, lung, heart) and skeletal muscle (i.e., biceps brachii, semitendinosus, and lamellar muscle) was quantified by two-step RT-qPCR. Tbp was amplified and used as an internal reference. The results of Sod1 mRNA knockdown after cODV-siRNA treatment in peripheral and skeletal muscle tissues are summarized in Tables 10 and 11, respectively. [Table 9]

[0418] [Table 10]

[0419] [Table 11]

[0420] Example 5: In vivo knockdown activity of cODV-siRNA 14 days after single ICV administration in C57BL / 6J mice To evaluate the persistence of cODV-siRNA in central nervous system (CNS) tissues, specified cODV-siRNAs (i.e., RD-13592, RD-13608, RD-13611, RD-13614, and RD-14794) (see Table 5) were administered to adult C57BL / 6J mice at 200 μg via ICV injection. Physiological saline was used as a solvent control to establish baseline Sod1 mRNA expression levels. Mice were sacrificed 14 days post-administration, and Sod1 mRNA knockdown in brain (frontal cortex, cerebellum, cerebrum) and spinal cord (cervical, thoracic, lumbar) tissues was quantified by two-step RT-qPCR. Geometric mean values ​​of Rpl13a and Hprt1 mRNA levels were used as internal references. The results of Sod1 mRNA knockdown after cODV-siRNA treatment in central nervous system tissues are summarized in Table 12. Monitoring of the animals' body weight after treatment revealed no adverse findings, supporting the general tolerability of cODV-siRNA treatment in C57BL / 6J mice (Figure 3).

[0421] To evaluate the persistence of cODV-siRNA in peripheral and skeletal muscle tissues, specified cODV-siRNAs (i.e., RD-13592, RD-13608, RD-13611, RD-13614, and RD-14794) were administered to adult C57BL / 6J mice by IV injection at a dose of 20 mg / kg. Physiological saline was used as a solvent control to establish baseline Sod1 mRNA expression levels. Mice were sacrificed 14 days post-administration, and Sod1 mRNA knockdown in peripheral tissues (i.e., liver, lung, and bladder) and skeletal muscle (i.e., semitendinosus and lamina) tissues was quantified by two-step RT-qPCR. Geometric mean values ​​of Rpl13a and Hprt1 mRNA levels were used as internal references. The results of Sod1 mRNA knockdown after cODV-siRNA treatment in peripheral and skeletal muscle tissues are summarized in Table 13. Post-treatment monitoring of animal body weight revealed no adverse findings supporting the generally well-tolerated nature of cODV-siRNA treatment in C57BL / 6J mice (Figure 4). [Table 12] [Table 13]

[0422] Example 6: In vivo knockdown activity of cODV-siRNA in retinal tissue of SD rats Adult SD rats were administered a 30 μg dose of designated cODV-siRNAs (i.e., RD-13184, RD-13185, RD-13592, RD-13596, RD-13600, RD-13604, RD-13608, RD-13611, RD-13615, RD-13619, and RD-13625) and a double-stranded siRNA control (i.e., RD-12556) via local intravitreal (IVT) injection into the left eye. Physiological saline was used as a solvent control to establish baseline Sod1 mRNA expression levels. Rats were sacrificed 14 days post-administration, and Sod1 knockdown was quantified by two-step RT-qPCR of retinal tissue. The results of Sod1 mRNA knockdown after cODV-siRNA treatment in retinal tissue are shown in Figures 5A and 5B.

[0423] Example 7: Design of a Novel cODV-siRNA Structure A series of cODV structures were designed and conjugated to double-stranded siRNAs via different linker designs. A list of these designs and cODV-siRNA variants is shown in Table 14. “No linker” indicates that the siRNA was not conjugated with a non-target site. All single-stranded oligonucleotide sequences, including those chemically modified with nucleate analogs, skeleton substitutions, linkers, and non-target sites, were synthesized as single molecules on a solid support. Subsequently, cODV double-stranded structures were prepared by annealing complementary single-stranded oligonucleotides. These compounds were chemically synthesized using the methods described in the “Materials and Methods” section. [Table 14] JPEG2026514403000029.jpg248170JPEG2026514403000030.jpg248170JPEG2026514403000031.jpg248170JPEG2026514403000032.jpg248170

[0424] Example 8: In vitro knockdown activity of cODV-siRNA in SK-N-AS cells and T98G cells To evaluate the knockdown activity of cODV-siRNA, specified cODV-siRNAs (i.e., RD-16989, RD-16978, RD-16102, and RD-16979) (see Table 14) were transfected into SK-N-AS cells and T98G cells for 24 hours at specified concentrations (i.e., 0.0001, 0.0002, 0.001, 0.004, 0.016, 0.063, 0.25, and 1). RD-16988 and RD-16990 were transfected as double-stranded controls. Figures 6A and 6B show SOD1 mRNA levels quantified by RT-qPCR in SK-N-AS cells. Figures 6C and 6D show SOD1 mRNA levels quantified by RT-qPCR in T98G cells. For each of the tested cODV-siRNAs that showed dose-dependent knockdown of SOD1 mRNA, EC50 values ​​were extrapolated to define efficacy in relation to maximal activity. EC50 values ​​after cODV-siRNA treatment in SK-N-AS cells and T98G cells were also measured. 50 The values ​​are summarized in Table 15.

[0425] [Table 15]

[0426] Example 9: In vivo knockdown activity of cODV-siRNA in hSOD1G93A mice To evaluate the in vivo knockdown activity of cODV-siRNA, 100 μg of specified cODV-siRNA (i.e., RD-16145 and RD-16978) was injected via ICV to reduce hSOD1 levels. G93A The drug was administered to mice. aCSF was administered as a solvent control group to establish a baseline expression level. hSOD1 G93AMice were sacrificed 14 days after administration. Figure 7 shows the remaining SOD1 mRNA levels quantified in brain (i.e., prefrontal cortex, cerebellum, and cerebrum), spinal cord, and peripheral (i.e., liver) tissues by RT-qPCR. Table 16 shows the SOD1 mRNA levels in brain, spinal cord, and peripheral tissues.

[0427] [Table 16]

[0428] Example 10: In vivo knockdown activity of cODV-siRNA in C57BL / 6J mice To evaluate the in vivo knockdown activity of cODV-siRNA, specified cODV-siRNAs (i.e., RD-16293, RD-16294, RD-16295, and RD-14794) were administered to C57BL / 6J mice at 200 μg via ICV injection. aCSF was administered as a solvent control to establish a baseline expression level. C57BL / 6J mice were euthanized 14 days after administration. Figure 8 shows the remaining Sod1 mRNA levels quantified in brain (i.e., prefrontal cortex, cerebellum, and cerebrum) and spinal cord (i.e., cervical, thoracic, and lumbar vertebrae) tissues by RT-qPCR. Sod1 mRNA levels in brain and spinal cord tissues are shown in Table 17.

[0429] [Table 17]

[0430] Example 11: In vitro knockdown activity of cODV-siRNA in N2a cells To evaluate the knockdown activity of cODV-siRNA, specified cODV-siRNAs (i.e., RD-18148, RD-18150, RD-18151, RD-18152, RD-18153, RD-18154, RD-18155, and RD-18156) were transfected into N2a cells at 0.1 nM for 24 hours. The residual Sod1 mRNA levels were quantified by two-step RT-qPCR. Figure 9 shows the residual Sod1 mRNA levels after cODV-siRNA treatment.

[0431] To further evaluate the knockdown activity of cODV-siRNA, specified cODV-siRNAs (i.e., RD-18151, RD-18317, RD-18318, RD-18319, RD-18320, RD-18321, RD-18322, RD-18323, RD-18153, RD-18325, RD-18326, RD-18327, RD-18329, RD-18150, and RD-18330) were transfected into N2a cells at 0.1 nM for 24 hours. The remaining Sod1 mRNA levels were quantified by two-step RT-qPCR. Figure 10 shows the remaining Sod1 mRNA levels after cODV-siRNA treatment.

[0432] Example 12: Synthesis of a linking compound for linking double-strand oligonucleotides List of compounds: [ka]

[0433] [ka]

[0434] Compounds 1 (Spacer-18 linker), 2 (Spacer-C6 linker), 3 (L6), 4 (Spacer-9 linker), 5 (Spacer-C3 linker), 6 (d spacer), and 7 (Spacer-C12 linker) are commercially available. Spacer-18 (HR-00214005), Spacer-C6 linker (HR-00214019), Spacer-9 linker (HR-00214009), Spacer-C3 linker (HR-00214004), d spacer (HR-00206013), and Spacer-C12 linker (HR-00214022) were purchased from Wuhu Huaren Technology Co., Ltd. (Anhui, China). The L6 linker was purchased from Hongene Biotech (Shanghai, China). Compounds 8 (spacer-L14 linker), 9 (spacer-L15 linker), 10 (spacer-L16 linker), 11 (C6x1 linker), 12 (C6x2 linker), 13 (C6x5 linker), 14 (C6x7 linker), L20 (L20 linker), and L42 (L42 linker) were synthesized using the following procedure. All of these compounds were used as monomers / spacers in oligonucleotide synthesis and are shown in Table 1.

