WO2024199445A1

WO2024199445A1 - Vehicles for delivering oligonucleotides and methods of use thereof

Info

Publication number: WO2024199445A1
Application number: PCT/CN2024/084814
Authority: WO
Inventors: Zubao GAN; Longcheng Li; Moorim KANG
Original assignee: Ractigen Therapeutics
Current assignee: Ractigen Therapeutics
Priority date: 2023-03-30
Filing date: 2024-03-29
Publication date: 2024-10-03
Anticipated expiration: 2025-09-30
Also published as: TW202538051A; EP4689116A1; CN121002179A

Abstract

Relates to nucleic acids, specifically as it relates to an oligonucleotide agent comprising a targeting oligonucleotide and a non-targeting moiety that is covalently tethered to the oligonucleotide and pharmaceutical use thereof.

Description

VEHICLES FOR DELIVERING OLIGONUCLEOTIDES AND METHODS OF USE THEREOF

FIELD

The present application relates to the technical field of nucleic acids, specifically as it relates to an oligonucleotide agent comprising a double-stranded RNA (dsRNA, duplex) and a non-targeting moiety that is covalently tethered to the dsRNA and pharmaceutical use thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to the filing date of Provisional Patent Application Serial No. PCT/CN2023/085065 filed March 30, 2023, the disclosure of which application is herein incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in computer readable format and is hereby incorporated by reference in its entirety.

BACKGROUND

Oligonucleotides are an innovative class of therapeutics that are garnering significant attention in the scientific community due to their versatility in addressing a multitude of diseases via a variety of mechanisms of action (MOA) . This category of therapeutics is bifurcated into single-stranded antisense oligonucleotides (ASOs) and double-stranded RNA molecules (dsRNAs) , each with distinct applications but in similar chemical composition.

The dsRNA molecules, which include small interfering RNA (siRNA) and small activating RNA (saRNA) , are exemplary of the analogous molecules within the dsRNA subclass. These molecules exhibit chemical similarities in terms of their structural framework, length, the strategies employed for chemical modification, and the configuration of their termini. Functionally, both siRNA and saRNA undergo a series of biological processes necessary for their MOA, which includes cellular internalization, evasion from endosomal or lysosomal pathways, and subsequent interaction with argonaute (AGO) proteins within the cytoplasm. This interaction leads to the cleavage and release of the non-functional "passenger" strand, while the functional "guide" strand remains bound to AGO to exert its MOA. This shared biological process suggests that delivery technologies for siRNA and saRNA may be interchangeable, offering a unified approach to the delivery of these therapeutic entities.

In comparison to ASOs, which can independently engage with cellular uptake mechanisms through targeted chemical modifications, dsRNA molecules like siRNA and saRNA require the support of drug delivery systems (DDS) to effectively enter target cells and elicit their therapeutic effects. Presently, a variety of dsRNA DDS platforms have been engineered to deliver dsRNAs, including polymer-based, lipid-based, and conjugate-based systems. However, the efficacy of these systems in reaching specific target organs, tissues, and cells is still limited, highlighting the necessity for innovative and advanced delivery solutions.

SUMMARY

The present application provides a novel oligonucleotide agent or oligonucleotide agent conjugate comprising a targeting oligonucleotide (e.g., a duplex RNA) and a non-targeting moiety that is conjugated to the targeting oligonucleotide. The oligonucleotide agent constitutes a system with “self-delivering” properties. The present inventors found surprisingly that, when the non-targeting moiety as disclosed herein is conjugated to the targeting oligonucleotide (e.g., dsRNA, including siRNA or saRNA) , favorable biodistribution and in vivo activity are obtained for local administration to selected tissues and systemic delivery across several organs/tissues including the liver, muscle, lung, kidney, bladder, brain, spinal cord, heart, eye, spleen, etc.

In some aspects, provided herein is an oligonucleotide agent comprising: (a) a double-stranded oligonucleotide comprising a sense strand and an antisense strand, wherein the antisense strand has complementarity to a target nucleic acid; and (b) a non-targeting moiety that comprises one or more components that are covalently linked by at least one phosphorothioate bond, wherein the double-stranded oligonucleotide is conjugated to the non-targeting moiety to form the oligonucleotide agent.

In certain embodiments, the double-stranded oligonucleotide is a siRNA or a saRNA.

The non-targeting moiety as disclosed herein may comprise one or more of the same or different components (or “units” ) that are covalently linked in tandem that forms a backbone of the non-targeting moiety. The non-targeting moiety may have a linear chain or branched chains. In some embodiments, the components are selected from substituted or unsubstituted alkyl, aralkyl, alkoxy, aryloxy, 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, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, wherein one or more methylenes are interrupted or terminated by O, S, S (O) , SO₂, N (R') ₂, C (O) , cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocyclic, and wherein each R’ is independently selected from hydrogen, substituted or unsubstituted alkyl, aryl, aralkyl, alkylaryl, alkoxy, aryloxy, acyl or aliphatic, which may be linear, branched, cyclic, polycyclic, unsaturated, hydroxylated, carbonylated, phosphorylated, and/or sulfured.

In some embodiments, at least one of the components or units is a nucleotide. The components of the non-targeting moiety may be natural or chemically modified. In some embodiments, the nucleotide comprised in the non-targeting moiety is chemically modified, and is selected from a group comprising 2′-fluoro-2′-deoxynucleoside (2′-F) , a 2′-O-methyl (2′-O-Me) , a 2′-O- (2-methoxyethyl) (2′-O-MOE) , locked nucleic acid (LNA) , bridged nucleic acid (BNA) , peptide nucleic acid (PNA) , 5’- (E) -vinylphosphonate, 5-methyl cytosine. In some embodiments, the non-targeting moiety does not comprise any nucleotide component. Specifically, the non-targeting moiety may comprise: (a) consecutively linked components, wherein none of the components is a nucleotide; (b) one or more nucleotides interspersed in components other than nucleotides; (c) one or more components other than nucleotides interspersed in nucleotides; or (d) a consecutive sequence of nucleotides and a consecutively linked sequence of components other than nucleotides.

In some embodiments, the one or more components or units of the non-targeting moiety 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) diisopropylphosphoramidite) ;

b) L4 or C6 (spacer-C6 linker) (6- (bis (4-methoxyphenyl) (phenyl) methoxy) hexyl (2-cyanoethyl) diisopropylphosphoramidite) ;

c) L6 (1, 1-bis (4-methoxyphenyl) -1-phenyl-2, 5, 8, 11, 14-pentaoxahexadecan-16-yl (2-cyanoethyl) diisopropylphosphoramidite) ;

d) L9 or S9 (spacer-9 linker) (2- (2- (2- (bis (4-methoxyphenyl) (phenyl) methoxy) ethoxy) ethoxy) ethyl (2-cyanoethyl) diisopropylphosphoramidite) ;

e) L10 or C3 (spacer-C3 linker) (3- (bis (4-methoxyphenyl) (phenyl) methoxy) propyl (2-cyanoethyl) diisopropylphosphoramidite) ;

f) L12 (d spacer) ( (2R, 3S) -2- ( (bis (4-methoxyphenyl) (phenyl) methoxy) methyl) tetrahydrofuranpar-3-yl (2-cyanoethyl) diisopropylphosphoramidite) ;

g) L13 or C12 (spacer-C12 linker) (12- (bis (4-methoxyphenyl) (phenyl) methoxy) dodecyl (2-cyanoethyl) diisopropylphosphoramidite) ;

h) L14 (spacer-L14 linker) ( ( (1r, 4r) -4- ( (bis (4-methoxyphenyl) (phenyl) methoxy) methyl) cyclohexyl) methyl (2-cyanoethyl) diisopropylphosphoramidite) ;

i) L15 (spacer-L15 linker) (4- (2- (bis (4-methoxyphenyl) (phenyl) methoxy) ethyl) phenethyl (2-cyanoethyl) diisopropylphosphoramidite) ;

j) L16 (spacer-L16 linker) (2- (1- (2- (bis (4-methoxyphenyl) (phenyl) methoxy) ethyl) cyclohexyl) ethyl (2-cyanoethyl) diisopropylphosphoramidite) ;

k) C6x1 ( (2S, 3S, 4S, 5S) -2- ( (bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -5-methoxy-4- (pent-4-yn-1-yloxy) tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite) ;

l) C6x2 ( (2S, 3S, 4S, 5S) -5- ( (bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -2-methoxy-4- (pent-4-yn-1-yloxy) tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite) ;

m) C6x5 (2- ( (2- (bis (4-methoxyphenyl) (phenyl) methoxy) ethyl) (pent-4-yn-1-yl) amino) ethyl (2-cyanoethyl) diisopropylphosphoramidite) ;

n) C6x7 ( (9H-fluoren-9-yl) methyl (4- ( (2S, 4R) -2- ( (bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -4- ( (bis (diisopropylamino) phosphanyl) oxy) pyrrolidin-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) diisopropylphosphoramidite.

In some embodiments, the one or more components or units of the non-targeting moiety comprises at least one phosphorothioate modification in the backbone. The components or units of the non-targeting moiety may be identical or different. For example, the non-targeting moiety may comprise m components (m is an integer in the range of e.g., 1-50) , wherein all m components are identical and covalently linked in tandem. In some other embodiments, m-1 components are identical while the remaining component is different. In some other embodiments, m-2 components are identical while the remaining components are different, and so on. Without wishing to be bound by any theory, there is no requirement to the positions of the specific components in the non-targeting moiety, as long as the components could provide a “scaffold” for the moiety which conjugates to the targeting oligonucleotide and chaperones the function of the targeting oligonucleotide.

The non-targeting moiety may be conjugated (i.e., covalently linked) to the targeting oligonucleotide directly or via a linker. In some embodiments, the non-targeting moiety is conjugated to a RNA duplex, such as a siRNA or saRNA, comprised of two complementary or partial complementary strands with one of the strands covalently linked to the non-targeting moiety. The RNA duplex targets at least one nucleic acid sequence (e.g., mRNA or DNA) and optionally is chemically modified using oligonucleotide chemistry technologies (e.g., 2’ fluoro, 2’-O-methyl, phosphorothioate, mesyl phosphoramidate or boranophosphate backbone, LNA, etc. ) conducive to in vivo activity, stability, and safety. The non-targeting moiety, unlike the RNA duplex, is unintentional or uncapable of specifically targeting any nucleic acid sequence in the subject to be administrated to. In the event that the non-targeting moiety unintentionally or unavoidably induces an "off-target" effect, specifically by interacting with a nucleic acid sequence in the subject, the oligonucleotide agent can still retain the "self-delivery" properties, thereby constituting an embodiment of the current invention as disclosed herein. The non-targeting moiety can be chemically-modified, e.g., comprising a phosphorothioate, mesyl phosphoramidate or boranophosphate bond in the backbone, a 2′-fluoro-2′-deoxynucleoside (2′-F) , a 2′-O-methyl (2′-O-Me) , a 2′-O- (2-methoxyethyl) (2′-O-MOE) , locked nucleic acid (LNA) , bridged nucleic acid (BNA) , peptide nucleic acid (PNA) , 5’- (E) -vinylphosphonate, 5-methyl cytosine, etc. as components, to impart physiochemical properties conducive to improve the agent’s bioavailability and delivery. As demonstrated herein, the non-targeting moiety presents certain benefits, for instance, cytoplasm protein binding, unconventional chemistries and modification patterns conducive to delivery, biodistribution, bioavailability, stability, cellular uptake, and other pharmacological properties without concerns of compromising duplex activity.

In some embodiments, the non-targeting moiety is linked to the targeting oligonucleotide via a linker. The linker connecting the targeting oligonucleotide and the non-targeting moiety may be selected from natural or unnatural nucleotides, ethlyglycol, carbohydrates, alkyl chains, or any other linkers that can be used to covalently connect any two oligonucleotides. On the other hand, the linker may also be deemed as part of the non-targeting moiety.

In some embodiments, the linkage between the targeting oligonucleotide and the non-targeting moiety and between adjacent components in the targeting oligonucleotide are selected from an ethylene glycol chain, an alkyl chain, an alkenyl chain, an alkynyl chain, a peptide, carbohydrates, thiol linkage, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, a tetrazole linkage, and a benzimidazole linkage.

Preferably, the non-targeting moiety comprises at least one phosphorothioate (PS) bond. In certain embodiments, the non-targeting moiety comprises at least one phosphorothioate (PS) bond in the backbone. In certain embodiments, the non-targeting moiety comprises 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, at least 30 or more phosphorothioate (PS) bonds. In certain embodiments, all the adjacent components in the non-targeting moiety are linked to each other via a PS bond.

In addition to the phosphorothioate bond (s) , the non-targeting moiety may comprise a phosphodiester bond, a mesyl phosphoramidate bond and/or a boranophosphate bond. For example, some of the adjacent components are linked to each other via a phosphorothioate bond, and some other adjacent components are linked to each other via a phosphodiester bond, a mesyl phosphoramidate bond and/or a boranophosphate bond.

In certain embodiments, one or more components of the non-targeting moiety are nucleotides, which may be those of a RNA, DNA, BNA, LNA or PNA. In certain embodiments, the non-targeting moiety comprises m components, wherein m is an integer in the range of 1-50.Further, among said m components, there are n nucleotides (natural or modified) , wherein n is an integer in the range of 0-49. The nucleotides of the non-targeting moiety may have a specific composition of A, G, C and/or U. One or more of the nucleotides may have 2’ Ome modification.

In certain embodiments, the sense strand of the double-stranded oligonucleotide in the oligonucleotide agent is at least 10 nucleotides in length. In certain embodiments of the present application, the sense strand has a nucleotide length ranging from 10-60 nucleotides.

In certain embodiments, the antisense strand of the double-stranded oligonucleotide in the oligonucleotide agent is at least 10 nucleotides in length. In certain embodiments, the antisense strand has a nucleotide length ranging from 10-60 nucleotides.

In certain embodiments, the chemical modification in the double-stranded oligonucleotide is an addition of a 5'-phosophate moiety at the 5’ end of the nucleotide sequence. In certain embodiments, the chemical modification is an addition of a 5’- (E) ‐vinylphosphonate moiety. In certain embodiments, the chemical modification is an addition of a 5-methyl cytosine at the 5’ end of the nucleotide sequence.

In some embodiments, the non-targeting moiety is conjugated to the double-stranded oligonucleotide directly or via a linker at the 3’ end of the double-stranded oligonucleotide. In some embodiments, the non-targeting moiety is conjugated to the double-stranded oligonucleotide directly or via a linker at the 5’ end of the double-stranded oligonucleotide. In some embodiments, the non-targeting moiety is conjugated to the double-stranded oligonucleotide directly or via a linker at the internal of the double-stranded oligonucleotide. In some embodiments, the double-stranded oligonucleotide is conjugated to the non-targeting moiety directly or via a linker at the internal of the non-targeting moiety.

In some embodiments, the double-stranded oligonucleotide comprises a sense strand and an antisense strand, and the non-targeting moiety is covalently conjugated to the sense strand, the antisense strand, or both the sense and the antisense strands of the double-stranded oligonucleotide directly or by a linker. In some embodiments, the non-targeting moiety is covalently conjugated to the 3’ end, the 5’ end, both the 3’ and the 5’ ends, or an internal nucleotide of the sense strand of the double-stranded oligonucleotide. In some embodiments, the non-targeting moiety is covalently conjugated to the 3’ end, the 5’ end, both the 3’ and the 5’ ends, or an internal nucleotide of the antisense strand of the double-stranded oligonucleotide. In some embodiments, the internal nucleotide in the sense or antisense strand of the double-stranded oligonucleotide is substituted by a linker, wherein the non-targeting moiety is covalently conjugated with the linker.

In some embodiments, more than one non-targeting moieties are covalently conjugated to the double-stranded oligonucleotide. In some embodiments, about 2-10 non-targeting moieties are covalently conjugated to the double-stranded oligonucleotide.

In some embodiments, more than one double-stranded oligonucleotides are covalently conjugated to the non-targeting moiety. In some embodiments, about 2-10 double-stranded oligonucleotides are covalently conjugated to the non-targeting moiety.

In some embodiments, the terminal component of the non-targeting moiety is directly linked to the double-stranded oligonucleotide through a phosphorothioate (PS) bond. In some other embodiments, the terminal component of the non-targeting moiety is linked to the double-stranded oligonucleotide via a linker, and the linker is covalently linked to the double-stranded oligonucleotide through a phosphorothioate (PS) bond.

In some embodiments, the linkage between the linker and the double-stranded oligonucleotide comprises a direct bond, or an oxygen or 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.

In some embodiments, the double-stranded RNA is designed for inhibiting expression of superoxide dismutase 1 (SOD1) in a cell. In some other embodiments, the dsRNA is designed for activating expression of survival motor neuron 2 (SMN2) protein in a cell.

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 sequences as set forth in any of SEQ ID NOs: 1, 3, 56 and 61, and the antisense strand comprises a nucleotide sequence that has partial or full complementarity with the first strand.

In some embodiments, the antisense strand comprises a nucleotide sequence that has partial complementarity with any of SEQ ID NOs: 1, 3, 56 and 61. In some further embodiments, the antisense strand comprises a nucleotide sequence that is at least 90%identical to the nucleotide sequences as set forth in any of SEQ ID NOs: 2, 4, 57 and 62.

In some embodiments, the sense strand of the double-stranded oligonucleotide has a nucleotide sequence that is at least 90%identical to the nucleotide sequence selected from the group of: 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) .

In some embodiments, the anti-sense strand of the double-stranded oligonucleotide has a nucleotide sequence that is at least 90%identical to the nucleotide sequence selected from the group of: 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) .

In some embodiments, the non-targeting moiety is further conjugated to one or more conjugation groups. In some embodiments, the double-stranded oligonucleotide is further conjugated to one or more conjugation groups. In some embodiments, the sense strand or the antisense strand of the double-stranded oligonucleotide is further conjugated to one or more conjugation groups.

In some embodiments, the conjugation groups are selected from one or more of a lipid, a fatty acid, a fluorophore, a ligand, a saccharide, a peptide, and an antibody. In some embodiments, the one or more conjugation groups are selected from a cell-penetrating peptide, polyethylene glycol, an alkaloid, a tryptamine, a benzimidazole, a quinolone, an amino acid, a cholesterol, glucose and N-acetylgalactosamine.

In some embodiments, each of the sense strand and the antisense strand independently has a nucleotide length ranging from 15-35 nucleotides.

In some embodiments, the oligonucleotide agent comprises a small interfering RNA (siRNA) , wherein the siRNA comprises a sense strand and an antisense strand to form a duplex structure, wherein the oligonucleotide agent is capable of inhibiting expression of targeted genes in a cell. Specifically, the targeted genes or proteins may be selected from but not limited to superoxide dismutase 1 (SOD1) .

In some embodiments, the oligonucleotide agent comprises a short activating RNA (saRNA) , wherein the saRNA comprises a sense strand and an antisense strand to form a duplex structure, wherein the oligonucleotide agent is capable of activating expression of targeted genes in a cell. Specifically, the targeted genes or proteins may be selected from but not limited to SMN2.

In some embodiments, the sense strand and the antisense strand of the siRNA have nucleotide sequences that are independently at least 85%homologous to the nucleotide sequence pairs selected from Table 5 or Table 14.

In some embodiments, the non-targeting moiety of the oligonucleotide agent improves the stability, bioavailability, biodistribution, and/or cellular uptake of the double-stranded oligonucleotide as compared to an oligonucleotide agent without the non-targeting moiety.

In some embodiments, the non-targeting moiety of the oligonucleotide agent increases the biodistribution of double-stranded oligonucleotide within one or more target tissues as compared to an oligonucleotide agent without the non-targeting moiety. In some embodiments, the one or more target tissues is selected from tissues of brain, spinal cord, muscle, spleen, lung, heart, liver, bladder, kidney and retina. In some embodiments, the one or more target tissues is selected from the group consisting of: prefrontal cortex, cerebellum, and cerebrum; cervical, thoracic and lumbar in spinal cord; heart, bicep, semitendinosus, platysma, and gluteus.

Also provided herein are vectors and cells comprising the oligonucleotide agents of the present disclosure. In some embodiments, the cell is a mammalian cell and is optionally a human cell. In some embodiments, the cell is a host cell. In some embodiments, the cell is in vitro. In some embodiments, the cell exists in a mammalian body.

Certain embodiments of the present application relate to a pharmaceutical composition comprising the oligonucleotide agent comprising: (a) a double-stranded oligonucleotide comprising a sense strand and an antisense strand, wherein the antisense strand has complementarity to a target nucleic acid; and (b) a non-targeting moiety comprising one or more components that are covalently linked via at least one PS bond, wherein the double-stranded oligonucleotide is conjugated to the non-targeting moiety to form the oligonucleotide agent. The target nucleic acids can be any target nucleic acid. The target nucleic acid includes, without limitation, a SOD1 gene, a SMN2 gene, etc.

In certain embodiments of the present application, the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier selected from an aqueous carrier, liposome or LNP, polymer, micelle, colloid, metal nanoparticle, non-metallic nanoparticle, bioconjugates, and polypeptide.

In certain embodiments, the pharmaceutical composition decreases or silences the transcription of the SOD1 gene or SOD1 protein.

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

Also provided herein are kits comprising the oligonucleotide agents or the pharmaceutical compositions of the present disclosure.

Certain embodiments relate to kits comprising a pharmaceutical composition of the present disclosure.

Certain embodiments relate to a method of decreasing or silencing the transcription of a SOD1 gene or protein, comprising administering to a subject a pharmaceutical composition of the present disclosure.

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 to a subject a pharmaceutical composition of the present disclosure. In certain embodiments, the subject has sporadic ALS (sALS) . In certain embodiments, the subject has familial ALS (fALS) .

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

Certain embodiments of the present application relate to a method for increasing or activating expression of SMN2 gene, comprising administering to a subject the pharmaceutical composition.

Certain embodiments of the present 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 to a subject a pharmaceutical composition of the present disclosure.

In certain embodiments, the pharmaceutical composition decreases or silences the expression of the SOD1 gene or protein.

In certain embodiments, the non-targeting moiety of the oligonucleotide agent improves the stability, bioavailability, biodistribution, and/or cellular uptake of the double-stranded oligonucleotide as compared to an oligonucleotide agent without the non-targeting moiety.

In certain embodiments, the non-targeting moiety of the oligonucleotide agent increases the biodistribution of double-stranded oligonucleotide within one or more target tissues as compared to an oligonucleotide agent without the non-targeting moiety.

In certain embodiments, the non-targeting moiety of the oligonucleotide agent increases the stability, biodistribution, bioavailability and activity of double-stranded oligonucleotide within two or more target cell types in a tissue as compared to an oligonucleotide agent without the non-targeting moiety. In certain embodiments, the one or more target tissues is selected from the tissues from brain, spinal cord, muscle, spleen, lung, heart, liver, bladder, and kidney. In certain embodiments, the one or more target tissues is selected from the group of: prefrontal cortex, cerebellum, and cerebrum; cervical, thoracic and lumbar in spinal cord; heart, bicep, semitendinosus, platysma, and gluteus.

Certain embodiments of the present application relate to a use of the oligonucleotide agent of the present disclosure, in manufacturing a medicament for treating or delaying the onset or progression of Amyotrophic lateral sclerosis (ALS) .

Certain embodiments of the present application relate to a use of the pharmaceutical composition of the present disclosure in manufacturing a medicament for treating or delaying the onset or progression of Amyotrophic lateral sclerosis (ALS) . In certain embodiments, the ALS comprises sporadic ALS (sALS) and/or familial ALS (fALS) .

Certain embodiments of the present application relate to the oligonucleotide agent of the present disclosure for use in treating or delaying the onset or progression of Amyotrophic lateral sclerosis (ALS) , optionally, the ALS comprises sporadic ALS (sALS) and/or familial ALS (fALS) .

Certain embodiments of the present application also relate to the pharmaceutical composition of the present disclosure for use in treating or delaying the onset or progression of Amyotrophic lateral sclerosis (ALS) , optionally, the ALS comprises sporadic ALS (sALS) and/or familial ALS (fALS) .

After continuous exploration, the inventors surprisingly found that nucleotide should not be a necessary component in a single-stranded vehicle for delivering dsRNA into a target cell, which represents a significant advancement in the field of nucleic acid-based therapeutics, offering a range of technical advantages over existing methodologies. The innovative aspects of this invention are characterized by the following beneficial effects:

1. Expanded delivery profile without sacrifice therapeutic efficacy: The technical solution of the present invention demonstrates equivalent or superior activity compared to its predecessors. It is noteworthy that the mechanism utilizing a non-targeting component, as detailed in this application, stands out as an exemplary approach in oligonucleotide delivery technology development, including dsRNAs such as siRNA and saRNA. This strategic design ensures that the targeting oligonucleotide maintains its efficacy while enabled with self-delivery properties brought by the non-targeting moiety, thereby expanding the therapeutic application of the oligonucleotide in various organs, tissues, and cells, especially central nerve system.

2. Cost-effective synthesis: The present invention employs a general chemical linker or spacer in its construction, as opposed to relying on costly chemically modified nucleotide monomers. This strategic choice has led to a significant reduction in the cost of chemical synthesis, making the production of the oligonucleotide agent more economically viable. This cost advantage is a critical factor for the commercialization and widespread adoption of the invention.

3. Enhanced therapeutic efficacy with target-specificity and minimized off-target effects: The present invention has been designed with a reduced or absent presence of nucleotides in the non-targeting moiety. This deliberate modification is theoretically sound and practically effective in diminishing the likelihood of non-specific base pairing, which can lead to off-target effects. By reducing these unintended interactions, the invention ensures a higher degree of specificity and therapeutic precision, thereby minimizing potential adverse effects and improving the overall efficacy of the treatment.

