Oligonucleotides comprising phosphoramidate inter-nucleotide bonds

Abstract

Provided herein are oligonucleotides comprising a phosphoramidite internucleotide bond having the formula: (I) wherein R1 is isopropyl, isobutyl, sec-butyl, C1-6 haloalkyl, C2-6 hydroxyalkyl, C2-8 alkylene-N (RN) 2, C0-2 alkylene-C3-8 cycloalkyl, 4-10 membered heterocycloalkyl having 1-3 ring heteroatoms selected from O, N and S, or 5-10 membered heteroaryl having 1-3 ring heteroatoms selected from O, N and S, with the proviso that the heterocycloalkyl group or the heteroaryl group is attached to sulfur via a carbocyclic atom, and the cycloalkyl group, the heterocycloalkyl group or the heteroaryl group is substituted with 0, 1, 2 or 3 R2 groups; each R2 is independently halo, CN, N (RN) 2, C1-3 alkyl, C1-3 haloalkyl, C1-3 alkoxy, oxo, CO2RN, or C (O) C1-3 alkyl; and each RN is independently H or a C1-3 alkyl group. Also provided are formulations comprising the oligonucleotides disclosed herein and methods of treating diseases and disorders using the oligonucleotides disclosed herein and formulations thereof.

Description

Background Technology

[0001] This disclosure generally relates to oligonucleotides comprising phosphoramidite internucleotide bonds, formulations thereof, and methods for preparing and using said oligonucleotides and said formulations.

[0002] In recent years, several drugs derived from nucleic acids have been approved for commercial use, including fomivirsen, patisiran, and givosiran, and many others are in clinical trials. However, the sensitivity of oligonucleotide drugs to nuclease digestion in vivo means there is a need to utilize non-natural nucleotides to improve the robustness of oligonucleotide-based drugs.

[0003] While various aspects of the structure of oligonucleotides can be modified, the phosphodiester nucleotide bonds present in natural oligonucleotides are an attractive modification target, and their alteration can modulate drug properties such as plasma stability, binding properties, solubility, cell penetration, and bioactivity of oligonucleotide drugs.

[0004] Therefore, there is a need for oligonucleotide therapeutic agents with modified nucleotide inter-bonds and improved properties. Summary of the Invention

[0005] This article provides oligonucleotides comprising 10 to 300 nucleotides, each nucleotide containing a sugar moiety, nucleobases, and internucleotide bonds, wherein at least one internucleotide bond is a PAX internucleotide bond having the structure of formula (I): (I), where R 1 It is isopropyl, isobutyl, sec-butyl, C 1-6 Haloalkyl, C 2-6 Hydroxyalkyl, C 2-8 Alkylene-N(R) N 2. C 0-2 Alkylene-C 3-8 Cycloalkyl, 4-10 membered heterocycloalkyl having 1-3 cyclic heteroatoms selected from O, N, and S, or 5-10 membered heteroaryl having 1-3 cyclic heteroatoms selected from O, N, and S, provided that the heterocycloalkyl or heteroaryl is attached to sulfur via a carbocyclic atom, and the heterocycloalkyl, heterocycloalkyl, or heteroaryl is surrounded by 0, 1, 2, or 3 R atoms. 2 Group substitution; each R 2 Independently halogenated, CN, N(R) N 2. C 1-3 Alkyl, C 1-3 Haloalkyl, C 1-3 Alkoxy, oxo, CO2R N or C(O)C 1-3Alkyl groups; and each R N Independently H or C 1-3 alkyl.

[0006] Formulations comprising the oligonucleotides disclosed herein and pharmaceutically acceptable excipients are also provided. Methods for treating a disease or condition in a patient in need are further provided, comprising administering to the patient a therapeutically effective amount of the oligonucleotides disclosed herein or formulations comprising the oligonucleotides disclosed herein. Kits are further provided comprising the oligonucleotides disclosed herein or formulations thereof, and optionally one or more containers, one or more additional therapeutic agents, a packaging insert with instructions for performing any of the methods disclosed herein, one or more syringes, one or more filter needles, and / or one or more needles for parenteral injection.

[0007] Unless otherwise defined, 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 disclosure pertains. This document describes methods and materials used in this disclosure; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of any conflict, this specification (including definitions) shall prevail. Attached Figure Description

[0008] Figure 1A and 1B The results of an editing efficiency study are presented, which compared the percentage of editing at the sites of interest in a PiZ mouse hepatocyte model. The data indicate that the oligonucleotides disclosed herein can be obtained through free intake (…). Figure 1A ) and transfection ( Figure 1B The editing efficiency was measured.

[0009] Figure 2 The percentage of E342K mutation edited by the oligonucleotides of this disclosure, as measured at 4, 7, and 14 days post-drug administration, is shown in the PiZ mouse model.

[0010] Figure 3A and 3B This illustrates the 42-mer disclosed herein ( Figure 3A ) and 30-mer ( Figure 3B Oligonucleotides, the effect of PA1 relative to the modified nucleotide bonds of this disclosure in the PiZ mouse model, as measured by A1AT expression in serum.

[0011] Figure 4A and 4BOligonucleotides A1-A4 (A4) are shown Figure 4A ) and A5-A7 ( Figure 4B The results of the stability test.

[0012] Figure 5 Results of an editing efficiency study are presented, which compared the percentage of editing at the sites of interest in a PiZ mouse hepatocyte model. The data demonstrate the editing efficiency of the disclosed oligonucleotides with L4 bonds against two controls, oligonucleotide A1 (A) and oligonucleotide A1a (B), as measured by transfection at concentrations ranging from 0.1 nM to 100 nM.

[0013] Figure 6 Results of an editing efficiency study are presented, which compared the percentage of editing at the sites of interest in a PiZ mouse hepatocyte model. The data demonstrate the editing efficiency of the disclosed oligonucleotides with L15 bonds against two controls, oligonucleotide A1 (A) and oligonucleotide A1a (B), as measured by transfection at concentrations ranging from 0.1 nM to 100 nM.

[0014] Figure 7 Results of an editing efficiency study are presented, which compared the percentage of editing at the sites of interest in a PiZ mouse hepatocyte model. The data demonstrate the editing efficiency of the oligonucleotides disclosed herein, as measured by transfection (A) and free uptake (B).

[0015] Figure 8 The results of an in vivo editing efficiency study performed in the liver of a mouse model are shown. Detailed Implementation

[0016] This document provides oligonucleotides comprising modified internucleotide bonds (such as phosphoramide bonds). It also provides formulations comprising oligonucleotides comprising modified internucleotide bonds (such as phosphoramide bonds). Furthermore, it provides therapeutic methods using modified internucleotide bonds (such as phosphoramide bonds) and their formulations, as well as methods for preparing oligonucleotides comprising modified internucleotide bonds (such as phosphoramide bonds) and their formulations.

[0017] Oligonucleotides are attractive therapeutic platforms; however, oligonucleotide therapeutics containing native phosphate ester bonds are readily degraded in vivo by, for example, endogenous nucleases. Several skeletal modification strategies have been envisioned to address this issue, including the introduction of phosphate thioester (PS) or methanesulfonamide (PA1) skeletal bonds into the oligonucleotide structure. In particular, PA1 bonds have been shown to improve nuclease stability and durability compared to PS bonds. Without being bound by any particular theory, it is thought that PA1-containing oligonucleotides exhibit enhanced interactions with serum proteins, resulting in longer pharmacokinetic (PK) profiles, and enhanced transmembrane translocation, thereby improving potency. L1 (isopropylsulfonamide) and L2 (cyclopropylsulfonamide) bonds are phosphoramidite bonds and are more hydrophobic than PA1. The enhanced hydrophobicity of L1 and L2-containing oligonucleotides can improve nuclease stability, protein binding, and cellular uptake properties. In addition, L1 and L2-modified oligonucleotides can improve in vivo editing activity and durability.

[0018] Oligonucleotides containing phosphoramide This article provides oligonucleotides containing modified internucleotide bonds (such as phosphoramide bonds).

[0019] The oligonucleotides disclosed herein comprise 10 to 300 nucleotides, each nucleotide containing a sugar moiety, nucleobases, and internucleotide bonds, wherein at least one internucleotide bond is a PAX internucleotide bond having the structure of formula (I): (I), in R 1 It is isopropyl, isobutyl, sec-butyl, C 1-6 Haloalkyl, C 2-6 Hydroxyalkyl, C 2-8 Alkylene-N(R) N 2. C 0-2 Alkylene-C 3-8 Cycloalkyl, 4-10 membered heterocycloalkyl having 1-3 cyclic heteroatoms selected from O, N, and S, or 5-10 membered heteroaryl having 1-3 cyclic heteroatoms selected from O, N, and S, provided that the heterocycloalkyl or heteroaryl is attached to sulfur via a carbocyclic atom, and the heterocycloalkyl, heterocycloalkyl, or heteroaryl is surrounded by 0, 1, 2, or 3 R atoms. 2 Group substitution; Each R 2 Independently halogenated, CN, N(R) N 2. C 1-3 Alkyl, C 1-3 Haloalkyl, C 1-3 Alkoxy, oxo, CO2R Nor C(O)C 1-3 Alkyl; and Each R N Independently H or C 1-3 alkyl.

[0020] In some cases, R 1 It is isopropyl. In some cases, R 1 It is isobutyl. In some cases, R 1 It is sec-butyl. In some cases, R 1 C 3-8 cycloalkyl, and in some cases, spiroC 7-8 Cycloalkyl. In some cases, R 1 It is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In some cases, R 1 It is cyclobutyl, cyclopentyl, or cyclohexyl. In some cases, R 1 It is cyclopropyl. In some cases, R 1 It is cyclobutyl. In some cases, R 1 It is cyclopentyl. In some cases, R 1 It is cyclohexyl. In some cases, R 1 It is spiro[3.3]heptyl. In some cases, R 1 C 1-6 Halogenated alkyl or C 1-6 Hydroxyalkyl. In some cases, R 1 C 1-6 Haloalkyl. In some cases, R 1 C 1-6 Fluoroalkyl groups. In some cases, R 1 It is CH2F or CHF2. In some cases, R 1 It is CH2F. In some cases, R 1 For CHF2. In some cases, R 1 C 1-6 Hydroxyalkyl. In some cases, R 1 C 2-8 Alkylene-N(R) N 2. In some cases, R 1 It is a 4-8 membered heterocyclic alkyl group. In some cases, R 1 It can be azahexacyclic butane, pyrrolidine, piperidine, oxacyclobutane, tetrahydrofuran, or tetrahydropyran. In some cases, R 1 It is a 5-10 heteroaryl group. In some cases, R 1 It may be furan, thiophene, thiazole, isoxazole, imidazole, pyridine, or pyrazine. In some cases, R 1 For those that have not been replaced. In some cases, R 1 By 1, 2 or 3 R2 Replacement. In some cases, R 1 By 1 R 2 Replacement. In some cases, R 1 By 2 R 2 Replacement. In some cases, R 1 3 Rs 2 replace.

[0021] In some cases, at least one R 2 For oxygenation, CO2R N Or halogenated. In some cases, at least one R 2 It can be oxidized, CO2H-substituted, or halogenated. In some cases, at least one R... 2 For oxygenation. In some cases, at least one R 2 It is CO2H. In some cases, at least one R 2 Halogenation. In some cases, at least one R 2 For CN, N(R) N )2C 1-3 Alkyl, C 1-3 Haloalkyl, C 1-3 Alkoxy or C(O)C 1-3 In some cases, at least one R N For H. In some cases, at least one R N C 1-3 Alkyl groups. In some cases, each R N For H. In some cases, each R N C 1-3 alkyl.

[0022] As used herein, the term "alkyl" or "alkylene" refers to a saturated straight-chain or branched hydrocarbon. The term C... n This indicates that the alkyl group has "n" carbon atoms. For example, a C4 alkyl group refers to an alkyl group with 4 carbon atoms. 1-6 Alkyl refers to an alkyl group having a number of carbon atoms covering the entire range (e.g., 1 to 6 carbon atoms) and all subranges (e.g., 1-6, 2-6, 1-5, 2-6, 1-4, 2-5, 1, 2, 3, 4, 5, and 6 carbon atoms). Specific examples include, but are not limited to, methyl, ethyl, isopropyl, n-propyl, sec-butyl, and tert-butyl.

[0023] As used herein, the term "alkoxy" refers to an O-alkyl group; As used in this article, the term "halogen" or "halogenated" refers to F, Cl, Br, or I.

[0024] As used herein, the term "haloalkyl" refers to an alkyl group as defined herein that has been substituted with one or more halogen atoms.

[0025] As used herein, the term "hydroxyalkyl" means an alkyl group as defined herein, such that it is substituted with one or more OH groups.