[0435] 1. Synthesis of Compound 8 In this example, compound 8 was prepared by the following procedure. [ka]

[0436] (1) Preparation of compound 16 from the starting compound ((1r,4r)-cyclohexane-1,4-diyl)dimethanol 15. [ka]

[0437] Under a nitrogen atmosphere, DMTrCl (23.48 g, 69.3 mmol, 1.0 eq) was slowly added to a solution of ((1r,4r)-cyclohexane-1,4-diyl)dimethanol 15 (10 g, 69.3 mmol, 1.0 eq) in anhydrous pyridine (200 mL). The reaction mixture was stirred at room temperature for 6 hours, then concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-3% MeOH / DCM) to obtain compound 16 (12.1 g, yield 39%) as a yellow oil. This product was characterized by mass spectrometry and 1H NMR. MW calc.: 446.25; MW Found: 303.09 [DMT] - , 144.15 [DMT off + H] + . 1 H NMR (400 MHz, CDCl3) δ 7.49 (d, J = 7.4 Hz, 2H), 7.37 (t, J= 6.0 Hz, 4H), 7.34 - 7.31 (m, 2H), 7.23 (dd, J = 4.7, 2.4 Hz, 1H), 6.88 - 6.85 (m, 4H), 3.83 (s, 6H), 3.53 - 3.49 (m, 2H), 2.93 (dd, J = 6.2, 3.8 Hz, 2H), 1.91 (dd, J = 13.0, 5.0 Hz, 4H), 1.63 (d, J = 3.3 Hz, 1H), 1.43 (s, 1H), 1.03 (dd, J = 12.7, 7.9 Hz, 4H).

[0438] (2) Preparation of compound 8 from compound 16. [ka]

[0439] Under a nitrogen atmosphere, compound 16 (3.4 g, 7.65 mmol, 1.0 eq) and diisopropylammonium tetrazolide (2.6 g, 15.3 mmol, 2.0 eq) were dissolved in anhydrous dichloromethane (DCM, 30 mL). 3-((bis(diisopropylamino)phosphanyl)oxy)propanitrile (4.6 g, 15.3 mmol, 2.0 eq.) was added at room temperature. The reaction mixture was stirred for 3 hours. The mixture was extracted twice with DCM, washed with brine, and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: siRNA 1-20%, Et3N 1%) to obtain compound 8 (3.65 g, yield 75%) as a yellow oil. This product was characterized by mass spectrometry and 1H NMR. MW calc.: 646.35; MW Found: 303.09 [DMT] - , 302.36 [DMT and one isopropyl off] + . 1 H NMR (400 MHz, CDCl3) δ 7.47 (d, J = 7.3 Hz, 2H), 7.35 (t, J= 6.0 Hz, 4H), 7.30 (d, J = 8.4 Hz, 2H), 7.24 - 7.20 (m, 1H), 6.87 - 6.83 (m, 4H), 3.94 - 3.83 (m, 2H), 3.82 (s, 6H), 3.64 (ddt, J = 13.6, 10.1, 6.8 Hz, 2H), 3.52 (dt, J = 9.7, 7.4 Hz, 1H), 3.43 (dt, J = 9.9, 7.1 Hz, 1H), 2.91 (d, J = 6.3 Hz, 2H), 2.67 (t, J = 6.5 Hz, 2H), 1.88 (dd, J = 24.8, 6.2 Hz, 4H), 1.64 - 1.55 (m, 2H), 1.22 (dd, J= 6.8, 3.2 Hz, 12H), 1.02 (t, J = 10.4 Hz, 4H). 2. Synthesis of Compound 9

[0440] In this example, compound 9 was prepared by the following procedure. [ka]

[0441] (1) Preparation of compound 18 from the starting compound 2,2'-(1,4-phenylene)bis(ethane-1-ol)17. [ka]

[0442] Under a nitrogen atmosphere, DMTrCl (6.1 g, 18.0 mmol, 1.0 eq) was slowly added to a solution of 2,2'-(1,4-phenylene)bis(ethane-1-ol) 17 (3 g, 18.0 mmol, 1.0 eq) in anhydrous pyridine (50 mL). The reaction mixture was stirred at room temperature for 6 hours, then concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-3% MeOH / DCM) to obtain compound 18 (3.74 g, 44% yield) as a yellow oil. This product was characterized by mass spectrometry and 1H NMR. MW calc.: 468.23; MW Found: 303.16 [DMT] - , 491.31 [M + Na] + . 1 1H NMR (400 MHz, CDCl3) δ 7.42 (dd, J = 5.3, 3.3 Hz, 2H), 7.33 - 7.29 (m, 4H), 7.28 (d, J = 7.8 Hz, 2H), 7.22 (s, 1H), 7.18 (s, 4H), 6.85 - 6.81 (m, 4H), 3.88 (dd, J = 8.3, 4.8 Hz, 2H), 3.81 (s, 6H), 3.31 (t, J = 7.0 Hz, 2H), 2.93 - 2.87 (m, 4H).

[0443] (2) Preparation of compound 9 from compound 18. [ka]

[0444] Under a nitrogen atmosphere, compound 18 (884 mg, 1.89 mmol, 1.0 eq) and diisopropylammonium tetrazolide (647 mg, 3.78 mmol, 2.0 eq) were dissolved in anhydrous DCM (10 mL) and 3-((bis(diisopropylamino)phosphanyl)oxy)propanitrile (1.14 g, 3.78 mmol, 2.0 eq) at room temperature. The reaction mixture was stirred for 3 hours. The mixture was extracted twice with DCM, washed with brine, and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: siRNA / Hexane 1-30%, Et3N 1%) to obtain compound 9 (292 mg, yield 23%) as a yellow oil. This product was characterized by mass spectrometry and 1H NMR. MW calc.: 668.34; MW Found: 303.11 [DMT] - . 1 H NMR (400 MHz, CDCl3) δ 7.41 - 7.38 (m, 2H), 7.29 (d, J = 7.9 Hz, 6H), 7.23 (d, J = 7.1 Hz, 1H), 7.18 - 7.14 (m, 4H), 6.85 - 6.80 (m, 4H), 3.96 - 3.82 (m, 2H), 3.81 (d, J= 6.8 Hz, 6H), 3.78 - 3.72 (m, 2H), 3.65 - 3.55 (m, 2H), 3.29 (t, J = 7.0 Hz, 2H), 2.92 (dt, J = 16.8, 7.1 Hz, 4H), 2.52 (td, J = 6.5, 2.9 Hz, 2H), 1.18 (dd, J = 14.4, 6.8 Hz, 12H).

[0445] 3. Synthesis of Compound 10 In this example, compound 10 was prepared by the following procedure. [ka]

[0446] (1) Preparation of compound 20 by reduction of the starting compound 2,2'-(cyclohexane-1,1-diyl)diacetic acid 19. [ka]

[0447] Under a nitrogen atmosphere and in an ice bath, compound 19 (10 g, 50 mmol, 1.0 eq) was dissolved in anhydrous THF (200 mL), to which LiAlH4 (5.7 g, 150 mmol, 3.0 eq) was added. The mixture was then transferred to room temperature after 10 minutes and stirred for approximately 1 hour. Next, the reaction mixture was transferred to an ice bath, and saturated potassium sodium tartrate aqueous solution (100 mL) was slowly added. After 30 minutes, the reaction mixture was extracted three times with Et2O, the organic phases were combined and washed with brine, dried over Na2SO4, and concentrated. This product 20 was characterized by mass spectrometry. MW calc.: 172.15; MW Found: 173.22 [M + H]+.

[0448] (2) Preparation of compound 21 from compound 20. [ka]

[0449] Under a nitrogen atmosphere, DMTrCl (4.7 g, 13.92 mmol, 0.8 eq) was slowly added to a solution of compound 20 (3 g, 17.4 mmol, 1.0 eq) in anhydrous pyridine (50 mL). The reaction mixture was stirred at room temperature for 6 hours and then concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-3% MeOH / DCM) to obtain compound 21 (3.0 g, yield 36%) as a yellow oil. This product was characterized by mass spectrometry and 1H NMR. MW calc.: 474.28; MW Found: 303.12 [DMT] - . 1H NMR (400 MHz, CDCl3) δ 7.46 - 7.41 (m, 2H), 7.35 - 7.30 (m, 4H), 7.28 (d, J = 2.3 Hz, 2H), 7.19 - 7.15 (m, 1H), 6.84 - 6.80 (m, 4H), 3.78 (s, 6H), 3.59 (dd, J = 9.5, 5.8 Hz, 2H), 3.11 (t, J = 7.2 Hz, 2H), 1.45 - 1.34 (m, 10H), 1.20 (dd, J= 6.8, 3.4 Hz, 4H).