4. Reduced cytotoxicity: Also discovered by the inventors are the lower cytotoxicity profile of the oligonucleotide agents. This reduction is explicitly observed and may be caused by involving less chemical modification to the nucleotide as compared to previous delivery vehicles. It is believed that cytotoxicity profile can be further improved by refined design and composition of the non-targeting moiety, as well as ameliorated forms of conjugation, which minimizes the potential for harm to healthy cells. This improvement is of paramount importance, as it translates to a better therapeutic index and a more favorable safety profile for patients receiving the treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are employed, and the accompanying drawings (also “figure” and “FIG. ” herein) , of which:

FIGs. 1A-1B show screening data for knockdown activity and cytotoxicity of 31 chain oligonucleotide delivery vehicle (cODV) -siRNA (cODV-siRNA) designs in primary mouse hepatocyte (PMH) . The indicated cODV-siRNAs (see Table 5) were transfected into PMH cells using RNAiMAX at 0.1 nM for 24 hours. Cells were transfected in the absence of an oligonucleotide as Mock treatments. dsCon2 duplex was transfected to serve as a non-targeting control. RD-12559 served as a positive control with known knockdown activity. FIG. 1A shows mouse Sod1 (i.e., Sod1) mRNA levels by each of the cODV-siRNAs as quantified by two step RT-qPCR using a gene specific primer set (shown in Table 4) . Geometric means of the mRNA levels of Hmbs and Hprt1 were used as an internal reference. The value (y-axis) shows the mean expression of Sod1 mRNA relative to Mock treatment after normalized to Hmbs and Hprt1 (mean ± SEM of two replicated transfection wells) . FIG. 1B shows cytotoxicity levels of 31 cODV-siRNAs at 0.1 nM in PMH cells by propidium iodide (PI) staining. A microplate reader system (Infinite M2000 Pro, Tecan) was used to detect the optical density (OD) of PI staining at 535 nm excitation and 615 nm emission wavelengths. The value (y-axis) shows the mean values of PI staining by each of the cODV-siRNAs relative to Mock (mean ± SEM of two replicated transfection wells) .

FIGs. 2A-2B show the knockdown activity and cytotoxicity of cODV-siRNA designs administrated to PMH cells by free uptake. The indicated cODV-siRNAs (see Table 5) were added to PMH cell culture media at 1000 nM for 3 days. RD-12559 was treated and served as a positive control with known knockdown activity. FIG. 2A shows relative Sod1 mRNA levels by each of the cODV-siRNAs as quantified by two step RT-qPCR using a gene specific primer set (shown in Table 4) . Geometric means of the mRNA levels of Hmbs and Hprt1 were used as an internal reference. The value (y-axis) shows the mean expression of Sod1 mRNA relative to Mock treatment after normalized to Hmbs and Hprt1 (mean ± SEM of two replicated wells) . FIG. 2B shows cytotoxicity levels of each of the cODV-siRNAs at 0.1 nM in PMH cells by PI staining. A microplate reader system was used to detect the OD of PI staining at 535 nm excitation and 615 nm emission wavelengths. The value (y-axis) shows the mean values of PI staining by each of the cODV-siRNAs relative to Mock (mean ± SEM of two replicated wells) .

FIG. 3 shows the body weight change of C57BL/6J mice following intracerebroventricular (ICV) injection with cODV-siRNAs. The indicated cODV-siRNAs (i.e., RD-13592, RD-13608, RD-13611, RD-13614 and RD-14794) were administered into adult C57BL/6J mice via ICV injection at 200 μg. Saline was injected as a vehicle control to establish baseline expression. Mice were sacrificed on day 14 post dosing. Body weight (g) change of C57BL/6J mice post dosing shows the mean of 3 animals per group (mean ± SEM of 3 animals per group) .

FIG. 4 shows the body weight change of C57BL/6J mice following tail vein (IV) injection with cODV-siRNAs. The indicated cODV-siRNAs (i.e., RD-13592, RD-13608, RD-13611, RD-13614 and RD-14794) were administered into adult C57BL/6J mice via IV injection at 20 mg/kg. Saline was injected as a vehicle control to establish baseline expression. Mice were sacrificed on day 14 post dosing. Body weight (g) change of C57BL/6J mice post dosing shows the mean of 3 animals per group (mean ± SEM of 3 animals per group) .

FIGs. 5A-5B show the in vivo knockdown activity of cODV-siRNAs on rat Sod1 mRNA expression via local intravitreous (IVT) injection in adult SD rats. The indicated cODV-siRNAs (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, ) were administered into adult SD rats via IVT injection at 30 μg. Saline was injected as a vehicle control to establish baseline expression. RD-12556 was injected as a duplex control. SD rats were sacrificed on day 14 post dosing. FIG. 5A and 5B show the rat Sod1 mRNA levels as quantified in retina via two step RT-qPCR using a rat gene specific primer set (shown in Table 4) . Gapdh was amplified as an internal reference. The value (y-axis) shows the mean expression of rat Sod1 mRNA relative to saline treatment after normalized to Gapdh (mean ± SEM of 2-3 animals per group) .

FIGs. 6A-6D show the knockdown activity of cODV-siRNAs on SOD1 mRNA expression levels in SK-N-AS and T98G cells. The indicated cODV-siRNAs (i.e., RD-16989, RD-16978, RD-16102 and RD-16979) were transfected into SK-N-AS and T98G cells at indicated concentrations (i.e., 0.0001, 0.0002, 0.001, 0.004, 0.016, 0.063, 0.25 and 1) for 24 hours. Cells were transfected in the absence of an oligonucleotide as Mock treatments (not shown) . dsCon2 duplex was transfected to serve as a non-targeting control (not shown) . RD-16988 and RD-16990 were transfected to serve as the duplex controls. Remaining human SOD1 (i.e., SOD1) mRNA levels shown in the figures are quantified by two-step RT-qPCR using a gene specific primer set. TBP was amplified as an internal reference. The values (y-axis) represent the remaining SOD1 mRNA level relative to Mock treatment after normalized to TBP (mean ± SEM of four replicated transfection wells) .

FIG. 7 shows the knockdown activity of cODV-siRNAs on SOD1 mRNA expression levels in human SOD1^G93A (hSOD1^G93A) mice. The indicated cODV-siRNAs (i.e., RD-16145 and RD-16978) were administered into hSOD1^G93A mice via ICV injection at 100 μg. Artificial cerebrospinal fluid (aCSF) was injected as a vehicle control to establish baseline expression. hSOD1^G93A mice were sacrificed on day 14 post dosing. Remaining SOD1 mRNA levels were quantified in tissues from the brain (i.e., frontal cortex, cerebellum and cerebrum) , spinal cord and periphery (i.e., liver) via two step RT-qPCR using a gene specific primer set. Mouse Rpl13a was amplified as an internal reference. Mean remaining SOD1 mRNA levels in the selected mouse tissues are shown relative to mRNA levels in the aCSF group after normalized to Rpl13a. The data represents mean ± SEM of 4 mice per group.

FIG. 8 shows the knockdown activity of cODV-siRNAs on Sod1 mRNA levels in C57BL/6J mice. The indicated cODV-siRNAs (i.e., RD-16293, RD-16294, RD-16295 and RD 14794) were administered into C57BL/6J mice via ICV injection at 200 μg. aCSF was injected as a vehicle control to establish baseline expression. C57BL/6J mice were sacrificed on day 14 post dosing. Remaining mouse Sod1 mRNA levels were quantified in tissues from the brain (i.e., frontal cortex, cerebellum and cerebrum) and spinal cord (i.e., cervical, thoracic and lumber) via two step RT-qPCR using a gene specific primer set. Mouse Rpl13a was amplified as an internal reference. Mean remaining Sod1 mRNA levels in the selected mouse tissues are shown relative to mRNA levels in the aCSF group after normalized to Rpl13a. The data represents mean ± SEM of 3 mice per group.

FIG. 9 shows the knockdown activity of cODV-siRNAs on Sod1 mRNA levels in Neuro-2a (N2a) cells. The indicated cODV-siRNAs (i.e., RD-18148, RD-18150, RD-18151, RD-18152, RD-18153, RD-18154, RD-18155 and RD-18156) were transfected in N2a cells at 0.1 nM for 24 hours. Cells were transfected in the absence of an oligonucleotide as Mock treatments. dsCon2M8 duplex was transfected to serve as a non-targeting control. Remaining Sod1 mRNA levels shown in the figures are quantified by two-step RT-qPCR using a gene specific primer set. Mouse Rpl13a was amplified as an internal reference. The values (y-axis) represent the remaining Sod1 mRNA level relative to Mock treatment after normalized to Rpl13a (mean ± SEM of four replicated transfection wells) .

FIG. 10 shows the knockdown activity of cODV-siRNAs on Sod1 mRNA levels in N2a cells. The indicated 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 in N2a cells at 0.1 nM for 24 hours. Cells were transfected in the absence of an oligonucleotide as Mock treatments. dsCon2M8 duplex was transfected to serve as a non-targeting control (not shown) . Remaining Sod1 mRNA levels shown in the figures are quantified by two-step RT-qPCR using a gene specific primer set. Mouse Rpl13a was amplified as an internal reference. The values (y-axis) represent the remaining Sod1 mRNA level relative to Mock treatment after normalized to Rpl13a (mean ±SEM of four replicated transfection wells) .

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

Before the present invention described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the exemplary methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

It must be noted that as used herein and in the appended claims, the singular forms "a" , "an" , and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "asample" includes a plurality of such samples and reference to "the molecule" includes reference to one or more molecules and equivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

DEFINITIONS

The term "oligonucleotide agent" or “oligonucleotide conjugate” can be used interchangeably and refers to a chimeric oligonucleotide molecule comprising a targeting oligonucleotide (s) and a non-targeting moiety capable of facilitating delivery of the targeting oligonucleotide (s) . The targeting oligonucleotide includes, but is not limited to, double-stranded nucleic acid molecules of DNA, RNA, or DNA/RNA hybrid, oligonucleotide strands containing regularly and irregularly alternating deoxyribosyl portions and ribosyl portions, as well as modified and naturally or unnaturally existing frameworks for such oligonucleotides. The targeting oligonucleotide as disclosed herein may be a small inhibiting nucleic acid molecule (siRNA) , a small activating nucleic acid molecule (saRNA) or an antisense oligonucleotide molecule (ASO) . Specifically, the oligonucleotide agent for inhibiting mRNA transcript level of target gene described herein is a non-targeting moiety conjugated siRNA molecule, and the oligonucleotide agent for activating transcription of target gene described herein is a non-targeting moiety conjugated saRNA molecule.

The term “non-targeting moiety” or “chain oligonucleotide delivery vehicle (cODV) ” refers to the portion of the oligonucleotide agent that is conjugated to the targeting oligonucleotide (directly or via a linker) , which is intended to facilitate in vivo delivery of the targeting oligonucleotide (s) and has no intentional gene targeting function. The non-targeting moiety comprises one or more components such as linkers, linking groups and nucleotides covalently linked together, which may be linear or branched. The components of the non-targeting moiety may further be chemically modified, e.g., in the backbone or in the branched chain of the component.

As used herein, the term "non-targeting" means that the referenced moiety which conjugates with the targeting oligonucleotide (e.g., siRNA, saRNA etc. ) does not specifically bind to the target sequence which the targeting oligonucleotide functions, or ideally any other nucleotide sequence in an animal cells. The targeting oligonucleotide disclosed herein is a nucleic acid sequence that specifically complements to the target sequence or the region thereof. In some cases, the “specifically complementary” may mean that the complementarity between the targeting oligonucleotide and the target sequence or the region thereof is at least about 95%. The non-targeting moiety is not intended to elicit biological activity via any known mechanism, nor intended to elicit activities indicative of ASO (e.g., “mixmer” or “gapmer” ) function onto a complementary nucleic acid sequence (i.e., mRNA) in a certain subject, an organ of the subject, a tissue of the subject, or a cell of the subject, when the oligonucleotide is administered. The non-targeting moiety is to facilitate in vivo biodistribution, cellular entry and intracellular function of the targeting oligonucleotide (e.g., siRNA, saRNA, and etc. ) to which it conjugates in certain subject, an organ of the subject, a tissue of the subject, a cell of the subject, or a cell nucleus of the subject, when the oligonucleotide conjugate is administered.

The term “backbone” , as used herein in reference to a chemical compound, generally refers to the longest carbon chain (may be modified to comprise other heteroatoms instead of carbon atoms) in the molecule or composed of carbon chain comprising functional groups. The linkages by functional groups in the backbone include but not limited to, -CH2-O-CH2-, CH2-O-P-O-, -O-P-O-CH2-, -CH2-NH-O-CH2-, -CH2-N (CH3) -O-CH2-, -CH2-O-N (CH3) -CH2-, -CH2-N (CH3) -N (CH3) -CH2-and -O-N (CH3) -CH2-CH2-. As shown herein, the backbone of the non-targeting moiety may be modified to comprise one or more phosphorothioate bonds.

The term “linker” , “chemical linker” , “spacer” , “component” or “unit” in the non-targeting moiety can be used interchangeably and as used herein refers to a molecule or a chemical group covalently joining two portions, including but not limited to, linkers commonly used for spacing two nucleotides, such as 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.; and any other chemical groups such as aliphatic or substituted aliphatic chains, linear or branched, that can be used for providing a scaffold; and chemical groups that connects two molecules, such as a disulfide bond. The term can also include a nucleic acid or nucleic acid mimic linker, a peptide linker, and the like. Linkers in the non-targeting moiety belong to the components or units of the non-targeting moiety in the present application.

As used herein, the terms “subject” and “individual” are used interchangeably herein to mean any living organism that may be treated with agents of the present application. The term “patient” means a human subject or individual, including disclosure infants, children and adults.

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

The effective amount may vary depending on such factors as the size and weight of the subject, the type of illness, or the particular agents of the application. For example, the choice of the agent of the application could affect what constitutes an “effective amount. ” One of ordinary skill in the art would be able to study the factors contained herein and make the determination regarding the effective amount of the agents of the application without undue experimentation.

The regime of administration may affect what constitutes an effective amount. The agent of the application can be administered to the subject either prior to or after the disease diagnosis or condition. Further, several divided dosages, as well as staggered dosages, can be administered daily or sequentially, or the dose can be continuously infused, or can be a bolus injection. Further, the dosages of the agent (s) of the application could be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

The terms “treat, ” “treated, ” “treating” , or “treatment” as used herein have the meanings commonly understood in the medical arts, and therefore do not require cure or complete remission, and include any beneficial or desired clinical results. Non-limiting examples of such beneficial or desired clinical results are prolonging survival as compared to expected survival without treatment, reduced symptoms including one or more of the followings: weakness and atrophy of proximal skeletal muscles, inability to sit or walk independently, difficulties in swallowing, breathing, etc.

As used herein, “preventing” or “delaying” a disease refers to inhibiting the full development of a disease.

The term “biological sample” refers to any tissue, cell, fluid, or other material derived from an organism (e.g., human subject) . In certain embodiments, the biological sample is serum or blood.

As used herein, the term "sequence identity" or "sequence homology" means that one oligonucleotide strand (sense or antisense) of, for example, an saRNA or siRNA has at least 80%similarity with a region on the coding strand or template strand of the promotor, or the sequence of a target gene. Unless indicated otherwise, the term “identity” and “homology” herein refer to nucleotides per se and does not take into account of their modifications, e.g. nucleotide A, mA (2’-OMe A) and fA (2’-fluoro A) are considered as the same nucleotides when calculating sequence identity or homology.

By "target sequence" is meant a sequence fragment to which the sense strand or antisense oligonucleotide of the siRNA or saRNA is homologous or complementary.

As used herein, the term "gapmer" refers to a short DNA antisense oligonucleotide (ASO) structure with modified RNA segments on both sides of the central DNA structure. In some embodiments, at least one of the modified RNA segments comprises one or more of modified nucleotides selected from locked nucleic acids (LNA) , and 2'-OMe or 2'-F modified nucleotides to increase affinity to the target, increase nuclease resistance, reduce immunogenicity, and/or decrease toxicity. In some embodiments, a gapmer comprises at least one nucleotide modified with a phosphorothioate (PS) group. In some embodiments, the gamper is designed to hybridize to a target piece of RNA and silence the gene transcript through the induction of RNase H cleavage.

As used herein, the term "mixmer" refers to an antisense oligonucleotide (ASO) characterized as a mixture of DNA and chemically modified nucleic acid analogs in structure. Optionally, a mixmer is composed of fully modified nucleotides or nucleic acid analogs. In some embodiments, a mixmer is designed to bind and mask complementary RNA sequence to sterically block proteins, factors, or other RNAs from interacting with targeted RNA. In some embodiments, a mixmer is designed to alter pre-mRNA splicing by displacing the spliceosome. In some embodiments, a mixmer is designed to bind and sequester microRNAs (miRNAs) in which it is adopt yet another name called an "antagomir" or an "anti-miR" .

As used herein, the terms "sense strand" of dsRNA (e.g., siRNA, saRNA) duplex refers to the strand having sequence homology or sequence identity with a fragment of the coding strand of the sequence of a target gene.

As used herein, the terms "antisense strand" of dsRNA (e.g., siRNA, saRNA) duplex refers to the strand having sequence complementary with the sense strand. Said antisense strand may interact with a target sequence to active or up-regulate gene expression, said target sequence may be a fragment of the coding strand of the sequence of a target gene.

As used herein, the term "first oligonucleotide strand" can be a sense strand or an antisense strand. For example, the sense strand of a saRNA refers to an oligonucleotide strand having homology with the coding strand of the promoter DNA sequence of the target gene of the saRNA. The sense strand of a siRNA refers to an oligonucleotide strand having homology with the mRNA sequence of the target gene of the siRNA. The antisense strand refers to an oligonucleotide strand complementary with the sense strand in the dsRNA.

As used herein, the term "second oligonucleotide strand" can also be a sense strand or an antisense strand. If the first oligonucleotide strand is a sense strand, the second oligonucleotide strand is an antisense strand; and if the first oligonucleotide strand is an antisense strand, the second oligonucleotide strand is a sense strand.

As used herein, the term "coding strand" refers to the DNA strand in the target gene that cannot be transcribed, the nucleotide sequence of which is identical to the sequence 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 described in the present disclosure refers to the promoter sequence on the same DNA strand as the DNA coding strand of the target gene.

As used herein, the term "template strand" refers to another strand of double-stranded DNA of a target gene that is complementary to the coding strand and that can be transcribed as a template into RNA that is complementary to the transcribed RNA base (A-U, G-C) . 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 described in the present disclosure refers to the promoter sequence on the same DNA strand as the DNA template strand of the target gene.

As used herein, the term "overhang" refers to an oligonucleotide strand end (5' or 3 ') with non-base paired nucleotide (s) resulting from another strand extending beyond one of the strands within the double stranded oligonucleotide. Single stranded regions extending beyond the 3' and/or 5' ends of the duplexes are referred to as overhangs. In certain embodiments, the overhang is from 0 to 6 nucleotides in length. It is understood that an overhang of 0 nucleotides means that there is no overhang.

The term “natural overhang” as used herein refers to an overhang which consists of one or more nucleotides identical to or complementary to the corresponding position on the target sequence. A natural overhang on a sense strand consists of one or more nucleotides identical to the corresponding position on the mRNA or DNA target. A natural overhang on an antisense strand consists of one or more nucleotides complementary to the corresponding position on the mRNA or DNA target.

As used herein, the terms "gene activation" , "activating gene expression" , “gene upregulation” and “upregulating gene expression” can be used interchangeably, and means an increase or upregulation in transcription, translation, expression or activity of a certain nucleic acid sequence as determined by measuring the transcription level, mRNA level, protein level, enzymatic activity, methylation state, chromatin state or configuration, translation level or the activity or state in a cell or biological system of a gene. These activities or states can be determined directly or indirectly. In addition, "gene activation" or "activating gene expression" refers to an increase in activity associated with a nucleic acid sequence, regardless the mechanism of such activation. For example, gene activation occurs at the transcriptional level to increase transcription into RNA and the RNA is translated into a protein, thereby increasing the expression of the protein.

As used herein, the terms "gene silencing" , "knockdown of gene expression" , “gene downregulation” and “downregulating gene expression” can be used interchangeably, and means a decrease or downregulation in transcription, translation, expression or activity of a certain nucleic acid sequence as determined by measuring the transcription level, mRNA level, protein level, enzymatic activity, methylation state, chromatin state or configuration, translation level or the activity or state in a cell or biological system of a gene. These activities or states can be determined directly or indirectly. In addition, "gene downregulation" or "downregulating gene expression" refers to a decrease in activity associated with a nucleic acid sequence, regardless the mechanism of such downregulation. For example, gene downregulation occurs at the transcriptional level to decrease or silence transcription into RNA and the RNA is not translated into a protein, thereby decreasing or silencing the expression of the protein.

As used herein, the terms “inhibition of gene expression “or “inhibiting gene expression” and “gene downregulation” or “down-regulating gene expression” can be used interchangeably, and mean an decrease in transcription, translation, expression or activity of a certain nucleic acid as determined by measuring the transcriptional level, mRNA level, protein level, enzymatic activity, methylation state, chromatin state or configuration, translation level or the activity or state in a cell or biological system of a gene. These activities or states can be determined directly or indirectly. In addition, “inhibition of gene expression “, “inhibiting gene expression” , “gene down-regulation” or “down-regulating gene expression” refers to a decrease in activity associated with a nucleic acid sequence, regardless of the mechanism of such inhibition. For example, inhibition of gene expression occurs at the transcriptional level to decrease transcription into RNA and the RNA is translated into a protein, thereby decreasing the expression of the protein.

As used herein, the terms "short interfering RNA" , "siRNA" and “silencing RNA” can be used interchangeably and refer to a ribonucleic acid molecule that can downregulate, knockdown, or silence target gene expression. It can be a double-stranded nucleic acid molecule. It interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, preventing translation. siRNA binds to target mRNA mainly in the cytoplasm to down-regulate gene expression post-transcriptionally via the RNA interference (RNAi) mechanism. siRNAs may be designed to target a gene’s mRNA sequence to silence its expression via the RNAi mechanism, such as SOD1, for maximizing treatment outcomes, e.g., for ALS patients. siRNAs are molecules having endogenous RNA bases or chemically modified nucleotides. The modifications do not abolish cellular activity, but rather impart increased stability and/or increased cellular potency. Examples of chemical modifications include phosphorothioate groups, 2'-deoxynucleotide, 2'-OCH₃-containing ribonucleotides, 2'-F-ribonucleotides, 2'-methoxyethyl ribonucleotides, combinations thereof and the like. The siRNA can have varying lengths (e.g., 10-200 bps) and structures (e.g., hairpins, single/double strands, bulges, nicks/gaps, mismatches) and are processed in cells to provide active gene silencing. A double-stranded siRNA can have the same number of nucleotides on each strand (blunt ends) or asymmetric ends (overhangs) . An overhang of 1-2 nucleotides, for example, can be present on the sense and/or the antisense strand, as well as present on the 5'-and/or the 3'-ends of a given strand. The length of the siRNA molecule is typically about 10 to about 60, about 10 to about 50, about 15 to about 30, about 17 to about 29, about 18 to about 28, about 19 to about 27, about 20 to about 26, about 21 to about 25, and about 22 to about 24 base pairs, and typically about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 23, about 25, about 30, about 40, or about 50 base pairs. In addition, the terms "small interfering RNA" , “silencing RNA” and "siRNA" also contain nucleic acids other than the ribonucleotide, including, but not limited to, modified nucleotides or analogues.

As used herein, the terms "small activating RNA" , "saRNA" and "small activating ribonucleic acid" can be used interchangeably and refer to a ribonucleic acid molecule that can upregulate target gene expression. It can be a double-stranded nucleic acid molecule composed of a first nucleic acid strand containing a ribonucleotide sequence with sequence homology with the non-coding nucleic acid sequence (such as a promoter and an enhancer) of a target gene and a second nucleic acid strand containing a nucleotide sequence complementary with the first strand. The saRNA can also be comprised of a synthesized or vector-expressed single-stranded RNA molecule that prone to form a hairpin structure by two complementary regions within the molecule, wherein the first region contains a ribonucleotide sequence having sequence homology with the target sequence of a promoter of a gene, and a ribonucleotide sequence contained in the second region is complementary with the first region. The length of the duplex region of the saRNA molecule is typically about 10 to about 60, about 10 to about 50, about 10 to about 40, about 12 to about 30, about 14 to about 28, about 16 to about 26, about 18 to about 24, and about 20 to about 22 base pairs, and typically about 10, about 13, about 15, about 17, about 18, about 19, about 20, about 21, about 22, about 25, about 30, about 40, about 50, or about 60 base pairs. In addition, the terms "small activating RNA" , "saRNA" and "small activating ribonucleic acid" also contain nucleic acids other than the ribonucleotide, including, but not limited to, modified nucleotides or analogues.

As used herein, the terms “isolated target site” , “target site” and “isolated polynucleotide" can be used interchangeably, and herein means a nucleic acid target site to which a siRNA/saRNA has complementarity or hybridizes to. For example, an isolated nucleic acid sequence of a target site can include a nucleic acid sequence to which a region of siRNAs/saRNAs has complementarity or hybridize to.

As used herein, the term “complementary” refers to the capability of forming base pairs between two oligonucleotide strands. The base pairs are generally formed through hydrogen bonds between nucleotides in the antiparallel oligonucleotide strands. The bases of the complementary oligonucleotide strands can be paired in the Watson-Crick manner (such as A to T, A to U, and C to G) or in any other manner allowing the formation of a duplex (such as Hoogsteen or reverse Hoogsteen base pairing) .