[0026] The term "oxo" refers to the =O group.

[0027] The term "cycloalkyl" refers to a non-aromatic monocyclic, fused, bridged, or spirocyclic system whose ring atom is carbon and may be saturated or have one or more unsaturated units. The carbocyclic ring may have three to eight carbon atoms. In some embodiments, the number of carbon atoms is four to six. A "fused" bicyclic system comprises two rings sharing two adjacent ring atoms. A bridged bicyclic group comprises two rings sharing three or four adjacent ring atoms. A spirocyclic bicyclic system shares one ring atom. Cycloalkyl groups may include cycloalkenyl groups. Specific examples include, but are not limited to, cyclohexyl, cyclopentyl, cyclobutyl, and cyclopropyl. The carbocyclic ring, as described herein, may be unsubstituted or substituted.

[0028] As used herein, the term "heterocyclic alkyl" refers to a non-aromatic monocyclic, fused, spirocyclic, or bridged cyclic system having four to ten ring atoms, which may be saturated or contain one or more unsaturated units, wherein one or more (e.g., one to three, or one, two, or three) ring atoms are heteroatoms selected from O, N, and S. Examples of heterocyclic alkyl groups include, but are not limited to, quinoline, piperidinyl, piperazine, pyrrolyl, pyrazolyl, imidazoyl, azirroheptanyl, diazacycloheptanyl, triazacycloheptanyl, azirroheptanyl, diazacycloheptanyl, triazacycloheptanyl, oxazolyl, isoxazolyl, thiazoyl, isothiazolyl, oxazolyl, oxazolyl, thiazoyl, benzimidazolone, tetrahydrofuranyl, tetrahydrothiophene, morpholino (including, for example, 3-morpholino, 4-morpholino), 2-thiomorpholino, 3-thiomorpholino, 4-thiomorpholino, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, and 4-pyrrolyl. 5mmune5dine-2-one, 1-tetrahydropiperazinyl, 2-tetrahydropiperazinyl, 3-tetrahydropiperazinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 1-pyrazolinyl, 3-pyrazolinyl, 4-pyrazolinyl, 5-pyrazolinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl, 2-thiazolyl, 3-thiazolyl, 4-thiazolyl, 1-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, indololinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, benzothioheteropentyl, benzodithiayl, 3-(1-alkyl)-benzimidazol-2-one, and 1,3-dihydro-imidazolyl-2-one. Heterocyclic alkyl groups are either unsubstituted or substituted as described herein.

[0029] The term "heteroaryl" refers to an aromatic heterocycle having five to ten members (e.g., five to six members), including monocyclic heteroaromatic rings and polycyclic aromatic rings, wherein the monocyclic aromatic ring is fused with one or more other aromatic rings. A heteroaryl group has one or more rings (e.g., one to three, one, two, or three) heteroatoms selected from O, N, and S. The scope of the term "heteroaryl" as used herein also includes groups in which the aromatic ring is "fused" with one or more non-aromatic rings (carbocyclic or heterocyclic), wherein the group or connecting point is located on the aromatic ring. Examples of heteroaryl groups include pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, imidazoleyl, pyrroleyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, or thiadiazolyl, including, for example, 2-furanyl, 3-furanyl, N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-isooxazolyl, 4-isooxazolyl, 5-isooxazolyl, 2-oxadiazolyl, 5-oxadiazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 3-pyrazolyl, 4-pyrazolyl, 1-pyrroleyl, 2-pyrroleyl. 3-Pyrroloyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 3-pyridazinyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-triazolyl, 5-triazolyl, tetrazolyl, 2-thienyl, 3-thienyl, isothiazolyl, 1,2,3-oxadiazolyl, 1,2,5-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,3-triazolyl, 1,2,3-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, pyrazinyl, and 1,3,5-triazinyl. The heteroaryl rings are either unsubstituted or substituted as described herein.

[0030] In some cases, oligonucleotides contain one PAX nucleotide bond. In others, oligonucleotides contain 2-10 PAX nucleotide bonds. The nucleotides described herein may also include other modified nucleotide bonds. In some cases, at least one nucleotide bond is a phosphate thioester (PS). In others, at least one nucleotide bond is a methanesulfonylaminophosphate (PA1).

[0031] The oligonucleotides disclosed herein contain 10-300 nucleotides. In some cases, oligonucleotides contain 25-100 nucleotides. In some cases, oligonucleotides contain 30-50 nucleotides.

[0032] The oligonucleotides disclosed herein can be used for any number of end uses. Example oligonucleotides include, but are not limited to, structural genes, genes including control and termination regions, self-replicating systems such as viral DNA or plasmid DNA, single-stranded and double-stranded RNAi agents and other RNA interference agents (RNAi agents or iRNA agents), shRNA, antisense oligonucleotides, ribozymes, microRNAs, microRNA mimics, supermir, aptamers, antimir, antagomir, adaptors, triplet-forming oligonucleotides, tRNA (transfer RNA), G-quadruplex oligonucleotides, RNA activators, immunostimulatory oligonucleotides, and decoy oligonucleotides. Given the intended activity and use of the oligonucleotides, the selection of characteristics for the oligonucleotides (in addition to the presence of one or more PAX bonds as disclosed herein) is within the skill of a person skilled in the art.

[0033] In some cases, the PAX internucleotide bonds of formula (I) have the structures shown in Table 1: Table 1 As used herein, the term "ADAR recruitment domain" refers to a nucleotide, i.e., a sequence that acts as the recruitment and binding region of an ADAR enzyme. Oligonucleotides including such ADAR recruitment domains may be referred to as 'axiomer AON' or 'self-circulating AON'. In some embodiments, stem-loop structures may act as recruitment domains of ADAR enzymes (e.g., ADAR recruitment domains), but oligonucleotides disclosed herein may also affect ADAR recruitment and activity against target adenosine in target RNA in the absence of such stem-loop structures. Therefore, in some embodiments, the oligonucleotides disclosed herein do not include stem-loop structures. Alternatively, in some embodiments, the oligonucleotides disclosed herein do include stem-loop structures. The ADAR recruitment domain portion may function to recruit endogenous ADAR enzymes present in the cell. Such ADAR recruitment domains do not require a conjugated entity or the presence of a modified recombinant ADAR enzyme. Alternatively, the ADAR recruitment portion may function to recruit recombinant ADAR fusion proteins that have been delivered to cells or subjects via an expression vector construct comprising a polynucleotide encoding an ADAR fusion protein. Such ADAR fusion proteins may include a deaminase domain of the ADAR1 or ADAR2 enzyme fused with another protein (e.g., fused with the MS2 bacterial phage capsid protein). The ADAR recruitment domain may be based on the nucleotide sequence of a natural substrate (e.g., GluR2 receptor precursor mRNA; such as the GluR2 ADAR recruitment domain), a Z-DNA structure, or a domain of another protein known to be recruited as part of an ADAR fusion protein, such as the MS2 ADAR recruitment domain known to be recognized via the dsRNA binding region of ADAR. The stem-loop structure of the ADAR recruitment domain may be an intermolecular stem-loop structure formed by two separate nucleic acid strands or an intramolecular stem-loop structure formed within a single nucleic acid strand.

[0034] In various cases, the oligonucleotides described herein comprise an ADAR recruitment moiety and a targeting moiety complementary to a target RNA having a target adenosine, wherein the “position 0” of the oligonucleotide of this disclosure is a nucleotide directly opposite the target adenosine, and the “central triplet” of the oligonucleotide is position -1, position 0, and position +1 of the oligonucleotide. In some cases, the internucleotide bond between position -1 and 0, and the internucleotide bond between position 0 and +1, are not phosphoramids (e.g., portions of formula (I), such as any of L1-L17). In some cases, the internucleotide bond between position +1 and +2 is a phosphoramid (e.g., portions of formula (I), such as any of L1-L17). In some cases, the internucleotide bond between position +4 and +5 is a phosphoramid (e.g., portions of formula (I), such as any of L1-L17). In some cases, the internucleotide bond between position +5 and +6 is a phosphoramid (e.g., portions of formula (I), such as any of L1-L17). In some cases, the internucleotide bond between positions +9 and +10 is phosphorusamide (e.g., a portion of formula (I), such as any of L1-L17). In some cases, the internucleotide bond between positions -31 and -30 is phosphorusamide (e.g., a portion of formula (I), such as any of L1-L17). In some cases, the internucleotide bond between positions -27 and -26 is phosphorusamide (e.g., a portion of formula (I), such as any of L1-L17). In some cases, the internucleotide bond between positions -25 and -24 is phosphorusamide (e.g., a portion of formula (I), such as any of L1-L17). In some cases, the internucleotide bond between positions -23 and -22 is phosphorusamide (e.g., a portion of formula (I), such as any of L1-L17). In some cases, the internucleotide bond between positions -19 and -18 is phosphorusamide (e.g., a portion of formula (I), such as any of L1-L17). In some cases, the internucleotide bond between positions -13 and -12 is phosphoramide (e.g., a portion of formula (I), such as any of L1-L17). In some cases, the internucleotide bond between positions -11 and -10 is phosphoramide (e.g., a portion of formula (I), such as any of L1-L17). In some cases, the internucleotide bond between positions -10 and -9 is phosphoramide (e.g., a portion of formula (I), such as any of L1-L17). In some cases, the internucleotide bond between positions -9 and -8 is phosphoramide (e.g., a portion of formula (I), such as any of L1-L17). In some cases, the internucleotide bond between positions -x and –(x-1) and the internucleotide bond between positions –(x-1) and –(x-2) are each phosphoramide (e.g., a portion of formula (I), such as any of L1-L17), where x is the terminal position at the 5' end of the oligonucleotide.In some cases, the internucleotide bond between positions +(y-1) and +y is a phosphoramidite (e.g., a portion of formula (I), such as any of L1-L17), where +y is the terminal position at the 3' end of the oligonucleotide. In some cases, the internucleotide bond between positions +(y-2) and +(y-1) is a phosphoramidite (e.g., a portion of formula (I), such as any of L1-L17). In some cases, the internucleotide bond between positions +(y-3) and +(y-2) is a phosphoramidite (e.g., a portion of formula (I), such as any of L1-L17).

[0035] In some embodiments, the oligonucleotides described herein may further include a 5' cap structure. In some embodiments, the 5' cap structure is a 2,2,7-trimethylguanosine cap.

[0036] Modified oligonucleotides This disclosure provides oligonucleotides comprising unmodified or modified nucleotides and combinations thereof. As used herein, “nucleoside” is defined as a compound containing a sugar moiety (e.g., pentose or ribose or other sugar moieties described herein) or a derivative thereof in combination with nucleobases described herein. “Nucleoside” is defined as a nucleoside comprising an internucleotide bond (e.g., a phosphate ester group or a modified phosphate ester group, such as a PAX bond of formula (I) described herein). Modified nucleotides can be synthesized by any useful method as described herein (e.g., chemically, enzymatically, or recombinantly synthesized to comprise one or more modified or non-natural nucleosides).

[0037] The oligonucleotides described herein may include various modifications derived from naturally occurring oligonucleotides. As used herein, when referring to an oligonucleotide, the term "chemically modified" or, where applicable, "chemically modified" means a modification relative to a nucleobase (e.g., adenosine (A), guanosine (G), uridine (U), thymidine (T), or cytidine (C)), a modification of a sugar moiety (e.g., ribose or deoxyribose), or a modification of an internucleotide bond at one or more of its positions. Generally, these terms are not intended to refer to modifications present in naturally occurring oligonucleotides. In some embodiments, one or more nucleotides of the oligonucleotides described herein are chemically modified to enhance stability or other beneficial properties. Without being bound by theory, certain modifications are believed to increase nuclease resistance and / or serum stability or decrease immunogenicity. For example, the oligonucleotides described herein may contain nucleotides found naturally in DNA or RNA (e.g., adenine, thymidine, guanosine, cytidine, uridine, or inosine), or may contain nucleotides having one or more chemical modifications to one or more components of the nucleotides described herein (e.g., nucleobases, sugars, or internucleotide bonds).

[0038] The modification can be various. In some embodiments, the region may contain one, two, or more (optionally different) nucleotide modifications. In some embodiments, modified oligonucleotides introduced into cells may exhibit reduced degradation in cells compared to unmodified oligonucleotides.

[0039] Modifications to the oligonucleotides disclosed herein include, but are not limited to, the modifications detailed below. Oligonucleotides may contain naturally occurring or non-naturally occurring modifications, or both.