[0450] (3) Preparation of compound 10 from compound 21. [ka]

[0451] Under a nitrogen atmosphere, compound 21 (1.3 g, 2.74 mmol, 1.0 eq) and diisopropylammonium tetrazolide (938 mg, 5.48 mmol, 2.0 eq) were dissolved in anhydrous DCM (15 mL) and 3-((bis(diisopropylamino)phosphanyl)oxy)propanitrile (1.65 g, 5.48 mmol, 2.0 eq.) at room temperature. The reaction mixture was stirred for 3 hours. The mixture was extracted twice with DCM, washed with brine, and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: siRNA / Hexane 1-20%, Et3N 1%) to obtain compound 10 (425 mg, yield 23%) as a yellow oil. This product was characterized by mass spectrometry and 1H NMR. MW calc.: 674.38; MW Found: 303.23 [DMT] - , 396.30 [DMT and one isopropyl off + Na] + . 1H NMR (400 MHz, CDCl3) δ 7.45 - 7.40 (m, 2H), 7.34 - 7.29 (m, 4H), 7.29 - 7.25 (m, 2H), 7.22 - 7.16 (m, 1H), 6.86 - 6.79 (m, 4H), 3.79 (s, 6H), 3.78 - 3.71 (m, 2H), 3.65 - 3.50 (m, 4H), 3.10 (t, J = 7.4 Hz, 2H), 2.58 (t, J = 6.6 Hz, 2H), 1.66 (t, J = 7.4 Hz, 2H), 1.51 (t, J= 7.6 Hz, 2H), 1.37 (dd, J = 18.4, 4.5 Hz, 6H), 1.22 (s, 4H), 1.17 (d, J= 6.8 Hz, 6H), 1.13 (d, J = 6.8 Hz, 6H).

[0452] 4. Synthesis of compounds 11 and 12 In this example, compounds 11 and 12 were prepared using the following procedure. [ka]

[0453] (1) Preparation of compounds 23 and 24 from the starting compound (2S,3R,4S,5S)-2-(hydroxymethyl)-5-methoxytetrahydrofuran-3,4-diol 22. [ka]

[0454] Compound 22 (4.8 g, 29 mmol, 1.0 eq) was dissolved in anhydrous DMF (200 mL) under a nitrogen atmosphere. The solution was cooled to 5°C, and NaH (1.54 g, 38.6 mmol, 60% dispersed in mineral oil, 1.3 eq) was added, followed by tetrabutylammonium bromide (TBAB) (1.87 g, 5.8 mmol, 0.2 eq) and 5-chloro-1-pentene (3.89 mL, 36.83 mmol, 1.27 eq). The reaction mixture was stirred overnight at 55°C. The reaction mixture was then filtered and concentrated under reduced pressure. Water (100 mL) was added to the mixture, and it was extracted three times with ethyl acetate. The organic phase was washed three times with saturated lithium chloride aqueous solution. After drying over anhydrous NaSO4, the mixture was concentrated under reduced pressure, and the resulting yellow oil was directly dissolved in anhydrous pyridine (100 mL) under a nitrogen atmosphere. DMTrCl (11.8 g, 34.8 mmol, 1.2 eq) was slowly added. The reaction mixture was stirred at room temperature for 6 hours and then concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-30% siRNA / hexane) to obtain compounds 23 (2.43 g, 15.7% yield) and 24 (1.49 g, 10% yield). These products were characterized by mass spectrometry and 1H NMR.

[0455] Compound 23 MW calc.: 532.25; MW Found: 555.8 [M + Na] + . 1H NMR (400 MHz, CDCl3) δ 7.50 (dd, J= 7.9, 3.8 Hz, 2H), 7.38 (dd, J = 8.2, 3.4 Hz, 4H), 7.27 (d, J = 6.7 Hz, 2H), 7.19 - 7.15 (m, 1H), 6.83 - 6.81 (m, 4H), 4.96 - 4.90 (m, 1H), 4.23 (s, 1H), 4.13 - 4.03 (m, 2H), 3.78 (s, 6H), 3.75 (d, J = 3.3 Hz, 2H), 3.71 - 3.62 (m, 2H), 3.38 (s, 3H), 3.17 (t, J = 5.0 Hz, 1H), 2.34 - 2.30 (m, 2H), 1.87 - 1.82 (m, 2H).

[0456] Compound 24 MW calc.: 532.25; MW Found: 303.4 [DMT] - 253.3 [DMT off + Na] + . 1 H NMR (400 MHz, CDCl3) δ 7.52 - 7.46 (m, 2H), 7.40 - 7.34 (m, 4H), 7.29 - 7.26 (m, 2H), 7.21 (dd, J = 8.3, 3.2 Hz, 1H), 6.83 (t, J = 5.6 Hz, 4H), 4.95 - 4.83 (m, 1H), 4.15 - 4.04 (m, 3H), 3.79 (s, 6H), 3.78 - 3.70 (m, 2H), 3.60 (dt, J = 8.5, 5.7 Hz, 1H), 3.54 - 3.48 (m, 1H), 3.37 (s, 3H), 3.20 - 3.15 (m, 1H), 2.80 - 2.65 (m, 1H), 2.31 - 2.15 (m, 2H), 1.73 (tdd, J = 9.5, 6.5, 2.7 Hz, 2H).

[0457] (2) Compound 23 is prepared from compound 11.

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[0458] Under a nitrogen atmosphere, compound 23 (300 mg, 0.56 mmol, 1.0 eq) and N,N-diisopropylethylamine (DIPEA) (139 μL, 0.84 mmol, 1.5 eq) were dissolved in anhydrous DCM (5 mL) and 3-((chloro(diisopropylamino)phosphanyl)oxy)propanenitrile (199 mg, 0.84 mmol, 1.5 eq) at room temperature. The reaction mixture was stirred for 1 hour. The mixture was extracted twice with DCM, washed with brine, and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: Â1-20%, Et3N 1%) to obtain compound 11 (196 mg, yield 48%) as a colorless oil. This product was analyzed by mass spectrometry. 1 Characterization was performed by 1H NMR. MW calc.: 732.35; MW Found: 733.2 [M+H] + . 1 H NMR (400 MHz, CDCl3) δ 7.53 - 7.48 (m, 2H), 7.38 (dd, J = 8.6, 6.4 Hz, 4H), 7.31 - 7.27 (m, 2H), 7.22 - 7.18 (m, 1H), 6.83 - 6.79 (m, 4H), 4.97 - 4.91 (m, 1H), 4.25 - 4.21 (m, 1H), 3.79 (s, 6H), 3.74 (dd, J = 6.0, 2.8 Hz, 2H), 3.70 - 3.61 (m, 2H), 3.58 - 3.45 (m, 4H), 3.41 (s, 3H), 3.10 (dd, J = 10.1, 5.2 Hz, 1H), 2.62 (dd, J = 6.5, 3.5 Hz, 1H), 2.39 - 2.26 (m, 4H), 1.95 (dd, J = 5.4, 2.6 Hz, 1H), 1.83 - 1.78 (m, 2H), 1.17 - 0.95 (m, 12H).

[0459] (3) Preparation of compound 12 from compound 24. [ka]

[0460] Under a nitrogen atmosphere, a solution of compound 24 (300 mg, 0.56 mmol, 1.0 eq) and DIPEA (139 μL, 0.84 mmol, 1.5 eq) in anhydrous DCM (5 mL) was mixed with 3-((chloro(diisopropylamino)phosphanyl)oxy)propanenitrile (199 mg, 0.84 mmol, 1.5 eq) at room temperature. The reaction mixture was stirred for 1 hour. The mixture was extracted twice with DCM, washed with brine, and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: siRNA / Hexane 1-20%, Et3N 1%) to obtain compound 12 (127 mg, yield 31%) as a colorless oil. This product was analyzed by mass spectrometry. 1 Characterization was performed by 1H NMR. MW calc.: 732.35; MW Found: 733.2 [M+H] + . 1H NMR (400 MHz, CDCl3) δ 7.53 - 7.49 (m, 2H), 7.38 (dd, J = 6.3, 2.6 Hz, 4H), 7.29 (d, J = 7.2 Hz, 2H), 7.22 - 7.19 (m, 1H), 6.85 - 6.81 (m, 4H), 5.02 (s, 1H), 4.20 (d, J = 8.2 Hz, 1H), 4.17 - 3.99 (m, 2H), 3.91 - 3.85 (m, 1H), 3.78 (s, 6H), 3.76 - 3.69 (m, 2H), 3.60 (ddd, J = 11.7, 8.0, 5.0 Hz, 3H), 3.41 (s, 3H), 3.12 - 3.07 (m, 1H), 2.64 (t, J = 6.4 Hz, 2H), 2.38 - 2.29 (m, 1H), 2.23 - 2.01 (m, 2H), 1.87 (t, J = 2.7 Hz, 1H), 1.68 (td, J = 13.6, 6.5 Hz, 2H), 1.25 - 1.14 (m, 12H).