Complementarity includes complete complementarity and incomplete complementarity. “Complete complementarity” or “100%complementarity” means that each nucleotide from the first oligonucleotide strand can form a hydrogen bond with a nucleotide at a corresponding position in the second oligonucleotide strand in the double-stranded region of the siRNA molecule, with no base pair being “mispaired” . “Incomplete complementarity” , “partial complementarity” , or “mismatch” means that not all the nucleotide units of the two strands are bound with each other by hydrogen bonds. For example, for two oligonucleotide strands each of 20 nucleotides in length in the double-stranded region, if only two base pairs in this double-stranded region can be formed through hydrogen bonds, the oligonucleotide strands have a complementarity of 10%. In the same example, if 18 base pairs in this double-stranded region can be formed through hydrogen bonds, the oligonucleotide strands have a complementarity of 90%. Substantial complementarity refers to at least about 75%, about 79%, about 80%, about 85%, about 90%, about 95%or 99%complementarity.

As used herein, the term "synthetic" refers to the manner in which oligonucleotides are synthesized, including any means capable of synthesizing or chemically modifying RNA, such as chemical synthesis, in vitro transcription, vector expression, and the like.

As used herein, the term “LNA” refers to a locked nucleic acid in which the 2′-oxygen and 4′-carbon atoms are joined by an extra bridge. As used herein, the term “BNA” refers to a 2'-O and 4'-aminoethylene bridged nucleic acid that can contain a five-membered or six-membered bridged structure with an N-O linkage. As used herein, the term “PNA” refers to a nucleic acid mimic with a pseudopeptide backbone composed of N- (2-aminoethyl) glycine units with the nucleobases attached to the glycine nitrogen via carbonyl methylene linkers.

Unless otherwise defined, all the technological and scientific terms used therein have the same meanings as those generally understood by those of ordinary skill in the art covering the present application.

Overview

Aspects of the present application include an oligonucleotide agent comprising an accessory moiety to provide improvements in efficient targeting one or more genes associated with a disease or condition, and improvements in the delivery, chemistry, biodistribution, bioavailability, and other pharmacological properties without compromising oligonucleotide activity.

The present application is based on investigations related to compositions and methods that, a targeting oligonucleotide (siRNA, saRNA, etc. ) , in combination with a non-targeting moiety, can activate/upregulate a gene expression and increase the amount of expression of full-length gene or protein, or knockout/silence a gene expression and decrease the amount of expression of full-length gene or protein, in order to improve therapeutic effects for genetic conditions. The term “chain oligonucleotide delivery vehicle (cODV) ” refers to the portion of the oligonucleotide agent that is conjugated to the targeting oligonucleotide (directly or via a linker) , which is intended to facilitate in vivo delivery of the targeting oligonucleotide (s) and has no intentional gene targeting function. The non-targeting moiety comprises one or more components such as linkers, linking groups and nucleotides covalently linked together, which may be linear or branched. The components of the non-targeting moiety may further be chemically modified, e.g., in the backbone or in the branched chain of the component.

The present inventors found that the non-targeting moiety did not interfere with siRNA knockdown activity or saRNA-induced gene activation. The present inventors also found that the length, compositions, modifications (e.g., 2’-Ome, 2’-MOE, 2’-F) , linking components, phosphorothioate (PS) backbone linkages of the non-targeting moiety had effects on dsRNA activity in vivo. When administered in either brain or spinal cord tissue as compared to a dsRNA without the non-targeting moiety, cODV-dsRNA shows an improved in vivo activity in the CNS via local injection.

Additional aspects of the present application include methods of treating Amyotrophic lateral sclerosis (ALS) by administering an effective amount of an oligonucleotide agent comprising a SOD1-targeting siRNA. The siRNA inhibits the expression of SOD1 gene through the RNAi silencing mechanism. The present inventors have developed SOD1 siRNAs with potent inhibitory effect, for use in the treatment of ALS.

Oligonucleotide Agents

Aspects of the present application include an oligonucleotide agent that includes a non-targeting moiety and a targeting double-strand oligonucleotide that is covalently linked, wherein the non-targeting moiety comprises one or more components that are covalently linked via at least one PS bond. The components may be selected from chemical linkers, nucleotides and other units that could provide as a “scaffold” for the moiety.

Aspects of the present application include an oligonucleotide agent that includes a non-targeting moiety and a targeting double-strand oligonucleotide that are covalently linked, wherein at least two adjacent linkers, two adjacent nucleotides, a spacer and an adjacent nucleotide in the non-targeting moiety is linked to each other via a phosphorothioate (PS) bond.

Aspects of the present application include an oligonucleotide agent that includes a non-targeting moiety and a targeting double-strand oligonucleotide that are covalently linked, wherein the non-targeting moiety comprises: (a) consecutively linked components, wherein none of the components is a nucleotide; (b) one or more nucleotides interspersed in components other than nucleotides; (c) one or more components other than nucleotides interspersed in nucleotides; or (d) a consecutive sequence of nucleotides and a consecutively linked sequence of components other than nucleotides. Preferably, the components other than nucleotides are linkers, such as the linkers shown in Table 1.

Aspects of the present application include an oligonucleotide agent that includes a non-targeting moiety and a targeting double-strand oligonucleotide that are covalently linked, wherein the non-targeting moiety does not comprise any nucleotides. In some embodiments, the non-targeting moiety comprises one or more nucleotides, which may be natural, synthetic or chemically modified.

Aspects of the present application include an oligonucleotide agent that includes a non-targeting moiety and a targeting double-strand oligonucleotide that are covalently linked, wherein the non-targeting moiety is capable of facilitating delivery of the double-stranded oligonucleotide in the central nervous system (CNS) .

In some embodiments, the oligonucleotide agent comprises a double-stranded oligonucleotide, wherein the double-stranded oligonucleotide comprises a sense strand and an antisense strand, wherein the antisense strand has complementarity to a target nucleic acid; and a non-targeting moiety comprising one or more linkers, one or more nucleotides or a hybrid of one or more linkers and nucleotides. The double stranded oligonucleotide and the non-targeting moiety are covalently linked directly or via a linker to form the oligonucleotide agent.

In some embodiments, the sense strand of the double stranded targeting oligonucleotide is covalently linked to the non-targeting moiety. In some embodiments, the antisense strand of the double stranded targeting oligonucleotide is covalently linked to the non-targeting moiety. In some embodiments, the oligonucleotide agent has a compound formula of:
O--L--M or O--M (Formula Ia) or
M--L--O or M--O (Formula Ib) , wherein:

O refers to the double-stranded oligonucleotide comprising a sense strand and an antisense strand, wherein the antisense strand has complementarity to a target nucleic acid (e.g., mammalian target nucleic acid) ; M refers to the non-targeting moiety comprising one or more components linked by at least one PS bond. L is an optional linker for covalently linking the double stranded oligonucleotide and the non-targeting moiety.

In some embodiments, the oligonucleotide agent has a compound formula of:

wherein

O is a double stranded oligonucleotide comprising a sense strand and an antisense strand, wherein the antisense strand has complementarity to a target nucleic acid; M refers to the non-targeting moiety 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 linking the double stranded oligonucleotide and the non-targeting moiety; and optional components Cx, Cy, and Cz, wherein Cx, Cy, and Cz are independently absence, or conjugation groups selected from one or more of a lipid, a fatty acid, a fluorophore, a ligand, a saccharide, a peptide, an antibody and any other commonly used conjugation groups. In some embodiments, the compound of Formula II comprises 1 conjugation group. In some embodiments, the compound of Formula II comprises 2 conjugation groups. In some embodiments, the compound of Formula II comprises 3 conjugation groups.

In some embodiments, the double-stranded oligonucleotide is a siRNA. In some embodiments, the double-stranded oligonucleotide is a saRNA.

In some embodiments, the 5’ end, the 3’ end, or an internal nucleotide of the non-targeting moiety, if present, is conjugated to a linking component. In some embodiments, the internal nucleotide in the sense or antisense strand of the double-stranded oligonucleotide is substituted by a linking component, wherein the single-stranded oligonucleotide is covalently conjugated with the linking component. In some embodiments, the non-targeting moiety is covalently conjugated to the sense strand, the antisense strand, or both the sense and antisense strands of the double-stranded oligonucleotide by a linking component.

In some embodiments, the non-targeting moiety is covalently conjugated to the 3’ end, or the 5’ end, or both the 3’ and 5’ ends, or an internal nucleotide of the sense strand of the double-stranded oligonucleotide. In some embodiments, the non-targeting moiety is covalently conjugated to the 3’ end, or the 5’ end, or both the 3’ and 5’ ends, or an internal nucleotide of the antisense strand of the double-stranded oligonucleotide.

Non-targeting moiety

Although some prior research proved that siRNAs capable of inhibiting SOD1 mRNA and decreasing SOD1 protein expression can be used to treat SOD1 protein-related diseases, e.g., for amyotrophic lateral sclerosis (ALS) patients, the inventors found there are two unsolved issues: 1) a lack of potency of SOD1 siRNA molecules and 2) a lack of efficient delivery method to deliver the siRNA molecule to cells of a target organ or tissue.

Similarly, for saRNA, there are also unsolved issues: 1) a lack of potency of saRNA molecules and 2) a lack of efficient delivery method to deliver the saRNA molecule to cells of a target organ or tissue.

Surprisingly, the current invention found, when the dsRNA agent, e.g., an siRNA or an saRNA, is conjugated to a non-targeting moiety as disclosed, bioavailability, biodistribution, and/or cellular uptake and in vivo potency of the dsRNA was significantly improved as compared to an oligonucleotide agent without the non-targeting moiety. Especially in some in vivo examples in the present application, the non-targeting moiety of the oligonucleotide agent increased the biodistribution of dsRNA within one, or two, or more target tissues as compared to an oligonucleotide agent without the non-targeting moiety.

“Delivering into a cell, ” when referring to a targeting double-strand oligonucleotide, e.g., a double-stranded RNA agent (dsRNA) such as a siRNA, a saRNA, or the like, means efficient uptake or absorption by the cell, as is understood by those skilled in the art. Absorption or uptake of an dsRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; a dsRNA can also be “introduced into a cell, ” wherein the cell is part of a living organism. In such an instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, dsRNA can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation, free uptake, and lipofection. Further approaches are described herein below which is not known in the art.

In general, the non-targeting moiety of the oligonucleotide agent is a single-stranded moiety that is in favor of the oligonucleotide agent by its delivery properties. Therefore, it does not target a nucleic acid sequence in a subject which the dsRNA is targeting, or a “natural” nucleic acid from the subject, such as a target nucleic acid of the dsRNA. In some embodiments, the moiety does not target a nucleic acid in a subject which the dsRNA is targeting. In some embodiments, the moiety does not have complementarity with a nucleic acid which the dsRNA is targeting. In some embodiments, the moiety does not have complementarity with a gene sequence or its mRNA transcript which the dsRNA is targeting.

In some embodiments, one or more components of the non-targeting moiety have a compound formula of:

wherein:

R1, R2, R3 and R4 are independently selected from H, 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, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, wherein one or more methylenes are interrupted or terminated by O, S, S (O) , SO₂, N (R') ₂, C (O) , cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocyclic. Each R’ may be independently selected from hydrogen, substituted or unsubstituted alkyl, aryl, aralkyl, alkylaryl, alkoxy, aryloxy, acyl or aliphatic, which may be linear, branched, cyclic, polycyclic, unsaturated, hydroxylated, carbonylated, phosphorylated, and/or sulfured. Preferably, one or more components of the non-targeting moiety have a phosphorothioate modification in the backbone. In some embodiments, R1, R2, R3 and R4 are independently selected from substituted or unsubstituted C1-C16 alkyl and aryl.

In some embodiments, the moiety comprises m components in which n are nucleotides, wherein m=1-50 and n=0-20. In some embodiments, the non-nucleotide components in the non-targeting moiety are selected from a group comprising 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 linkers that can be used for spacing two nucleotides. In some embodiments, the non-targeting moiety does not comprise any nucleotides and comprises at least 1 linker, at least 2 linkers, at least 3 linkers, at least 4 linkers, at least 5 linkers, at least 6 linkers, at least 7 linkers, at least 8 linkers, at least 9 linkers, at least 10 linkers, at least 11 linkers, at least 12 linkers, at least 13 linkers, at least 14 linkers, at least 15 linkers, at least 16 linkers, at least 17 linkers, at least 18 linkers, at least 19 linkers, at least 20 linkers, at least 21 linkers, at least 22 linkers, at least 23 linkers, at least 24 linkers, at least 25 linkers, at least 26 linkers, at least 27 linkers, at least 28 linkers, at least 29 linkers, at least 30 linkers or more. The linkers may be same or different and 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 among others. The moiety may comprise a combination of one or more linkers selected from 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, L42 linker. Same linkers can either be consecutively linked in tandem or interspersed by different linkers.

In some embodiments, the moiety comprises zero, one or more S9 linkers and one or more linkers selected from 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 moiety comprises one or more spacer-18 linker and one or more linkers selected from 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 moiety comprises one or more spacer-C6 linker and one or more linkers selected from 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 moiety comprises one or more L6 linker and one or more linkers selected from 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 moiety comprises one or more spacer-C3 linker and one or more linkers selected from 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 moiety comprises one or more L12 (d spacer) and one or more linkers selected from 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 moiety comprises one or more spacer-C12 linker and one or more linkers selected from 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 moiety comprises one or more spacer-L14 linker and one or more linkers selected from 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 moiety comprises one or more spacer-L15 linker and one or more linkers selected from 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 moiety comprises one or more spacer-L15 linker and one or more linkers selected from 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 moiety comprises one or more spacer-L16 linker and one or more linkers selected from 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 moiety comprises one or more C6x1 linker and one or more linkers selected from 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 moiety comprises one or more C6x2 linker and one or more linkers selected from 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 moiety comprises one or more C6x5 linker and one or more linkers selected from 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 moiety comprises one or more C6x7 linker and one or more linkers selected from 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 moiety comprises one or more L20 linker and one or more linkers selected from 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 moiety comprises one or more L42 linker and one or more linkers selected from 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.

In some specific embodiments, the moiety comprises 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 L10 linkers or more L10 linkers. Further, the moiety comprises a S9 linker, which may be internal in the moiety or at the terminal.

In some specific embodiments, in addition to one linker that is linked to the targeting oligonucleotide, the moiety further comprises 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 L12 linkers or more L12 linkers.

In certain embodiments, the non-targeting moiety comprises 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, or S9- (L10) ₃₀, among others. The linkage between S9 and L10 and/or between two L10s may be substituted by a PS bond.

In certain embodiments, the non-targeting moiety comprises 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, among others. The linkage between S9 and L12 and/or between two L12s may be substituted by a PS bond.

In certain embodiments, the non-targeting moiety comprises 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, among others. The linkage between L20 and L12 and/or between two L12s may be substituted by a PS bond.

In certain embodiments, the non-targeting moiety comprises 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 among others. The linkage between L42 and L12 and/or between two L12s may be substituted by a PS bond.

In certain embodiments, the non-targeting moiety comprises 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) ₂₄, S9- (L10) ₂₆, S9- (L10) ₂₈, or S9- (L10) ₃₀, wherein at least one phosphodiester bond between two adjacent linkers is substituted by a phosphorothioate (PS) , mesyl phosphoramidate or boranophosphate bond.

In certain embodiments, the non-targeting moiety comprises S9- (L12) ₂, S9- (L12) ₄, S9- (L12) ₆, S9- (L12) ₈, S9- (L12) ₁₀, S9- (L12) ₁₂, S9- (L12) ₁₄, S9- (L12) ₁₆, S9- (L12) ₁₈, S9- (L12) ₂₀, S9- (L12) ₂₂, S9- (L12) ₂₄, S9- (L12) ₂₆, S9- (L12) ₂₈, or S9- (L12) ₃₀, wherein at least one phosphodiester bond between two adjacent linkers is substituted by a phosphorothioate (PS) , mesyl phosphoramidate or boranophosphate bond.

In certain embodiments, the non-targeting moiety comprises L20- (L12) ₆, L20- (L12) ₁₂ or L20- (L12) ₁₈, wherein at least one phosphodiester bond between two adjacent linkers is substituted by a phosphorothioate (PS) , mesyl phosphoramidate or boranophosphate bond.

In certain embodiments, the non-targeting moiety comprises L42- (L12) ₆ or L42- (L12) ₁₂, wherein at least one phosphodiester bond between two adjacent linkers is substituted by a phosphorothioate (PS) , mesyl phosphoramidate or boranophosphate bond.

Optionally, the moiety is conjugated to the targeting oligonucleotide via a linker or linking component. The linkage between the linker and the terminal nucleotide in the targeting oligonucleotide may be substituted with phosphorothioate (PS) bond. In some embodiments, the moiety comprises one or more nucleotides in addition to one or more linkers. The one or more nucleotides may be interspersed by the linkers or may be consecutively linked to form a nucleotide sequence. For example, the linker linking the targeting oligonucleotide to the non-targeting moiety is spacer-9 linker. The linker between the targeting oligonucleotide and the moiety may also be considered as belonging to the non-targeting moiety.

In some embodiments, the moiety can modulate the biodistribution, bioavailability, and/or cell-uptake of the oligonucleotide agent within the target tissue or cell of interest. In some embodiments, efficacy, activity, pharmacokinetics, and/or pharmacodynamics of the oligonucleotide agent throughout the entire central nervous system are improved as compared to an oligonucleotide agent without the non-targeting moiety. In some embodiments, efficacy, activity, pharmacokinetics, and/or pharmacodynamics of the oligonucleotide agent in particular regions of the brain and spinal cord are improved. In some embodiments, endosomal escape and/or lysosomal escape of the oligonucleotide agent are improved.

The moiety may comprise a series of linkers and/or nucleotides that are modified in order to further increase the capacity of the oligonucleotide agent to deliver a targeting oligonucleotide. In some embodiments, the moiety comprises one or more of a chemically modified nucleotide, or at least one phosphodiester bond between two adjacent linkers or two adjacent nucleotides or a linker and an adjacent nucleotide in the moiety is substituted by a phosphorothioate or boranophosphate bond. The chemical modifications of the moiety includes, without limitation, modification of the 2’-OH of the ribose in the nucleotide, the modification or the absence of a base in the nucleotide, the locking or bridging of a nucleic acid, a nucleotide being a peptide nucleic acid, a nucleotide being a deoxyribonucleotide (DNA) , a nucleotide having a 5'-phosophate moiety, a nucleotide having a 5’- (E) ‐vinylphosphonate moiety, a nucleotide having a 5-methyl cytosine moiety, etc. Chemical modifications that may be found in moieties are also further described below.

The moiety may comprise at least one phosphodiester bond substituted with phosphorothioate (PS) bond on the backbone of the nucleotide sequence. In some embodiments, the moiety comprises multiple PS backbone modifications, e.g., at least 2 PS, at least 3 PS, at least 4 PS, at least 5 PS, at least 6 PS, or greater than 6 PS backbone modifications. In some embodiments, the moiety comprises 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%phosphodiester bond substituted with phosphorothioate (PS) bond on the backbone. As a non-limiting example, a moiety comprising 14 linkers may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 PS backbone modifications. In some embodiments, a moiety comprising m linkers and n nucleotides may comprise m+n-1 PS modifications.

The moiety may also have a specific composition of nucleotides and non-nucleotide chemical groups. Therefore, aspects of the present application further relate to an oligonucleotide agent capable of inhibiting the expression of superoxide dismutase 1 (SOD1) comprising a small interfering RNA (siRNA) , and a moiety.

The moiety may also have a specific composition of nucleotides and non-nucleotide chemical groups. Therefore, aspects of the present application further relate to an oligonucleotide agent capable of activating the expression of SMN2 comprising a saRNA, and a moiety.

In some embodiments, the oligonucleotide agent comprising one or more conjugated moieties to enhance the biodistribution and cell-uptake of the oligonucleotide agent in particular tissues of the oligonucleotide agent, and increase permeability of the oligonucleotide agent and passage through membranes, such as the blood brain barrier.

In some embodiments, the dsRNA and the moiety are covalently linked, with or without one or more linking components, to form the oligonucleotide agent.

In another aspect of the present application, an oligonucleotide agent comprising a siRNA and a non-targeting moiety is provided. In some embodiments, the oligonucleotide agent comprises a non-targeting moiety tethered to a 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.

In another aspect of the present application, an oligonucleotide agent comprising a saRNA and a non-targeting moiety is provided. In some embodiments, the oligonucleotide agent comprises a non-targeting moiety tethered to a 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.

In some embodiments, the moiety comprises one or more nucleotides. The nucleotides may be randomly selected. The nucleotides may be those of RNA, DNA, BNA, LNA PNA, or combinations thereof.

In some embodiments, the moiety interacts with one or more of: proteins in the plasma membrane, plasma proteins, peptides, ligands, lipids, fatty acids, saccharides, proteoglycan and zwitterionic phosphocholines. Such interaction of the moiety provides for increased biodistribution and enrichment of the targeting double stranded oligonucleotide of the oligonucleotide agent for local delivery to various target issues and cells of interest. Additionally, such interaction reduces or eliminates cytotoxicity of the oligonucleotide agent, confirming strong ‘on-target’ activity without overt effects on cell viability.

In certain embodiments, the protein interacting with moiety is selected from one or more of: serum albumin, IgG, Apolipoprotein A-I, 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.

In yet other embodiments, the protein interacting with moiety is selected from one or more of: 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.

In certain embodiments, the interaction of the moiety is through direct binding or mediated by one or more conjugated ligands which is covalently linked to the moiety or the double stranded oligonucleotide, or both. In some embodiments, the one or more conjugated ligands comprise a lipid, a fatty acid, a fluorophore, a saccharide, a peptide, an antibody, and any other commonly used conjugation ligands.

In certain embodiments, the conjugating ligands are selected from one or more of a cell-penetrating peptide, polyethylene glycol, an alkaloid, a tryptamine, a benzimidazole, a quinolone, an amino acid, a cholesterol, glucose and N-acetylgalactosamine. In certain embodiments, the one or more conjugation ligands is a fatty acid.

In some embodiments, the oligonucleotide agent comprising the one or more conjugation ligands enhances the biodistribution of the oligonucleotide agent in particular tissues, reduce or eliminate cytotoxicity of the oligonucleotide agent, and increase permeability of the oligonucleotide agent and passage through membranes, such as the blood brain barrier.

In certain embodiments, the moiety comprises a chemically modified nucleotide sequence.

In some embodiments, the oligonucleotide agent of the present application comprises more than one non-targeting moieties, for example, 2, 3, 4, 5, 6, 7, 9, 10 moieties, covalently linked to a dsRNA, with or without one or more linkers in between the moieties and dsRNA. The amount of moiety, upon need, can vary from 2 to 10, or 2 to 100, or 2 to 1,000, or 2 to 10,000, linked to dsRNA via a multivalent linker, for example, a polymeric linker, in branch or liner form. In some embodiments, multiple moieties are covalently linked to 2 or more dsRNAs, for example, 2, 3, 4, 5, 6, 7, 9, 10 or more dsRNAs, including saRNAs and/or siRNAs, in one agent.

In some embodiments, the oligonucleotide agent of the present application comprises one moiety and multiple dsRNAs, for example, 2, 3, 4, 5, 6, 7, 9, 10 dsRNAs, linked with or without one or more linkers in between the moiety and dsRNAs. The amount of dsRNA, including saRNAs and/or siRNAs, upon need, can vary from 2 to 10, or 2 to 100, or 2 to 1,000, or 2 to 10,000, linked to moiety via a multivalent linker, for example, a polymeric linker, in branch or liner form.

Targeting Oligonucleotide

In some embodiments, the targeting 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 deemed useful. dsRNAs that find use in the present disclosure include, without limitation, siRNA, saRNA, etc.

In some embodiments, the double-stranded oligonucleotide comprises a sense strand and an antisense strand, the antisense strand having complementarity to a target nucleic acid. In some embodiments, the antisense strand having complementarity to a target nucleic acid is located in a promoter sequence. In some embodiments, the antisense strand having complementarity to a target nucleic acid is located in a coding or template sequence of a gene. In some embodiments, one of the sense or antisense strands has complementarity to a target nucleic acid which is a gene transcript, e.g., a mRNA or a pre-mRNA.

In some embodiments, the dsRNA comprises a sense strand that is at least 17 contiguous nucleotides. In some embodiments, the dsRNA comprises a sense strand that is at least 18 contiguous nucleotides. In some embodiments, the dsRNA comprises a sense strand that is at most 60 contiguous nucleotides.

In some embodiments, the sense strand and the antisense strand independently has a length ranging from 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 the antisense strand independently has 10-100 nucleotides in length (e.g., 10-20 nucleotides, 10-50 nucleotides, 10-90 nucleotides, 20-95 nucleotides, 30-70 nucleotides, 40-80 nucleotides, 50-100 nucleotides, 10-40 nucleotides, 10-30 nucleotides) . In some embodiments, the sense strand is 10-60 nucleotides in length (e.g., 10-20 nucleotides, 10-50 nucleotides, 10-40 nucleotides, 10-30 nucleotides) . In some embodiments, the sense strand has a nucleotide length ranging from 27-41 nucleotides. In some embodiments, the antisense strand is 19-30 nucleotides in length. In some embodiments, the antisense strand is 18-26 nucleotides in length.