[0040] The oligonucleotides disclosed herein may include any modifications as described in detail below, such as to sugars, nucleobases, or internucleotide bonds (e.g., to phosphate ester, phosphodiester, or phosphodiester backbones). For example, one or more atoms of a pyrimidine or purine nucleobase may be replaced or substituted with an optionally substituted amino group, an optionally substituted thiol group, an optionally substituted alkyl group (e.g., methyl or ethyl), or a halogen (e.g., chlorine or fluorine). In some embodiments, the modification (e.g., one or more modifications) is present in each of the sugar and the internucleotide bond. Modifications according to this disclosure may be modifications that convert ribonucleic acid (RNA) into deoxyribonucleic acid (DNA), threonucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or hybrids thereof. Additional modifications are described herein.

[0041] Non-natural and / or modified nucleotides can be introduced into oligonucleotides during or after chain synthesis to achieve desired functions or properties. Modifications can be located at nucleotide bonds, nucleobases, or sugars. Modifications can be introduced at the ends of the chain or elsewhere in the chain via chemical synthesis or polymerases. Any region of an oligonucleotide can be chemically modified.

[0042] Nucleotide bonds The oligonucleotides disclosed herein comprise one or more internucleotide bonds. In all cases, at least one internucleotide bond in the oligonucleotides disclosed herein is a PAX internucleotide bond having a structure of formula (I) as described herein. The remaining internucleotide bonds in the disclosed oligonucleotides may be unmodified or natural internucleotide bonds and / or alternative internucleotide bonds. Unmodified or natural internucleotide bonds may be phosphate ester bonds. Alternative internucleotide bonds include non-natural internucleotide bonds and / or modified natural internucleotide bonds. Examples of alternative internucleotide bonds are known in the art, including but not limited to thiophosphate, borophosphate, phosphotriester, thiophosphate, or aminophosphate bonds and other variations of the phosphate ester backbone. In some embodiments, the oligonucleotides disclosed herein comprise at least one alternative internucleotide bond.

[0043] In some cases, the phosphate ester group (PO) of the natural nucleotide interbonding bond can be replaced by a thiophosphate ester (PS) group or a borophosphate ester (PB) group, or the 3',5'-phosphodiester bond of the natural nucleotide interbonding bond can be replaced by a 2',5'- bond, or the ester bond can be replaced by an amide bond, etc. Some embodiments include oligonucleotides having a heteroatomic backbone, and particularly those described in U.S. Patent No. 5,489,677, which are described as -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- [referred to as methylene (methylimino) or MMI backbone], -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -N(CH3)-CH2-CH2- [wherein the natural phosphate diester backbone is represented as -OPO-CH2-] heteroatomic backbones, and those described in U.S. Patent No. 5,602,240, which are described above. In some embodiments, the oligonucleotides characterized herein have the morpholine backbone structure of U.S. Patent No. 5,034,506, cited above. In some embodiments, the internucleotide bonds within the continuous nucleotide sequence are alternative internucleotide bonds. In some embodiments, the internucleotide bonds within the continuous nucleotide sequence are aminophosphate bonds, such as PAX internucleotide bonds having the structure of formula (I) disclosed herein. In some embodiments, the alternative internucleotide bonds are stereochemically pure alternative aminophosphate bonds. In some embodiments, the alternative internucleotide bonds are Sp aminophosphate bonds. In other embodiments, the alternative internucleotide bonds are Rp aminophosphate bonds. In all cases, the oligonucleotides disclosed herein comprise at least one PAX internucleotide bond having the structure of formula (I) disclosed herein.

[0044] Other alternative chemical components of the oligonucleotides described herein include 5' phosphate esters or 5' phosphate ester mimics, such as the 5'-terminal phosphate ester or phosphate ester mimic of the oligonucleotide. Suitable phosphate ester mimics are disclosed, for example, in U.S. Patent Publication No. 2012 / 0157511, the entire contents of which are incorporated herein by reference.

[0045] nucleobases The oligonucleotides described herein may also include one or more nucleobases (generally referred to simply as "bases" in the art), including one or more standard nucleobases and / or alternative nucleobases (e.g., non-natural nucleobases or natural nucleobases containing modifications or substitutions). In some cases, at least one nucleotide in the oligonucleotides disclosed herein does not have a nucleobase—that is, it is a baseless oligonucleotide.

[0046] Standard nucleobases include purine bases adenine (A) and guanine (G) and pyrimidine bases thymine (T), cytosine (C), and uracil (U). Alternative nucleobases include other non-natural / synthetic nucleobases described herein, as well as natural nucleobases, including those containing modifications or substitutions.

[0047] In some instances, the oligonucleotide contains at least one non-natural nucleobase. In some instances, the oligonucleotide contains at least one modified natural nucleobase. The term "modified" or, where appropriate, "modified" refers to structural and / or chemical modifications made relative to the A, G, U, T, or C nucleobases, nucleosides, and / or nucleotides. The nucleotides in the oligonucleotides of this disclosure may comprise non-standard nucleotides, such as non-naturally occurring nucleotides ("non-natural nucleotides") or chemically synthesized nucleotides. One or more atoms of the nucleobase may be replaced or substituted with an optionally substituted amino group, an optionally substituted thiol, an optionally substituted alkyl group (e.g., methyl or ethyl), or a halogen (e.g., chlorine or fluorine).

[0048] Non-limiting examples of synthetic and natural nucleobases that can serve as alternative nucleobases include 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 5-carboxycytosine, pyrrolocytosine, dideoxycytosine, uracil, 5-methoxyuracil, 5-hydroxydeoxyuracil, dihydrouracil, 4-thiouracil, pseudouracil, 1-methyl-pseudouracil, deoxyuracil, and 5-hydroxybutyl- 2'-Deoxyuracil, xanthine, hypoxanthine, 7-denitroxanthine, thienoguanine, 8-aza-7-denitroguanine, 7-methylguanine, 7-denitroguanine, 6-aminomethyl-7-denitroguanine, 8-aminoguanine, 2,2,7-trimethylguanine, 8-methyladenine, 8-azaadenine, 7-methyladenine, 7-denitroadenine, 3-denitroadenine, 2, 6-Diaminopurine, 2-aminopurine, 7-deazo-8-aza-adenine, 8-amino-adenine, thymine, dideoxythymine, 5-nitroindole, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and Cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 4-thiouracil, 8-halogenated, 8-amino, 8-thio, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halogenated, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 8-azaguanine and 8-azaadenine and 3-deazoguanine. In some embodiments, the nucleobase portion is modified by changing the purine or pyrimidine to a modified purine or pyrimidine, such as a substituted purine or substituted pyrimidine, such as a “substitute nucleobase” selected from the following: isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiazo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil, 5-thiazo-uracil, 2-thiouracil, pseudouracil, 1-methylpseudouracil, 5-methoxyuracil, 2'-thio-thymidine, hypoxanthine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine.Other nucleobases include those disclosed in U.S. Patent No. 3,687,808; those disclosed in "Modified Nucleosides" in *Biochemistry, Biotechnology and Medicine*, Herdewijn, P. (ed.), Wiley-VCH, 2008; those disclosed in *The Concise Encyclopedia of Polymer Science and Engineering*, pp. 858-859, Kroschwitz, J. L. (ed.), John Wiley & Sons, 1990; those disclosed in *Angewandte Chemie*, International Edition, 30:613, Englisch et al. (1991); and those disclosed in Chapter 15, "Antisense Research and Applications." Nucleotides disclosed in *Applications*, pp. 289-302, Crooke, ST and Lebleu, B., CRC Press, 1993; *Accounts of Chemical Research*, Vol. 45, p. 2055, Hirao et al. (2012); and *Current Protocols in Nucleic Acid Chemistry*, Supplement 37, 1.4.1, Bergstrom (2009). Certain of these nucleotides can be used specifically to enhance the binding affinity of the oligonucleotides described herein. These nucleotides include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitution has been shown to increase the stability of nucleic acid duplexes by 0.6–1.2 °C (Sanghvi, YS, Crooke, ST and Lebleu, B., eds., *Antisense Research and Applications*, CRC Press, Boca Raton, 1993, pp. 276–278), and is an exemplary base substitution.

[0049] In other embodiments, for each position, the nucleobases in the oligonucleotide may be independently selected from adenine, uridine, guanine, or cytidine, or analogs of adenine, uridine, guanine, or cytidine, such as modified adenine, uridine, guanine, or cytidine. Non-limiting examples of adenine, uridine, guanine, and cytidine analogs and modified adenine, uridine, guanine, and cytidine include N6-methyladenine, N1-methyladenine, N6-2'-O-dimethyladenine, pseudouridine, N1-methylpseudouridine, 5-iodouridine, 4-thiouridine, 2-thiouridine, 5-methyluridine, pseudoisocytosine, 5-methoxycytosine, 2-thiocytosine, 5-hydroxycytosine, N4-methylcytosine, 5-hydroxymethylcytosine, hypoxanthine, N1-methylguanine, O6-methylguanine, 1-methyl-guanosine, N2-methyl-guanosine, N2,N2-dimethyl-guanosine, 2-methyl-guanosine, N7-methyl-guanosine, 1-methyl-guanosine, N2,N7-dimethyl-guanosine, and isoguanine. For example, uridine (U) can be replaced by pseudouridine (ψ), 2-thiouridine (s2U), dihydrouridine (D), 5-bromo-U, 5-iodo-U, etc. Purines can be replaced by 2,6-diaminopurine.

[0050] Representative U.S. patents teaching the preparation of the aforementioned nucleobases and other nucleobases include, but are not limited to, U.S. Patent Nos. 3,687,808, 4,845,205, 5,130,30, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, 5,552,540, 5,587,469, and 5,594,121. Nos. 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of these documents are hereby incorporated herein by reference.