[0461] 5. Synthesis of Compound 13 In this example, compound 13 was prepared by the following procedure. [ka]

[0462] (1) Preparation of compound 26 from the starting compound diethanolamine 25. [ka]

[0463] Under ice bath conditions, anhydrous potassium carbonate (47.1 g, 341 mmol, 5.0 eq) was added to a solution of diethanolamine 25 (7.16 g, 68 mmol, 1.0 eq) in 110 mL of MeCN while vigorously stirring. After stirring for 30 minutes, 5-chloro-1-pentene (7.2 mL, 68 mmol, 1.0 eq) was added dropwise over 5 minutes. The reaction mixture was then stirred at 60°C for 3 days. The resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-8% MeOH / DCM) to obtain compound 26 (2.85 g, yield 24%). This product was analyzed by mass spectrometry. 1 Characterization was performed by 1H NMR. MW calc.: 171.13; MW Found: 172.3 [M + H] + . 1 H NMR (400 MHz, CDCl3) δ 3.64 - 3.55 (m, 4H), 3.13 (s, 1H), 2.67 - 2.52 (m, 6H), 2.24 (td, J = 6.9, 2.6 Hz, 2H), 1.73 - 1.61 (m, 2H).

[0464] (2) Preparation of compound 27 from compound 26. [ka]

[0465] To a solution of compound 26 (2.8 g, 16.35 mmol, 1.0 eq) in DCM (30 mL), Et3N (2.27 mL, 16.35 mmol, 1.0 eq) was added while stirring. Next, DMTrCl (4.43 g, 13.08 mmol, 0.8 eq) was slowly added. The reaction mixture was stirred at room temperature for 6 hours and then concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, gradient eluent: 1-3% MeOH / DCM) to obtain compound 27 (2.98 g, yield 48%). This product was analyzed by mass spectrometry. 1 Characterization was performed by 1H NMR. MW calc.: 473.26; MW Found: 474.2 [M + H] + . 1H NMR (400 MHz, CDCl3) δ 7.44 (d, J = 7.5 Hz, 2H), 7.32 (t, J = 5.9 Hz, 4H), 7.29 - 7.24 (m, 2H), 7.20 (t, J = 7.3 Hz, 1H), 6.83 (t, J= 5.8 Hz, 4H), 3.78 (s, 6H), 3.53 (t, J = 5.2 Hz, 2H), 3.18 (t, J= 5.8 Hz, 2H), 2.69 (t, J = 5.8 Hz, 2H), 2.63 - 2.53 (m, 4H), 2.17 (td, J= 7.0, 2.6 Hz, 2H), 1.90 (t, J = 2.6 Hz, 1H), 1.65 (p, J = 7.0 Hz, 2H).

[0466] (3) Preparation of compound 13 from compound 27. [ka]

[0467] Under a nitrogen atmosphere, a solution of compound 27 (1.23 g, 2.6 mmol, 1.0 eq) and Et3N (1.81 mL, 13 mmol, 5.0 eq) in anhydrous DCM (20 mL) was mixed with 3-((chloro(diisopropylamino)phosphanyl)oxy)propanenitrile (1.85 g, 7.8 mmol, 3.0 eq) at room temperature. The reaction mixture was stirred for 30 minutes. The mixture was extracted twice with DCM, washed with brine, and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: siRNA / Hexane 1-30%, Et3N 1%) to obtain compound 13 (1.12 g, yield 64%) as a yellow oil. This product was analyzed by mass spectrometry. 1 Characterization was performed by 1H NMR. MW calc.: 673.36; MW Found: 633.2 [one isopropyl off + 1H] + . 1H NMR (400 MHz, CDCl3) δ 7.46 - 7.42 (m, 2H), 7.35 - 7.30 (m, 4H), 7.29 - 7.23 (m, 2H), 7.22 - 7.16 (m, 1H), 6.84 - 6.77 (m, 4H), 3.79 (d, J= 5.1 Hz, 1H), 3.78 (s, 6H), 3.62 (qdd, J = 17.0, 9.1, 4.9 Hz, 4H), 3.13 (t, J = 6.4 Hz, 2H), 2.75 - 2.70 (m, 3H), 2.57 (td, J = 6.7, 1.6 Hz, 4H), 2.18 (td, J = 7.1, 2.6 Hz, 2H), 2.04 (s, 1H), 1.87 (t, J= 2.6 Hz, 1H), 1.62 (p, J = 7.1 Hz, 2H), 1.25 (t, J = 7.1 Hz, 1H), 1.16 (dd, J = 11.4, 6.8 Hz, 12H).

[0468] 6. Synthesis of Compound 14 In this example, compound 14 was prepared by the following procedure. [ka]

[0469] (1) Preparation of compound 29 from the starting compound Fmoc-L-hydroxyproline 28. [ka]

[0470] To a solution of Fmoc-L-hydroxyproline 28 (13.3 g, 37.6 mmol, 1.0 eq) in anhydrous THF (250 mL), borane-methyl sulfide complex (8.0 mL, 80 mmol, 2.1 eq at 10 M in THF) was slowly added at room temperature. The reaction mixture was stirred at room temperature for 5 minutes, then heated under reflux for approximately 1 hour. Methanol (15 mL) was carefully added to the reaction mixture, and after refluxing for 15 minutes, the reaction mixture was concentrated under reduced pressure. The crude product was then evaporated three times with methanol (100 mL each). The crude product 29 was used directly in the next step without further purification.

[0471] (2) Preparation of compound 30 from compound 29. [ka]

[0472] To a solution of compound 29 (37.6 mmol, 1.0 eq) in anhydrous pyridine (200 mL), DMTrCl (14 g, 41.4 mmol, 1.1 eq) was slowly added under ice bath. The reaction mixture was stirred overnight under a nitrogen atmosphere and then concentrated under reduced pressure. The crude product was dissolved in dry MeCN (300 mL), and Et3N (15.6 mL, 113 mmol, 3.0 eq) was added to the mixture and heated at 60°C for 4 hours. After concentration under reduced pressure, the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-8% of MeOH / DCM) to obtain the desired product 30 (7.57 g, 48% yield) as a yellow solid. This product was analyzed by mass spectrometry and 1 Characterization was performed by 1H NMR. MW calc.: 419.21; MW Found: 303.2 [DMT] - . 1H NMR (400 MHz, CDCl3) δ 7.41 (d, J = 7.4 Hz, 2H), 7.30 (d, J = 8.8 Hz, 4H), 7.28 - 7.22 (m, 2H), 7.18 (t, J = 7.2 Hz, 1H), 6.80 (d, J= 8.8 Hz, 4H), 4.34 (s, 1H), 3.75 (d, J = 11.1 Hz, 6H), 3.60 (dd, J= 12.7, 6.7 Hz, 1H), 3.10 - 2.92 (m, 5H), 2.86 (d, J = 11.5 Hz, 1H), 1.85 (dd, J = 13.5, 7.1 Hz, 1H), 1.63 (ddd, J = 13.7, 7.9, 5.9 Hz, 1H).

[0473] (3) Preparation of compound 31 from compound 30. [ka]

[0474] Compound 30 (500 mg, 1.19 mmol, 1.0 eq) was dissolved in 5 mL of DCM, and then 4-(((((9H-fluoren-9-yl)methoxy)amino)butanoic acid (465 mg, 1.43 mmol, 1.2 eq), HBTU (903 mg, 2.38 mmol, 2.0 eq), and DIPEA (671 μL, 4.05 mmol, 3.4 eq) were added to the reactant under a nitrogen atmosphere. The reaction mixture was stirred overnight at room temperature. Next, 10 mL of H2O was added to the reactant, and the mixture was extracted with DCM (3 x 10 mL), the organic phases were combined, dried over Na2SO4, and concentrated. The resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-5% MeOH / DCM) to obtain the target product 31 (740 mg, yield 85%) as a yellow solid. This product was analyzed by mass spectrometry and 1 Characterization was performed by 1H NMR. MW calc.: 726.33; MW Found: 425.2 [DMT off + 1H] + . 1 1H NMR (400 MHz, CDCl3) δ 7.74 (d, J = 7.5 Hz, 2H), 7.58 (d, J= 7.4 Hz, 2H), 7.38 (t, J = 7.4 Hz, 2H), 7.32 - 7.23 (m, 7H), 7.24 - 7.10 (m, 4H), 6.88 - 6.72 (m, 4H), 4.52 - 4.25 (m, 4H), 4.18 (t, J = 6.8 Hz, 1H), 3.78 (s, 6H), 3.52 - 3.38 (m, 3H), 3.27 - 3.10 (m, 3H), 2.42 - 2.19 (m, 2H), 2.04 (dd, J = 13.6, 7.5 Hz, 1H), 1.84 (d, J = 6.5 Hz, 1H), 1.69 (ddd, J = 13.6, 9.1, 4.5 Hz, 1H), 1.48 - 1.27 (m, 2H).