The double-stranded oligonucleotide may comprise a sequence that is modified in order to further increase the stability and/or the ability of the double-stranded oligonucleotide to modulate gene expression. In some embodiments, the sequence of the double-stranded oligonucleotide comprises one or more of a chemically modified nucleotide, or at least one phosphodiester bond between two adjacent nucleotides in the oligonucleotide sequence is substituted by a phosphorothioate or boranophosphate bond. The chemical modifications of the double-stranded oligonucleotide includes, without limitation, modification of the 2’-OH of the ribose in the nucleotide, the modification or the absence of a base in the nucleotide, the locking or bridging of a nucleic acid, a nucleotide being a peptide nucleic acid, a nucleotide being a deoxyribonucleotide (DNA) , a nucleotide having a 5'-phosophate moiety, a nucleotide having a 5’- (E) ‐vinylphosphonate moiety, a nucleotide having a 5-methyl cytosine moiety, etc. Chemical modifications that may be found in double-stranded oligonucleotides are also further described below.

Exemplary short interfering RNA (siRNA)

Embodiments of the present application are based in part on the disclosure that an oligonucleotide agent (for example, a siRNA, also referred to as “SOD1 gene siRNA” , “SOD1 siRNA” , or “siSOD1” herein) is capable of inhibiting or downregulating the expression of a SOD1 gene in a cell. The decrease in functional SOD1 gene transcript following administration with an oligonucleotide agent of the present application can achieve a significant decrease or downregulation in the levels of SOD1 mRNA and SOD1 protein in a cell or CNS of a mammal.

In particular, the inventors disclose that the functional oligonucleotide agents capable of inhibiting expression of superoxide dismutase 1 (SOD1) comprising a siRNA, wherein the siRNA comprises a sense strand and an antisense strand forming a double strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 10 contiguous nucleotides, with 0, 1, 2 or 3 mismatches, having at least 85%nucleotide sequence complementarity or homology to a portion of the nucleotide sequence of SOD1 mRNA.

In some embodiments, the differences or mismatches are located in the middle or 3’ terminus of the oligonucleotide sequence of the siRNA. Methods and principles of siRNA molecule design are well known to those skilled in the art and are described in detail in, 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 herein incorporated by reference in their entireties.

As a beneficial consequence, a target sequence (e.g., an isolated nucleic acid sequence comprising the target sequence) , upon interacting with the siRNA, can inhibit/downregulate the SOD1 mRNA transcript by at least 10%as compared to a baseline level of SOD1 mRNA. Based at least in part on these discoveries, the present application features siRNA, compositions, and pharmaceutical compositions for inhibiting/downregulating the SOD1 mRNA transcript by at least 10%as compared to baseline levels of SOD1 mRNA. In some embodiments, the siRNA inhibits or downregulates the SOD1 mRNA more than 10%. For instance, the siRNA inhibits or downregulates SOD1 mRNA by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, or greater than 100%as compared to baseline levels of SOD1 mRNA.

Also provided herein are methods for preventing or treating a disease or condition induced by over-expression of SOD1 protein, a SOD1 gene mutation, and/or high or abnormal SOD1 level in an individual comprising administering to the individual any of the siRNA, compositions, and/or pharmaceutical compositions described herein.

In some embodiments, the antisense strand disclosed herein is capable of interacting with a target nucleic acid sequence of a mRNA of a SOD1 gene in a sequence specific manner, meaning that the antisense strand is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding. In some embodiments, an antisense strand has a nucleotide sequence that, when written in the 5' to 3' direction, comprises the reverse complement of the target portion of a target nucleic acid to which it is targeted. In certain such embodiments, an antisense strand has a nucleotide sequence that, when written in the 5' to 3' direction, comprises the reverse complement of the target portion in a fragment of a SOD1 gene transcript.

Exemplary small activating RNA (saRNA)

Embodiments of the present application are based in part on the disclosure that an oligonucleotide agent (for example, saRNA, also referred to as “SMN2 gene saRNA” , “SMN2 saRNA” , or “saSMN2” herein) is capable of activating or upregulating the expression of a SMN2 gene in a cell. The decrease in functional SMN2 gene transcript following administration with an oligonucleotide agent of the present application can achieve a significant increase or upregulation in the levels of SMN2 mRNA and SMN2 protein in a cell or CNS of a mammal.

In particular, the inventors disclose that the functional oligonucleotide agents capable of activating expression of SMN2 comprising a saRNA, wherein the saRNA comprises a sense strand and an antisense strand forming a double strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 10 contiguous nucleotides, with 0, 1, 2 or 3 mismatches, having at least 85%nucleotide sequence complementarity or homology to a portion of the nucleotide sequence of SMN2 mRNA.

In some embodiments, the differences or mismatches are located in the middle or 3’ terminus of the oligonucleotide sequence of the saRNA. Methods and principles of saRNA molecule design are well known to those skilled in the art and are described in detail in, 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 herein incorporated by reference in their entireties. Chemical modifications

All nucleotides comprised in the oligonucleotides or non-targeting moiety (if any) described herein may be natural, i.e., non-chemically modified, nucleotides or at least one nucleotide may be a chemically modified nucleotide. Non-limiting examples of the chemical modification include one or more of a combination of the following: a) modification of a phosphodiester bond between two nucleotides, two linkers, a nucleotide and a linker in the oligonucleotide agent; b) modification of 2'-OH of the ribose in the nucleotide; c) modification of a base in the nucleotide; d) at least one nucleotide in the oligonucleotide sequence being a locked nucleic acid, and e) at least one nucleotide in the oligonucleotide sequence being a deoxyribonucleotide (DNA) .

In some embodiments, the nucleotides or oligonucleotides of the present application are chemically modified to enhance stability or other beneficial characteristics. The nucleic acids featured in the present application may be synthesized and/or modified by conventional methods, such as those described in “Current protocols in nucleic acid chemistry, ” Beaucage, S.L. et al. (Edrs. ) , John Wiley &Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, (a) end modifications, e.g., 5’ end modifications (phosphorylation, conjugation, inverted linkages, etc. ) 3’ end modifications (conjugation, DNA nucleotides, inverted linkages, etc. ) , (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides) , or conjugated bases, (c) sugar modifications (e.g., at the 2’ position or 4’ position) or replacement of the sugar, as well as (d) backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of siRNA molecules that can be used in this present application include but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. In some embodiments, RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. In some embodiments, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be oligonucleosides. In some embodiments, the modified oligonucleotide will have a phosphorus atom in its internucleoside backbone.

Chemical modifications of nucleotides or linkers in the present disclosure are well known to those skilled in the art, and modifications of the phosphodiester bond refer to modifications of oxygen in the phosphodiester bond, including phosphorothioate modifications and boronated phosphate modifications. The modifications disclosed herein stabilize an oligonucleotide structure, maintaining high specificity and high affinity for base pairing. The modifications disclosed herein also stabilize the non-targeting moiety structure and maintain its delivering accessory properties including bioavailability, biodistribution, and/or cellular uptake of the oligonucleotide agent in various tissues prefrontal cortex, cerebellum, spinal cord (e.g., cervical, thoracic, lumber) , muscle, liver, and kidney.

In some embodiments, the chemical modification is to substitute the phosphodiester bond with phosphorothioate (PS) bond on the backbone of the oligonucleotide agent disclosed herein. In some embodiments, the oligonucleotide agent disclosed herein comprises at least one PS backbone modification. In some embodiments, the non-targeting moiety comprises at least one PS backbone modification. In some embodiments, the oligonucleotide agent comprises at least 2 PS, at least 3 PS, at least 4 PS, at least 5 PS, at least 6 PS, or greater than 6 PS backbone modifications. In some embodiments, about 90%to about 95%of the phosphodiester backbone bond of the non-targeting moiety are substituted with phosphorothioate (PS) bond. In some embodiments, the oligonucleotide agent comprises at least one PS backbone modification on 5’ end, 3’ end or internal site of the sense strand of the dsRNA. In some embodiments, the oligonucleotide agent comprises at least one PS backbone modification on 5’ end, 3’ end or internal site of the antisense strand of the dsRNA. In some embodiments, the oligonucleotide agent comprises at least one PS backbone modification on 5’ end, 3’ end or internal site of the non-targeting moiety.

In some embodiments, the nucleotides or oligonucleotides of the present application includes at least one chemically modified nucleotide which is modified at 2'-OH in pentose of a nucleotide, i.e., the introduction of certain substituents at the hydroxyl position of the ribose, such as 2'-fluoro modification, 2'-oxymethyl modification, 2'-oxyethylidene methoxy modification, 2, 4'-dinitrophenol modification, locked nucleic acid (LNA) , 2'-amino modification or 2'-deoxy modification, e.g., a 2’-deoxy-2’-fluoro modified nucleotide, a 2’-deoxy-modified nucleotide.

In some embodiments, the nucleotides or oligonucleotides of the present application includes at least one chemically modified nucleotide which is modified at the base of the nucleotide, e.g., 5 '-bromouracil modification, 5’-iodouracil modification, N-methyluracil modification, or 2, 6-diaminopurine modification.

In some embodiments, the chemical modification of the nucleotides or oligonucleotides in the present application is an addition of a (E) ‐vinylphosphonate moiety at the 5’ end of the sense or antisense sequence. In some embodiments, the chemical modification of the at least one chemically modified nucleotide is an addition of a 5-methyl cytosine moiety at the 5’ end of the sense or antisense sequence.

In some embodiments, the nucleotides or oligonucleotides in the present application are modified at the base of the nucleotide, e.g., 5 '-bromouracil modification, 5'-iodouracil modification, N-methyluracil modification, or 2, 6-diaminopurine modification. In some embodiments, at least one oligonucleotide in the oligonucleotide agent includes at least one modified nucleotide, e.g., a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide. In some embodiments, the first and second dsRNAs include “endo-light” modification with 2′-O-methyl modified nucleotides and nucleotides comprising a 5′-phosphorothioate group.

Modified backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3’-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3’-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3’-5’ linkages, 2’-5’ linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3’-5’ to 5’-3’ or 2’-5’ to 5’-2’. Various salts, mixed salts and free acid forms are also included.

Non-limiting examples of preparation of the phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 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. Pat. RE39464, which are herein specifically each incorporated by reference in their entireties.

In some embodiments, the nucleotides or oligonucleotides comprise one or more of RNA, DNA, BNA, LNA or peptide nucleic acid (PNA) .

In other RNA mimetics suitable or contemplated for use in siRNAs, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA) . In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion, S or O atoms of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

In some embodiments, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or least about 95%, or about 100%nucleotides of the non-targeting moiety, if present, are chemically modified nucleotides.

In some embodiments, the sense strand and the antisense strand of the oligonucleotide agent independently comprise 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%nucleotides which are chemically modified nucleotides.

These modifications may increase the bioavailability of the oligonucleotides, increase affinity for the target sequence, and enhance resistance to nuclease hydrolysis in a cell.

In addition, to facilitate entry of the oligonucleotides into a cell, lipophilic groups such as cholesterol may be introduced at the ends of the sense or antisense strands of the oligonucleotides on the basis of the above modifications to facilitate action through a cell membrane composed of lipid bilayers and gene promoter regions within the nuclear membrane and nucleus.

In some embodiments, the oligonucleotide agents of the present application which, upon contact with a cell, are effective in deactivating or downregulating the expression of one or more genes in the cell, preferably by 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%) .

In some embodiments, the oligonucleotide agents of the present application which, upon contact with a cell, are effective in activating or upregulating the expression of one or more genes in the cell, preferably by 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%) .

One aspect of the application provides a cell comprising an oligonucleotide agent of the present application or a nucleic acid encoding the oligonucleotide agent of the present application. In one embodiment, the cell is a mammalian cell, preferably a human cell. Such cells may be ex vivo, such as cell lines or cell lines, and the like, or may be present in mammalian bodies, such as humans, including infants, children or adults.

In some embodiments, the at least one chemical modified oligonucleotide is in the non-targeting moiety. In some embodiments, the at least one chemical modified oligonucleotide is in the targeting double stranded oligonucleotide. In certain embodiments, the at least one chemical modified oligonucleotide is in the non-targeting moiety and the targeting double stranded oligonucleotide.

Covalent Linkage or Conjugation

Aspects of the present application include an oligonucleotide agent comprising a double-stranded targeting oligonucleotide and a non-targeting moiety that are covalently linked.

In some embodiments, a double-stranded targeting oligonucleotide and a non-targeting moiety are covalently linked by a linking component.

In some embodiments, the double-stranded oligonucleotide and the non-targeting moiety are linked with a covalent linker. In some embodiments, the linker is a disulfide linker. Various combinations of strands can be linked, e.g., the dsRNA sense strand and the non-targeting moiety are covalently linked or the dsRNA antisense strand and the non-targeting moiety are covalently linked.

In some embodiments, the sense strand of the double stranded targeting oligonucleotide is covalently linked to the non-targeting moiety. In some embodiments, the antisense strand of the double stranded targeting oligonucleotide is covalently linked to the non-targeting moiety.

In some embodiments, any of the oligonucleotides and linkers in the oligonucleotide agent of the present application includes a linking component.

Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR¹, C (O) , C (O) O, C (O) NR¹, SO, SO₂, SO₂NH or a chain of atoms, such as 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, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, where one or more methylenes can be interrupted or terminated by O, S, S (O) , SO₂, N (R') ₂, C (O) , cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R¹ is hydrogen, acyl, aliphatic or substituted aliphatic. Each R’ is independently selected from hydrogen, substituted or unsubstituted alkyl, aryl, aralkyl, alkylaryl, alkoxy, aryloxy, acyl or aliphatic, which may be linear, branched, cyclic, polycyclic, unsaturated, hydroxylated, carbonylated, phosphorylated, and/or sulfured.

Without limitations, various types of linker functionality can be included in the subject conjugates, including but not limited to cleavable linkers, and non-cleavable linkers, as well as reversible linkers and irreversible linkers.

In some embodiments, the linker is a cleavable linker. Cleavable linkers are those that rely on processes inside a target cell to liberate the two parts the linker is holding together, e.g., the non-targeting moiety and the dsRNA, as reduction in the cytoplasm, exposure to acidic conditions in a lysosome or endosome, or cleavage by specific enzymes (e.g., proteases) within the cell. As such, cleavable linkers allow the dsRNA to be released in its original form after the conjugate has been internalized and processed inside a target cell. Cleavable linkers include, but are not limited to, those whose bonds can be cleaved by enzymes (e.g., peptide linkers) ; reducing conditions (e.g., disulfide linkers) ; or acidic conditions (e.g., hydrazones and carbonates) .

In some embodiments, the linking component is selected from one or more of ethylene glycol chain, an alkyl chain, a peptide, nucleic acid, carbohydrates, thiol linkage, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, and a carbamate. In some embodiments, the linking component includes, but is not limited to:

f) L12 (d spacer) ( (2R, 3S) -2- ( (bis (4-methoxyphenyl) (phenyl) methoxy) methyl) tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite) ;

m) C6x5 (2- ( (2- (bis (4-methoxyphenyl) (phenyl) methoxy) ethyl) (pent-4-yn-1-yl) amino) ethyl (2-cyanoethyl) diisopropylphosphoramidite) ; and

In certain embodiments, the linking component comprises a compound structure shown in Table 1. In certain embodiments, one or more of the linking components are linked in tandem to constitute the non-targeting moiety. In certain embodiments, the linking component conjugates to a nucleotide in the non-targeting moiety or the double-stranded oligonucleotide. In certain embodiments, the linking component conjugates at a nucleoside position selected from 5’-phosphate, 3’, base and 2’-H/OH of a nucleotide in the single-stranded oligonucleotide or the double-stranded oligonucleotide. In certain embodiments, the linking component is Spacer phosphoramidite 18 (Phosphoramidous acid, N, N-bis (1-methylethyl) -, 19, 19-bis (4-methoxyphenyl) -19-phenyl-3, 6, 9, 12, 15, 18-hexaoxanonadec-1-yl 2-cyanoethyl ester) .

In some embodiments, the double-stranded targeting oligonucleotide and the non-targeting moiety are covalently linked by a phosphodiester bond. In some embodiments, the double-stranded targeting oligonucleotide and the non-targeting moiety are covalently linked by a phosphorothioate bond.

In some embodiments, the double-stranded targeting oligonucleotide comprises a sense strand that is covalently linked to the non-targeting moiety. In some embodiments, the double-stranded targeting oligonucleotide comprises an antisense strand that is covalently linked to the non-targeting moiety.

In some embodiments, the double-stranded targeting oligonucleotide and the non-targeting moiety are covalently linked by one or more nucleotides.

In some embodiments, the double-stranded targeting oligonucleotide and the non-targeting moiety are covalently linked by one or more linkers.

Non-limiting examples of covalent linkers can be found in U.S. Patent Application Publication No.: 20200332292, which is hereby incorporated by reference in its entirety. The covalent linker can join the double-stranded targeting oligonucleotide and the non-targeting moiety. In some embodiments, the covalent linker can join two sense strands, two antisense strands, one sense and one antisense strand, two sense strands and one antisense strand, two antisense strands and one sense strand, two sense and two antisense strands, an antisense strand and single-stranded oligonucleotide, a sense strand and inactivated oligonucleotide, and the like.

In certain embodiments, the covalent linker includes a nucleic acid (e.g., RNA and/or DNA) and/or a peptide. The linker can be single-stranded, double-stranded, partially single-strands, or partially double-stranded. In some embodiments the linker includes a disulfide bond. The linker can be cleavable or non-cleavable.

In certain embodiments, the covalent linker includes, e.g., dTsdTuu= (5′-2′deoxythymidyl-3′-thiophosphate-5′-2′deoxythymidyl-3′-phosphate-5′-uridyl-3′-phosphate-5′-uridyl-3′-phosphate) ; rUsrU (a thiophosphate linker: 5′-uridyl-3′-thiophosphate-5′-uridyl-3′-phosphate) ; an 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′deoxythyrnidyl-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.

When the covalent linker is a RNA, the RNA linker may be composed of any combination of nucleotides. The combination of nucleotides may be adenine, uracil, guanosine, cytosine, or any combination thereof. The RNA linker may be any length. In some embodiments, the RNA linker is 2-50 nucleotides in length. When the RNA linker is 2-50 nucleotides in length, the RNA linker may be any intervening length such as 5-10, 10-15, or 15-20 nucleotides in length. In some embodiments, the covalent linker includes a polyRNA, such as poly (5′-adenyl-3′-phosphate-AAAAAAAA) or poly (5′ -cytidyl-3′-phosphate-5′-uridyl-3′-phosphate-CUCUCUCU) ) , e.g., X_n single-stranded poly RNA linker wherein n is an integer from 2-50 inclusive, preferable 4-15 inclusive, most preferably 7-8 inclusive. Modified nucleotides or a mixture of nucleotides can also be present in said polyRNA linker. When the covalent linker is a DNA, the DNA linker may be composed of any combination of nucleotides. The combination of nucleotides may be adenine, thymine, guanosine, cytosine, or any combination thereof. The DNA linker may be any length. In some embodiments, the DNA linker is 1- 50 nucleotides in length. When the DNA linker is 1-50 nucleotides in length, the DNA linker may be any intervening length such as 5-10, 10-15, or 15-20 nucleotides in length. The covalent linker can be a polyDNA, such as poly (5′-2′deoxythymidyl-3′-phosphate-TTTTTTTT) , e.g., wherein n is an integer from 2-50 inclusive, preferable 4-15 inclusive, most preferably 7-8 inclusive. Modified nucleotides or a mixture of nucleotides can also be present in said polyDNA linker. a single-stranded polyDNA linker wherein n is an integer from 2-50 inclusive, preferable 4-15 inclusive, most preferably 7-8 inclusive. Modified nucleotides or a mixture of nucleotides can also be present in said polyDNA linker.

In some embodiments, the covalent linker includes a disulfide bond, optionally a bis-hexyl-disulfide linker. In one embodiment, the disulfide linker is

In some embodiments, the covalent linker includes a peptide bond, e.g., include amino acids. In one embodiment, the covalent linker is a 1-10 amino acid long linker, preferably comprising 4-5 amino acids, optionally X-Gly-Phe-Gly-Y wherein X and Y represent any amino acid.

In some embodiments, the covalent linker includes HEG, a hexaethylenglycol linker.

Orientation and position of covalent linkage

Aspects of the present application include covalently linking the double-stranded targeting oligonucleotide and the non-targeting moiety, to form an oligonucleotide agent. In some embodiments, the orientation of the linkage and positioning of the double-stranded targeting oligonucleotide and the non-targeting moiety may enhance stability, oligonucleotide activity, or other beneficial characteristics, such as maximized target gene output, increased or decreased activity or expression (e.g., mRNA expression, protein expression, etc. ) of one or more target genes.

In some embodiments, the non-targeting moiety is covalently linked to a 3’ end of the sense or antisense strand of the double-stranded target oligonucleotide; b) the non-targeting moiety is covalently linked to a 5’ end of the sense or antisense strand of the double-stranded targeting oligonucleotide; or c) the non-targeting moiety is covalently linked to an internal nucleotide between the 5’ end and the 3’ end of the sense or antisense strand of the double-stranded targeting oligonucleotide. In some embodiments, the internal nucleotide of the sense or antisense strand of the double-stranded targeting oligonucleotide is located at nucleotide position 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 from 5’ end of the sense or antisense strand; or located at nucleotide position 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 from 3’ end of the sense or antisense strand.

In some embodiments, an internal nucleotide of the sense or antisense strand of the double-stranded targeting oligonucleotide is substituted by one or more linking component or spacer which is covalently linked to the non-targeting moiety on its 5’ end or 3’ end (i.e., internal conjugated) . In some embodiments, the internal conjugated cODV has enhanced potency as compared to the 3’ or 5’ end conjugated cODV (i.e., non-targeting moiety conjugated on 3’ or 5’ end of the sense or antisense strand of the double stranded oligonucleotide) .

In certain embodiments, the 5’ end of the single-stranded oligonucleotide is conjugated to a linking component. In some embodiments, the 3’ end of the single-stranded oligonucleotide is conjugated to a linking component.

In certain embodiments, the linking component or spacer comprises a compound shown in Table 1.

Agents that decrease/upregulate the expression of a target gene or protein

Various double stranded oligonucleotides can be designed for many target genes, such as those associated with a specific disease or disorder. In some embodiments, the oligonucleotide agent decreases the expression of a SOD1 gene or protein. Administration of the oligonucleotide agent to a patient treats or delays the onset of ALS, such as familial or sporadic ALS or Leu Lou Gehrig's disease.

Various double stranded oligonucleotides can be designed for many target genes, such as those associated with a specific disease or disorder. In some embodiments, the oligonucleotide agent upregulates the expression of a SMN2 gene or protein. Administration of the oligonucleotide agent to a patient treats or delays the progression or the severity of Spinal Muscular Atrophy (SMA) disease.

The administration may be performed in any route of administration deemed useful. In some embodiments, the administration route is locally at the site of a central nervous system location. In some embodiments, the administration route is systemic.

In certain embodiments, a double-stranded targeting oligonucleotide of the oligonucleotide agent that decreases the expression of the target gene or protein is an siRNA. The siRNA deactivates or downregulates the expression of the target gene, its mRNA transcript or protein in a cell in which the gene, its mRNA transcript or protein is abnormally or over-expressed.

In certain embodiments, a double-stranded targeting oligonucleotide of the oligonucleotide agent that upregulates the expression of the target gene or protein is an saRNA. The saRNA activates or upregulates the expression of the target gene, its mRNA transcript or protein in a cell in which the gene, its mRNA transcript or protein is abnormally or under-expressed.

In certain embodiments of the present application, the siRNA comprises a sense nucleic acid strand and an antisense nucleic acid strand, the sense nucleic acid strand comprising at least one region that is complementary to at least one region on the antisense nucleic acid strand to form a double-stranded nucleic acid structure capable of deactivating expression of the protein in a cell.

In certain embodiments of the present application, the saRNA comprises a sense nucleic acid strand and an antisense nucleic acid strand, the sense nucleic acid strand comprising at least one region that is complementary to at least one region on the antisense nucleic acid strand to form a double-stranded nucleic acid structure capable of activating expression of the protein in a cell.

In certain embodiments of the present application, the sense nucleic acid strand and the antisense nucleic acid strand are located on two different nucleic acid strands. In certain embodiments of the present application, the sense nucleic acid fragment and the antisense nucleic acid fragment are located on the same nucleic acid strand, forming a hairpin single-stranded nucleic acid molecule, wherein the complementary regions of the sense nucleic acid fragment and the antisense nucleic acid fragment form a double-stranded nucleic acid structure.

In some embodiments, the oligonucleotide agent has a nucleotide sequence of the antisense strand 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 SEQ ID NO: 4 that has complementarity with a fragment of the cODV structured sense strand of any of SEQ ID NOs: 6-39.

In some embodiments, the oligonucleotide agent has a nucleotide sequence of the antisense strand 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 SEQ ID NO: 57 that has complementarity with a fragment of the cODV structured sense strand of any of SEQ ID NOs: 60.

In some embodiments, the oligonucleotide agent has a nucleotide sequence of the antisense strand 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 SEQ ID NO: 62 that has complementarity with a fragment of the cODV structured sense strand of any of SEQ ID NOs: 64.

In some embodiments, the oligonucleotide agent has a nucleotide sequence of the antisense strand 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 SEQ ID NO: 4 that has complementarity with a fragment of the cODV structured sense strand of any of SEQ ID NOs: 39, 65, 66 and 67.

In some embodiments, the oligonucleotide agent has a nucleotide sequence of the antisense strand 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 SEQ ID NO: 69 that has complementarity with a fragment of the cODV structured sense strand of any of SEQ ID NOs: 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86 and 87.

In some embodiments, an siRNA includes a nucleotide sequence of a sense strand 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 the nucleotide sequence selected from: SEQ ID NOs: 1, 3, 56 and 61. In some embodiments, an siRNA includes a nucleotide sequence of an antisense strand 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 the nucleotide sequence selected from the group of: SEQ ID NOs: 2, 4, 57 and 62.