[0051] Other nucleobases present in the oligonucleotides disclosed herein include, but are not limited to, the following: 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine; 2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonylcarbamoyladenosine; N6-glycylcarbamoyladenosine; N6-isopentenyladenosine; N6-methyladenosine; N6-threonylcarbamoyladenosine; 1,2'-O-dimethyladenosine; 1-methyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladenosine (phosphate); 2-methyladenosine; 2-methylthio-N6-isopentenyladenosine; 2-methylthio-N6- Hydroxy-n-valine-carbamoyl adenosine; 2'-O-methyl adenosine; 21-O-ribosyl adenosine (phosphate); isopentenyl adenosine; N6-(cis-hydroxyisopentenyl) adenosine; N6,2'-O-dimethyl adenosine; N6,2'-O-dimethyl adenosine; N6,N6,2'-O-trimethyl adenosine; N6,N6-dimethyl adenosine; N6-acetyl adenosine; N6-hydroxy-n-valine-carbamoyl adenosine; N6-methyl-N6-threonyl-carbamoyl adenosine; 2-methyl adenosine; 2-methylthio-N6-isopentenyl adenosine; 7-deazo-adenosine; N1-methyl-adenosine; N6,N6(dimethyl)adenine; N6-cis-hydroxy-isopentenyl-adenine; α-thio-adenine; 2(amino)adenine; 2(aminopropyl)adenine; 2(methylthio)N6(isopentenyl)adenine; 2-(alkyl)adenine; 2-(aminoalkyl)adenine; 2-(aminopropyl)adenine; 2-(halogenated)adenine; 2-(halogenated)adenine; 2-(propyl)adenine; 2'-amino-2'-deoxy-ATP; 2' -azido-2'-deoxy-ATP; 2'-deoxy-2'-a-aminoadenosine TP; 2'-deoxy-2'-a-azido-adenosine TP; 6(alkyl)adenine; 6(methyl)adenine; 6-(alkyl)adenine; 6-(methyl)adenine; 7(deadenine); 8(alkenyl)adenine; 8(alkynyl)adenine; 8(amino)adenine; 8(thioalkyl)adenine; 8-(alkenyl)adenine; 8-(alkyl)adenine Purine; 8-(alkynyl)adenine; 8-(amino)adenine; 8-(halogenated)adenine; 8-(hydroxy)adenine; 8-(thioalkyl)adenine; 8-(thiol)adenine; 8-azido-adenine; azaadenine; deadenine; N6-(methyl)adenine; N6-(isopentyl)adenine; 7-deaden-8-azaa-adenine; 7-methyladenine; 1-deadenine TP; 2'-fluoro-N6-Bz-deoxyadenine TP ; 2'-OMe-2-amino-ATP; 2'-O-methyl-N6-Bz-deoxyadenosine TP; 2'-α-ethynyladenosine TP; 2-aminoadenosine; 2-aminoadenosine TP; 2-amino-ATP; 2'-α-trifluoromethyladenosine TP; 2-azidoadenosine TP; 2'-β-ethynyladenosine TP; 2-bromoadenosine TP; 2'-β-trifluoromethyladenosine TP; 2-chloroadenosine TP; 2'-deoxy-2',2'-Difluoroadenosine TP; 2'-Deoxy-2'-α-mercaptoadenosine TP; 2'-Deoxy-2'-α-thiomethoxyadenosine TP; 2'-Deoxy-2'-β-aminoadenosine TP; 2'-Deoxy-2'-β-azidoadenosine TP; 2'-Deoxy-2'-β-bromoadenosine TP; 2'-Deoxy-2'-β-chloroadenosine TP; 2'-Deoxy-2'-β-fluoroadenosine TP; 2'-Deoxy-2'-β-iodoadenosine TP; 2'-Deoxy-2'-β-mercaptoadenosine TP; 2'-Deoxy-2'-β-thiomethoxyadenosine TP; 2-Fluoroadenosine TP; 2-Iodoadenosine TP; 2'-Deoxy-2'-β-mercaptoadenosine TP; 2'-Deoxy-2'-β-thiomethoxyadenosine TP; 2-Fluoroadenosine TP; 2-Iodoadenosine 2-Mercaptoadenosine TP; 2-Methoxyadenosine TP; 2-Methylthioadenosine; 2-Trifluoromethyladenosine TP; 3-Denitro-3-bromoadenosine TP; 3-Denitro-3-chloroadenosine TP; 3-Denitro-3-fluoroadenosine TP; 3-Denitro-3-iodoadenosine TP; 3-Denitroadenosine TP; 4'-Azide-adenosine TP; 4'-Carbocyclic adenosine TP; 4'-Ethynyladenosine TP; 5'-Hy-adenosine TP; 8-Zaza-ATP; 8-bromoadenosine TP; 8-Trifluoromethyladenosine TP; 9-Denitroadenosine TP; 2-Aminopurine; 7-Denitro-2,6-di Aminopurine; 7-deazo-8-aza-2,6-diaminopurine; 7-deazo-8-aza-2-aminopurine; 2,6-diaminopurine; 7-deazo-8-aza-adenine; 7-deazo-2-aminopurine; 2-thiocytidine; 3-methylcytidine; 5-formylcytidine; 5-hydroxymethylcytidine; 5-methylcytidine; N4-acetylcytidine; 2'-O-methylcytidine; 21-O-methylcytidine; 5,2'-O-dimethylcytidine; 5-formyl-2'-O-methylcytidine; Lysicillin; N4,2'-O-dimethylcytidine; N4-acetyl-2'-O-methylcytidine Glycoside; N4-methylcytidine; N4,N4-dimethyl-2'-OMe-cytidine TP; 4-methylcytidine; 5-aza-cytidine; pseudoisocytidine; pyrrolo-cytidine; α-thio-cytidine; 2-(thio)cytosine; 2'-amino-2'-deoxy-CTP; 2'-azido-2'-deoxy-CTP; 2'-deoxy-2'-α-aminocytidine TP; 2'-deoxy-2'-α-azido-cytidine TP; 3(deazo)5(aza)cytidine; 3(methyl)cytidine; 3-(alkyl)cytidine; 3-(deazo)5(aza)cytidine; 3-(methyl)cytidine; 4,21-O-dimethylcytidine; 5-(halo)cytosine; 5-(methyl)cytosine; 5-(propynyl)cytosine; 5-(trifluoromethyl)cytosine; 5-(alkyl)cytosine; 5-(alkynyl)cytosine; 5-(halo)cytosine; 5-(propynyl)cytosine; 5-(trifluoromethyl)cytosine; 5-bromo-cytosine; 5-iodo-cytosine; 5-propynylcytosine; 6-(azo)cytosine; 6-aza-cytosine; aza-cytosine; deazocytosine; N4-(acetyl)cytosine; 1-methyl-1-deazo-pseudocytosine; 1-methyl-pseudocytosine; 2-methoxy-5-methylcytosine; 2-methoxy-cytosine; 2-thio-5-methylcytosine; 4-methoxy-1-methyl-pseudocytosine Glycoside; 4-methoxy-pseudoisocytidine; 4-thio-1-methyl-1-denitro-pseudoisocytidine; 4-thio-1-methyl-pseudoisocytidine; 4-thio-pseudoisocytidine; 5-aza-zebularine; 5-methyl-zebularine; pyrrolo-pseudoisocytidine; zebularine; (E)-5-(2-bromo-vinyl)cytidine TP; 2,2'-anhydride-cytidine TP hydrochloride; 2'-fluoro-N4-Bz-cytidine TP; 2'-fluoro-N4-acetyl-cytidine TP; 2'-O-methyl-N4-acetyl-cytidine TP; 2'-O-methyl-N4-Bz-cytidine TP; 2'-a-ethynylcytidine TP; 2'-a-trifluoromethylcytidine TP; 2'-b- 2'-β-trifluoromethylcytidine TP; 2'-deoxy-2',2'-difluorocytidine TP; 2'-deoxy-2'-α-mercaptocytidine TP; 2'-deoxy-2'-α-thiomethoxycytidine TP; 2'-deoxy-2'-β-aminocytidine TP; 2'-deoxy-2'-β-azidocytidine TP; 2'-deoxy-2'-β-bromocytidine TP; 2'-deoxy-2'-β-chlorocytidine TP; 2'-deoxy-2'-β-fluorocytidine TP; 2'-deoxy-2'-β-iodocytidine TP; 2'-deoxy-2'-β-mercaptocytidine TP; 2'-deoxy-2'-β-thiomethoxycytidine TP; 21-O-methyl-5-(1-propynyl)cytidine TP; 3'-ethynylcytidine TP; 4'-azidocytidine TP; 4'-carbocyclic cytidine TP; 4'-ethynylcytidine TP; 5-(1-propynyl)arsyl-cytidine TP; 5-(2-chloro-phenyl)-2-thiocytidine TP; 5-(4-amino-phenyl)-2-thiocytidine TP; 5-aminoallyl-CTP; 5-cyanocytidine TP; 5-ethynylarsyl-cytidine TP; 5-ethynylcytidine TP; 5'-homocytidine TP; 5-methoxycytidine TP; 5-trifluoromethylcytidine TP; N4-aminocytidine TP; N4-benzoylcytidine TP; pseudoisocytidine; 7-methylguanosine; N2,2'-O-dimethylguanosine; N2-methylguanosine; Wyomerosine; 1,2'-O-dimethylguanosine; 1-methylguanosine; 2'-O-methylguanosine; 2'-O-ribosylguanosine (phosphate); 2'-O-methylguanosine; 2'-O-ribosylguanosine (phosphate); 7-aminomethyl-7-denitroguanosine; 7-cyano-7-denitroguanosine; archapurin; methylwyoside; N2,7-dimethylguanosine; N2,N2,2'-O-trimethylguanosine; N2,N2,7-trimethylguanosine; N2,N2-dimethylguanosine; N2,7,2'-O-trimethylguanosine; 6-thio-guanosine; 7-denitro-guanosine; 8-oxo- Guanosine; N1-methyl-guanosine; α-thio-guanosine; 2(propyl)guanine; 2-(alkyl)guanine; 2'-amino-2'-deoxy-GTP; 2'-azido-2'-deoxy-GTP; 2'-deoxy-2'-α-aminoguanosine TP; 2'-deoxy-2'-α-azido-guanosine TP; 6-methyl)guanine; 6-(alkyl)guanine; 6-(methyl)guanine; 6-methyl-guanosine; 7-(alkyl)guanine; 7-(deoxy)guanine; 7-(methyl)guanine; 7-(alkyl)guanine; 7-(deoxy)guanine; 7 -(methyl)guanine; 8(alkyl)guanine; 8(alkynyl)guanine; 8(halo)guanine; 8(thioalkyl)guanine; 8-(alkenyl)guanine; 8-(alkyl)guanine; 8-(alkynyl)guanine; 8-(amino)guanine; 8-(halo)guanine; 8-(hydroxy)guanine; 8-(thioalkyl)guanine; 8-(thio)guanine; azaguanine; denitroguanine; N(methyl)guanine; N-(methyl)guanine; 1-methyl-6-thio-guanosine; 6-methoxy-guanosine; 6-thio-7-denitro-8-azaguanosine -Guanine; 6-Thio-7-Denitro-Guanine; 6-Thio-7-Methyl-Guanine; 7-Denitro-8-aza-Guanine; 7-Methyl-8-oxo-Guanine; N2,N2-Dimethyl-6-Thio-Guanine; N2-Methyl-6-Thio-Guanine; 1-Me-GTP; 2'Fluoro-N2-Isobutyl-Guanine TP; 2'O-Methyl-N2-Isobutyl-Guanine TP; 2'-α-Ethynyl-Guanine TP; 2'-α-Trifluoromethyl-Guanine TP; 2'-β-Ethynyl-Guanine TP; 2'-β-Trifluoromethyl-Guanine TP; 2'-Deoxy-2'2'-Difluoroguanosine TP; 2'-Deoxy-2'-α-mercaptoguanosine TP; 2'-Deoxy-2'-α-thiomethoxyguanosine TP; 2'-Deoxy-2'-β-aminoguanosine TP; 2'-Deoxy-2'-β-azidoguanosine TP; 2'-Deoxy-2'-β-bromoguanosine TP; 2'-Deoxy-2'-β-chloroguanosine TP; 2'-Deoxy-2'-β-fluoroguanosine TP; 2'-Deoxy-2'-β-iodoguanosine TP; 2'-Deoxy-2 '-β-mercaptoguanosine TP; 2'-deoxy-2'-β-thiomethoxyguanosine TP; 4'-azidoguanosine TP; 4'-carbocyclic guanosine TP; 4'-ethynylguanosine TP; 5'-homo-guanosine TP; 8-bromo-guanosine TP; 9-deazoguanosine TP; N2-isobutyl-guanosine TP; 1-methylinosine; inosine; 1,2'-O-dimethylinosine; 2'-O-methylinosine; 7-methylinosine; 2'-O-methylinosine; epox yqueuosine); galactosyl-thymidine; mannosyl-thymidine; thymidine; allylamino-thymidine; azathymidine; deazothymidine; deoxy-thymidine; 2'-O-methyluridine; 2-thiouridine; 3-methyluridine; 5-carboxymethyluridine; 5-hydroxyuridine; 5-methyluridine; 5-taurate methyl-2-thiouridine; 5-taurate methyluridine; dihydrouridine; pseudouridine; (3-(3-amino-3-carboxypropyl)uridine; 1-methyl-3-(3-amino)uridine 5-(carboxypropyl)-1-methyl ...6-Dihydro-uridine; 5-Aminomethyl-2-thiouridine; 5-Carbamoylmethyl-2'-O-methyluridine; 5-Carbamoylmethyluridine; 5-Carboxyhydroxymethyluridine; 5-Carboxyhydroxymethyluridine methyl ester; 5-Carboxymethylaminomethyl-2'-O-methyluridine; 5-Carboxymethylaminomethyl-2-thiouridine; 5-Carboxymethylaminomethyl-2-thiouridine; 5-Carboxymethylaminomethyluridine; 5-Carboxymethylaminomethyluridine; 5-Carbamoylmethyluridine TP; 5-Methoxycarbonylmethyl-2'-O-methyluridine; 5-Methoxycarbonylmethyl-2-thiouridine; 5-Methoxycarbonyl 5-Methyluridine; 5-Methyluridine; 5-Methoxyuridine; 5-Methyl-2-thiouridine; 5-Methylaminomethyl-2-selenouridine; 5-Methylaminomethyluridine; 5-Methyldihydrouridine; 5-Hydroxyacetic acid-uridine TP; 5-Hydroxyacetic acid-methyl ester-uridine TP; N1-Methyl-pseudouracil; N1-Ethyl-pseudouracil; Uridine 5-Hydroxyacetic acid; Uridine 5-Hydroxyacetic acid methyl ester; 3-(3-amino-3-carboxypropyl)-uridine TP; 5-(isopentenylaminomethyl)-2-thiouridine TP; 5-(isopentenylaminomethyl)-2'-O- Methyluridine TP; 5-(isopentenylaminomethyl O)uridine TP; 5-propynyluracil; α-thio-uridine; 1-(aminoalkylamino-carbonylethynyl)-2-(thio)-pseudouracil; 1-(aminoalkylamino-carbonylethynyl)-2,4-(dithio)pseudouracil; 1-(aminoalkylamino-carbonylethynyl)-4-(thio)pseudouracil; 1-(aminoalkylamino-carbonylethynyl)-pseudouracil; 1-(aminocarbonylethynyl)-2-(thio)pseudouracil; 1-(aminocarbonylethynyl)-2,4-(dithio)pseudouracil; 1-(aminocarbonylethynyl)-4-(thio)pseudouracil ; 1-(aminocarbonylethynyl)-pseudouracil; 1-substituted 2-(thio)-pseudouracil; 1-substituted 2,4-(dithio)pseudouracil; 1-substituted 4-(thio)pseudouracil; 1-substituted pseudouracil; 1-(aminoalkylamino-carbonylethynyl)-2-(thio)-pseudouracil; 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine TP; 1-methyl-3-(3-amino-3-carboxypropyl)pseudoUTP; 1-methyl-pseudoUTP; 1-ethyl-pseudoUTP; 2-(thio)pseudouracil; 2'deoxyuridine; 2'fluorouridine; 2-(thio)uracil; 2,4-(dithio)pseudouracil; 2'methyl, 2'amino, 2'azido, 2'fluoro-guanosine; 2'-amino-2'-deoxy-UTP; 2'-azido-2'-deoxy-UTP; 2'-azido-deoxyuridine TP; 2'-O-methylpseudouridine; 2'-deoxyuridine; 2'-fluorouridine; 2'-deoxy-2'-a-aminouridine TP; 2'-deoxy-2'-a-azidouridine TP; 2-methylpseudouridine; 3(3amino-3-carboxypropyl)uracil; 4-(thio)pseudouracil; 4-(thio)pseudouracil; 4-(thio)uracil; 4-thiouracil Pyridine; 5(1,3-diazol-1-alkyl)uracil; 5(2-aminopropyl)uracil; 5(aminoalkyl)uracil; 5(dimethylaminoalkyl)uracil; 5(guanidinylalkyl)uracil; 5(methoxycarbonylmethyl)-2-(thio)uracil; 5(methoxycarbonyl-methyl)uracil; 5(methyl)2(thio)uracil; 5(methyl)2,4(dithio)uracil; 5(methyl)4(thio)uracil; 5(methylaminomethyl)-2(thio)uracil; 5(methylaminomethyl)-2,4(dithio)uracil; 5(methylaminomethyl)-4(thio)uracil; 5-(propynyl)uracil; 5-(trifluoromethyl)uracil; 5-(2-aminopropyl)uracil; 5-(alkyl)-2-(thio)pseudouracil; 5-(alkyl)-2,4-(dithio)pseudouracil; 5-(alkyl)-4-(thio)pseudouracil; 5-(alkyl)pseudouracil; 5-(alkyl)uracil; 5-(alkynyl)uracil; 5-(allylamino)uracil; 5-(cyanoalkyl)uracil; 5-(dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil; 5-(guanidinylalkyl)uracil; 5-(halogenated)uracil; 5-(1,3-diazole) -1-alkyl)uracil; 5-(methoxy)uracil; 5-(methoxycarbonylmethyl)-2-(thio)uracil; 5-(methoxycarbonyl-methyl)uracil; 5-(methyl)2-(thio)uracil; 5-(methyl)2,4-(dithio)uracil; 5-(methyl)4-(thio)uracil; 5-(methyl)-2-(thio)pseudoruracil; 5-(methyl)-2,4-(dithio)pseudoruracil; 5-(methyl)-4-(thio)pseudoruracil; 5-(methyl)pseudoruracil; 5-(methylaminomethyl)-2-(thio)uracil; 5-(methylaminomethyl)-2,4-(dithio)uracil; 5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil; 5-(trifluoromethyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine; 5-iodo-uridine; 5-uracil; 6-(azo)uracil; 6-(azo)uracil; 6-aza-uridine; allylamino-uracil; aza-uridine; deazouracil; N3-(methyl)uracil; pseudoUTP-1-2-acetic acid; pseudouracil; 4-thio-pseudoUTP; 1-carboxymethyl-pseudouridine; 1-methyl-1-deazo- Pseudouridine; 1-Propyno-uridine; 1-Tauratemethyl-1-methyl-uridine; 1-Tauratemethyl-4-thio-uridine; 1-Tauratemethyl-pseudouridine; 2-Methoxy-4-thio-pseudouridine; 2-Thio-1-methyl-1-deazo-pseudouridine; 2-Thio-1-methyl-pseudouridine; 2-Thio-5-aza-uridine; 2-Thio-dihydropseudouridine; 2-Thio-dihydrouridine; 2-Thio-pseudouridine; 4-Methoxy-2-thio-pseudouridine; 4-Methoxy-pseudouridine; 4-Thio-1-methyl-pseudouridine; 4-Thio- Pseudouridine; 5-aza-uridine; dihydropseudouridine; (±)1-(2-hydroxypropyl)pseudouridine TP; (2R)-1-(2-hydroxypropyl)pseudouridine TP; (2S)-1-(2-hydroxypropyl)pseudouridine TP; (E)-5-(2-bromo-vinyl)arsyluridine TP; (E)-5-(2-bromo-vinyl)arsyluridine TP; (Z)-5-(2-bromo-vinyl)arsyluridine TP; (Z)-5-(2-bromo-vinyl)uridine TP; 1-(2,2,2-trifluoroethyl)-pseudoUTP; 1-(2,2,3,3) 1-(2,2-diethoxyethyl) pseudouridine TP; 1-(2,4,6-trimethylbenzyl) pseudouridine TP; 1-(2,4,6-trimethyl-benzyl) pseudouridine TP; 1-(2,4,6-trimethyl-benzyl) pseudouridine