[0475] (4) Preparation of compound 14 from compound 31. [ka]

[0476] Under a nitrogen atmosphere, a solution of compound 31 (400 mg, 0.55 mmol, 1.0 eq) and Et3N (382 μL, 2.75 mmol, 5.0 eq) in anhydrous DCM (5 mL) was mixed with 3-((chloro(diisopropylamino)phosphanyl)oxy)propanenitrile (390 mg, 1.65 mmol, 3.0 eq) at room temperature. The reaction mixture was stirred for 1 hour. The mixture was extracted twice with DCM, washed with brine, and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-3% MeOH / DCM, 1% Et3N) to obtain compound 14 (420 mg, yield 82%) as a yellow oil. This product was analyzed by mass spectrometry and 1 Characterization was performed by 1H NMR. MW calc.: 926.44; MW Found: 403.3 [DMT and Fmoc off + 1H] + . 1H NMR (400 MHz, CDCl3) δ 7.75 (d, J = 7.5 Hz, 2H), 7.56 (d, J = 7.4 Hz, 2H), 7.36 (t, J = 7.4 Hz, 2H), 7.31 - 7.22 (m, 7H), 7.25 - 7.12 (m, 4H), 6.88 - 6.70 (m, 4H), 4.53 - 4.27 (m, 4H), 4.17 (t, J = 6.8 Hz, 1H), 3.78 (s, 6H), 3.51 - 3.39 (m, 3H), 3.26 - 3.11 (m, 3H), 3.05 (t, J = 6.4 Hz, 2H), 2.57 (td, J = 6.7, 1.6 Hz, 4H), 2.41 - 2.18 (m, 2H), 2.05 (dd, J = 13.6, 7.5 Hz, 1H), 1.85 (d, J= 6.5 Hz, 1H), 1.67 (ddd, J = 13.6, 9.1, 4.5 Hz, 1H), 1.47 - 1.28 (m, 2H), 1.16 (dd, J = 11.4, 6.8 Hz, 12H).

[0477] 7. Synthesis of compound L20 In this example, the following formula is prepared using Compound L20.

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[0478] (1) Preparation of compound A18

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[0479] Under a nitrogen atmosphere, compound A12 (2.84 g, 27.6 mmol, 1.1 eq) was added to a solution of compound A17 (5 g, 25.12 mmol, 1.0 eq) and K2CO3 (3.8 g, 27.6 mmol, 1.1 eq) in anhydrous DMF (40 mL). The reaction mixture was stirred overnight at room temperature, after which cold water (100 mL) was added. The mixture was extracted three times with ethyl acetate, and the organic phase was washed three times with saturated LiCl aqueous solution and once with brine. The organic phase was then dried over anhydrous Na2SO4 and concentrated under reduced pressure to form compound A18 as a yellow oil, which was used directly in the next step without further purification. This product was characterized by mass spectrometry. MW calc.: 282.12; MW Found: 283.86 [M + H] + .

[0480] (2) Preparation of compound A19 [ka]

[0481] Under a nitrogen atmosphere, DMTrCl (8.51 g, 27.63 mmol, 1.1 eq) was added to a solution of compound A18 (25.12 mmol, 1.0 eq) and triethylamine (TEA, 3.81 g, 37.68 mmol, 1.5 eq) in DCM (50 mL). The reaction mixture was stirred at room temperature for 4 hours and then concentrated under reduced pressure. Next, saturated aqueous NaHCO3 (50 mL) was added, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, washed with brine, dried over Na2SO4, and concentrated. The resulting residue A19 was used directly in the next step without further purification. This product was characterized by mass spectrometry. MW calc.: 584.25; MW.Found: 303.12 [DMT] - .

[0482] (3) Preparation of compound A20 [ka]

[0483] Under ice bath conditions, a solution of compound A19 (25.12 mmol, 1.0 eq) in THF / H2O (9:1, 111 mL) was mixed with HCOONH4 (9.51 g, 150.72 mmol, 6.0 eq) and Zn powder (9.86 g, 150.72 mmol, 6.0 eq). After stirring for 10 minutes, the reaction mixture was removed from the ice bath and stirred overnight at room temperature. The reaction mixture was then filtered and concentrated under reduced pressure. Water (100 mL) was added to the mixture, and the mixture was extracted three times with ethyl acetate. The organic phase was washed once with brine. After drying on anhydrous Na2SO4 and concentration under reduced pressure, the resulting product A20 was used directly in the next step without further purification. Compound A20 was characterized by mass spectrometry. MW calc.: 554.28; MW Found: 253.19 [M - DMT + H] + .

[0484] (4) Preparation of compound A22 [ka]

[0485] Under a nitrogen atmosphere, a solution of compound A20 (25.12 mmol, 1.0 eq) in EtOH (140 mL) was mixed with 5-hydroxypentanal A4 (2.57 g, 25.12 mmol, 1.0 eq) and AcOH (5.8 mL, 100.48 mmol, 4.0 eq). The reaction mixture was stirred at 80°C for 6 hours and then concentrated under reduced pressure. Next, saturated aqueous NaHCO3 (100 mL) was added, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, washed with brine, dried over Na2SO4, and concentrated. The resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-5% MeOH / DCM) to obtain compound A22 (9.4 g, 60% yield). This product was characterized by mass spectrometry and 1H NMR. MW calc.: 636.32; MW Found: 637.51 [M + H] + . 1H NMR (400 MHz, CDCl3) δ 8.41 (d, J = 1.2 Hz, 1H), 7.94 (dd, J = 8.5, 1.5 Hz, 1H), 7.43 - 7.37 (m, 2H), 7.30 - 7.25 (m, 6H), 7.24 (d, J = 1.6 Hz, 1H), 7.22 - 7.16 (m, 1H), 6.83 - 6.78 (m, 4H), 4.08 (dd, J = 10.1, 4.7 Hz, 2H), 3.92 (s, 3H), 3.77 (s, 6H), 3.67 (t, J = 6.1 Hz, 2H), 3.04 (t, J = 6.3 Hz, 2H), 2.88 (t, J = 7.2 Hz, 2H), 1.78 - 1.70 (m, 4H), 1.66 - 1.61 (m, 2H), 1.53 - 1.41 (m, 2H), 1.25 (t, J = 7.1 Hz, 2H).

[0486] (5) Preparation of compound L20 [ka]

[0487] Compound A22 (2 g, 3.14 mmol, 1.0 eq) and diisotropyruammonium tetrazolide (1.61 g, 9.43 mmol, 3.0 eq) were dissolved in anhydrous DCM (20 mL) under a nitrogen atmosphere, and 3-((bis(diisopropylamino)phosphin)oxy)propanitrile (2.85 g, 9.43 mmol, 3.0 eq) was added at room temperature. The reaction mixture was stirred for 6 hours. The mixture was extracted twice with DCM, washed with brine, and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-5% MeOH / DCM, 1% Et3N) to obtain compound L20 (2.4 g, 93% yield). This product was characterized by mass spectrometry and 1H NMR. MW calc.: 836.43; MW Found: 303.70 [DMT] - . 1H NMR (400 MHz, CDCl3) δ 8.41 (d, J = 1.2 Hz, 1H), 7.94 (dd, J = 8.5, 1.5 Hz, 1H), 7.42 - 7.37 (m, 2H), 7.30 - 7.26 (m, 7H), 7.23 - 7.16 (m, 1H), 6.83 - 6.78 (m, 4H), 4.10 (t, J = 7.4 Hz, 2H), 3.93 (s, 3H), 3.78 (s, 6H), 3.64 - 3.49 (m, 5H), 3.04 (t, J = 6.3 Hz, 2H), 2.91 - 2.85 (m, 2H), 2.65 - 2.55 (m, 3H), 2.00 (dt, J = 15.3, 7.6 Hz, 2H), 1.82 - 1.76 (m, 4H), 1.64 (dd, J = 14.2, 6.7 Hz, 2H), 1.51 - 1.44 (m, 2H), 1.17 - 1.13 (m, 12H).

[0488] 8.Synthesis of compound L42 In this example, the following formula is prepared using Compound L42.

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[0489] (1) Preparation of compound A2

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[0490] Under a nitrogen atmosphere, a methanol solution containing iodine (11.3 g of iodine in 50 mL of MeOH, 44.7 mmol, 1.0 eq) was added to a solution of A1 (6 g, 44.7 mmol, 1.0 eq) in dry MeOH (50 mL). The reaction mixture was stirred at room temperature for 15 hours. TLC showed that A1 was completely consumed. The reaction was quenched with a 10% NaHCO3 solution, and DCM (200 mL) was added. The DCM layer was washed twice with H2O (200 mL) and once with brine. The mixture was then concentrated under reduced pressure, and the resulting residue A2 was used directly in the next step without further purification. 1 It was characterized by 1H NMR. 1 H NMR (400 MHz, CDCl3) δ 3.64 (t, J = 6.5 Hz, 4H), 2.73 - 2.65 (m, 4H), 1.75 - 1.65 (m, 4H), 1.59 (dd, J = 14.0, 6.8 Hz, 4H), 1.45 - 1.35 (m, 8H).