In addition, to facilitate entry of the siRNA into a cell, chemical conjugation groups other than the non-targeting moiety disclosed herein may be introduced at the ends of the sense or antisense strands of the siRNA on the basis of the above modifications to facilitate action through a cell membrane composed of lipid bilayers and mRNA regions within the nuclear membrane and nucleus.

In addition, to facilitate entry of the saRNA into a cell, chemical conjugation groups other than the non-targeting moiety disclosed herein may be introduced at the ends of the sense or antisense strands of the saRNA on the basis of the above modifications to facilitate action through a cell membrane composed of lipid bilayers and mRNA regions within the nuclear membrane and nucleus.

In some embodiments, siRNAs disclosed in the present application are covalently attached to one or more conjugate groups. In some embodiments, conjugate groups modify one or more properties of the attached oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge and clearance. In some embodiments, conjugate groups impart a new property on the attached oligonucleotide, e.g., fluorophores or reporter groups that enable detection of the oligonucleotide. Certain conjugate groups and conjugate moieties have been described previously, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556) , cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060) , a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N. Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3, 2765-2770) , a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538) , an aliphatic chain, e.g., do-decan-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) , a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1, 2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783) , a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides &Nucleotides, 1995, 14, 969-973) , or adamantane acetic acid a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237) , an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937) , a tocopherol group (Nishina et al., Molecular Therapy Nucleic Acids, 2015, 4, e220; and Nishina et al., Molecular Therapy, 2008, 16, 734-740) , or a GalNAc cluster (e.g., WO2014/179620) .

In some embodiments, the siRNA/saRNA of the present application relates to the sense strand or the antisense strand of the siRNA/saRNA that is conjugated to one or more conjugation groups selected from: intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates, vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins, fluorophores, and dyes.

In some embodiments, a conjugate group comprises an active drug substance, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (S) - (+) -pranoprofen, carprofen, dansylsarcosine, 2, 3, 5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.

In some embodiments, the siRNA/saRNA of the present application is conjugated to one or more conjugation groups selected from: a lipid, a fatty acid, a fluorophore, a ligand, a saccharide, a peptide, and an antibody.

In some embodiments, the siRNA/saRNA of the present application relates to the sense strand or the antisense strand of the siRNA/saRNA that is conjugated to one or more conjugation groups selected from a cell-penetrating peptide, polyethylene glycol, an alkaloid, a tryptamine, a benzimidazole, a quinolone, an amino acid, a cholesterol, glucose and N-acetylgalactosamine.

In some embodiments, the siRNA/saRNA conjugated to one or more conjugation groups disclosed in the embodiments is directly contacted, transferred, delivered or administrated to a cell or a subject.

Methods of modulating gene expression

In some aspects, oligonucleotide agents of the present application can be useful for therapeutic approaches to treating diseases such as spinal muscular atrophy (SMA) or ALS.

By non-limiting embodiments, the present application provides a method of decreasing or silencing the levels of mRNA transcript of a SOD1 gene or SOD1 protein in a cell or individual, comprising administering to a subject a pharmaceutical composition disclosed herein.

In some embodiments, the non-targeting moiety of the oligonucleotide agent improves the stability, bioavailability, biodistribution, and/or cellular uptake of the siRNA/saRNA as compared to an oligonucleotide agent without the non-targeting moiety. In some embodiments, the non-targeting moiety of the oligonucleotide agent increases the biodistribution of siRNA/saRNA within one or more target tissues as compared to an oligonucleotide agent without the non-targeting moiety. In some embodiments, the non-targeting moiety of the oligonucleotide agent increases the biodistribution of siRNA/saRNA within two or more target tissues as compared to an oligonucleotide agent without the non-targeting moiety.

In some embodiments, the one or more target tissues is selected from tissues of brain, spinal cord, muscle, spleen, lung, heart, liver, bladder, and kidney. In some embodiments, the one or more target tissues is selected from the group of: prefrontal cortex, cerebellum, and cerebrum; cervical, thoracic and lumbar in spinal cord; heart, bicep, semitendinosus, platysma, and gluteus.

In some embodiments, the oligonucleotide agent of the present application achieves a decrease in full-length protein that is less than the amount achieved by administration of the same amount of double stranded oligonucleotide such as a siRNA substance without an cODV structure used individually, with higher potency, reduced toxicity, or unwanted side effects. In some embodiments, the oligonucleotide agent of the present application achieves a decrease in full-length protein that is less than the additive effect of treatment with the same amount of the double-stranded targeting oligonucleotide used individually.

In some embodiments, the oligonucleotide agent of the present application achieves an increase in full-length protein that is more than the amount achieved by administration of the same amount of double stranded oligonucleotide such as a saRNA substance without an cODV structure used individually, with higher potency, reduced toxicity, or unwanted side effects.

In any of the embodiments provided herein, such cells may be ex vivo, such as cell lines, and the like, or may be present in mammalian bodies, such as humans. In some embodiments, the human is a subject or individual suffering from a SOD1 protein related condition or ALS.

Another aspect of the application relates to the use of an oligonucleotide agent of the present application, a nucleic acid encoding two or more oligonucleotides of the oligonucleotide agent of the present application or a composition comprising the oligonucleotide agent of the present application or a nucleic acid encoding two or more oligonucleotides of the oligonucleotide agent of the present for the preparation of a medicament for the treatment or delaying the onset of an SMN-deficiency-related condition or ALS. The subject may be a mammal, such as a human. The subject may be an infant, a child or an adult.

In certain embodiments, the oligonucleotide agent of the present application achieves a decrease in full-length SOD1 protein that is less than the amount achieved by administration of the same amount of double stranded oligonucleotide substance used individually, with reduced toxicity or unwanted side effects. In certain embodiments, the oligonucleotide agent of the present application achieves a decrease in full-length SOD1 protein that is less than the additive effect of treatment with the same amount of the double stranded targeting oligonucleotide used individually.

In certain embodiments, the effect of the oligonucleotide agent of the present application achieves a greater clinical improvement compared to the effect of the same amount of either substance used individually. In certain embodiments, the effect of the oligonucleotide agent achieves a greater than additive clinical improvement compared to the effect of the same amount of double-stranded oligonucleotide used individually.

In any of the embodiments provided herein, such oligonucleotide agent, nucleic acids encoding the oligonucleotide agent of the present application, or compositions comprising such oligonucleotide agent or nucleic acids encoding oligonucleotide agent of the present application may be introduced directly into a cell, or may be produced intracellularly upon introduction of a nucleotide sequence encoding the oligonucleotide agent into a cell, preferably a mammalian cell, more preferably a human cell. Such cells may be ex vivo, such as cell lines, and the like, or may be present in mammalian bodies, such as humans. In some embodiments, the human is a patient or individual suffering from a SMN-deficiency-related condition or ALS. In certain embodiments, a nucleic acid encoding an oligonucleotide agent or a composition comprising the aforementioned oligonucleotide agent or a nucleic acid encoding an oligonucleotide agent of the application, in respective amounts sufficient to effect treatment of ALS.

In certain embodiments, the baseline measurement is obtained from a biological sample, as defined herein, obtained from an individual prior to administering the therapy described herein. In certain embodiments, the biological sample is peripheral blood mononuclear cells, blood plasma, serum, skin tissue, cerebrospinal fluid (CSF) .

Cells comprising siRNA

After contacting a cell, the oligonucleotide agent disclosed herein can effectively inhibit or downregulate the expression of a target gene in a cell, for example downregulate the expression by at least 10% (e.g., as compared to baseline transcription) .

In some embodiments, the present application relates to a cell comprising the oligonucleotide agent disclosed herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell, such as a human cell in various tissues in organs including brain, spinal cord, muscle, spleen, lung, heart, liver, bladder, and kidney. In some embodiments, the cell in target tissues is selected from the group of: prefrontal cortex, cerebellum, and cerebrum; cervical, thoracic and lumbar in spinal cord; heart, bicep, semitendinosus, platysma, and gluteus muscles.

The cell disclosed herein may be in vitro, or ex vivo, such as a cell line or a cell strain, or may exist in a mammalian body, such as a human body.

In some embodiments, the cell is from a CNS tissue of a subject suffering from ALS. In some embodiments, the cell is from a subject suffering from ALS.

Compositions of an oligonucleotide agent

Another aspect of the present application provides a pharmaceutical composition comprising the double stranded targeting oligonucleotide and the non-targeting single-stranded oligonucleotide as described in the present application.

The present application provides a composition or pharmaceutical composition capable of downregulating the level of SOD1 mRNA transcript by the mechanism of action (MoA) of RNA interference, comprising the oligonucleotide agent disclosed herein, to treat or prevent onset of a SOD1 related disease (particularly ALS) .

In some embodiments, the present application relates to a composition or pharmaceutical composition comprising the siRNA/saRNA of the present application. In some embodiments, the present application relates to a composition or pharmaceutical composition comprising the siRNA/saRNA and the non-targeting moiety as described herein. In some embodiments, the present application relates to a composition or pharmaceutical composition comprising the siRNA/saRNA and the non-targeting moiety covalently linked by a linking component as described herein.

In some embodiments, the pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier. In one embodiment, the pharmaceutically acceptable carrier includes one or more of an aqueous carrier, liposome or LNP, polymer, micelle, colloid, metal nanoparticle, non-metallic nanoparticle, bioconjugates (e.g., GalNAc) , and polypeptide.

In some embodiments, the composition comprises 1-150 nM of the oligonucleotide agent of the present application.

Another aspect of the present application relates to the use of the oligonucleotide agent as described herein, a nucleic acid encoding the oligonucleotides agent as described herein, or a composition comprising such oligonucleotide agent or a nucleic acid encoding the oligonucleotide agent as described herein, where the double-stranded targeting oligonucleotide and the single-stranded oligonucleotide are covalently linked, for the preparation of one or more compositions for modulate the expression of one or more genes or proteins expressed by a cell.

Another embodiment provides pharmaceutical compositions or medicaments comprising the agents of the present application and a therapeutically inert carrier, diluent or pharmaceutically acceptable excipient, as well as methods of using the agents of the present application to prepare such compositions and medicaments.

For the oligonucleotide agent compositions of the present application, the delivery can be optionally through parenteral infusions including intrathecal, intramuscular, intravenous, intraarterial, intraperitoneal, intravesical, intracerebroventricular, intravitreal or subcutaneous administration; or through oral administration, intranasal administration, inhaled administration, vaginal administration, or rectal administration.

A typical formulation is prepared by mixing an agent of the present application and a carrier or excipient. Suitable carriers and excipients are well known to those skilled in the art and are described in detail in, e.g., Ansel H. C. et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems (2004) Lippincott, Williams &Wilkins, Philadelphia; Gennaro A. R. et al., Remington: The Science and Practice of Pharmacy (2000) Lippincott, Williams &Wilkins, Philadelphia; and Rowe R.C, Handbook of Pharmaceutical Excipients (2005) Pharmaceutical Press, Chicago.

Compositions of the present application are formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.

In another aspect, the application provides use of the oligonucleotide agent, according to any one of the embodiments described herein, or a composition according to any one of the embodiments described herein, in the manufacture of a medicament for the treatment of gene or protein-related condition in an individual. The use according to certain embodiments, the condition can include a SMN-deficiency-related condition that comprises ALS. The use according to certain embodiments, the condition can include a SMN-deficiency-related condition that comprises a hereditary neuromuscular disease, preferably spinal muscular atrophy. In other embodiments, the condition can include an immune-related condition, such as cancer. Also provided is the use according to certain embodiments wherein the individual is a mammal, preferably a human.

Dose regiments and route of administration

Aspects of the present application relate to a pharmaceutical composition comprising the oligonucleotide agent of the present application. In some embodiments, the pharmaceutical composition comprising the oligonucleotide agent of the present application and a pharmaceutically acceptable carrier, a therapeutically inert carrier, diluent or pharmaceutically acceptable excipient. The pharmaceutical composition disclosed herein is to be developed into a medicament preventing or treating the SOD1 protein related condition or ALS.

Kits

In another aspect, any of the compositions described herein can be provided in one or more kits, optionally including instructions for use of the compositions. That is, the kit can include a description of use of an oligonucleotide agent or composition in any method described herein. A "kit, " as used herein, typically defines a package, assembly, or container (such as an insulated container) including one or more of the components or embodiments of the application, and/or other components associated with the application, for example, as previously described. Any of the agents or components of the kit may be provided in liquid form (e.g., in solution) , or in solid form (e.g., a dried powder, frozen, etc. ) .

In summary, the present invention offers a technologically superior approach to nucleic acid-based therapeutics, with improved self-delivery properties, enhanced efficacy, reduced cytotoxicity, cost-effective synthesis, and minimized off-target effects. These advantages position the invention as a promising candidate for further development and potential commercialization, with the ultimate goal of improving patient outcomes through safer and more effective treatments.

Exemplary embodiments

The present application provides the following particular embodiments:

Embodiment 1: An oligonucleotide agent comprising a targeting oligonucleotide conjugated to a non-targeting moiety capable of facilitating delivery of the targeting oligonucleotide, wherein the non-targeting moiety comprises one or more units that are covalently linked in tandem to form a backbone of the non-targeting moiety, and at least two adjacent units are linked via a phosphorothioate (PS) bond; wherein each unit in the non-targeting moiety is selected from chemical linkers and nucleotides; and

wherein the non-targeting moiety comprises:

one or more chemical linkers interspersed in nucleotides; one or more nucleotides interspersed in chemical linkers; a consecutive nucleotide sequence and a consecutively linked sequence of chemical linkers; or a consecutively linked sequence of chemical linkers without any nucleotide;

wherein at least one phosphodiester bond between two adjacent nucleotides, between two adjacent linkers or between a nucleotide and an adjacent linker is substituted by a phosphorothioate (PS) , mesyl phosphoramidate or boranophosphate bond.

Embodiment 2: The oligonucleotide agent according to Embodiment 1, wherein the chemical linkers are selected from the following:

Embodiment 3: The oligonucleotide agent according to Embodiment 1, wherein the chemical linkers are chemical groups selected from the following: 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, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, wherein one or more methylenes are interrupted or terminated by O, S, S (O) , SO₂, N (R') ₂, C (O) , cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocyclic, and wherein each R’ is independently selected from hydrogen, substituted or unsubstituted alkyl, aryl, aralkyl, alkylaryl, alkoxy, aryloxy, acyl or aliphatic which may be linear or branched.

Embodiment 4: The oligonucleotide agent according to any one of Embodiments 1-3, wherein the non-targeting moiety comprises m same or different chemical linkers and n same or different nucleotides, wherein m is an integer in the range of 1-40 and n is an integer in the range of 0-30. Embodiment 5: The oligonucleotide agent according to any one of Embodiments 1-4, wherein the non-targeting moiety comprises a consecutively linked sequence of chemical linkers as shown in formula (linker1) _x- (linker2) _y, wherein linker1 is a first chemical linker and linker2 is a second chemical linker different from the first chemical linker, and x and y are integers with 0<x+y<50, for example, the non-targeting moiety comprises 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,

wherein at least one phosphodiester bond between two adjacent linkers is substituted by a phosphorothioate (PS) , mesyl phosphoramidate or boranophosphate bond.

Embodiment 6: The oligonucleotide agent according to any of Embodiments 1-5, wherein the non-targeting moiety comprises 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 the backbone.

Embodiment 7: The oligonucleotide agent according to any of Embodiments 1-5, wherein the non-targeting moiety comprises 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, 40 or more phosphorothioate (PS) bonds in the backbone.

Embodiment 8: The oligonucleotide agent according to any of Embodiments 1-7, wherein the targeting oligonucleotide is an antisense oligonucleotide, or a double-stranded oligonucleotide comprising a sense strand and an antisense strand, such as a small interfering RNA (siRNA) or a small activating RNA (saRNA) .

Embodiment 9: The oligonucleotide agent according to Embodiment 8, wherein the non-targeting moiety is conjugated to the sense strand or the antisense strand of the double-stranded oligonucleotide. Embodiment 10: The oligonucleotide agent according to any one of Embodiments 1-9, wherein if present, the nucleotide (s) of the non-targeting moiety are non-chemically modified nucleotides, or at least one nucleotide is a chemically modified nucleotide.

Embodiment 11: The oligonucleotide agent according to any one of Embodiments 1-10, wherein all nucleotides of the targeting oligonucleotide are non-chemically modified nucleotides, or at least one nucleotide is a chemically modified nucleotide, or at least one phosphodiester bond between two adjacent nucleotides in the targeting oligonucleotide is substituted by a phosphorothioate, mesyl phosphoramidate or boranophosphate bond.

Embodiment 12: The oligonucleotide agent according to any one of Embodiments 10-11, wherein the chemically modified nucleotide comprises one or more of the following modifications:

a) modification of 2'-OH of the ribose in the nucleotide;

b) modification or absence of a base moiety on the nucleoside ring in the nucleotide;

c) a nucleotide being a locked or bridged nucleic acid, and

d) a nucleotide being a deoxyribonucleotide (DNA) .

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

Embodiment 14: The oligonucleotide agent according to any of Embodiments 1-13, wherein if present, the nucleotides in the non-targeting moiety are selected from the group of RNA, DNA, bridged nucleic acid (BNA) , locked nucleic acid (LNA) and peptide nucleic acid (PNA) . Embodiment 15: The oligonucleotide agent according to any one of Embodiments 10-11, wherein the at least one chemically modified nucleotide is a nucleotide having an addition of a 5'-phosophate, 5-methyl cytosine or 5’- (E) ‐vinylphosphonate.

Embodiment 16: The oligonucleotide agent according to any one of Embodiments 1-15, wherein the targeting oligonucleotide and the non-targeting moiety are directly conjugated, for example, via a phosphorothioate (PS) bond.

Embodiment 17: The oligonucleotide agent according to Embodiment 16, wherein the terminal unit or an internal unit of the non-targeting moiety is conjugated to the targeting oligonucleotide.

Embodiment 18: The oligonucleotide agent according to any of Embodiments 1-17, wherein the non-targeting moiety is conjugated to the 3’ end, the 5’ end, both the 3’ and the 5’ ends, or an internal nucleotide of the sense strand or antisense strand of the double-stranded oligonucleotide.

Embodiment 19: The oligonucleotide agent according to any of Embodiments 1-18, wherein the non-targeting moiety is selected from:

1) one chemical linker without any nucleotide, and the chemical linker is conjugated with the end of the targeting oligonucleotide via a phosphorothioate (PS) ;

2) a consecutively linked sequence of chemical linkers without any nucleotide, and the consecutively linked sequence of chemical linkers is conjugated with one end of the targeting oligonucleotide, optionally via one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20) deoxyribonucleotides (DNA) ;

3) two consecutively linked sequences of chemical linkers without any nucleotide, and the consecutively linked sequences of chemical linkers are respectively conjugated with both ends of the targeting oligonucleotide, optionally via one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20) deoxyribonucleotides (DNA) ;

4) one or more consecutive nucleotide sequences and a consecutively linked sequence of chemical linkers, and the nucleotide (s) being interspersed in chemical linkers, preferably every single nucleotide being interspersed in two chemical linkers, or one or more consecutive nucleotide sequences being interspersed in chemical linkers;

wherein at least one phosphodiester bond between two adjacent nucleotides, between two adjacent linkers or between a nucleotide and an adjacent linker is substituted by a phosphorothioate (PS) .

Embodiment 20: The oligonucleotide agent according to any one of Embodiments 1-19, wherein the internal nucleotide in the sense or antisense strand of the double-stranded oligonucleotide is substituted by a linking component, wherein the non-targeting moiety is conjugated to the linking component.

Embodiment 21: The oligonucleotide agent according to any one of Embodiments 1-20, wherein more than one (e.g., 2-10) non-targeting moieties are conjugated to the double-stranded oligonucleotide, or more than one (e.g., 2-10) double-stranded oligonucleotides are conjugated to the non-targeting moiety.

Embodiment 22: The oligonucleotide agent according to Embodiment 20, wherein the linking component is selected from one or more of an ethylene glycol chain, an alkyl chain, an alkenyl chain, an alkynyl chain, a peptide, carbohydrates, thiol linkage, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, a tetrazole linkage, and a benzimidazole linkage.

Embodiment 23: The oligonucleotide agent according to any one of Embodiments 1-22, wherein the non-targeting moiety and/or the double-stranded oligonucleotide is conjugated to one or more conjugation groups.

Embodiment 24: The oligonucleotide agent according to Embodiment 23, wherein the one or more conjugation groups is selected from a lipid, a fatty acid, a fluorophore, a ligand, a saccharide, a peptide, and an antibody, optionally, the one or more conjugation groups is selected from a cell-penetrating peptide, polyethylene glycol, an alkaloid, a tryptamine, a benzimidazole, a quinolone, an amino acid, a cholesterol, glucose and N-acetylgalactosamine.

Embodiment 25: The oligonucleotide agent of any one of Embodiments 1-24, wherein the oligonucleotide agent comprises a nucleotide sequence of the sense strand that is at least 90%identical to the nucleotide sequence as set forth in any of SEQ ID NOs: 1, 3, 56 and 61.

Embodiment 26: The oligonucleotide agent of any one of Embodiments 1-25, wherein the oligonucleotide agent comprises a nucleotide sequence of antisense strand that is at least 90%identical to the nucleotide sequence as set forth in any of SEQ ID NOs: 2, 4, 57, 62 and 69.

Embodiment 27: The oligonucleotide agent of any one of Embodiments 1-24, comprising the sequence of the sense strand as set forth in any of SEQ ID NOs: 6-39, 60, 64, 65, 66, 67, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86 and 87.

Embodiment 28: The oligonucleotide agent according to any one of Embodiments 1-27, wherein the non-targeting moiety of the oligonucleotide agent improves the stability, bioavailability, biodistribution, and/or cellular uptake of the targeting oligonucleotide as compared to an oligonucleotide agent without the non-targeting moiety.

Embodiment 29: The oligonucleotide agent according to any one of Embodiments 1-28, wherein the non-targeting moiety of the oligonucleotide agent increases the biodistribution of the targeting oligonucleotide within one or more target tissues as compared to an oligonucleotide agent without the non-targeting moiety.

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

Embodiment 31: The oligonucleotide agent according to Embodiment 29, wherein the one or more target tissues is selected from the group consisting of: prefrontal cortex, cerebellum, and cerebrum; cervical, thoracic and lumbar in spinal cord; heart, bicep, semitendinosus, platysma, and gluteus.

Embodiment 32: A vector, comprising the oligonucleotide agent of any one of Embodiments 1-31.

Embodiment 33: A cell, comprising the oligonucleotide agent of any one of Embodiments 1-31.

Embodiment 34: The cell according to Embodiment 33, wherein the cell is a mammalian cell, optionally a human cell.

Embodiment 35: The cell according to any one of Embodiments 33-34, wherein the cell is a host cell.

Embodiment 36: The cell according to any one of Embodiments 33-35, wherein the cell is in vitro, or exists in a mammalian body.

Embodiment 37: A pharmaceutical composition, comprising the oligonucleotide agent of any one of Embodiments 1-30 and/or the cell of any one of Embodiments 33-36.

Embodiment 38: The pharmaceutical composition according to Embodiment 37, wherein the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier selected from an aqueous carrier, liposome or LNP, polymer, micelle, colloid, metal nanoparticle, non-metallic nanoparticle, bioconjugates, and polypeptide.

Embodiment 39: The pharmaceutical composition according to any one of Embodiments 37-38, wherein the pharmaceutical composition inhibits the SOD1 gene expression or decreases the SOD1 protein.

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

Embodiment 41: A kit, comprising the oligonucleotide agent of any one of Embodiments 1-31 or the pharmaceutical composition of any one of Embodiments 37-40.

Embodiment 42: A method of inhibiting the SOD1 gene expression or decreasing the SOD1 protein, comprising administering to a subject a pharmaceutical composition of any one of Embodiments 37-39.

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

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

Embodiment 45: A method of activating the expression of an SMN2 gene or increasing the SMN2 protein, the method comprising administering to a subject a pharmaceutical composition of any one of Embodiments 37-38 and 40.

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

Embodiment 47: The method according to any one of Embodiments 42-46, wherein the non-targeting moiety of the oligonucleotide agent improves the stability, bioavailability, biodistribution, and/or cellular uptake of the double-stranded oligonucleotide as compared to an oligonucleotide agent without the non-targeting moiety.

Embodiment 48: The method according to any one of Embodiments 42-47, wherein the non-targeting moiety of the oligonucleotide agent increases the biodistribution of double-stranded oligonucleotide within one or more target tissues as compared to an oligonucleotide agent without the non-targeting moiety.

Embodiment 49: The method according to any one of Embodiments 42-48, wherein the non-targeting moiety of the oligonucleotide agent increases the biodistribution of double-stranded oligonucleotide within two or more target cell types in a tissue as compared to an oligonucleotide agent without the non-targeting moiety.

Embodiment 50: Use of the oligonucleotide agent of any one of Embodiments 1-31 or the pharmaceutical composition of any of Embodiments 37-40 in manufacturing a medicament for treating or delaying the onset or progression of Amyotrophic lateral sclerosis (ALS) .

Embodiment 51: The oligonucleotide agent of any one of Embodiments 1-31 or the pharmaceutical composition of any of Embodiments 37-40 for use in treating or delaying the onset or progression of Amyotrophic lateral sclerosis (ALS) .

Embodiment 52: A kit comprising a container comprising the oligonucleotide agent of any one of Embodiments 1-31.