TP; 1-(2,4,6-trimethyl-phenyl) pseudouridine TP; 1-(2-amino-2-carboxyethyl) pseudouridine TP; 1-(2-amino-ethyl) pseudouridine TP; 1-(2-hydroxyethyl) pseudouridine TP; 1-(2-methoxyethyl) pseudouridine TP; 1-(3,4-bis-trifluoromethoxybenzyl) pseudouridine TP; 1-(3,4-Dimethoxybenzyl) pseudouridine TP; 1-(3-amino-3-carboxypropyl) pseudoUTP; 1-(3-amino-propyl) pseudoUTP; 1-(3-cyclopropyl-prop-2-ynyl) pseudouridine TP; 1-(4-amino-4-carboxybutyl) pseudoUTP; 1-(4-amino-benzyl) pseudoUTP; 1-(4-amino-butyl) pseudoUTP; 1-(4-amino-phenyl) pseudoUTP; 1-(4-azidobenzyl) pseudouridine TP; 1-(4-bromobenzyl) pseudouridine TP; 1-(4-chlorobenzyl) pseudouridine TP; 1-(4-fluorobenzyl) pseudouridine TP; 1-(4-iodobenzyl) pseudouridine TP; 1-(4-methanesulfonylbenzyl) 1-(4-methoxybenzyl)-pseudouridine TP; 1-(4-methoxy-benzyl)-pseudoUTP; 1-(4-methoxy-phenyl)-pseudoUTP; 1-(4-methylbenzyl)-pseudouridine TP; 1-(4-methyl-benzyl)-pseudoUTP; 1-(4-nitrobenzyl)-pseudouridine TP; 1-(4-nitro-benzyl)-pseudoUTP; 1-(4-nitro-phenyl)-pseudoUTP; 1-(4-thiomethoxybenzyl)-pseudouridine TP; 1-(4-trifluoromethoxybenzyl)-pseudouridine TP; 1-(4-trifluoromethylbenzyl)-pseudouridine TP; 1-(5-amino-pentyl)-pseudoUTP; 1-(6-amino-hexyl)-pseudoUTP; 1,6-Dimethyl-pseudo-UTP; 1-[3-(2-{2-[2-(2-aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudo-uridine TP; 1-13-[2-(2-aminoethoxy)-ethoxy]-propionyl}pseudo-uridine TP; 1-acetyl-pseudo-uridine TP; 1-alkyl-6-(1-propynyl)-pseudo-UTP; 1-alkyl-6-(2-propynyl)-pseudo-UTP; 1-alkyl-6 -Allyl-pseudoUTP; 1-alkyl-6-ethynyl-pseudoUTP; 1-alkyl-6-homoallyl-pseudoUTP; 1-alkyl-6-vinyl-pseudoUTP; 1-allyl-pseudouridine TP; 1-aminomethyl-pseudoUTP; 1-benzoyl-pseudouridine TP; 1-benzyloxymethyl-pseudouridine TP; 1-benzyl-pseudoUTP; 1-biotinyl-PEG2-pseudouridine TP; 1-biotinyl-pseudouridine TP; 1-butyl - Pseudo-UTP; 1-Cyanomethylpseudouridine TP; 1-Cyclobutylmethyl-pseudo-UTP; 1-Cyclobutyl-pseudo-UTP; 1-Cycloheptylmethyl-pseudo-UTP; 1-Cycloheptyl-pseudo-UTP; 1-Cyclohexylmethyl-pseudo-UTP; 1-Cyclohexyl-pseudo-UTP; 1-Cyclooctylmethyl-pseudo-UTP; 1-Cyclooctyl-pseudo-UTP; 1-Cyclopentylmethyl-pseudo-UTP; 1-Cyclopentyl-pseudo-UTP; 1-Cyclopropylmethyl-pseudo-UTP ; 1-Cyclopropyl-pseudo-UTP; 1-Ethyl-pseudo-UTP; 1-Hexyl-pseudo-UTP; 1-Homoallyl-pseudo-uridine TP; 1-Hydroxymethyl-pseudo-uridine TP; 1-Isopropyl-pseudo-UTP; 1-Me-2-Thio-pseudo-UTP; 1-Me-4-Thio-pseudo-UTP; 1-Me-α-Thio-pseudo-UTP; 1-Methanesulfonylmethyl-pseudo-uridine TP; 1-Methoxymethyl-pseudo-uridine TP; 1-Methyl-6-(2,2,2-Trifluoroethyl) pseudo-UTP; 1-Methyl-6-(4-morpholino)-pseudo-UTP; 1-Methyl-6-(4-thiomorpholino)-pseudo-UTP; 1-Methyl-6-(substituted phenyl)-pseudo-UTP; 1-Methyl-6-amino-pseudo-UTP; 1-Methyl-6-azido-pseudo-UTP; 1-Methyl-6-bromo-pseudo-UTP; 1-Methyl-6-butyl-pseudo-UTP; 1-Methyl-6-chloro-pseudo-UTP; 1-Methyl-6-cyano-pseudo-UTP; 1-Methyl-6-dimethylamino-pseudo-UTP; 1- Methyl-6-ethoxy-pseudo-UTP; 1-Methyl-6-ethylcarboxylic acid-pseudo-UTP; 1-Methyl-6-ethyl-pseudo-UTP; 1-Methyl-6-fluoro-pseudo-UTP; 1-Methyl-6-formyl-pseudo-UTP; 1-Methyl-6-hydroxyamino-pseudo-UTP; 1-Methyl-6-hydroxy-pseudo-UTP; 1-Methyl-6-iodo-pseudo-UTP; 1-Methyl-6-isopropyl-pseudo-UTP; 1-Methyl-6-methoxy-pseudo-UTP; 1-Methyl-6-methylamino-pseudo-UTP; 1-Methyl-6-phenyl-pseudo-UTP ; 1-Methyl-6-propyl-pseudo-UTP; 1-Methyl-6-tert-butyl-pseudo-UTP; 1-Methyl-6-trifluoromethoxy-pseudo-UTP; 1-Methyl-6-trifluoromethyl-pseudo-UTP; 1-morpholinomethyl-pseudo-uridine TP; 1-pentyl-pseudo-UTP; 1-phenyl-pseudo-UTP; 1-neovaleryl-pseudo-uridine TP; 1-propynyl-pseudo-uridine TP; 1-propyl-pseudo-UTP; 1-propynyl-pseudo-uridine; 1-p-tolyl-pseudo-UTP; 1-tert-butyl-pseudo-UTP; 1-thiomethoxymethyl-pseudo-uridine TP; 1- Thiomorpholinomethyl pseudouridine TP; 1-trifluoroacetyl pseudouridine TP; 1-trifluoromethyl-pseudoUTP; 1-vinyl pseudouridine TP; 2,2'-anhydride-uridine TP; 2'-bromo-deoxyuridine TP; 2'-F-5-methyl-2'-deoxy-UTP; 2'-OMe-5-Me-UTP; 2'-OMe-pseudoUTP; 2'-a-ethynyluridine TP; 2'-a-trifluoromethyluridine TP; 2'-b-ethynyluridine TP; 2'-b-trifluoromethyluridine TP; 2'-deoxy-2',2'-Difluorouridine TP; 2'-Deoxy-2'-α-mercaptouridine TP; 2'-Deoxy-2'-α-Thiomethoxyuridine TP; 2'-Deoxy-2'-β-aminouridine TP; 2'-Deoxy-2'-β-azidouridine TP; 2'-Deoxy-2'-β-bromouridine TP; 2'-Deoxy-2'-β-chlorouridine TP; 2'-Deoxy-2'-β-fluorouridine TP; 2'-Deoxy-2'-β-iodouridine TP; 2'-Deoxy-2'-β-mercaptouridine TP; 2'-Deoxy-2'-β-thiomethoxyuridine TP; 2-Methoxy-4-thio-uridine ; 2-Methoxyuridine; 2'-O-methyl-5-(1-propynyl)uridine TP; 3-alkyl-pseudo-UTP; 4'-azidouridine TP; 4'-carbocyclic uridine TP; 4'-ethynyluridine TP; 5-(1-propynyl)arsour-uridine TP; 5-(2-furanyl)uridine TP; 5-cyanouridine TP; 5-dimethylaminouridine TP; 5'-homouridine TP; 5-iodo-2'-fluoro-deoxyuridine TP; 5-phenylethynyluridine TP; 5-trideutermethyl-6-deuterated uridine TP; 5-trifluoromethyl-uridine TP; 5-vinylarsour-uridine TP; 6-(2,2,2-Trifluoroethyl)-pseudo-UTP; 6-(4-morpholino)-pseudo-UTP; 6-(4-thiomorpholino)-pseudo-UTP; 6-(substituted phenyl)-pseudo-UTP; 6-amino-pseudo-UTP; 6-azido-pseudo-UTP; 6-bromo-pseudo-UTP; 6-butyl-pseudo-UTP; 6-chloro-pseudo-UTP; 6-cyano-pseudo-UTP; 6-dimethylamino-pseudo-UTP; 6-ethoxy-pseudo-UTP; 6-ethylcarboxylic acid-pseudo-UTP; 6-ethyl-pseudo-UTP; 6-fluoro-pseudo-UTP; 6-Formyl-pseudo-UTP; 6-Hydroxy-pseudo-UTP; 6-Hydroxy-pseudo-UTP; 6-Iodo-pseudo-UTP; 6-Isopropyl-pseudo-UTP; 6-Methoxy-pseudo-UTP; 6-Methyl-pseudo-UTP; 6-Phenyl-pseudo-UTP; 6-Phenyl-pseudo-UTP; 6-Propyl-pseudo-UTP; 6-Tert-Butyl-pseudo-UTP; 6-Trifluoromethoxy-pseudo-UTP; 6-Trifluoromethyl-pseudo-UTP; α-Thio-pseudo-UTP; Pseudouridine 1-(4-Methylbenzenesulfonic acid) TP; Pseudouridine 1-(4-methylbenzoic acid) TP; Pseudouridine TP 1-[3-(2-ethoxy)]propionic acid; Pseudouridine TP 1-[3-12-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy1]propionic acid; Pseudouridine TP 1-[3-{24242-12(2-ethoxy)-ethoxy1-ethoxy]-ethoxy)-ethoxy1}]propionic acid; Pseudouridine TP 1-[3-12-(2-[2-ethoxy]-ethoxy)-ethoxypropionic acid; Pseudouridine TP 143-12-(2-ethoxy)-ethoxypropionic acid; Pseudouridine TP 1-methylphosphate; Pseudouridine TP Diethyl 1-methylphosphate; pseudo-UTP-N1-3-propionic acid; pseudo-UTP-N1-4-butyric acid; pseudo-UTP-N1-5-valeric acid; pseudo-UTP-N1-6-hexanoic acid; pseudo-UTP-N1-7-heptanoic acid; pseudo-UTP-N1-methyl-p-benzoic acid; pseudo-UTP-N1-p-benzoic acid; huaitin; hydroxyhuaitin; isoweiosin; peroxyhuaitin; undermodified hydroxyhuaitin; 4-Demethylwyoside; 2,6-(diamino)purine; 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl; 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 1,3,5-(triaza)-2,6-(dioxa)-naphthalene; 2-(amino)purine; 2,4,5-(trimethyl)phenyl; 2'methyl, 2'amino, 2'azido, 2'fluoro-cytidine; 21-methyl, 2'amino, 2'azido, 2'fluoro-adenine; 2'methyl, 2'amino, 2'azido, 2'fluoro-uridine; 2'-amino-2'-deoxyribose; 2-amino-6-chloropurine; 2-aza-inulinosyl; 2'-azido-2'-deoxyribose; 2'-fluoro-2'-deoxyribose; 2'-fluoro-modified base; 2'-O-methyl-ribose; 2-oxo-7-aminopyridinopyrimidine-3-yl; 2-oxo-pyridinopyrimidine-3-yl; 2-pyridinone; 3-nitropyrrole; 3-(methyl)-7-(propyne) 3-(methyl)isoquinolone; 4-(fluoro)-6-(methyl)benzimidazole; 4-(methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl; 5-nitroindol; 5-substituted pyrimidine; 5-(methyl)isoquinolone; 5-nitroindol; 6-(aza)pyrimidine; 6-(azo)thymine; 6-(methyl)-7-(aza)indolyl; 6-chloro-purine; 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl; 7-(aminoalkylhydroxy)-1-(aza)-2- (Thio)-3-(aza)-phenoxazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(aza)indolyl; 7-(guanidinylalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-(guanidinylalkylhydroxy)- 1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl; 7-(guanidinylalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazine-1-yl; 7-(guanidinylalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazine-1-yl; 7-(guanidinylalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazine-1-yl; 7-(propynyl)isoquinolone; 7-(propynyl)isoquinolone, propynyl-7-(aza)indolyl; 7-deadenyl-inulinosyl; 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl; 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazine-1-yl; 9-(methyl)-imidazolium-pyridyl; aminoindolyl; anthracene; bis-in-situ (aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; bis-in-situ substituted 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; difluorotolyl; hypoxanthine; imidazolium-pyridyl; inulinosyl; isoquinolone; isoguanosine; N2-substituted purine; N6-methyl-2-amino-purine; N6-substituted purine; N-alkylated derivatives; naphthyl; nitrobenzimidazolyl; nitroimidazolyl; nitroindazolyl; nitropyrazolyl; nubularine; O6-substituted purine; O-alkylated Derivatives; in situ -(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; in situ substituted 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; oxo-meta-mycin TP; para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; para-substituted 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; pentaphenyl; phenanthrene; phenyl; propynyl-7-(aza)indolyl; pyrene; pyridopyrimidin-3-yl; pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-one-3-yl; pyrrolopyrimidyl; pyrrolopyrazinyl; stilbeneyl; substituted 1,2,4-Triazole; Tetraphenyl; Tuberculin; Xanthine; Xanthine-5'-TP; 2-Thio-Zebraline; 5-aza-2-Thio-Zebraline; 7-Denitro-2-Amino-Purine; Pyridine-4-ketoribonucleoside; 2-Amino-ribonucleoside-TP; Metamycin A TP; Metamycin B TP; Pyrrolidone TP; 2'-OH-Adenosine TP; 2'-OH-Adenosine TP; 2'-OH-Adenosine TP; 2'-OH-Adenosine TP; 2'-OH-Adenosine TP; 2'-Adenosine TP; 5-(2-Methoxycarbonylvinyl)uridine TP; and N6-(19-Amino-pentazododecyl)adenosine TP. In some embodiments, the alternative nucleobase is selected from the group consisting of: pseudouridine (ψ), 2-thiouridine (s2U), 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deazo-pseuuridine, 2-thio-1-methyl-pseuuridine, 2-thio-5-aza-uridine, 2-thio-dihydropseuuridine, 2-thio-dihydrouridine, 2-thio-pseuuridine, 4-methyl 2-Thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, 21-O-methyluridine, 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C) ), α-thioguanosine, α-thio-adenosine, 5-cyanouraside, 4'-thiouridine, 7-deazo-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), and 2,6-diaminopurine (I), 1-methyl-inosine (m1I), wyoside (imG), methyl wyoside (mimG), 7-deazo-guanosine, 7- Cyano-7-deazo-guanosine (preQO), 7-aminomethyl-7-deazo-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 2,8-dimethyl adenosine, 2-geranylthiouridine, 2-lysicrysine, 2-selenouridine, 3-(3-amino-3-carboxypropyl)-5,6-Dihydrouridine, 3-(3-amino-3-carboxypropyl)pseudouridine, 3-methylpseudouridine, 5-(carboxyhydroxymethyl)-2'-O-methyluridine methyl ester, 5-aminomethyl-2-geranylthiouridine, 5-aminomethyluridine, 5-carboxymethyl-2-thiouridine, 5-carboxymethyl-2-thiouridine, 5-carboxymethyl-2-geranylthiouridine, 5-carboxymethyl-2-geranylthiouridine, 5-carboxymethyl-2-seranylthiouridine, 5-cyanomethyluridine, 5-hydroxycytidine, 5-methylaminomethyl-2-geranylthiouridine, 7-aminocarboxypropyl-dehydrouridine Methylwyoside, 7-aminocarboxypropylwyoside, 7-aminocarboxypropylwyoside methyl ester, 8-methyladenosine, N4,N4-dimethylcytidine, N6-formyladenosine, N6-hydroxymethyladenosine, agmatidine, cyclic N6-threonylcarbamoyladenosine, glutamyl-pigmentoside, under-methylated hydroxywyoside, N4,N4,2'-O-trimethylcytidine, geranylated 5-methylaminomethyl-2-thiouridine, geranylated 5-carboxymethylaminomethyl-2-thiouridine, Qbase, preQObase, preQ1base, and combinations of two or more thereof. In some embodiments, the alternative nucleobase is selected from the group consisting of: pseudouridine, 1-methyl-pseudouridine, 1-ethyl-pseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0052] In some embodiments, the nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having modified uracil include pseudouridine (ψ), pyridine-4-ketoribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m3U), and 5-methoxy-uridine (mo5U). 5-Oxyacetic acid uridine (cmo5U), 5-Oxyacetic acid methyl uridine (mcmo5U), 5-Carboxymethyl-uridine (cm5U), 1-Carboxymethyl-pseudouridine, 5-Carboxyhydroxymethyl-uridine (chm5U), 5-Carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-Methoxycarbonylmethyl-uridine (mcm5U), 5-Methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-Aminomethyl-2-thio-uridine (nm5s2U), 5-Methylaminomethyl-uridine (mnm5U), 5-Methylaminomethyl- 2-Thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurylmethyl-uridine (τm5U), 1-taurylmethyl-pseudouridine, 5-taurylmethyl-2-thio-uridine (τm5s2U), 1-taurylmethyl-4- Thio-pseudouridine, 5-methyl-uridine (m5U, i.e., deoxythymidine with nucleobase), 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deazo-pseudouridine, 2-thio-1-methyl-1-deazo-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-Dihydrouridine, 5-Methyl-Dihydrouridine (m5D), 2-Thio-Dihydrouridine, 2-Thio-Dihydropseudouridine, 2-Methoxy-uridine, 2-Methoxy-4-Thio-uridine, 4-Methoxy-pseudouridine, 4-Methoxy-2-Thio-pseudouridine, N1-Methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-Methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3) kv), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'- O-methyluridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm5Um), 3,2'-O-dimethyluridine (m3Um), and 5-(isopentenylaminomethyl)-2'-O-methyluridine (inm5Um), 1-thiouridine, deoxythymidine, 2'-F-arasugar-uridine, 2'-F-uridine, 2'-OH-arasugar-uridine, 5-(2-carbonmethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)]uridine.