[0491] (2) Preparation of compound A3 [ka]

[0492] Under a nitrogen atmosphere, DMTrCl (4.5 g, 13.41 mmol, 0.6 eq) was added to a solution of crude A2 (22.35 mmol, 1.0 eq) in dried pyridine (60 mL). The reaction mixture was stirred at room temperature for 15 hours. TLC showed that A2 was completely consumed. The reaction mixture was concentrated under vacuum to obtain a crude residue, to which DCM (100 mL) was added. The DCM layer was washed twice with H2O (200 mL) and once with brine. The mixture was then concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: ethyl acetate / hexane 1-50%) to obtain compound A3 (5.5 g, yield 43%) as a yellow oil. 1 It was characterized by 1H NMR. 1H NMR (400 MHz, CDCl3) δ 7.43 (d, J = 7.5 Hz, 2H), 7.32 (d, J = 8.8 Hz, 4H), 7.27 (d, J= 8.5 Hz, 2H), 7.20 (t, J = 7.2 Hz, 1H), 6.82 (d, J = 8.8 Hz, 4H), 3.79 (s, 6H), 3.63 (d, J = 2.1 Hz, 2H), 3.04 (t, J = 6.5 Hz, 2H), 2.67 (q, J = 7.4 Hz, 4H), 1.76 - 1.63 (m, 4H), 1.63 - 1.50 (m, 4H), 1.46 - 1.32 (m, 8H).

[0493] (3) Preparation of compound L42 [ka]

[0494] Under a nitrogen atmosphere, a solution of compound A3 (2 g, 3.5 mmol, 1.0 eq) and diisotropylammonium tetrazolide (1.2 g, 7.0 mmol, 2.0 eq) in anhydrous DCM (15 mL) was mixed with 3-((bis(diisopropylamino)phosphin)oxy)propanitrile (2.1 g, 7.0 mmol, 2.0 eq) at room temperature. The reaction mixture was stirred for 6 hours. The mixture was extracted twice with DCM, washed with brine, and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: ethyl acetate / hexane 1-50%, 1% Et3N) to obtain compound L42 (2.0 g, yield 74%) as a yellow oil. 1 It was characterized by 1H NMR. 1H NMR (400 MHz, CDCl3) δ 7.46 - 7.40 (m, 2H), 7.35 - 7.29 (m, 4H), 7.27 (d, J = 6.6 Hz, 2H), 7.19 (t, J = 7.2 Hz, 1H), 6.89 - 6.75 (m, 4H), 3.79 (s, 6H), 3.66 - 3.55 (m, 4H), 3.04 (t, J = 6.5 Hz, 2H), 2.75 - 2.57 (m, 6H), 1.76 - 1.58 (m, 8H), 1.47 - 1.24 (m, 10H), 1.18 (dd, J= 6.7, 4.6 Hz, 12H).

[0495] All the compounds used in this example are shown in Table 1.

[0496] All references cited herein are incorporated by reference to the same extent as individual publications, database entries (e.g., Genbank sequences or GeneID entries), patent applications, or patents are specifically and individually indicated to be incorporated by reference in whole for all purposes. Such references are intended by the applicant in accordance with 37 U.S.R.C. 1.57(b)(1) and relate to individual publications, database entries (e.g., Genbank sequences or GeneID entries), patent applications, or patents, each of which is clearly identified in accordance with 37 U.S.R.C. 1.57(b)(2), even if such reference is not adjacent to a specific reference. Where references are found herein, they do not diminish the general references. References herein are not intended to acknowledge that the references are appropriate prior art, nor do they imply any acknowledgment of the content or date of these publications or documents.

[0497] While this application has been specifically shown and described with reference to preferred embodiments and various alternative embodiments, it will be understood by those skilled in the art that various modifications in form and detail can be made therewith without departing from the spirit and scope of this application.

Claims

1. An oligonucleotide agent comprising a target oligonucleotide conjugated to a non-target site capable of promoting the delivery of the target oligonucleotide, wherein the non-target site comprises one or more covalently bonded structural units in series to form its backbone, with at least two adjacent structural units linked by phosphorothioate (PS) bonds, and each structural unit of the non-target site is selected from a chemical linker and a nucleotide, and the non-target site is It consists of one or more chemical linkers interposed between nucleotides, one or more nucleotides interposed between chemical linkers, a sequence of consecutive nucleotides and a sequence of chemical linkers continuously linked, or a sequence of chemical linkers that does not contain any nucleotides. An oligonucleotide agent in which at least one phosphodiester bond between two adjacent nucleotides, between two adjacent linkers, or between a nucleotide and an adjacent linker is replaced with a phosphorothioate (PS) bond, a mesylphosphoramide bond, or a boranophosphate bond.

2. The aforementioned chemical linker a) L1 or S18 (Spacer-18 Linker) (1,1-Bis(4-methoxyphenyl)-1-phenyl-2,5,8,11,14,17-Hexaoxanonadecane-19-yl(2-cyanoethyl)diisopropylphosphorumidite), b) L4 or C6 (spacer-C6 linker) (6-(bis(4-methoxyphenyl)(phenyl)methoxy)hexyl(2-cyanoethyl)diisopropylphosphorumidite), c), L6(1,1-bis(4-methoxyphenyl)-1-phenyl-2,5,8,11,14-pentaoxahexadecane-16-yl (2-cyanoethyl)diisopropylphosphorumidite), d), L9 or S9 (spacer-9 linker) (2- (2- (2- (bis(4-methoxyphenyl) (phenyl)methoxy)ethoxy)ethoxy)ethyl (2-cyanoethyl)diisopropylphosphorumidite), e), L10 or C3 (spacer-C3 linker) (3-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl(2-cyanoethyl)diisopropylphosphorumidite), f), L12(d spacer) ((2R, 3S)-2- ((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphorumidite), g), L13 or C12 (spacer-C12 linker) (12- (bis(4-methoxyphenyl) (phenyl)methoxy)dodecyl (2-cyanoethyl)diisopropylphosphorumidite), h), L14 (spacer-L14 linker) (((1r, 4r)-4- ((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)cyclohexyl)methyl (d-cyanoethyl)diisopropylphosphorumidite), i) L15 (Spacer-L15 Linker) (4-(2-(Bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)phenethyl(2-cyanoethyl)diisopropylphosphorumidite), j), L16 (spacer-L16 linker) (2- (1- (2- (bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)cyclohexyl)ethyl (2-cyanoethyl)diisopropylphosphorumidite), k), C6x1((2S, 3S, 4S, 5S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-methoxy-4-(pento-4-in-1-yloxy)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphorumidite), l), C6x2((2S, 3S, 4S, 5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2-methoxy-4-(pento-4-in-1-yloxy)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphorumidite), m), C6x5(2-((2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(pento-4-in-1-yl)amino)ethyl(2-cyanoethyl)diisopropylphosphorumidite), n) , C6x7((9H-Fluoren-9-yl)methyl(4-((2S,4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((bis(diisopropylamino)phosphanyl)oxy)pyrrolidine-1-yl)-4-oxobutyl)carbamate), o), L20 methyl 1-(5-(bis(4-methoxyphenyl)(phenyl)methoxy)pentyl)-2-(4-(((2-cyanoethoxy)(diisopropylamino)phosphanyl)oxy)butyl)-1H-benzo[d]imidazole-5-carboxylate, and The oligonucleotide agent according to claim 1, selected from p) and L42 6-((6-(bis(4-methoxyphenyl)(phenyl)methoxy)hexyl)disulfanyl)hexyl(2-cyanoethyl)diisopropylphosphoramidite.

3. The chemical linker is a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, an arylalkyl, an arylalkenyl, an arylalkynyl, a heteroarylalkyl, a heteroarylalkenyl, a heteroarylalkynyl, a heterocyclic alkyl, a heterocyclic alkenyl, a heterocyclic alkynyl, an aryl, a heteroaryl, a heterocyclic, a cycloalkyl, a cycloalkenyl, an alkylarylalkyl, an alkylarylalkenyl, an alkylarylalkynyl, an alkenylarylalkyl, an alkenylarylalkenyl, an alkenylarylalkynyl, an alkenylarylalkynyl, an alkenylarylalkynyl, an alkenylarylalkynyl, an alkenylarylalkynyl, an alkyl hetero A chemical group selected from arylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkylheterocyclicalkyl, alkylheterocyclicalkenyl, alkylheterocyclicalkenyl, alkylheterocyclicalkenyl, alkenylheterocyclicalkenyl, alkenylheterocyclicalkenyl, alkenylheterocyclicalkenyl, alkenylheterocyclicalkenyl, alkenylheterocyclicalkenyl, alkenylheterocyclicalkenyl, alkenylheterocyclicalkenyl, alkylaryl, alkenylaryl, alkenylaryl, alkylheteroaryl, alkenylheteroaryl, and alkynylheteroaryl, where one or more methylene groups are O, S, S(O), SO 2 , N(R') 2 The oligonucleotide agent according to claim 1, wherein the oligonucleotide is interrupted or terminated by C(O), a cleavable linking group, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, or a substituted or unsubstituted heterocyclic, where each R' is independently selected from hydrogen, a substituted or unsubstituted alkyl, aryl, aralkyl, alkylaryl, alkoxy, aryloxy, acyl, or an aliphatic group that may be linear or branched.