The present application further provides the following particular embodiments:

Embodiment 1a: An oligonucleotide agent comprising a targeting oligonucleotide conjugated to a non-targeting moiety capable of facilitating delivery of the targeting oligonucleotide, wherein the non-targeting moiety comprises one or more units that are covalently linked in tandem to form a backbone of the non-targeting moiety, and at least two adjacent units are linked via a phosphorothioate (PS) bond.

Embodiment 2a: The oligonucleotide agent according to Embodiment 1a, wherein each unit in the non-targeting moiety is selected from chemical groups, chemical linkers and nucleotides.

Embodiment 3a: The oligonucleotide agent according to Embodiment 2a, wherein the chemical groups are selected from the following: 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, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, wherein one or more methylenes are interrupted or terminated by O, S, S (O) , SO₂, N (R') ₂, C (O) , cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocyclic, and wherein each R’ is independently selected from hydrogen, substituted or unsubstituted alkyl, aryl, aralkyl, alkylaryl, alkoxy, aryloxy, acyl or aliphatic which may be linear or branched.

Embodiment 4a: The oligonucleotide agent according to Embodiment 2a or 3a, wherein the chemical linkers are selected from the following:

n) C6x7 ( (9H-fluoren-9-yl) methyl (4- ( (2S, 4R) -2- ( (bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -4- ( (bis (diisopropylamino) phosphanyl) oxy) pyrrolidin-1-yl) -4-oxobutyl) carbamate) .

Embodiment 5a: The oligonucleotide agent according to any one of Embodiments 1a-4a, wherein the non-targeting moiety comprises:

a) one or more chemical linkers interspersed in nucleotides;

b) one or more nucleotides interspersed in chemical linkers;

c) a consecutive nucleotide sequence and a consecutively linked sequence of chemical linkers; or

d) a consecutively linked sequence of chemical linkers without any nucleotide;

Embodiment 6a: The oligonucleotide agent according to Embodiment 5a, wherein the non-targeting moiety comprises m same or different chemical linkers and n same or different nucleotides, wherein m is an integer in the range of 1-40 and n is an integer in the range of 0-30.

Embodiment 7a: The oligonucleotide agent according to Embodiment 6a, wherein the non-targeting moiety comprises a consecutively linked sequence of chemical linkers as shown in formula (linker1) _x- (linker2) _y, wherein linker1 is a first chemical linker and linker2 is a second chemical linker different from the first chemical linker, and x and y are integers with 0<x+y<50, for example, the non-targeting moiety comprises 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, wherein at least one phosphodiester bond between two adjacent linkers is substituted by a phosphorothioate (PS) , mesyl phosphoramidate or boranophosphate bond.

Embodiment 8a: The oligonucleotide agent according to any of Embodiments 1a-7a, wherein the non-targeting moiety comprises 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 the backbone.

Embodiment 9a: The oligonucleotide agent according to any of Embodiments 1a-7a, wherein the non-targeting moiety comprises 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 the backbone.

Embodiment 10a: The oligonucleotide agent according to any of Embodiments 1a-9a, wherein the targeting oligonucleotide is an antisense oligonucleotide, or a double-stranded oligonucleotide comprising a sense strand and an antisense strand, such as a small interfering RNA (siRNA) or a small activating RNA (saRNA) .

Embodiment 11a: The oligonucleotide agent according to Embodiment 10a, wherein the non-targeting moiety is conjugated to the sense strand or the antisense strand of the double-stranded oligonucleotide.

Embodiment 12a: The oligonucleotide agent according to any one of Embodiments 2a-11a, wherein if present, the nucleotide (s) of the non-targeting moiety are non-chemically modified nucleotides, or at least one nucleotide is a chemically modified nucleotide.

Embodiment 13a: The oligonucleotide agent according to any one of Embodiments 1a-12a, wherein all nucleotides of the targeting oligonucleotide are non-chemically modified nucleotides, or at least one nucleotide is a chemically modified nucleotide, or at least one phosphodiester bond between two adjacent nucleotides in the targeting oligonucleotide is substituted by a phosphorothioate, mesyl phosphoramidate or boranophosphate bond.

Embodiment 14a: The oligonucleotide agent according to any one of Embodiments 12a-13a, wherein the chemically modified nucleotide comprises one or more of the following modifications:

a) modification of 2'-OH of the ribose in the nucleotide;

c) a nucleotide being a locked or bridged nucleic acid, and

d) a nucleotide being a deoxyribonucleotide (DNA) .

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

Embodiment 16a: The oligonucleotide agent according to any of Embodiments 2a-15a, wherein if present, the nucleotides in the non-targeting moiety are selected from the group of RNA, DNA, bridged nucleic acid (BNA) , locked nucleic acid (LNA) and peptide nucleic acid (PNA) .

Embodiment 17a: The oligonucleotide agent according to any one of Embodiments 12a-13a, wherein the at least one chemically modified nucleotide is a nucleotide having an addition of a 5'-phosophate, 5'-methyl cytosine or 5’- (E) ‐vinylphosphonate.

Embodiment 18a: The oligonucleotide agent according to any one of Embodiments 1a-17a, wherein the targeting oligonucleotide and the non-targeting moiety are directly conjugated, for example, via a phosphorothioate (PS) bond.

Embodiment 19a: The oligonucleotide agent according to Embodiment 18a, wherein the terminal unit or an internal unit of the non-targeting moiety is conjugated to the targeting oligonucleotide. Embodiment 20a: The oligonucleotide agent according to any of Embodiments 10a-19a, wherein the non-targeting moiety is conjugated to the 3’ end, the 5’ end, both the 3’ and the 5’ ends, or an internal nucleotide of the sense strand or antisense strand of the double-stranded oligonucleotide. Embodiment 21a: The oligonucleotide agent according to any one of Embodiments 1a-20a, wherein the internal nucleotide in the sense or antisense strand of the double-stranded oligonucleotide is substituted by a linking component, wherein the non-targeting moiety is conjugated to the linking component.

Embodiment 22a: The oligonucleotide agent according to any one of Embodiments 10a-21a, wherein more than one (e.g., 2-10) non-targeting moieties are conjugated to the double-stranded oligonucleotide, or more than one (e.g., 2-10) double-stranded oligonucleotides are conjugated to the non-targeting moiety.

Embodiment 23a: The oligonucleotide agent according to Embodiment 21a, wherein the linking component is selected from one or more of an ethylene glycol chain, an alkyl chain, an alkenyl chain, an alkynyl chain, a peptide, carbohydrates, thiol linkage, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, a tetrazole linkage, and a benzimidazole linkage. Embodiment 24a: The oligonucleotide agent according to any one of Embodiments 1a-23a, wherein the non-targeting moiety and/or the double-stranded oligonucleotide is conjugated to one or more conjugation groups.

Embodiment 25a: The oligonucleotide agent according to Embodiment 24a, wherein the one or more conjugation groups is selected from a lipid, a fatty acid, a fluorophore, a ligand, a saccharide, a peptide, and an antibody, optionally, the one or more conjugation groups is selected from a cell-penetrating peptide, polyethylene glycol, an alkaloid, a tryptamine, a benzimidazole, a quinolone, an amino acid, a cholesterol, glucose and N-acetylgalactosamine.

Embodiment 26a: The oligonucleotide agent of any one of Embodiments 1a-25a, wherein the oligonucleotide agent comprises a nucleotide sequence that is at least 90%identical to the nucleotide sequence as set forth in any of SEQ ID NOs: 1, 3 and 56-66.

Embodiment 27a: The oligonucleotide agent of any one of Embodiments 1a-26a, wherein the oligonucleotide agent comprises a nucleotide sequence that is at least 90%identical to the nucleotide sequence as set forth in any of SEQ ID NOs: 2, 4 and 67-77.

Embodiment 28a: The oligonucleotide agent of any one of Embodiments 1a-25a, comprising the sequence as set forth in any of SEQ ID NOs: 5-40.

Embodiment 29a: The oligonucleotide agent according to any one of Embodiments 1a-28a, wherein the non-targeting moiety of the oligonucleotide agent improves the stability, bioavailability, biodistribution, and/or cellular uptake of the targeting oligonucleotide as compared to an oligonucleotide agent without the non-targeting moiety.

Embodiment 30a: The oligonucleotide agent according to any one of Embodiments 1a-29a, wherein the non-targeting moiety of the oligonucleotide agent increases the biodistribution of the targeting oligonucleotide within one or more target tissues as compared to an oligonucleotide agent without the non-targeting moiety.

Embodiment 31a: The oligonucleotide agent according to Embodiment 30a, wherein the one or more target tissues is selected from tissues of brain, spinal cord, muscle, spleen, lung, heart, liver, bladder, and kidney.

Embodiment 32a: The oligonucleotide agent according to Embodiment 30a, wherein the one or more target tissues is selected from the group consisting of: prefrontal cortex, cerebellum, and cerebrum; cervical, thoracic and lumbar in spinal cord; heart, bicep, semitendinosus, platysma, and gluteus.

Embodiment 33a: A chemical compound for conjugation to a targeting oligonucleotide, wherein the chemical compound comprises one or more units that are covalently linked in tandem to form a backbone of the chemical compound, and at least two adjacent units are linked via a phosphorothioate (PS) bond, wherein each unit is selected from chemical groups, chemical linkers and nucleotides.

Embodiment 34a: The chemical compound according to Embodiment 33a, wherein the chemical linkers 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 and any other linkers that can be used for spacing two nucleotides.

Embodiment 35a: A vector, comprising the oligonucleotide agent of any one of Embodiments 1a-32a.

Embodiment 36a: A cell, comprising the oligonucleotide agent of any one of Embodiments 1a-32a.

Embodiment 37a: The cell according to Embodiment 36a, wherein the cell is a mammalian cell, optionally a human cell.

Embodiment 38a: The cell according to any one of Embodiments 36a-37a, wherein the cell is a host cell.

Embodiment 39a: The cell according to any one of Embodiments 36a-38a, wherein the cell is in vitro, or exists in a mammalian body.

Embodiment 40a: A pharmaceutical composition, comprising the oligonucleotide agent of any one of Embodiments 1a-32a and/or the cell of any one of Embodiments 36a-39a.

Embodiment 41a: The pharmaceutical composition according to Embodiment 40a, wherein the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier selected from an aqueous carrier, liposome or LNP, polymer, micelle, colloid, metal nanoparticle, non-metallic nanoparticle, bioconjugates, and polypeptide.

Embodiment 42a: The pharmaceutical composition according to any one of Embodiments 40a-41a, wherein the pharmaceutical composition inhibits the SOD1 gene expression or decreases the SOD1 protein.

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

Embodiment 44a: A kit, comprising the oligonucleotide agent of any one of Embodiments 1a-32a or the pharmaceutical composition of any one of Embodiments 40a-43a.

Embodiment 45a: A method of inhibiting the SOD1 gene expression or decreases the SOD1 protein, comprising administering to a subject a pharmaceutical composition of any one of Embodiments 40a-42a.

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

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

Embodiment 48a: A method of activating the expression of an SMN2 gene or increasing the SMN2 protein, the method comprising administering to a subject a pharmaceutical composition of any one of Embodiments 40a-41a and 43a.

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

Embodiment 50a: The method according to any one of Embodiments 45a-49a, wherein the non-targeting moiety of the oligonucleotide agent improves the stability, bioavailability, biodistribution, and/or cellular uptake of the double-stranded oligonucleotide as compared to an oligonucleotide agent without the non-targeting moiety.

Embodiment 51a: The method according to any one of Embodiments 45a-50a, wherein the non-targeting moiety of the oligonucleotide agent increases the biodistribution of double-stranded oligonucleotide within one or more target tissues as compared to an oligonucleotide agent without the non-targeting moiety.

Embodiment 52a: The method according to any one of Embodiments 45a-51a, wherein the non-targeting moiety of the oligonucleotide agent increases the biodistribution of double-stranded oligonucleotide within two or more target cell types in a tissue as compared to an oligonucleotide agent without the non-targeting moiety.

Embodiment 53a: A use of the oligonucleotide agent of any one of Embodiments 1a-32a or the pharmaceutical composition of any of Embodiments 40a-43a in manufacturing a medicament for treating or delaying the onset or progression of Amyotrophic lateral sclerosis (ALS) .

Embodiment 54a: The oligonucleotide agent of any one of Embodiments 1a-32a or the pharmaceutical composition of any of Embodiments 40a-43a for use in treating or delaying the onset or progression of Amyotrophic lateral sclerosis (ALS) .

Embodiment 55a: A kit comprising a container comprising the oligonucleotide agent of any one of Embodiments 1a-32a.

EXAMPLES

The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc. ) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair (s) ; kb, kilobase (s) ; pl, picoliter (s) ; s or sec, second (s) ; min, minute (s) ; h or hr, hour (s) ; aa, amino acid (s) ; nt, nucleotide (s) ; i.m., intramuscular (ly) ; i. p., intraperitoneal (ly) ; s. c., subcutaneous (ly) ; i. c. v. or icv or ICV, intracerebroventricular and the like.

Materials and methods

General methods

Starting materials, reagents and solvents for organic synthesis were purchased from commercial sources and used as received unless stated otherwise. Purification of reactions products was performed by column chromatography using silica gel (200-300 mesh) and eluting with hexane/ethyl acetate, DCM/MeOH. Thin layer chromatography (TLC) was carried out using precoated silica Gel GF plates and visualized using KMnO4 stains. 1H-NMR spectra were recorded at 400 or 500 MHz (Varian) using CDCl3 with TMS. Mass spectra (MS) were recorded on LC/MS (Agilent Technologies 1260 Infinity II/6120 Quadrupole) and a time-of-flight mass spectrometer by ESI or matrix assisted laser desorption/ionization (MALDI) .

Oligonucleotide Synthesis

(1) Single-stranded synthesis

The oligonucleotides used were synthesized on a K&ADNA synthesizer (K&ALaborgeraete GbR, Schaafheim, Germany) by using solid phase technique. Briefly, during solid phase synthesis, phosphoramidite monomers including various linkers and conjugations (0.1M in acetonitrile or dichloromethane) , were added sequentially onto a solid support to generate the desired full-length oligonucleotides. Each cycle of base addition consisted of four chemical reactions including detritylation, coupling, oxidation/thiolation and capping.

Detritylations were performed using 3%dichloroacetic acid (DCA) in DCM for 45 seconds and capping was done with a 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 . Sulfurizations were carried out with 0.1 M solution of xanthane 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. Phosphoramidite coupling times were 360 s for all amidites.

Deprotection I (Nucleobase Deprotection) : After completion of synthesis, the solid support was then transferred to a screw-cap microcentrifuge tube. For 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℃ to 65℃ for 15 min and then allowed to cool to room temperature. The cleavage solution was collected and evaporated to dryness in a speedvac.

Deprotection II (Removal of 2’-TBDMS Group) : The crude RNA oligonucleotide, still carrying the 2’-TBDMS groups, 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 ensure complete dissolution. The bottle was heated in an oven at 60℃ to 65℃ for 3 to 3.5 hours. The tube was removed from the oven and cooled to room temperature. The solution containing the completely desilylated oligonucleotide was cooled on dry ice. 2 mls of ice-cold n-butanol (-20℃) were carefully added in 0.5 ml portions to precipitate the oligonucleotide. The precipitate was filtered, washed with 1 ml ice-cold n-butanol, and subsequently dissolved in 2M TEAA (triethylammonium acetate) . The crude oligonucleotides were then purified by exchange (IEX) HPLC using a source 15Q column. Purity of the fractions were analyzed by ion exchange (IEX) HPLC using Column DNA Pac^TM PA100. Following the generation of desalted purified single-stranded solutions, a duplex was made by annealing two complimentary single-stranded oligonucleotides, which were subsequently lyophilized to powder.

(2) Single-stranded purification

The purification of oligonucleotides was performed on an AKTA explorer 10 equipped with a Source 15Q 4.6/100 PE column using the following conditions: buffer A: (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) , B: (10 mM Tris-HCl, 1 mM EDTA, 2M NaCl, pH 7.5) , gradient: 10%B to 60%B in 25 min, flow rate: 1 ml/min. The pure oligonucleotides were collected and desalting by a HiPrep 26/10 Desalting column.

(3) Annealing to form duplex

For duplex, after the generation of desalted purified single-stranded solutions, sense strand and antisense strand were mixed by equal volumes at equimolar concentration in the tube. Place the tube in a heat block at 95℃ for 5 min and then cool to room temperature then subsequently lyophilized to powder.

These linker entities mentioned in this application are used as amidtes also following oligonucleotide synthesis protocol.

RP-HPLC and ESI-MS

Oligonucleotides were analyzed via reverse phase chromatography (i.e., RP-HPLC) (Waters XBridge oligonucleotide BEH C18 130A) using an acetonitrile grant and detection wavelength of 260 nm to qualify oligonucleotide purity. Electrospray ionization mass spectrometry (ESI-MS) was performed on desalted oligonucleotides resuspended in water/acetonitrile (50: 50) containing 1%(vol/vol) triethylamine in negative ion mode.

Electrophoretic mobility shift assay (EMSA)

To assess in vitro protein binding activity of cODV oligonucleotides, oligonucleotides were diluted to 1 μM using 1× PBS, and 10 μL of the diluted oligonucleotides was mixed with equal volume of plasma from C57BL/6J mice (Code ID: 201, Beijing Vital River Laboratory Animal Technology Co., Ltd. ) at a 0.5 μM final concentration (20 μL total volume) . Each of sample was centrifuged at 1000 g for 30 seconds and subsequently incubated at 37℃ for 1 hour. Then 2 μL 10 × loading buffer (Code No. 9157, TaKaRa, Japan) was pipetted into each of mixture sample to give 22 μL stock solution. 10 μL stock solution was loaded into a 4%agarose gel well and separated by electrophoresis at 120 volts for 60 min. The same amount of the PBS diluted oligonucleotide without mixing with plasma was also loaded into a well next to their plasma mixed sample to serve as an input control and a size reference. Mice plasma without mixing with oligonucleotides served a negative control. After electrophoresis, the gel was documented using a ChemiDoc MP system to quantify the intensity of each oligonucleotide band. The oligonucleotide band from plasma mixed oligonucleotide sample with the same migration rate on the gel as their input was regarded as unbound fraction. For each sample, an %unbound fraction was calculated following the Formula IV:

Unbound fraction (%) = (oligonucleotide band intensity of plasma-mixed sample) /(oligonucleotide band intensity of input) × 100

(Formula IV)

Primary mouse hepatocyte (PMH) isolation

C57BL/6J mice (Beijing Vital River Laboratory Animal Technology Co., Ltd. ) were anesthetized with isoflurane and perfused by initial flushing reagent and digestion reagent successively. The liver was placed into a 10 cm dish and torn apart using forceps in culture medium. The cell suspension was collected by filtering through a 70-75-micron membrane in 50 mL conical tube, followed by centrifuging at 4℃ for 2 minutes at 100 × g in a swinging-arm centrifuge. 20 mL cold PBS was pipetted to wash cells after removing the supernatant (Repeat this step twice) . Cells with at least 80%viability were allowed to proceed the assay. Cells were seeded to the cell culture plates which coated collagen Ι 4 -12 hours in advance, yield a final confluence of 90-95%and started the assay.

Cell culture and treatment

PMH cells were cultured in modified Willian’s Medium E (WME) medium (A12176-01, Gibco, Thermo Fisher Scientific, Carlsbad, CA) 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) supplemented with 10%bovine 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) supplemented with 10%bovine calf serum and 1%penicillin/streptomycin. Neuro-2a cells (N-2a, BNCC338529, Beijing, China) were cultured in EMEM medium (Gibco, Thermo Fisher Scientific, Carlsbad, CA) supplemented with 10%bovine calf serum and 1%penicillin/streptomycin. All cell lines were cultured in a humidified atmosphere of 5%CO₂ and 37℃. Transfections were carried out using Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA) in growth media according to the manufacture’s protocol. Cells were transfected in the absence of an oligonucleotide as Mock treatments. dsCon2 and dsCon2M8 duplexes were transfected as non-targeting controls. Oligonucleotides were individually added into medium contained PMH cells at 1000 nM without the use of any additional transfection reagent for 3 days for free uptake.

RNA isolation and two step reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

(1) RNA isolation and two-step RT-qPCR

Total cellular RNA was isolated from treated cells using an RNeasy Plus Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Animal tissues were isolated using the MagPure Total RNA Micro LQ kit (Magen, R6621, Guangzhou, Guangdong, China) in conjunction with the auto-pure96 machine (ALLSHENG, Hangzhou, Zhejiang, China) . The resultant RNA (～1 μg) was reverse transcribed into cDNA by using a PrimeScript^TM RT reagent kit with gDNA Eraser (Takara, RR047A, Shlga, Japan) . The resultant cDNA was amplified in a Roche LightCycler 480 Multiwell Plate 384 (Roche, ref: 4729749001, US) using TB Premix Ex Taq^TM II (Takara, RR820A, Shlga, Japan) reagents and primers specifically for amplified target genes of interest. Melting curves were performed after amplification to confirm primer specificity.

Reaction conditions were as follows: reverse transcription reaction (stage 1) : 42℃ for 5 min, 95℃ for 10 sec; PCR reaction (stage 2) : 95℃ for 5 sec, 60℃ for 30 sec, 72℃ for 10 sec, 40 cycles of amplification; and melting curve (stage 3) . PCR reaction conditions were shown in Table 2 and Table 3. Primer sequences are listed in Table 4.

Table 2. RT reaction

Table 3. RT-qPCR reaction

Table 4. Primer sequences for RT-PCR.

1) One reference gene for two step RT-qPCR

To calculate the relative expression levels (E_rel) of target gene mRNA in an dsRNA-transfected sample relative to control treatment (Mock, aCSF or saline) , the Ct values of the target gene and the internal reference gene were substituted into Formula V,
E_rel=2^(CtTm-CtTs)/2^(CtRm-CtRs)
(Formula V)

wherein CtTm was the Ct value of the target gene from the control-treated sample; CtTs was the Ct value of the target gene from the dsRNA-treated sample; CtRm was the Ct value of the internal reference gene from the control-treated sample; CtRs was the Ct value of the internal reference gene from the dsRNA-treated sample.

2) Two reference genes for two step RT-qPCR

To calculate the relative expression levels (E_rel) of target gene mRNA in an dsRNA-transfected sample relative to control treatment (Mock, aCSF or saline) , the Ct values of the target gene and the two internal reference genes were substituted into Formula VI,
E_rel=2^(CtTm-CtTs)/( (2^{(CtR1m-CtR1s)}*2^{(CtR2m-CtR2s)} ) ^(1/2) ) (Formula VI)

wherein CtT_m was the Ct value of the target gene from the control-treated sample; CtT_s was the Ct value of the target gene from the dsRNA-treated sample; CtR1_m was the Ct value of the internal reference gene 1 from the control-treated sample; CtR1_s was the Ct value of the internal reference gene 1 from the dsRNA-treated sample; CtR2_m was the Ct value of the internal reference gene 2 from the control-treated sample; and CtR2_s was the Ct value of the internal reference gene 2 from the dsRNA-treated sample.

Propidium iodide (PI) staining

PMH cells were cultured in 96-well plates following siRNA treatment for 24 hours or 3 days. Cells were washed with cold PBS and lysed using 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 μM PI. Plates were incubated on ice for 5 minutes prior to measuring optical density (OD) on a microplate reader system (Infinite M2000 Pro, TECAN) at 535 nm excitation and 615 nm emission wavelengths.

Animal procedures

All animal procedures were conducted by certified laboratory personnel using protocols consistent with local and state regulations and approved by the Institutional Animal Care and Use Committee. C57BL/6 mice (4～5-week-old) were purchased from JOINN Biologics (Suzhou, Jiangsu, China) . Sprague-Dawley (SD) rats (six-week-old) imported from the experimental animal center of Nantong University (SCXK2019-0001, Nantong, Jiangsu, China) as specific pathogen free rats. SD rats were administered by intravitreous (IVT) injection into the left eye. Parental transgenic hSOD1^G93A mice (Strain ID #004435) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and imported into China via Nantong University (Nantong City, Jiangsu Province, China) . Mice were delivered to the animal facility at 6 weeks of age and subsequently bred domestically at Nantong University who supplied the animals for this study. Formulations for in vivo studies were prepared fresh prior to use by dissolving aliquots of lyophilized oligonucleotide into saline or aCSF to create stock solutions for dilution to the intended treatment concentrations.

Intracerebroventricular (ICV) injection

Avertin (1.2%) was prepared fresh and sterilized via 0.2-micron filter. Mice were dosed at 0.30-0.35 ml per 10 g body weight via intraperitoneal (IP) injection in a stereotaxic apparatus to rapidly induce anesthesia for up to 30 minutes. An approximate 11.5 mm incision was made in the animal’s scalp and a 25-gauge needle attached to a Hamilton syringe containing the appropriate siRNA or saRNA formulation was placed at bregma level. The needle was moved to the appropriate anterior/posterior and medial/lateral coordinates (0.2 mm anterior/posterior and 1 mm to the right medial/lateral) . A total of 10 μL was injected into the lateral ventricle at an approximate rate of 1 μl/s. Following treatment, the needle was slowly withdrawn, and the wound sutured close.

Tail vein (IV) injection

Mice were exposed to an infrared lamp for 2-3 min to dilate the veins, and then held in the restrainer to straighten the tail. The tail was wiped with 75%alcohol and the needle was inserted 2 to 4 mm parallel to the tail vein into the lumen, keeping the bevel of the needle upwards. The preformed solution was injected slowly and should be free of resistance if administered correctly. The recommended injection volume for test article is 200 μg and the injection rate don’ t exceed 5 ml/min. At the end of administration, the injection site is pressed firmly with a cotton swab or finger to prevent backflow of the administration solution and/or blood.