[0053] In some embodiments, the nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having modified cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudocytidine, 3-methylcytidine (m3C), N4-acetylcytidine (ac4C), 5-formylcytidine (f5C), N4-methylcytidine (m4C), 5-methylcytidine (m5C), 5-halocytidine (e.g., 5-iodocytidine), 5-hydroxymethylcytidine (hm5C), 1-methyl-pseudocytidine, pyrrolo-cytidine, pyrrolo-pseudocytidine, 2-thiocytidine (s2C), 2-thio-5-methylcytidine, 4-thio-pseudocytidine, 4-thio-1-methyl-pseudocytidine, 4-thio-1-methyl-1-deazo-pseudocytidine, 1-methyl-1-deazo-pseudocytidine, zebralin, 5-aza-zebralin Larin, 5-methyl-zabralin, 5-aza-2-thio-zabralin, 2-thio-zabralin, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudo-cytidine, 4-methoxy-1-methyl-pseudo-cytidine, lysicidine (k2C), α-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl-cytidine (m) 5Cm), N4-acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (f5Cm), N4,N4,2'-trimethyl-cytidine (m42Cm), 1-thio-cytidine, 2'-F-arasacchar-cytidine, 2'-F-cytidine and 2'-OH-arasacchar-cytidine.

[0054] In some embodiments, the nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having modified adenine include 2-amino-purine, 2,6-diamino-purine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deadenine, 7-deadenine-8-aza-adenosine, 7-deadenine-2-amino-purine, 7-deadenine-8-aza-2-amino-purine, 7-deadenine-2,6-diamino-purine, 7-deadenine-8-aza-2,6- Diaminopurine, 1-methyl-adenosine (m1A), 2-methyl-adenosine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-glycylcarbamoyl-adenosine (g6A), N6-threonylamino N6-methyl-N6-threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxyn-valine-carbamoyl-adenosine (hn6A), 2-methylthio-N6-hydroxyn-valine-carbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-adenosine, 2-methylthio-adenosine, 2-methoxy-adenosine α-Thio-adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m62Am), 1,2'-O-dimethyl-adenosine (miAm), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-arabinose-adenosine, 2'-F-adenosine, 2'-OH-arabinose-adenosine, and N6-(19-amino-pentaenodecyl)-adenosine.