4. The oligonucleotide agent according to any one of claims 1 to 3, wherein the non-target site is composed of m identical or different chemical linkers and n identical or different nucleotides, where m is an integer from 1 to 40 and n is an integer from 0 to 30.

5. The aforementioned non-target site is (linker1). x -(linker2) y It consists of a sequence of consecutively linked chemical linkers shown by the formula, where linker1 is the first chemical linker, linker2 is a second chemical linker different from the first chemical linker, and x and y are integers satisfying 0 < x + y < 50. For example, the non-target site is S9、(S9) 2 、(S9) 3 、(S9) 4 、(S9) 5 、(S9) 6 、(S9) 7 、(S9) 8 、(S9) 9 、(S9) 10 、(S9) 11 、(S9) 12 、(S9) 13 、(S9) 14 、(S9) 15 、(S9) 16 、(S9) 17 、(S9) 18 、(S9) 19 、(S9) 20 、(S9) 21 、(S9) 22 、(S9) 23 、(S9) 24 、(S9) 25 、(S9) 26 、(S9) 27 、(S9) 28 、(S9) 29 、 (S9) 30 、 L10、(L10) 2 、(L10) 3 、(L10) 4 、(L10) 5 、(L10) 6 、(L10) 7 、(L10) 8 、(L10) 9 、(L10) 10 、(L10) 11 、(L10) 12 、(L10) 13 、(L10) 14 、(L10) 15 、(L10) 16 、(L10) 17 、(L10) 18 、(L10) 19 、(L10) 20 、(L10) 21 、(L10) 22 、(L10) 23 、(L10) 24 、(L10) 25 、(L10) 26 、(L10) 27 、(L10) 28 、(L10) 29 、(L10) 30 、 L12, (L12)2, (L12)3, (L12)4, (L12)5, (L12)6, (L12)7, (L12)8, (L12)9, (L12)10, (L12)11, (L12)12, (L12)13, (L12)14, (L12)15, (L12)1 6, (L12)17, (L12)18, (L12)19, (L12)20, (L12)21, (L12)22, (L12)23, (L12)24, (L12)25, (L12)26, (L12)27, (L12)28, (L12)29, (L12)30, S9-L10、S9-(L10) 2 、S9-(L10) 3 、S9-(L10) 4 、S9-(L10) 5 、S9-(L10) 6 、S9-(L10) 7 、S9-(L10) 8 、S9-(L10) 9 、S9-(L10) 10 、S9-(L10) 11 、S9-(L10) 12 、S9-(L10) 13 、S9-(L10) 14 、S9-(L10) 15 、S9-(L10) 16 、S9-(L10) 17 、S9-(L10) 18 、S9-(L10) 19 、S9-(L10) 20 、S9-(L10) 21 、S9-(L10) 22 、S9-(L10) 23 、S9-(L10) 24 、S9-(L10) 25 、S9-(L10) 26 、S9-(L10) 27 、S9-(L10) 28 、S9-(L10) 29 、S9-(L10) 30 、 S9-L12、S9-(L12) 2 、S9-(L12) 3 、S9-(L12) 4 、S9-(L12) 5 、S9-(L12) 6 、S9-(L12) 7 、S9-(L12) 8 、S9-(L12) 9 、S9-(L12) 10 、S9-(L12) 11 、S9-(L12) 12 、S9-(L12) 13 、S9-(L12) 14 、S9-(L12) 15 、S9-(L12) 16 、S9-(L12) 17 、S9-(L12) 18 、S9-(L12) 19 、S9-(L12) 20 、S9-(L12) 21 、S9-(L12) 22 、S9-(L12) 23 、S9-(L12) 24 、S9-(L12) 25 、S9-(L12) 26 、S9-(L12) 27 、S9-(L12) 28 、S9-(L12) 29 、S9-(L12) 30 、 L20、(L20) 2 、(L20) 3 、(L20) 4 、(L20) 5 、(L20) 6 、(L20) 7 、(L20) 8 、(L20) 9 、(L20) 10 、(L20) 11 、(L20) 12 、(L20) 13 、(L20) 14 、(L20) 15 、(L20) 16 、(L20) 17 、(L20) 18 、(L20) 19 、(L20) 20 、(L20) 21 、(L20) 22 、(L20) 23 、(L20) 24 、(L20) 25 、(L20) 26 、(L20) 27 、(L20) 28 、(L20) 29 、(L20) 30 、 L42、(L42) 2 (L42) 3 (L42) 4 (L42) 5 (L42) 6 (L42) 7 (L42) 8 (L42) 9 (L42) 10 (L42) 11 (L42) 12 (L42) 13 (L42) 14 (L42) 15 (L42) 16 (L42) 17 (L42) 18 (L42) 19 (L42) 20 (L42) 21 (L42) 22 (L42) 23 (L42) 24 (L42) 25 (L42) 26 (L42) 27 (L42) 28 (L42) 29 (L42) 30 、 L20-L12, L20-(L12) 2 L20-(L12) 3 L20-(L12) 4 L20-(L12) 5 L20-(L12) 6 L20-(L12) 7 L20-(L12) 8 L20-(L12) 9 L20-(L12) 10 L20-(L12) 11 L20-(L12) 12 L20-(L12) 13 L20-(L12) 14 L20-(L12) 15 L20-(L12) 16 L20-(L12) 17 L20-(L12) 18 L20-(L12) 19 L20-(L12) 20 L20-(L12) 21 L20-(L12) 22 L20-(L12) 23 L20-(L12) 24 L20-(L12) 25 L20-(L12) 26 L20-(L12) 27 L20-(L12) 28 L20-(L12) 29 L20-(L12) 30 ,or L42-L12、L42-(L12) 2 、L42-(L12) 3 、L42-(L12) 4 、L42-(L12) 5 、L42-(L12) 6 、L42-(L12) 7 、L42-(L12) 8 、L42-(L12) 9 、L42-(L12) 10 、L42-(L12) 11 、L42-(L12) 12 、L42-(L12) 13 、L42-(L12) 14 、L42-(L12) 15 、L42-(L12) 16 、L42-(L12) 17 、L42-(L12) 18 、L42-(L12) 19 、L42-(L12) 20 、L42-(L12) 21 、L42-(L12) 22 、L42-(L12) 23 、L42-(L12) 24 、L42-(L12) 25 、L42-(L12) 26 、L42-(L12) 27 、L42-(L12) 28 、L42-(L12) 29 、or L42-(L12) 30 、 It consists of one of the following: Hereinafter, at least one phosphodiester bond between two adjacent linkers is substituted with a phosphorothioate (PS) bond, a mesylphosphoramidate bond, or a boranophosphate bond, as described in any one of claims 1 to 4.

6. The oligonucleotide agent according to any one of claims 1 to 5, wherein the non-target site contains 1 to about 50, about 2 to about 48, about 3 to about 46, about 4 to about 44, about 5 to about 42, about 6 to about 40, about 7 to about 38, about 8 to about 36, about 9 to about 34, about 10 to about 32, about 11 to about 30, about 12 to about 28, about 13 to about 26, about 14 to about 24, about 15 to about 22, about 16 to about 20, or about 17 to about 18 phosphorothioate (PS) bonds in its skeleton.

7. The oligonucleotide agent according to any one of claims 1 to 5, wherein the non-target site contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 or more phosphorothioate (PS) bonds in its skeleton.

8. The oligonucleotide agent according to any one of claims 1 to 7, wherein the target oligonucleotide is an antisense oligonucleotide, or a double-stranded oligonucleotide (e.g., siRNA or saRNA) comprising a sense strand and an antisense strand.

9. The oligonucleotide agent according to claim 8, wherein the non-target site is conjugated to the sense strand or antisense strand of the double-stranded oligonucleotide.

10. The oligonucleotide agent according to any one of claims 1 to 9, wherein if a nucleotide is present at the non-target site, the nucleotide is either an unchemically modified nucleotide or at least one nucleotide is a chemically modified nucleotide.

11. The oligonucleotide agent according to any one of claims 1 to 10, wherein all nucleotides of the target oligonucleotide are unchemically modified nucleotides, or at least one nucleotide is chemically modified, or at least one phosphodiester bond between two adjacent nucleotides in the target oligonucleotide is substituted with a phosphorothioate (PS) bond, a mesylphosphoramidate bond, or a boranophosphate bond.

12. The aforementioned chemically modified nucleotide a) Modification of the 2'-OH group of ribose in a nucleotide, b) Modification or non-modification of the base portion on the nucleoside ring in the nucleotide, c) that one nucleotide is a locked nucleic acid or a cross-linked nucleic acid, and d) Each nucleotide is a deoxyribonucleotide (DNA), The oligonucleotide agent according to claim 10 or 11, comprising one or more of the above.