Intravitreous (IVT) injection

SD rats were housed in animal facility of Ractigen (Nantong, Jiangsu, China) and fed for at least three days prior to the intravitreous (IVT) injection of compounds. SD rats were anesthetized in an isoflurane (RWD, R510-22-16) induction chamber (5%isoflurane in 100%medical oxygen, 2 L/min) until they had no response to toe pinches. SD rats were transferred to the experimental operating platform and positioned for delivery of isoflurane (2%isoflurane in 100%medical oxygen, 1.5 L/min) using a homemade face mask during the procedure of IVT injection. Before the IVT injection of compounds, one drop of 0.5%alcaine as topical anesthetics was applied to the injected eye (left eye) . An anterior chamber paracentesis was performed using a 30-gauge needle, followed by approximately 5 μL aqueous humor were outflowed. Each compound for corresponding group was dissolved in 4 μL normal saline and loaded into a 30-gauge needle for IVT injection. The compounds were administered by inserting at a 45° angle of the needle through the sclera into the vitreous body, then injected into the posterior chamber keeping for 5 seconds to avoid the leaking. At the end of administration, the injected eye was administered with antibiotics to prevent infection after the IVT injection.

Statistical analysis

Differences between groups of continuous variables were compared using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons. A P value of less than 0.05 was considered statistically significant between two groups. *represents p < 0.05, **represents p < 0.01, ***represents p < 0.001, ****represents p < 0.0001.

Example 1. Design of various chain oligonucleotide delivery vehicle (cODV) structures

A series of cODV structures were designed and conjugated to a duplex siRNA (siSOD1, RD-12556) via different linker designs, resulting in 35 cODV-siRNA variants (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) . The designs and cODV-siRNA variants are listed in Table 5. “No linker” indicates the siRNA is not conjugated to any non-targeting moiety. All single-stranded oligonucleotide sequences with chemical modifications including nucleic acid analogs, backbone substitutions, linkers, and non-targeting moieties were synthesized on solid-support as single molecular entities. cODV duplexes were subsequently created by annealing complimentary single-stranded oligonucleotides. These compounds were chemically synthesized using the methods as described in the Materials and Methods section.

Example 2. Assessing protein binding of cODV duplexes by EMSA

It is hypothesized that self-delivery capability of cODV-siRNAs is due to increased protein binding endowed by the linker and/or the numbers of linker components in the structure. To test this idea, each of cODV-siRNAs was mixed with C57BL/6J mice plasma (mostly albumin) at a 0.5 μM final concentration at 37℃ for 1 hour as described in the Materials and Methods section, and then separated by 4%agarose gel electrophoresis to quantify changes in mobility shift. The same compound without plasma was also separated by the gel to serve as an input control. RD-11810 served as a duplex control without a cODV conjugate. For each compound, oligonucleotide band intensity was quantified using ChemiDoc MP system, and the %of unbound protein fraction of cODV-siRNA was calculated as the ratio of band intensity of free oligonucleotide band of the plasma mixed sample over the band intensity of input sample. A smaller number of %of unbound fraction indicates greater protein binding capacity. The calculated %of unbound fraction relative to RD-11810 (as the non-cODV conjugate control with a value of 100%) was summarized in Table 6. As shown in Table 6, all cODV-siRNA structures exhibited a reduced unbound fraction compared to a siRNA duplex without the cODV structure (RD-11810) , indicating that the conjugation to a non-targeting moiety increased protein binding to the conjugated cODV-siRNA structures. The decrease in unbound fraction ranged from 92%(RD-13592) to the lowest 22% (RD-13619)

Table 6. Unbound protein fraction of cODV-siRNAs after incubating with C57BL/6J mice plasma

Example 3. In vitro delivery activity and cytotoxicity of cODV-siRNAs in PMH cells

To test in vitro delivery activity of the cODV-siRNAs, the indicated cODV-siRNAs (see Table 5) were transfected into PMH cells with RNAiMAX at 0.1 nM for 24 hours. Cells were transfected in the absence of an oligonucleotide as Mock treatments. dsCon2 and RD-12559 served as a non-targeting duplex control and a positive control with known knockdown activity, respectively. mRNA levels of Sod1 were quantified by two step RT-qPCR using a gene specific primer set. As shown in FIG. 1A, cODV-siRNAs resulted in potent knockdown on the Sod1 mRNA expression after 24 hours treatment as compared to the positive control of RD-12559 (dotted line) . Table 7 summarized the Sod1 mRNA knockdown levels following cODV-siRNA treatments in PMH cells.

Table 7. Sod1 mRNA knockdown levels following cODV-siRNA treatments in PMH cells

Note: "SEM" represents Standard Error of the Mean.

Untoward cytotoxicity of the 31 cODV compounds was assessed 24 hours after treatment by PI staining. As shown in FIG. 1B, with the exception of 7 cODV-siRNAs with slightly decreased cell viability (RD-13616, RD-13617, RD-13618, RD-13619, RD-13623, RD-13624 and RD-13625) , all siRNAs exhibited minimal changes in cell viability at 0.1 nM, indicating these cODV designs have minimal in vitro cytotoxicity.

To further test in vitro delivery activity of the newly designed cODV-siRNAs, free uptake assay was conducted in which the cODV-siRNAs (see Table 5) were directly added to freshly isolated PMH cells at 1000 nM without using a transfection reagent for 3 days. RD-12559 served as a positive control with known knockdown activity. mRNA levels of Sod1 were quantified by two step RT-qPCR using a gene specific primer set. As shown in FIG. 2A, cODV-siRNAs presented modest knockdown on the Sod1 mRNA expression after a treatment duration of 3 days as compared to the positive control of RD-12559 (dotted line) . Table 8 summarized the Sod1 mRNA knockdown levels following cODV-siRNA treatments in PMH cells.

Table 8. Sod1 mRNA knockdown levels following cODV-siRNA treatments in PMH cells

Note: "SEM" represents Standard Error of the Mean.

Untoward cytotoxicity of the indicated cODV-siRNAs was assessed 72 hours post-treatment by PI staining. As shown in FIG. 2B, all cODV-siRNAs showed minimal changes in cell viability, suggesting that these cODV designs have minimal cytotoxicity.

Example 4. In vivo knockdown activity of cODV-siRNAs 7 days after a single ICV or IV dose treatment in C57BL/6J mice

To test cODV-siRNA activity in CNS and peripheral tissues, adult C57BL/6J mice were administered with cODV-siRNAs (i.e., RD-13184 and RD-13185) and a duplex siRNA control (i.e., RD-12556) at 200 μg via ICV injection. Saline served as a vehicle control to establish baseline expression levels of Sod1 mRNA. Mice were sacrificed on day 7 post dosing and knockdown of Sod1 mRNA was quantified in brain (i.e., cerebellum) , spinal cord (i.e., cervical, thoracic and lumber) and peripheral (i.e., liver) tissues via two step RT-qPCR. Tbp was amplified and used as an internal reference. The results of Sod1 mRNA knockdown following cODV-siRNA treatment in CNS and peripheral tissues are summarized in Table 9.

To test cODV-siRNA activity in peripheral and skeletal muscle tissues, adult C57BL/6J mice were administered with indicated cODV-siRNAs (i.e., RD-13184 and RD-13185) and a duplex siRNA control (i.e., RD-12556) at 20 mg/kg via IV injection. Saline served as a vehicle control to establish baseline expression levels of Sod1 mRNA. Mice were sacrificed on day 7 post dosing and knockdown of Sod1 mRNA was quantified in peripheral (i.e., liver, spleen, lung and heart) and skeletal muscle (i.e., bicep, semitendinosus and platysma) tissues via two step RT-qPCR. Tbp was amplified and used as an internal reference. The results of Sod1 mRNA knockdown following cODV-siRNA treatment in peripheral and skeletal muscle tissues are summarized in Table 10 and Table 11, respectively.

Example 5. In vivo knockdown activity of cODV-siRNAs 14 days after a single ICV dose in C57BL/6J mice

To test the durability of cODV-siRNAs in CNS tissues, the indicated cODV-siRNAs (i.e., RD-13592, RD-13608, RD-13611, RD-13614 and RD-14794) (see Table 5) were administered into adult C57BL/6J mice at 200 μg via ICV injection. Saline served as a vehicle control to establish baseline expression levels of Sod1 mRNA. Mice were sacrificed on day 14 post dosing and knockdown of Sod1 mRNA was quantified in brain (i.e., frontal cortex, cerebellum and cerebrum) and spinal cord (i.e., cervical, thoracic and lumber) tissues via two step RT-qPCR. Geometric means of the mRNA levels of Rpl13a and Hprt1 were used as an internal reference. The results of Sod1 mRNA knockdown following cODV-siRNA treatment in CNS tissues are summarized in Table 12. Monitoring animal body weight following treatment revealed no adverse finding supporting cODV-siRNA treatment was generally well-tolerated in C57BL/6J mice (FIG. 3) .

To test the durability of cODV-siRNA in peripheral and skeletal muscle tissues, the indicated cODV-siRNAs (i.e., RD-13592, RD-13608, RD-13611, RD-13614 and RD-14794) were administered into adult C57BL/6J mice at 20 mg/kg via IV injection. Saline served as a vehicle control to establish baseline expression levels of Sod1 mRNA. Mice were sacrificed on day 14 post dosing and knockdown of Sod1 mRNA was quantified in peripheral (i.e., liver, lung and bladder) and skeletal muscle (i.e., semitendinosus and platysma) tissues via two step RT-qPCR. Geometric means of the mRNA levels of Rpl13a and Hprt1 were used as an internal reference. The results of Sod1 mRNA knockdown following cODV-siRNA treatment in peripheral and skeletal muscle tissues are summarized in Table 13. Monitoring animal body weight following treatment revealed no adverse finding supporting cODV-siRNA treatment was generally well-tolerated in C57BL/6J mice (FIG. 4) .

Example 6. In vivo knockdown activity of cODV-siRNAs in retinal tissue of SD rats

To test in vivo knockdown activity of cODV-siRNAs in the eye, adult SD rats were treated with indicated 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 duplex siRNA control (i.e., RD-12556) at a 30 μg dose via local IVT injection into the left eye. Saline served as a vehicle control to establish baseline expression levels of Sod1 mRNA. Rats were sacrificed on day 14 post dosing and Sod1 knockdown was quantified via two step RT-qPCR in retinal tissue. The results of Sod1 mRNA knockdown following cODV-siRNA treatment in retina tissue are shown in FIG. 5A and 5B.

Example 7. Design of novel cODV-siRNA structures

A series of cODV structures were designed and conjugated to a duplex siRNA via different linker designs. The designs and cODV-siRNA variants are listed in Table 14. “No linker” indicates the siRNA is not conjugated to any non-targeting moiety. All single-stranded oligonucleotide sequences with chemical modifications including nucleic acid analogs, backbone substitutions, linkers, and non-targeting moieties were synthesized on solid-support as single molecular entities. cODV duplexes were subsequently created by annealing complimentary single-stranded oligonucleotides. These compounds were chemically synthesized using the methods as described in the Materials and Methods section.

Example 8. In vitro knockdown activity of cODV-siRNAs in SK-N-AS and T98G cells

To assess the knockdown activity of cODV-siRNAs, the indicated cODV-siRNAs (i.e., RD-16989, RD-16978, RD-16102 and RD-16979) (see Table 14) were transfected into SK-N-AS and T98G cells at indicated concentrations (i.e., 0.0001, 0.0002, 0.001, 0.004, 0.016, 0.063, 0.25 and 1) for 24 hours. RD-16988 and RD-16990 were transfected to serve as the duplex controls. FIG. 6A and 6B showed the SOD1 mRNA levels as quantified by RT-qPCR in SK-N-AS cells. FIG. 6C and 6D showed the SOD1 mRNA levels as quantified by RT-qPCR in T98G cells. EC₅₀ values were extrapolated to define potency in context to maximal activity for each of the tested cODV-siRNAs that demonstrated dose-dependent knockdown of SOD1 mRNA. The resulting EC₅₀ values following cODV-siRNA treatment in SK-N-AS and T98G cells are summarized in Table 15.

Table 15. EC₅₀ values following siRNA treatment in both SK-N-AS and T98G cells

Example 9. In vivo knockdown activity of cODV-siRNAs in hSOD1^G93A mice

To assess the in vivo knockdown activity of cODV-siRNAs, the indicated cODV-siRNAs (i.e., RD-16145 and RD-16978) were administered into hSOD1^G93A mice via ICV injection at 100 μg. aCSF was injected as a vehicle control to establish baseline expression. hSOD1^G93A mice were sacrificed on day 14 post dosing. FIG. 7 showed the remaining SOD1 mRNA levels as quantified in tissues from the brain (i.e., frontal cortex, cerebellum and cerebrum) , spinal cord and periphery (i.e., liver) via RT-qPCR. The SOD1 mRNA levels in tissues from the brain, spinal cord and periphery are shown in Table 16.

Table 16. SOD1 mRNA levels in CNS and liver tissues 14 days after a single ICV dose in hSOD1^G93A mice

Note: "-" represents not available. "SEM" represents Standard Error of the Mean.

Example 10. In vivo knockdown activity of cODV-siRNAs in C57BL/6J mice

To assess the in vivo knockdown activity of cODV-siRNAs, the indicated cODV-siRNAs (i.e., RD-16293, RD-16294, RD-16295 and RD 14794) were administered into C57BL/6J mice via ICV injection at 200 μg. aCSF was injected as a vehicle control to establish baseline expression. C57BL/6J mice were sacrificed on day 14 post dosing. FIG. 8 showed the remaining Sod1 mRNA levels as quantified in tissues from the brain (i.e., frontal cortex, cerebellum and cerebrum) and spinal cord (i.e., cervical, thoracic and lumber) via RT-qPCR. The Sod1 mRNA levels in tissues from the brain and spinal cord are shown in Table 17.

Table 17. Sod1 mRNA levels in CNS tissues 14 days after a single ICV dose in C57BL/6J mice

Example 11. In vitro knockdown activity of cODV-siRNAs in N2a cells

To assess the knockdown activity of cODV-siRNAs, the indicated cODV-siRNAs (i.e., RD-18148, RD-18150, RD-18151, RD-18152, RD-18153, RD-18154, RD-18155 and RD-18156) were transfected in N2a cells at 0.1 nM for 24 hours. Remaining Sod1 mRNA levels were quantified by two-step RT-qPCR. FIG. 9 showed the remaining Sod1 mRNA levels following cODV-siRNA treatments.

To further assess the knockdown activity of cODV-siRNAs, the indicated 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 in N2a cells at 0.1 nM for 24 hours. Remaining Sod1 mRNA levels were quantified by two-step RT-qPCR. FIG. 10 showed the remaining Sod1 mRNA levels following cODV-siRNA treatments.

Example 12. Synthesis of linking component compounds for linking duplex oligonucleotide

Compound list:

Compounds 1 (spacer-18 linker) , 2 (spacer-C6 linker) , 3 (L6) , 4 (spacer-9 linker) , 5 (spacer-C3 linker) , 6 (d spacer) , 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) are purchased from Wuhu Huaren Science and Technology Co., Ltd (Anhui, China) . L6 linker is purchased from Hongene Biotech (Shanghai, China) . Compounds 8 (spacer-L14 linker) , 9 (spacer-L15 linke) , 10 (spacer-L16 linker) , 11 (C6x1 linker) , 12 (C6x2 linker) , 13 (C6x5 linker) , 14 (C6x7 linker) , L20 (L20 linker) , L42 (L42 linker) are synthesized by using the following procedures. All these compounds were applied as monomer/spacer in oligonucleotide synthesis and listed in Table 1.

1. Compound 8 synthesis

Compound 8 was prepared in this Example by using the following procedures.

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

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) under nitrogen atmosphere was added DMTrCl (23.48 g, 69.3 mmol, 1.0 eq) slowly. The reaction mixture was stirred at room temperature for 6 h then concentrated under reduced pressure, the resultant residue was purified by flash chromatography (silica gel, gradient eluent: 1-3%of MeOH/DCM) to provide compound 16 (12.1 g, 39%yield) as yellow oil. The product was characterized with mass spectrometry and ¹H NMR. MW calc.: 446.25; MW Found: 303.09 [DMT] ^-, 144.15 [DMT off + H] ⁺. ¹H 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) .

(2) Preparation of compound 8 from compound 16.

To a solution of compound 16 (3.4 g, 7.65 mmol, 1.0 eq) and diisopropylammonium tetrazolide (2.6 g, 15.3 mmol, 2.0 eq) in anhydrous Dichloromethane (DCM, 30 mL) under nitrogen atmosphere was added 3- ( (bis (diisopropylamino) phosphaneyl) oxy) propanenitrile (4.6 g, 15.3 mmol, 2.0 eq. ) at room temperature . The reaction mixture was stirred for 3 h. The mixture was extracted two times with DCM, then washed with brine and dried with anhydrous Na₂SO₄. The organic layer was concentrated under reduced pressure and the resultant residue was purified with flash chromatography (silica gel, gradient eluent: 1-20%of EtOAc/Hexane, 1%Et₃N) to provide compound 8 (3.65 g, 75%yield) as yellow oil. The product was characterized with mass spectrometry and ¹H NMR. MW calc.: 646.35; MW Found: 303.09 [DMT] ^-, 302.36 [DMT and one isopropyl off] ⁺. ¹H NMR (400 MHz, CDCl₃) δ7.47 (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. Compound 9 synthesis

Compound 9 was prepared in this Example by using the following procedures.

(1) Preparation of compound 18 from the starting compound 2, 2'- (1, 4-phenylene) bis (ethan-1-ol) 17.

To a solution of 2, 2'- (1, 4-phenylene) bis (ethan-1-ol) 17 (3 g, 18.0 mmol, 1.0 eq) in anhydrous pyridine (50 mL) under nitrogen atmosphere was added DMTrCl (6.1 g, 18.0 mmol, 1.0 eq) slowly. The reaction mixture was stirred at room temperature for 6 h then concentrated under reduced pressure, the resultant residue was purified by flash chromatography (silica gel, gradient eluent: 1-3%of MeOH/DCM) to provide compound 18 (3.74 g, 44%yield) as yellow oil. The product was characterized with mass spectrometry and ¹H NMR. MW calc.: 468.23; MW Found: 303.16 [DMT] ^-, 491.31 [M + Na] ⁺. 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) .

(2) Preparation of compound 9 from compound 18.

To a solution of compound 18 (884 mg, 1.89 mmol, 1.0 eq) and diisopropylammonium tetrazolide (647 mg, 3.78 mmol, 2.0 eq) in anhydrous DCM (10 mL) under nitrogen atmosphere was added 3- ( (bis (diisopropylamino) phosphaneyl) oxy) propanenitrile (1.14 g, 3.78 mmol, 2.0 eq. ) at room temperature. The reaction mixture was stirred for 3 h. The mixture was extracted two times with DCM, then washed with brine and dried with anhydrous Na₂SO₄. The organic layer was concentrated under reduced pressure and the resultant residue was purified with flash chromatography (silica gel, gradient eluent: 1-30%of EtOAc/Hexane, 1%Et₃N) to provide compound 9 (292 mg, 23%yield) as yellow oil. The product was characterized with mass spectrometry and ¹H NMR. MW calc.: 668.34; MW Found: 303.11 [DMT] ^-. ¹H 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) .

3. Compound 10 synthesis

Compound 10 was prepared in this Example by using the following procedures.

(1) Preparation of compound 20 from the reduction of starting compound 2, 2'- (cyclohexane-1, 1-diyl) diacetic acid 19.

To a solution of compound 19 (10 g, 50 mmol, 1.0 eq) in anhydrous THF (200 mL) under nitrogen atmosphere and ice bath was added LiAlH₄ (5.7 g, 150 mmol, 3.0 eq) . The mixture was then transferred to room temperature after 10 minutes and stirred for about 1 h. Then the reaction was transferred to ice bath, saturated potassium sodium tartrate solution (100 mL) was added slowly into the mixture. After 30 minutes, the reaction was extracted 3 times by Et₂O, then the organic phase was combined and washed by brine, dried over Na₂SO₄, and concentrated. The crude product 20 was characterized with mass spectrometry. MW calc.: 172.15; MW Found: 173.22 [M + H] +.

(2) Preparation of compound 21 from compound 20.

To a solution of compound 20 (3 g, 17.4 mmol, 1.0 eq) in anhydrous pyridine (50 mL) under nitrogen atmosphere was added DMTrCl (4.7 g, 13.92 mmol, 0.8 eq) slowly. The reaction mixture was stirred at room temperature for 6 h then concentrated under reduced pressure. The resultant residue was purified by flash chromatography (silica gel, gradient eluent: 1-3%of MeOH/DCM) to provide compound 21 (3.0 g, 36%yield) as yellow oil. The product was characterized with 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) .

(3) Preparation of compound 10 from compound 21.

To a solution of compound 21 (1.3 g, 2.74 mmol, 1.0 eq) and diisopropylammonium tetrazolide (938 mg, 5.48 mmol, 2.0 eq) in anhydrous DCM (15 mL) under nitrogen atmosphere was added 3- ( (bis (diisopropylamino) phosphaneyl) oxy) propanenitrile (1.65 g, 5.48 mmol, 2.0 eq. ) at room temperature. The reaction mixture was stirred for 3 h. The mixture was extracted two times with DCM, then washed with brine and dried with anhydrous Na₂SO₄. The organic layer was concentrated under reduced pressure and the resultant residue was purified with flash chromatography (silica gel, gradient eluent: 1-20%of EtOAc/Hexane, 1%Et₃N) to provide compound 10 (425 mg, 23%yield) as yellow oil. The product was characterized with 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) δ7.45 –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) .

4. Compounds 11 and 12 synthesis

Compounds 11 and 12 were prepared in this Example by using the following procedures.

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

Compound 22 (4.8 g, 29 mmol, 1.0 eq) was dissolved in anhydrous DMF (200 mL) under nitrogen atmosphere. The solution was cooled to 5 ℃, and NaH (1.54 g, 38.6 mmol, 60%dispersion in mineral oil, 1.3 eq) was subsequently added, followed by the addition of Tetrabutylammonium bromide (TBAB) (1.87 g, 5.8 mmol, 0.2 eq) and 5-chloro-1-pentyne (3.89 mL, 36.83 mmol, 1.27 eq) . The reaction mixture was allowed to stir overnight at 55 ℃. Then the reaction liquid was filtered and concentrated under reduced pressure. After that, water (100 mL) was added into the mixture, then extracted 3 times by ethyl acetate, the organic phase was washed 3 times by saturated lithium chloride solution. After dried by anhydrous NaSO₄ and concentrated under reduced pressure, the yellow oil formed then was directly dissolved in anhydrous pyridine (100 mL) under nitrogen atmosphere. DMTrCl (11.8 g, 34.8 mmol, 1.2 eq) was added slowly. The reaction mixture was stirred at room temperature for 6 h then concentrated under reduced pressure. The resultant residue was purified by flash chromatography (silica gel, gradient eluent: 1-30%of EtOAc/Hexane) to provide compound 23 (2.43 g, 15.7%yield) and 24 (1.49 g, 10%yield) . The products were characterized with mass spectrometry and ¹H NMR.

Compound 23 MW calc.: 532.25; MW Found: 555.8 [M + Na] ⁺. ¹H NMR (400 MHz, CDCl₃) δ7.50 (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) .

Compound 24 MW calc.: 532.25; MW Found: 303.4 [DMT] ^-, 253.3 [DMT off + Na] ⁺. ¹H 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) .

(2) Preparation of compound 11 from compound 23.

To a solution of compound 23 (300 mg, 0.56 mmol, 1.0 eq) and N, N-Diisopropylethylamine (DIPEA) (139 μL, 0.84 mmol, 1.5 eq) in anhydrous DCM (5 mL) under nitrogen atmosphere was added 3- ( (chloro (diisopropylamino) phosphaneyl) oxy) propanenitrile (199 mg, 0.84 mmol, 1.5 eq) at room temperature. The reaction mixture was stirred for 1 h. The mixture was extracted two times with DCM, then washed with brine and dried with anhydrous Na₂SO₄. The organic layer was concentrated under reduced pressure and the resultant residue was purified with flash chromatography (silica gel, gradient eluent: 1-20%of EtOAc/Hexane, 1%Et₃N) to provide compound 11 (196 mg, 48%yield) as colorless oil. The product was characterized with mass spectrometry and ¹H NMR. MW calc.: 732.35; MW Found: 733.2 [M+H] ⁺. ¹H 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) .

(3) Preparation of compound 12 from compound 24.

To 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) under nitrogen atmosphere, was added 3- ( (chloro (diisopropylamino) phosphaneyl) oxy) propanenitrile (199 mg, 0.84 mmol, 1.5 eq) at room temperature. The reaction mixture was stirred for 1 h. The mixture was extracted two times with DCM, then washed with brine and dried with anhydrous Na₂SO₄. The organic layer was concentrated under reduced pressure and the resultant residue was purified with flash chromatography (silica gel, gradient eluent: 1-20%of EtOAc/Hexane, 1%Et₃N) to provide compound 12 (127 mg, 31%yield) as colorless oil. The product was characterized with mass spectrometry and 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) .

5. Compound 13 synthesis

Compound 13 was prepared in this Example by using the following procedures.

(1) Preparation of compound 26 from the starting compound diethanolamine 25.

To a solution of diethanolamine 25 (7.16 g, 68 mmol, 1.0 eq) in 110 mL of MeCN under ice bath was added anhydrous potassium carbonate (47.1 g, 341 mmol, 5.0 eq) with vigorous stirring. After 30 minutes of stirring, dropwise addition of 5-chloro-1-pentyne (7.2 mL, 68 mmol, 1.0 eq) was carried out over 5 minutes. The reaction was then left to stir for 3 days at 60 ℃. Then the reaction liquid was filtered and concentrated under reduced pressure. the resultant residue was purified by flash chromatography (silica gel, gradient eluent: 1-8%of MeOH/DCM) to provide compound 26 (2.85 g, 24%yield) . The product was characterized with mass spectrometry and ¹H NMR. MW calc.: 171.13; MW Found: 172.3 [M + H] ⁺. ¹H 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) .