[0055] In some embodiments, the nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having modified guanine include inosine (I), 1-methyl-inosine (miI), wyoside (imG), methyl wyoside (mimG), 4-demethyl-wyoside (imG-14), isowyoside (imG2), weitingoside (yW), peroxyweitingoside (o2yW), hydroxyweitingoside (OhyW), undermodified hydroxyweitingoside (OhyW*), 7-deazo-guanosine, piracetamine (Q), epoxypiracetamine (oQ), and galactosylpiracetamine. (galQ), mannosyl-guanosine (manQ), 7-cyano-7-deazo-guanosine (preQo), 7-aminomethyl-7-deazo-guanosine (preQi), archapurin (G+), 7-deazo-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deazo-guanosine, 6-thio-7-deazo-8-aza-guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine N2-Methyl-guanosine (m2G), N2,N2-dimethyl-guanosine (m22G), N2,7-dimethyl-guanosine (m2'7G), N2,N2,7-dimethyl-guanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m22Gm), 1-methyl-2'-O-methyl-guanosine (miGm), N2,7-dimethyl-2'-O-methyl-guanosine (m2'7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine, 2'-O-ribosylguanosine (phosphate) (Gr(p)), 1-thio-guanosine, O6-methyl-guanosine, 2'-F-arose-guanosine and 2'-F-guanosine.

[0056] Non-limiting further examples of suitable alternative nucleosides, nucleosides, and nucleotides for the oligonucleotides disclosed herein include those disclosed in WO 2020 / 154342, WO 2020 / 154344, and WO 2020 / 154343, each of which is incorporated herein by reference in its entirety.

[0057] In some embodiments, oligonucleotides are uniformly modified for specific modifications (e.g., completely modified, modified throughout the entire sequence). For example, oligonucleotides can be uniformly modified with 1-methyl-pseuuridine, meaning that all uridine residues in the oligonucleotide sequence are replaced by 1-methyl-pseuuridine. Similarly, oligonucleotides can be uniformly modified for any type of nucleobase present in the sequence by substituting it with an alternative nucleobase (as described above).

[0058] The oligonucleotides of this disclosure can be partially or completely modified along the entire length of the molecule. For example, one or more or all or a given type of nucleobase (e.g., purine or pyrimidine, or any one or more or all of A, G, U, T, or C) can be uniformly modified in the oligonucleotides of this disclosure or in a given predetermined sequence region (e.g., in mRNA with or without a polyA tail). In some embodiments, all nucleotides X in the oligonucleotides of this disclosure (or in a given sequence region thereof) are modified nucleotides, wherein X can be any or a combination of nucleotides A, G, U, C, A+G, A+U, A+C, G-HU, G-FC, U+C, A+G-HU, A+G-FC, G-HU+C, or A+G+C.

[0059] In some embodiments, the oligonucleotide contains 1% to 100% of modified nucleobases (relative to the total nucleotide content, or relative to one or more types of nucleotides, i.e., any one or more of A, G, U, T and / or C) or any intermediate percentage (e.g., 1% to 5%, 1% to 10%, 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 10% to 10% to 10% to 100%). 95%, 10% to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100% of modified nucleobases. It should be understood that any remaining percentages are explained by the presence of standard nucleobases A, G, T, U, and / or C.

[0060] Oligonucleotides may contain at least 0% and at most 100% of modified nucleosides, or any intermediate percentage, such as at least 1% modified nucleosides, at least 5% modified nucleosides, at least 10% modified nucleosides, at least 25% modified nucleosides, at least 50% modified nucleosides, at least 80% modified nucleosides, or at least 90% modified nucleosides. For example, oligonucleotides may contain modified pyrimidines, such as modified uracil or cytosine. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the uracil in the oligonucleotide is replaced by modified uracil (e.g., 5-substituted uracil). The modified uracil may be replaced by a compound having a single unique structure, or by a plurality of compounds having different structures (e.g., two, three, four, or more unique structures). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the cytosine in the oligonucleotide is replaced by a modified cytosine (e.g., 5-substituted cytosine). The modified cytosine may be replaced by a compound having a single unique structure or by a plurality of compounds having different structures (e.g., two, three, four, or more unique structures).

[0061] In some embodiments, the oligonucleotides described herein comprise at least one modified nucleotide, such as at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen modified nucleotides. In other embodiments, the oligonucleotides comprise one to ten modified nucleotides, such as two to nine modified nucleotides, three to eight modified nucleotides, four to seven modified nucleotides, or six or seven modified nucleotides. In some embodiments, the oligonucleotides comprise a combination of at least two (e.g., two, three, four, or more) of the aforementioned modified nucleotides.

[0062] Sugar portion The oligonucleotides described herein may also include one or more sugar moieties, including one or more standard sugar moieties and / or alternative sugar moieties (e.g., non-natural sugar moieties, sugar analogs, or natural sugar moieties containing modifications or substitutions).

[0063] The standard sugar moiety includes ribose and deoxyribose. In some embodiments, the oligonucleotides disclosed herein comprise ribose or a modified ribose moiety. In some embodiments, the sugar moiety in the oligonucleotide may be ribose optionally modified with 2'-O-methyl, 2'-O-MOE, 2'-F, 2'-amino, 2'-O-propyl, 2'-aminopropyl, or 2'-OH. In some embodiments, the oligonucleotides disclosed herein comprise deoxyribose or a modified deoxyribose moiety. Alternative sugar moieties include other non-natural and natural sugars described herein, as well as sugar analogs, including those comprising modified or substituted sugar moieties.

[0064] The alternative sugar moiety also includes a substituted sugar moiety. The oligonucleotides disclosed herein may comprise a substituted furanose moiety having one or more of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-ynyl; or O-alkyl-O-alkyl; wherein the alkyl, alkenyl, and ynyl groups may be substituted or unsubstituted C1 to C2 groups. 10 Alkyl or C2 to C 10 Alkenyl and ynyl groups. Exemplary suitable modifications include -O[(CH2)] n O] m CH3, -O(CH2) n OCH3, -O(CH2) n -NH2、-O(CH2) n CH3, -O(CH2) n -ONH2 and -O(CH2) n -ON[(CH2) n CH3]2, wherein n and m are 1 to about 10. In other embodiments, the oligonucleotides disclosed herein may comprise a substituted furanose moiety having one or more of the following at the 2' position: C1 to C 10 Alkyl groups, substituted C1 to C1 10Alkyl groups (e.g., substituted with one or more of OH, halogen, amino, alkoxy, or thiol, or combinations thereof), alkylaryl, aralkyl, O-alkylaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocyclic alkyl, heterocyclic alkylaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intercalator, group for improving the pharmacokinetic properties of oligonucleotides, or group for improving the pharmacodynamic properties of oligonucleotides, and other substituents having similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-O-MOE), i.e., alkoxy-alkoxy. 2'-O-MOE nucleoside endows oligonucleotides with several beneficial properties, including but not limited to increased nuclease resistance, improved pharmacokinetic properties, reduced nonspecific protein binding, reduced toxicity, reduced immunostimulatory properties, and enhanced target affinity compared to unmodified oligonucleotides.

[0065] Another exemplary alternative sugar moiety comprises a 2'-dimethylaminoethoxy group, i.e., -O(CH2)2ON(CH3)2, also known as 2'-DMAOE, as described in the examples below herein, and a 2'-dimethylaminoethoxyethoxy group (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-(CH2)2-O-(CH2)2-N(CH3)2. Further exemplary alternative sugar moiety includes: 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides (both the R and S isomers of these three families); 2'-alkoxyalkyl groups; and 2'-NMA (N-methylacetamide).

[0066] Other alternative forms include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the sugar moiety of the oligonucleotide, specifically at the 3' position of the sugar on the 3' terminal nucleotide or the 5' position of the sugar on the 2'-5' linked oligonucleotide. The oligonucleotide can also have sugar analogs or simulants, such as replacing the cyclobutyl moiety of the pentofuranosyl sugar. Representative U.S. patents teaching the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957, 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, 5,514,785, 5,519,134, and 5,567. Nos. 811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, some of which are commonly owned with this application. The entire contents of each of the foregoing items are hereby incorporated herein by reference.

[0067] The oligonucleotides disclosed herein may include one or more bicyclic sugar moieties. A “bicyclic sugar” is a furanyl ring modified by a two-atom bridging. A “bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety comprising a bridge connecting the two carbon atoms of the sugar ring, thereby forming a bicyclic system. In some embodiments, the bridge connects the 4' and 2' carbons of the sugar ring. Therefore, in some embodiments, the oligonucleotides disclosed herein may include one or more locked nucleosides. A locked nucleoside is a nucleoside having a modified ribose moiety comprising an additional bridge connecting the 2' and 4' carbons. In other words, a locked nucleoside is a nucleoside comprising a bicyclic sugar moiety comprising 4'-CH2-O-2'. This structure effectively “locks” the ribose in a 3'-inner conformation. Adding locked nucleosides to oligonucleotides has been shown to increase oligonucleotide stability in serum and reduce off-target effects (Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides used in the oligonucleotides of this disclosure include, but are not limited to, nucleosides comprising a bridge between the 4' and 2' ribosyl ring atoms. In some embodiments, the oligonucleotides of this disclosure comprise one or more bicyclic nucleosides comprising a 4' to 2' bridge. Examples of such 4' to 2'-bridged bicyclic nucleotides include, but are not limited to, 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (also referred to as "bound ethyl" or "cEt") and 4'-CH(CH2OCH3)-O-2' (and their analogues; see, for example, U.S. Patent No. 7,399,845); 4'- C(CH3)(CH3)-O-2' (and its analogues; see, for example, U.S. Patent No. 8,278,283); 4'-CH2-N(OCH3)-2' (and its analogues; see, for example, U.S. Patent No. 8,278,425); 4'-CH2-ON(CH3)2-2' (see, for example, U.S. Patent Publication No. 2004 / 0171570); 4'-CH2-N(R)-O-2', wherein R is H, Cl-C 12Alkyl groups or protecting groups (see, for example, U.S. Patent No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (see, for example, Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and the like; see, for example, U.S. Patent No. 8,278,426). The entire contents of each of the foregoing items are hereby incorporated by reference. In some embodiments, at least one sugar moiety of the alternative sugar moiety is a BNA (e.g., LNA), such as at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight sugar moiety of the alternative sugar moiety are BNAs. In still other embodiments, all alternative sugar moiety are BNAs.

[0068] Other representative U.S. patents and U.S. patent publications teaching the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; and others. The entire contents of each of the aforementioned U.S. patents, namely U.S. Patent No. 2008 / 0039618 and U.S. Patent No. 2009 / 0012281, are hereby incorporated herein by reference.

[0069] Any of the aforementioned bicyclic nucleotides having one or more stereochemical sugar configurations can be prepared, including, for example, α-L-ribofuranosyl and β-D-ribofuranosyl (see WO 99 / 14226).

[0070] The oligonucleotides disclosed herein can also be modified to include one or more restricted ethyl nucleotides. As used herein, "restricted ethyl nucleotide" or "cEt" refers to a locked nucleic acid comprising a bicyclic sugar moiety containing a 4'-CH(CH3)-O-2' bridge. In one embodiment, the restricted ethyl nucleotide is in the S conformation referred to herein as "S-cEt".

[0071] The oligonucleotides disclosed herein may also include one or more "conformation-restricted nucleotides" ("CRNs"). A CRN is a nucleotide analog with a linker having a linker connecting the C2' and C4' carbons of the ribose or the C3 and -C5' carbons of the ribose. A CRN locks the ribose ring into a stable conformation and increases hybridization affinity to mRNA. The linker is long enough to place oxygen in the optimal position for achieving stability and affinity, resulting in fewer ribose ring wrinkles.

[0072] Representative publications teaching the preparation of the CRN mentioned above include, but are not limited to, U.S. Patent Publication No. 2013 / 0190383; and PCT Publication WO 2013 / 036868, the entire contents of each of which are hereby incorporated herein by reference.