13. The oligonucleotide agent according to claim 12, wherein the chemically modified nucleotide has a 2'-OH ribose modification selected from 2'-fluoro-2'-deoxynucleoside (2'-F) modification, 2'-O-methyl (2'-O-Me) modification, and 2'-O-(2-methoxyethyl) (2'-O-MOE) modification.

14. If a nucleotide is present at the non-target site, the nucleotide is selected from the group consisting of RNA, DNA, cross-linked nucleic acid (BNA), locked nucleic acid (LNA), and peptide nucleic acid (PNA), according to any one of claims 1 to 13.

15. The oligonucleotide agent according to any one of claims 10 or 11, wherein the at least one chemically modified nucleotide is a nucleotide to which a 5'-phosphate group, 5-methylcytosine, or 5'-(E)-vinylphosphonate has been added.

16. The oligonucleotide agent according to any one of claims 1 to 15, wherein the target oligonucleotide and the non-target site are directly conjugated, for example, via a phosphorothioate (PS) bond.

17. The oligonucleotide agent according to claim 16, wherein the terminal unit or internal unit of the non-target site is conjugated to the target oligonucleotide.

18. The oligonucleotide agent according to any one of claims 1 to 17, wherein the non-target site is conjugated to the 3' end, 5' end, both the 3' and 5' ends, or one internal nucleotide of the sense strand or antisense strand of the double-stranded oligonucleotide.

19. The aforementioned non-target site is 1) A single chemical linker, which does not contain nucleotides, is conjugated to the terminal of the target oligonucleotide via a phosphorothioate (PS) bond. 2) A sequence of nucleotide-free, sequentially linked chemical linkers conjugated to one end of the target oligonucleotide, for example, via 1 to 20 deoxyribonucleotides (DNA) of any choice. 3) Two sequences of nucleotide-free, sequentially linked chemical linkers, conjugated to both ends of each target oligonucleotide, for example, via 1 to 20 deoxyribonucleotides (DNA) as optional, and 4) A sequence comprising one or more consecutive nucleotide sequences and a sequence of consecutively linked chemical linkers, wherein the nucleotides are interposed between the chemical linkers (preferably, each nucleotide is interposed between two chemical linkers, or one or more consecutive nucleotide sequences are interposed between chemical linkers). Selected from, Herein, at least one phosphodiester bond between two adjacent nucleotides, between two adjacent linkers, or between a nucleotide and an adjacent linker is substituted with a phosphorothioate (PS) bond, as described in any one of claims 1 to 18.

20. The oligonucleotide agent according to any one of claims 1 to 19, wherein the internal nucleotides of the sense strand or antisense strand of the double-stranded oligonucleotide are substituted with a linking component, and the non-target site is conjugated to the linking component.

21. An oligonucleotide agent according to any one of claims 1 to 20, wherein two or more (e.g., 2 to 10) non-target sites are conjugated to the double-stranded oligonucleotide, or two or more (e.g., 2 to 10) double-stranded oligonucleotides are conjugated to the non-target sites.

22. The oligonucleotide agent according to claim 20, wherein the linking component comprises one or more selected from ethylene glycol chains, alkyl chains, alkenyl chains, alkynyl chains, peptides, carbohydrates, thiol bonds, phosphodiesters, phosphorothioates, phosphoramidates, amides, carbamates, tetrazole bonds, and benzimidazole bonds.

23. The oligonucleotide agent according to any one of claims 1 to 22, wherein the non-target site and / or the double-stranded oligonucleotide is conjugated to one or more conjugation groups.

24. The oligonucleotide agent according to claim 23, wherein the one or more conjugation groups are selected from lipids, fatty acids, fluorescent dyes, ligands, sugars, peptides, and antibodies, and optionally the one or more conjugation groups are selected from cell-permeable peptides, polyethylene glycol, alkaloids, tryptamine, benzimidazole, quinolone, amino acids, cholesterol, glucose, and N-acetylgalactosamine.

25. The oligonucleotide agent according to any one of claims 1 to 24, wherein the nucleotide sequence of the sense strand has at least 90% identity with a nucleotide sequence such as those shown in SEQ ID NO: 1, 3, 56, and 61.

26. The oligonucleotide agent according to any one of claims 1 to 25, wherein the nucleotide sequence of the antisense strand has at least 90% identity with a nucleotide sequence such as those shown in SEQ ID NO: 2, 4, 57, 62, and 69.

27. An oligonucleotide agent according to any one of claims 1 to 24, comprising a sense chain sequence such as those shown in SEQ ID NO: 6-39, 60, 64, 65, 66, 67, or 70-87.

28. The oligonucleotide agent according to any one of claims 1 to 27, wherein, compared to an oligonucleotide agent without the non-target-directing portion, the non-target-directing portion of the oligonucleotide agent improves the stability, bioavailability, in vivo distribution and / or intracellular uptake of the target-directing oligonucleotide.

29. Compared to an oligonucleotide agent that does not have the aforementioned non-target site, the non-target site of the oligonucleotide agent increases the in vivo distribution of the target oligonucleotide in one or more target tissues, according to any one of claims 1 to 28.

30. The oligonucleotide agent according to claim 29, wherein the one or more target tissues are selected from the brain, spinal cord, muscle, spleen, lung, heart, liver, bladder, and kidney.

31. The oligonucleotide agent according to claim 29, wherein the one or more target tissues are selected from the group consisting of the prefrontal cortex, cerebellum, and cerebrum, the cervical vertebrae, thoracic vertebrae, and lumbar vertebrae of the spinal cord, and the heart, biceps brachii muscle, semitendinosus muscle, platysma muscle, and gluteus maximus muscle.

32. A vector comprising an oligonucleotide agent according to any one of claims 1 to 31.

33. A cell comprising an oligonucleotide agent according to any one of claims 1 to 31.

34. The cell according to claim 33, which is a mammalian cell and optionally a human cell.

35. A host cell, as described in claim 33 or 34.

36. A cell according to any one of claims 33 to 35, which exists outside of a living organism or within a mammal.

37. A pharmaceutical composition comprising an oligonucleotide agent according to any one of claims 1 to 30 and / or cells according to any one of claims 33 to 36.

38. The pharmaceutical composition according to claim 37, comprising at least one pharmaceutically acceptable carrier selected from aqueous carriers, liposomes or LNPs, polymers, micelles, colloids, metal nanoparticles, nonmetal nanoparticles, bioconjugates, and polypeptides.

39. The pharmaceutical composition according to claim 37 or 38, which inhibits the expression of the SOD1 gene or reduces the SOD1 protein.

40. A pharmaceutical composition according to claim 37 or 38, which activates the expression of the SMN2 gene or increases the SMN2 protein.

41. A kit comprising an oligonucleotide agent according to any one of claims 1 to 31 or a pharmaceutical composition according to any one of claims 37 to 40.

42. A method for inhibiting the expression of the SOD1 gene or reducing the SOD1 protein, comprising administering a pharmaceutical composition according to any one of claims 37 to 39 to a subject.

43. A method for treating or delaying the onset or progression of amyotrophic lateral sclerosis (ALS) in a subject, comprising administering to the subject a pharmaceutical composition according to any one of claims 37 to 39.

44. The method according to claim 43, wherein the subject is a patient with sporadic ALS (sALS) or familial ALS (fALS).

45. A method for activating the expression of the SMN2 gene or increasing the SMN2 protein, comprising administering to a subject a pharmaceutical composition according to any one of claims 37, 38, and 40.

46. A method for treating or delaying the onset or progression of spinal muscular atrophy (SMA), comprising administering a pharmaceutical composition according to any one of claims 37 to 40 to a subject.

47. The method according to any one of claims 42 to 46, wherein, compared to an oligonucleotide agent that does not have the non-target site, the non-target site of the oligonucleotide agent improves the stability, bioavailability, in vivo distribution and / or intracellular uptake of the double-stranded oligonucleotide.

48. The method according to any one of claims 42 to 47, wherein, compared to an oligonucleotide agent that does not have the aforementioned non-target site, the non-target site of the oligonucleotide agent increases the in vivo distribution of the double-stranded oligonucleotide in one or more target tissues.

49. The method according to any one of claims 42 to 48, wherein, compared to an oligonucleotide agent that does not have the aforementioned non-target site, the non-target site of the oligonucleotide agent increases the in vivo distribution of the double-stranded oligonucleotide in two or more target cells in the tissue.

50. Use of an oligonucleotide agent according to any one of claims 1 to 31 or a pharmaceutical composition according to any one of claims 37 to 40 in the manufacture of a pharmaceutical product for treating or delaying the onset or progression of amyotrophic lateral sclerosis (ALS).

51. An oligonucleotide agent according to any one of claims 1 to 31 or a pharmaceutical composition according to any one of claims 37 to 40, for use in treating or delaying the onset or progression of amyotrophic lateral sclerosis (ALS).

52. A kit comprising a container containing an oligonucleotide agent according to any one of claims 1 to 31.

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