(2) Preparation of compound 27 from the compound 26.

To a solution of compound 26 (2.8 g, 16.35 mmol, 1.0 eq) in 30 ml of DCM was added Et₃N (2.27 mL, 16.35 mmol, 1.0 eq) with stirring. Then the DMTrCl (4.43 g, 13.08 mmol, 0.8 eq) was added slowly. The reaction mixture was stirred at room temperature for 6 h then concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, gradient eluent: 1-3%of MeOH/DCM) to provide compound 27 (2.98 g, 48%yield) . The product was characterized with mass spectrometry and 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) .

(3) Preparation of compound 13 from the compound 27.

To 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) under nitrogen atmosphere, was added 3- ( (chloro (diisopropylamino) phosphaneyl) oxy) propanenitrile (1.85 g, 7.8 mmol, 3.0 eq) at room temperature. The reaction mixture was stirred for 30 min. The mixture was extracted two times with DCM, then washed with brine and dried with anhydrous Na₂SO₄. The organic layer was concentrated under reduced pressure and the resultant residue was purified with flash chromatography (silica gel, gradient eluent: 1-30%of EtOAc/Hexane, 1%Et₃N) to provide compound 13 (1.12 g, 64%yield) as yellow oil. The product was characterized with mass spectrometry and ¹H NMR. MW calc.: 673.36; MW Found: 633.2 [one isopropyl off + H] ⁺. 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) .

6. Compound 14 synthesis

Compound 14 was prepared in this Example by using the following procedures.

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

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

(2) Preparation of compound 30 from the compound 29.

To a solution of compound 29 (37.6 mmol, 1.0 eq) in anhydrous pyridine (200 mL) , was added DMTrCl (14 g, 41.4 mmol, 1.1 eq) slowly at ice bath. The reaction was stirred under nitrogen atmosphere overnight and then concentrated under reduced pressure. The crude product was dissolved in dry MeCN (300 mL) then the mixture was added Et₃N (15.6 mL, 113 mmol, 3.0 eq) and heated to 60 ℃ for 4 h. After concentrated under reduced pressure, the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-8%of MeOH/DCM) to provide the desired product 30 (7.57 g, 48%yield) as yellow solid. The product was characterized with mass spectrometry and 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) .

(3) Preparation of compound 31 from the compound 30.

Compound 30 (500 mg, 1.19 mmol, 1.0 eq) was dissolved in 5 mL DCM, then 4- ( ( ( (9H-fluoren-9-yl) methoxy) carbonyl) amino) butanoic acid (465 mg, 1.43 mmol, 1.2 eq) , HBTU (903 mg, 2.38 mmol, 2.0 eq) and DIPEA (671 uL, 4.05 mmol, 3.4 eq) were added into the reaction under nitrogen atmosphere. The reaction mixture was stirred overnight at room temperature. Then 10 mL H₂O was added into the reaction, the mixture was extracted by DCM (3*10 mL) and the organic phase was combined, dried over Na₂SO₄, and concentrated. The resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-5%of MeOH/DCM) to provide the desired product 31 (740 mg, 85%yield) as yellow solid. The product was characterized with mass spectrometry and ¹H NMR. MW calc.: 726.33; MW Found: 425.2 [DMT off + H] ⁺. 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) .

(4) Preparation of compound 14 from the compound 31.

To 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) under nitrogen atmosphere, was added 3- ( (chloro (diisopropylamino) phosphaneyl) oxy) propanenitrile (390 mg, 1.65 mmol, 3.0 eq) at room temperature. The reaction mixture was stirred for 1 h. The mixture was extracted two times with DCM, then washed with brine and dried with anhydrous Na₂SO₄. The organic layer was concentrated under reduced pressure and the resultant residue was purified with flash chromatography (silica gel, gradient eluent: 1-3%of MeOH/DCM, 1%Et₃N) to provide compound 14 (420 mg, 82%yield) as yellow oil. The product was characterized with mass spectrometry and ¹H NMR. MW calc.: 926.44; MW Found: 403.3 [DMT and Fmoc off + H] ⁺. 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) .

7. Compound L20 synthesis

Compound L20 was prepared in this Example by using the following procedures.

(1) The preparation of compound A18

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

(2) The preparation of compound A19

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) under nitrogen atmosphere, was added DMTrCl (8.51 g, 27.63 mmol, 1.1 eq) . The reaction mixture was stirred at room temperature for 4 h, after which it was concentrated under reduced pressure. Then a saturated NaHCO₃ solution (50 mL) was added, the mixture was extracted three times with ethyl acetate, then the organic phase was combined and washed with brine, dried over Na₂SO₄, and concentrated. The resultant residue A19 was directly used in the next step without further purification. The product was characterized with mass spectrometry. MW calc.: 584.25; MW. Found: 303.12 [DMT] ^-.

(3) The preparation of compound A20

To a solution of compound A19 (25.12 mmol, 1.0 eq) in THF/H₂O (9: 1, 111 mL) , under ice bath, was added HCOONH₄ (9.51 g, 150.72 mmol, 6.0 eq) and Zn powder (9.86 g, 150.72 mmol, 6.0 eq) . After stirred for 10 minutes, the reaction mixture was moved from the ice bath and stirred at room temperature overnight. Then the reaction mixture was filtered and concentrated under reduced pressure. After that, water (100 mL) was added into the mixture, then the mixture was extracted three times with ethyl acetate, the organic phase was washed one time with brine. After dried over anhydrous Na₂SO₄ and concentrated under reduced pressure, the product A20 formed then was directly used in the next step without further purification. The compound A20 was characterized with mass spectrometry. MW calc.: 554.28; MW Found: 253.19 [M –DMT + H] ⁺.

(4) The preparation of compound A22

To a solution of compound A20 (25.12 mmol, 1.0 eq) in EtOH (140 mL) under nitrogen atmosphere, was added 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 ℃ for 6 h, and then concentrated under reduced pressure. Then a saturated NaHCO₃ solution (100 mL) was added, the mixture was extracted three times with ethyl acetate, then the organic phase was combined and washed with brine, dried over Na₂SO₄, and concentrated. The resultant residue was purified with flash chromatography (silica gel, gradient eluent: 1-5%of MeOH/DCM) to provide compound A22 (9.4 g, 60%yield) . The product was characterized with 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) .

(5) The preparation of compound L20

The compound A22 (2 g, 3.14 mmol, 1.0 eq) and Diisoropyl ammonium tetrazolide (1.61 g, 9.43 mmol, 3.0 eq) were dissolved in anhydrous DCM (20 mL) under nitrogen atmosphere was added 3- ( (Bis (diisopropylamino) phosphino) oxy) propanenitrile (2.85 g, 9.43 mmol, 3.0 eq) at room temperature. The reaction mixture was stirred for 6 h. The mixture was extracted two times with DCM, then washed with brine and dried with anhydrous Na₂SO₄. The organic layer was concentrated under reduced pressure and the resultant residue was purified with flash chromatography (silica gel, gradient eluent: 1-5%of MeOH/DCM, 1%Et₃N) to provide compound L20 (2.4 g, 93%yield) . The product was characterized with 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) .

8. Compound L42 synthesis

Compound L42 was prepared in this Example by using the following procedures.

(1) The preparation of compound A2

To a solution of A1 (6 g, 44.7 mmol, 1.0 eq) in dry MeOH (50 mL) , under nitrogen atmosphere, was added methanol solution with iodine (11.3 g iodine in 50 mL MeOH, 44.7 mmol, 1.0 eq) . The reaction mixture was stirred at room temperature for 15 h. TLC showed A1 was consumed completely. The reaction was quenched with 10%NaHCO₃ solution and then DCM (200 mL) was added. The DCM layer was washed by H₂O (200 mL) two times and one time by brine. Then concentrated under reduced pressure and the resultant residue A2 was directly used in next step without further purification. The resultant residue A2 was characterized with ¹H NMR. ¹H 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) .

(2) The preparation of compound A3

To a solution of crude A2 (22.35 mmol, 1.0 eq) in dry pyridine (60 mL) , under nitrogen atmosphere, was added DMTrCl (4.5 g, 13.41 mmol, 0.6 eq) . The reaction mixture was stirred at room temperature for 15 h. TLC showed A2 was consumed completely. The reaction mixture was concentrated in vacuo to give crude residue and then added DCM (100 mL) . The DCM layer was washed by H₂O (200 mL) two times and one time by brine. Then concentrated under reduced pressure and the resultant residue was purified with flash chromatography (silica gel, gradient eluent: 1-50%of Ethyl acetate/Hexane) to provide compound A3 (5.5 g, 43%yield) as yellow oil. The product was characterized with 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) .

(3) The preparation of compound L42

To a solution of compound A3 (2 g, 3.5 mmol, 1.0 eq) and Diisoropyl ammonium tetrazolide (1.2 g, 7.0 mmol, 2.0 eq) in anhydrous DCM (15 mL) under nitrogen atmosphere was added 3- ( (Bis (diisopropylamino) phosphino) oxy) propanenitrile (2.1 g, 7.0 mmol, 2.0 eq) at room temperature. The reaction mixture was stirred for 6 h. The mixture was extracted two times with DCM, then washed with brine and dried with anhydrous Na₂SO₄. The organic layer was concentrated under reduced pressure and the resultant residue was purified with flash chromatography (silica gel, gradient eluent: 1-50%of Ethyl acetate/Hexane, 1%Et₃N) to provide compound L42 (2.0 g, 74%yield) as yellow oil. The product was characterized with 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) .

All compounds in this example are listed in Table 1.

All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g., Genbank sequences or GeneID entries) , patent application, or patent, was specifically and individually indicated incorporated by reference in its entirety, for all purposes. This statement of incorporation by reference is intended by Applicants, pursuant to 37 C.F.R. §1.57 (b) (1) , to relate to each and every individual publication, database entry (e.g., Genbank sequences or GeneID entries) , patent application, or patent, each of which is clearly identified in compliance with 37 C.F.R. §1.57 (b) (2) , even if such citation is not immediately adjacent to a dedicated statement of incorporation by reference. The inclusion of dedicated statements of incorporation by reference, if any, within the specification does not in any way weaken this general statement of incorporation by reference. Citation of the references herein is not intended as an admission that the reference is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents.

While the application has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it is understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the application.

Claims

An oligonucleotide agent comprising a targeting oligonucleotide conjugated to a non-targeting moiety capable of facilitating delivery of the targeting oligonucleotide, wherein the non-targeting moiety comprises one or more units that are covalently linked in tandem to form a backbone of the non-targeting moiety, and at least two adjacent units are linked via a phosphorothioate (PS) bond; wherein each unit in the non-targeting moiety is selected from chemical linkers and nucleotides; and wherein the non-targeting moiety comprises:

one or more chemical linkers interspersed in nucleotides; one or more nucleotides interspersed in chemical linkers; a consecutive nucleotide sequence and a consecutively linked sequence of chemical linkers; or a consecutively linked sequence of chemical linkers without any nucleotide;

wherein at least one phosphodiester bond between two adjacent nucleotides, between two adjacent linkers or between a nucleotide and an adjacent linker is substituted by a phosphorothioate (PS) , mesyl phosphoramidate or boranophosphate bond.
The oligonucleotide agent according to claim 1, wherein the chemical linkers 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) diisopropylphosphoramidite) ;

b) L4 or C6 (spacer-C6 linker) (6- (bis (4-methoxyphenyl) (phenyl) methoxy) hexyl (2-cyanoethyl) diisopropylphosphoramidite) ;

c) L6 (1, 1-bis (4-methoxyphenyl) -1-phenyl-2, 5, 8, 11, 14-pentaoxahexadecan-16-yl (2-cyanoethyl) diisopropylphosphoramidite) ;

d) L9 or S9 (spacer-9 linker) (2- (2- (2- (bis (4-methoxyphenyl) (phenyl) methoxy) ethoxy) ethoxy) ethyl (2-cyanoethyl) diisopropylphosphoramidite) ;

e) L10 or C3 (spacer-C3 linker) (3- (bis (4-methoxyphenyl) (phenyl) methoxy) propyl (2-cyanoethyl) diisopropylphosphoramidite) ;

f) L12 (d spacer) ( (2R, 3S) -2- ( (bis (4-methoxyphenyl) (phenyl) methoxy) methyl) tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite) ;

g) L13 or C12 (spacer-C12 linker) (12- (bis (4-methoxyphenyl) (phenyl) methoxy) dodecyl (2-cyanoethyl) diisopropylphosphoramidite) ;

h) L14 (spacer-L14 linker) ( ( (1r, 4r) -4- ( (bis (4-methoxyphenyl) (phenyl) methoxy) methyl) cyclohexyl) methyl (2-cyanoethyl) diisopropylphosphoramidite) ;

i) L15 (spacer-L15 linker) (4- (2- (bis (4-methoxyphenyl) (phenyl) methoxy) ethyl) phenethyl (2-cyanoethyl) diisopropylphosphoramidite) ;

j) L16 (spacer-L16 linker) (2- (1- (2- (bis (4-methoxyphenyl) (phenyl) methoxy) ethyl) cyclohexyl) ethyl (2-cyanoethyl) diisopropylphosphoramidite) ;

k) C6x1 ( (2S, 3S, 4S, 5S) -2- ( (bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -5-methoxy-4- (pent-4-yn-1-yloxy) tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite) ;

l) C6x2 ( (2S, 3S, 4S, 5S) -5- ( (bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -2-methoxy-4- (pent-4-yn-1-yloxy) tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite) ;

m) C6x5 (2- ( (2- (bis (4-methoxyphenyl) (phenyl) methoxy) ethyl) (pent-4-yn-1-yl) amino) ethyl (2-cyanoethyl) diisopropylphosphoramidite) ;

n) C6x7 ( (9H-fluoren-9-yl) methyl (4- ( (2S, 4R) -2- ( (bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -4- ( (bis (diisopropylamino) phosphanyl) oxy) pyrrolidin-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) diisopropylphosphoramidite.
The oligonucleotide agent according to claim 1, wherein the chemical linkers are chemical groups selected from the following: 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, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, wherein one or more methylenes are interrupted or terminated by O, S, S (O) , SO₂, N (R') ₂, C (O) , cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocyclic, and wherein each R’ is independently selected from hydrogen, substituted or unsubstituted alkyl, aryl, aralkyl, alkylaryl, alkoxy, aryloxy, acyl or aliphatic which may be linear or branched.
The oligonucleotide agent according to any one of claims 1-3, wherein the non-targeting moiety comprises m same or different chemical linkers and n same or different nucleotides, wherein m is an integer in the range of 1-40 and n is an integer in the range of 0-30.
The oligonucleotide agent according to any one of claims 1-4, wherein the non-targeting moiety comprises a consecutively linked sequence of chemical linkers as shown in formula (linker1) _x- (linker2) _y, wherein linker1 is a first chemical linker and linker2 is a second chemical linker different from the first chemical linker, and x and y are integers with 0<x+y<50,

for example, the non-targeting moiety comprises 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,

wherein at least one phosphodiester bond between two adjacent linkers is substituted by a phosphorothioate (PS) , mesyl phosphoramidate or boranophosphate bond.
The oligonucleotide agent according to any of claims 1-5, wherein the non-targeting moiety comprises 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 the backbone.
The oligonucleotide agent according to any of claims 1-5, wherein the non-targeting moiety comprises 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, 40 or more phosphorothioate (PS) bonds in the backbone.
The oligonucleotide agent according to any of claims 1-7, wherein the targeting oligonucleotide is an antisense oligonucleotide, or a double-stranded oligonucleotide comprising a sense strand and an antisense strand, such as a small interfering RNA (siRNA) or a small activating RNA (saRNA) .
The oligonucleotide agent according to claim 8, wherein the non-targeting moiety is conjugated to the sense strand or the antisense strand of the double-stranded oligonucleotide.
The oligonucleotide agent according to any one of claims 1-9, wherein if present, the nucleotide (s) of the non-targeting moiety are non-chemically modified nucleotides, or at least one nucleotide is a chemically modified nucleotide.
The oligonucleotide agent according to any one of claims 1-10, wherein all nucleotides of the targeting oligonucleotide are non-chemically modified nucleotides, or at least one nucleotide is a chemically modified nucleotide, or at least one phosphodiester bond between two adjacent nucleotides in the targeting oligonucleotide is substituted by a phosphorothioate, mesyl phosphoramidate or boranophosphate bond.
The oligonucleotide agent according to any one of claims 10-11, wherein the chemically modified nucleotide comprises one or more of the following modifications:

a) modification of 2'-OH of the ribose in the nucleotide;

b) modification or absence of a base moiety on the nucleoside ring in the nucleotide;

c) a nucleotide being a locked or bridged nucleic acid, and

d) a nucleotide being a deoxyribonucleotide (DNA) .
The oligonucleotide agent according to claim 12, wherein the chemically modified nucleotide has a 2’-OH ribose modification selected from: a 2′-fluoro-2′-deoxynucleoside (2′-F) modification, a 2′-O-methyl (2′-O-Me) modification, and a 2′-O- (2-methoxyethyl) (2′-O-MOE) modification.
The oligonucleotide agent according to any of claims 1-13, wherein if present, the nucleotides in the non-targeting moiety are selected from the group of RNA, DNA, bridged nucleic acid (BNA) , locked nucleic acid (LNA) and peptide nucleic acid (PNA) .
The oligonucleotide agent according to any one of claims 10-11, wherein the at least one chemically modified nucleotide is a nucleotide having an addition of a 5'-phosophate, 5-methyl cytosine or 5’- (E) ‐vinylphosphonate.
The oligonucleotide agent according to any one of claims 1-15, wherein the targeting oligonucleotide and the non-targeting moiety are directly conjugated, for example, via a phosphorothioate (PS) bond.
The oligonucleotide agent according to claim 16, wherein the terminal unit or an internal unit of the non-targeting moiety is conjugated to the targeting oligonucleotide.
The oligonucleotide agent according to any of claims 1-17, wherein the non-targeting moiety is conjugated to the 3’ end, the 5’ end, both the 3’ and the 5’ ends, or an internal nucleotide of the sense strand or antisense strand of the double-stranded oligonucleotide.
The oligonucleotide agent according to any of claims 1-18, wherein the non-targeting moiety is selected from:

1) one chemical linker without any nucleotide, and the chemical linker is conjugated with the end of the targeting oligonucleotide via a phosphorothioate (PS) ;

2) a consecutively linked sequence of chemical linkers without any nucleotide, and the consecutively linked sequence of chemical linkers is conjugated with one end of the targeting oligonucleotide, optionally via one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20) deoxyribonucleotides (DNA) ;

3) two consecutively linked sequences of chemical linkers without any nucleotide, and the consecutively linked sequences of chemical linkers are respectively conjugated with both ends of the targeting oligonucleotide, optionally via one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20) deoxyribonucleotides (DNA) ;

4) one or more consecutive nucleotide sequences and a consecutively linked sequence of chemical linkers, and the nucleotide (s) being interspersed in chemical linkers, preferably every single nucleotide being interspersed in two chemical linkers, or one or more consecutive nucleotide sequences being interspersed in chemical linkers;

wherein at least one phosphodiester bond between two adjacent nucleotides, between two adjacent linkers or between a nucleotide and an adjacent linker is substituted by a phosphorothioate (PS) .
The oligonucleotide agent according to any one of claims 1-19, wherein the internal nucleotide in the sense or antisense strand of the double-stranded oligonucleotide is substituted by a linking component, wherein the non-targeting moiety is conjugated to the linking component.
The oligonucleotide agent according to any one of claims 1-20, wherein more than one (e.g., 2-10) non-targeting moieties are conjugated to the double-stranded oligonucleotide, or more than one (e.g., 2-10) double-stranded oligonucleotides are conjugated to the non-targeting moiety.
The oligonucleotide agent according to claim 20, wherein the linking component is selected from one or more of an ethylene glycol chain, an alkyl chain, an alkenyl chain, an alkynyl chain, a peptide, carbohydrates, thiol linkage, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, a tetrazole linkage, and a benzimidazole linkage.
The oligonucleotide agent according to any one of claims 1-22, wherein the non-targeting moiety and/or the double-stranded oligonucleotide is conjugated to one or more conjugation groups.
The oligonucleotide agent according to claim 23, wherein the one or more conjugation groups is selected from a lipid, a fatty acid, a fluorophore, a ligand, a saccharide, a peptide, and an antibody, optionally, the one or more conjugation groups is selected from a cell-penetrating peptide, polyethylene glycol, an alkaloid, a tryptamine, a benzimidazole, a quinolone, an amino acid, a cholesterol, glucose and N-acetylgalactosamine.
The oligonucleotide agent of any one of claims 1-24, wherein the oligonucleotide agent comprises a nucleotide sequence of the sense strand that is at least 90%identical to the nucleotide sequence as set forth in any of SEQ ID NOs: 1, 3, 56 and 61.
The oligonucleotide agent of any one of claims 1-25, wherein the oligonucleotide agent comprises a nucleotide sequence of antisense strand that is at least 90%identical to the nucleotide sequence as set forth in any of SEQ ID NOs: 2, 4, 57, 62 and 69.
The oligonucleotide agent of any one of claims 1-24, comprising the sequence of the sense strand as set forth in any of SEQ ID NOs: 6-39, 60, 64, 65, 66, 67, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86 and 87.
The oligonucleotide agent according to any one of claims 1-27, wherein the non-targeting moiety of the oligonucleotide agent improves the stability, bioavailability, biodistribution, and/or cellular uptake of the targeting oligonucleotide as compared to an oligonucleotide agent without the non-targeting moiety.
The oligonucleotide agent according to any one of claims 1-28, wherein the non-targeting moiety of the oligonucleotide agent increases the biodistribution of the targeting oligonucleotide within one or more target tissues as compared to an oligonucleotide agent without the non-targeting moiety.
The oligonucleotide agent according to claim 29, wherein the one or more target tissues is selected from tissues of brain, spinal cord, muscle, spleen, lung, heart, liver, bladder, and kidney.
The oligonucleotide agent according to claim 29, wherein the one or more target tissues is selected from the group consisting of: prefrontal cortex, cerebellum, and cerebrum; cervical, thoracic and lumbar in spinal cord; heart, bicep, semitendinosus, platysma, and gluteus.
A vector, comprising the oligonucleotide agent of any one of claims 1-31.
A cell, comprising the oligonucleotide agent of any one of claims 1-31.
The cell according to claim 33, wherein the cell is a mammalian cell, optionally a human cell.
The cell according to any one of claims 33-34, wherein the cell is a host cell.
The cell according to any one of claims 33-35, wherein the cell is in vitro, or exists in a mammalian body.
A pharmaceutical composition, comprising the oligonucleotide agent of any one of claims 1-30 and/or the cell of any one of claims 33-36.
The pharmaceutical composition according to claim 37, wherein the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier selected from an aqueous carrier, liposome or LNP, polymer, micelle, colloid, metal nanoparticle, non-metallic nanoparticle, bioconjugates, and polypeptide.
The pharmaceutical composition according to any one of claims 37-38, wherein the pharmaceutical composition inhibits the SOD1 gene expression or decreases the SOD1 protein.
The pharmaceutical composition according to any one of claims 37-38, wherein the pharmaceutical composition activates the expression of the SMN2 gene or increases SMN2 protein.
A kit, comprising the oligonucleotide agent of any one of claims 1-31 or the pharmaceutical composition of any one of claims 37-40.
A method of inhibiting the SOD1 gene expression or decreasing the SOD1 protein, comprising administering to a subject a pharmaceutical composition of any one of claims 37-39.
A method for treating or delaying the onset or progression of Amyotrophic lateral sclerosis (ALS) in a subject, the method comprising: administering to a subject a pharmaceutical composition of any one of claims 37-39.
The method according to claim 43, wherein the subject has sporadic ALS (sALS) or familial ALS (fALS) .
A method of activating the expression of an SMN2 gene or increasing the SMN2 protein, the method comprising administering to a subject a pharmaceutical composition of any one of claims 37-38 and 40.
A method for treating or delaying the onset or progression of spinal muscular atrophy (SMA) in a subject, the method comprising: administering to a subject a pharmaceutical composition of any one of claims 37-40 and 40.
The method according to any one of claims 42-46, wherein the non-targeting moiety of the oligonucleotide agent improves the stability, bioavailability, biodistribution, and/or cellular uptake of the double-stranded oligonucleotide as compared to an oligonucleotide agent without the non-targeting moiety.
The method according to any one of claims 42-47, wherein the non-targeting moiety of the oligonucleotide agent increases the biodistribution of double-stranded oligonucleotide within one or more target tissues as compared to an oligonucleotide agent without the non-targeting moiety.
The method according to any one of claims 42-48, wherein the non-targeting moiety of the oligonucleotide agent increases the biodistribution of double-stranded oligonucleotide within two or more target cell types in a tissue as compared to an oligonucleotide agent without the non-targeting moiety.
Use of the oligonucleotide agent of any one of claims 1-31 or the pharmaceutical composition of any of claims 37-40 in manufacturing a medicament for treating or delaying the onset or progression of Amyotrophic lateral sclerosis (ALS) .
The oligonucleotide agent of any one of claims 1-31 or the pharmaceutical composition of any of claims 37-40 for use in treating or delaying the onset or progression of Amyotrophic lateral sclerosis (ALS) .
A kit comprising a container comprising the oligonucleotide agent of any one of claims 1-31.