[0073] In some embodiments, the oligonucleotides disclosed herein comprise one or more monomers that are UNA (unlocked nucleic acid) nucleotides. An UNA is an unlocked acyclic nucleic acid in which any bonds of the sugar have been removed, resulting in unlocked “sugar” residues. In one instance, UNA also encompasses monomers in which the bond between C1' and C4' has been removed (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons). In another instance, the C2'-C3' bond of the sugar (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Molecular Biology Systems, 2009, 10, 1039, which are hereby incorporated by reference).

[0074] Representative U.S. publications teaching the preparation of UNA include, but are not limited to, U.S. Patent No. 8,314,227; and U.S. Patent Publication Nos. 2013 / 0096289, 2013 / 0011922, and 2011 / 0313020, the entire contents of each of which are hereby incorporated herein by reference.

[0075] The sugar moiety can also be modified with a cyclopropane ring to produce tricyclic deoxyribonucleic acid (tricyclic DNA). The sugar moiety can be, for example, 1,5'-anhydrohexitol, threose for the production of threononucleotides (TNA), or arabinose for the production of arabinonucleotides.

[0076] The sugar moiety can also be non-carbohydrate, such as cyclohexene used to produce cyclohexene nucleic acid (CeNA) or ethylene glycol used to produce ethylene glycol nucleic acid (GNA). Potential stabilizing modifications to the ends of nucleotide molecules can include N-(acetamidocahexanoyl)-4-hydroxyproline (Hyp-C6-NHAc), N-(hexanoyl-4-hydroxyproline) (Hyp-C6), N-(acetyl-4-hydroxyproline) (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocahexanoyl)-4-hydroxyproline (Hyp-C6-amino), 2-docosyl-uridine-3''-phosphate, reverse base dT (idT), etc. The disclosure of this modification can be found in PCT Publication WO 2011 / 005861.

[0077] The exemplary oligonucleotides of this disclosure include any combination of standard and / or alternative sugar moieties located at various positions, and may also include DNA or RNA oligonucleotides. Incorporation of modified sugar moieties into the oligonucleotides of this disclosure can enhance the affinity of the oligonucleotides for target nucleic acids.

[0078] In some embodiments, for each position, the sugar moiety may be independently selected from ribose and deoxyribose, and / or may contain modifications such as, but not limited to, 2'-O-alkyl, 2'-O-methoxyethyl, 2'-O-allyl, 2'-O-alkylamine, 2'-fluororibose, 2'-deoxyribose, and locked nucleic acid (LNA). In some embodiments, the oligonucleotide comprises one or more modified sugar moieties, for example, 2'-modified modified sugar moieties. In some embodiments, the oligonucleotide described herein comprises one or more 2'-modified sugar moieties independently selected from the group consisting of: 2'-O-alkyl-RNA (e.g., 2'-O-methyl-RNA), 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA, 2'-amino-DNA, 2'-fluoro-DNA, ANA, 2'-fluoro-ANA, and bridging nucleic acid (“BNA”, e.g., locked nucleic acid “LNA”) moieties. In some embodiments, one or more modified sugar moieties are BNAs. In some cases, at least one sugar moiety is ribose or modified ribose. In some cases, at least one sugar moiety is ribose. In some cases, at least one sugar moiety is modified ribose. In some cases, at least one sugar moiety is independently 2'-methoxy-ribose, 2'-MOE-ribose, 5'-methyl-2'-deoxyribose, 2'-deoxy-2'-fluororibose, 2'-fluoro-arabinose, 2-methoxy-arabinose, 2'-deoxyribose, locked nucleic acid (LNA), or deoxyhexose. In some cases, each sugar moiety is independently 2'-methoxy-ribose, 2'-MOE-ribose, 5'-methyl-2'-deoxyribose, 2'-deoxy-2'-fluororibose, 2'-fluoro-arabinose, 2-methoxy-arabinose, 2'-deoxyribose, locked nucleic acid (LNA), or deoxyhexose.

[0079] In some embodiments, the oligonucleotides described herein include phosphoryldiamine morpholine oligomers (PMOs) in which the deoxyribose moiety is replaced by a morpholine ring and the charged phosphodiester subunit bonds are replaced by uncharged phosphodiamid bonds, as described in Summerton et al., *Antisense Nucleic Acid Drug Dev.* 1997, 7:63-70. In some instances, the oligonucleotides include a modified sugar moiety comprising bicyclic sugar derivatives (LNA, ENA, CLNA, CENA, AENA, etc.), acyclic sugar analogs (UNA, PNA, etc.), or analogs containing sugars other than ribose or deoxyribose (e.g., pyranose rings (ANA, HNA)).

[0080] In some embodiments, the oligonucleotide comprises at least one modified sugar moiety, such as at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, or at least sixteen modified sugar moietyes. In other embodiments, the oligonucleotide comprises one to ten modified sugar moietyes, such as two to nine modified sugar moietyes, such as three to eight modified sugar moietyes, such as four to seven modified sugar moietyes, such as six or seven modified sugar moietyes. In some embodiments, the oligonucleotide contains 1% to 100% of a modified sugar moiety (relative to the total sugar moiety content, or relative to one or more types of sugar moiety, such as ribose, deoxyribose, or derivatives thereof) or any intermediate percentage (e.g., 1% to 5%, 1% to 10%, 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 90%). Modified sugar moieties (95%, 10%, 10%, 100%, 20%, 25%, 20%, 50%, 20%, 60%, 20%, 70%, 20%, 80%, 20%, 90%, 20%, 95%, 20%, 100%, 50%, 60%, 50%, 70%, 80%, 90%, 70%, 95%, 70%, 100%, 80%, 90%, 95%, 80%, 100%, 90%, 95%, 90%, 100%, and 95% to 100%). It should be understood that any remaining sugar moieties in the oligonucleotide are standard sugar moieties, i.e., ribose or deoxyribose moieties.

[0081] In some embodiments, the sugar portion has the structure of any one of formulas IA-VA: Where N 1 It is a hydrogen or nucleobase; R 1A It is a hydroxyl, halogen, or C1-C6 alkoxy group; R 2A It can be hydrogen, hydroxyl, halogen, or C1-C6 alkoxy; R 3A It can be hydrogen, hydroxyl, halogen, or C1-C6 alkoxy; R 4A It is hydrogen, hydroxyl, halogen, or C1-C6 alkoxy; and R5A It can be hydrogen, hydroxyl, halogen, or C1-C6 alkoxy.

[0082] In some embodiments, the sugar portion has the structure of any of formulas IB-VIB: Where N 1 It is a hydrogen or nucleobase; R 12 It is hydrogen, hydroxyl, fluorine, halogen, C1-C6 alkyl, C1-C6 heteroalkyl, or C1-C6 alkoxy; and R 13 It is hydrogen or C1-C6 alkyl.

[0083] In some embodiments, the sugar portion has the structure of any of formulas IC-IVC: Where N 1 It is a hydrogen or nucleobase; R 6C It can be hydrogen, hydroxyl, or halogen; R 7C It can be hydrogen, hydroxyl, halogen, or C1-C6 alkoxy; R 8C It is hydrogen or halogen; R 9C It is hydrogen or hydroxyl, halogen or C1-C6 alkoxy; R 10C It is hydrogen or halogen; and R 11C It is hydrogen or hydroxyl, halogen or C1-C6 alkoxy.

[0084] Non-limiting further examples of suitable sugars for the oligonucleotides disclosed herein include those disclosed in WO 2020 / 154342, WO 2020 / 154344 and WO 2020 / 154343, each of which is incorporated herein by reference in its entirety.

[0085] Oligonucleotides conjugated with other parts In some embodiments, the oligonucleotides disclosed herein comprise one or more additional chemical moieties (or ligands). Various additional chemical moieties, such as targeting moieties, carbohydrate moieties, lipid moieties, etc., are known in the art and can be used according to this disclosure to modulate the properties and / or activity of the provided oligonucleotides, such as stability, half-life, activity, delivery, pharmacodynamic properties, and pharmacokinetic properties. In some embodiments, certain additional chemical moieties facilitate the delivery of the oligonucleotide to desired cells, tissues, and / or organs, including but not limited to cells of the central nervous system. In some embodiments, certain additional chemical moieties facilitate the internalization of the oligonucleotide. In some embodiments, certain additional chemical moieties increase the stability of the oligonucleotide. In some embodiments, this disclosure provides techniques for incorporating various additional chemical moieties into the oligonucleotide. In some embodiments, the additional chemical moieties are or comprise small molecule moieties. In some embodiments, the small molecule is a ligand for a protein (e.g., a receptor). In some embodiments, the small molecule binds to a peptide. In some embodiments, the small molecule is an inhibitor of the peptide. In some embodiments, the additional chemical moieties are or comprise peptide moieties (e.g., antibodies). In some embodiments, the additional chemical portion is or comprises a nucleic acid portion, such as a nucleic acid portion that forms a duplex or other secondary structure with the original oligonucleotide chain (before conjugation) or a portion thereof. In some embodiments, the nucleic acid is or comprises an oligonucleotide targeting the same or different targets and can exert its activity through the same or different mechanisms. In some embodiments, the nucleic acid is or comprises an RNAi agent. In some embodiments, the nucleic acid is or comprises a miRNA agent. In some embodiments, the nucleic acid is or comprises an RNase H-dependent agent. In some embodiments, the nucleic acid is or comprises gRNA. In some embodiments, the nucleic acid is or comprises an aptamer. In some embodiments, the additional chemical portion is or comprises a carbohydrate portion as described herein. Many useful agents, such as small molecules, peptides, carbohydrates, nucleic acid agents, etc., can be conjugated to the oligonucleotides herein according to this disclosure. In some embodiments, the provided oligonucleotide may comprise two or more additional chemical portions, wherein said additional chemical portions may be the same or different.

[0086] The oligonucleotides described herein may be chemically linked to one or more ligands, portions, or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotides.Such fractions include, but are not limited to, lipid fractions, such as cholesterol fractions (Letsinger et al., (1989) *Proceedings of the National Academy of Sciences of the United States of America*, 86:6553-6556); bile acids (Manoharan et al., (1994) *Biorg. Med. Chem. Let.*, 4:1053-1060); thioethers, for example, beryl-S-triphenylmethanethiol (Manoharan et al., (1992) *Annals of the New York Academy of Sciences*, 660:306-309; Manoharan et al., (1993) *Biorg. Med. Chem. Let.*, 3:2765-2770); thiocholesterol (Oberhauser et al., (1992) *Nucleic Acid Research*, ...). 20:533-538); aliphatic chains, for example, dodecanediol or undecyl residues (Saison-Behmoaras et al., (1991) EMBO J, 10:1111-1118; Kabanov et al., (1990) FEBS Letters, 259:327-330; Svinarchuk et al., (1993) Biochimie, 75:49-54); phospholipids, for example, di-hexadecyl-rac-glycerol or 1,2-di-O-hexadecyl-rac-glycerol-3-phosphonate triethyl-ammonium (Manoharan et al., (1995) Tetrahedron Letters, 36:3651-3654; Shea et al., (1990) Nucleic Acid Research, 18:3777-3783; polyamine or polyethylene glycol chain (Manoharan et al., (1995) Nucleosides & Nucleotides, 14:969-973) or adamantane acetic acid (Manoharan et al., (1995) Tetrahedral Letters, 36:3651-3654); palmityl moiety (Mishra et al., (1995) Biochim. Biophys. Acta, 1264:229-237); or octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., (1996) J. Pharmacol. Exp. Ther., 277:923-937).

[0087] In some embodiments, ligands alter the distribution, targeting, or lifetime of the oligonucleotide agent to which they are incorporated. In some embodiments, for example, ligands provide enhanced affinity for selected targets (e.g., molecules, cells, or cell types) and compartments (e.g., cellular or organ compartments, tissues, organs, or body regions) compared to species in which such ligands are absent.

[0088] Ligands can include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine, or hyaluronic acid); or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, for example, synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolic acid) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymers, or polyphosphonazines. Examples of polyamines include: polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, pseudopeptide-polyamines, dendritic polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helical peptides.

[0089] The ligand may also include a targeting moiety, such as a cell or tissue target, like a lectin, glycoprotein, lipid, or protein, such as an antibody that binds to a specific cell type (e.g., kidney cells). The targeting moiety can be thyroid-stimulating hormone, melanocyte-stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrates, polylactose, polygalactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polymannose, polyfucose, glycosylated polyamino acids, polygalactose, transferrin, bisphosphonates, polyglutamate, polyaspartate, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, vitamin A, biotin, or RGD peptide or RGD peptide mimics.

[0090] Other examples of ligands include dyes, intercalating agents (e.g., acridine), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), and lipophilic molecules (e.g., cholesterol, bile acids, adamantane acetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O-hexadecylglycerol, geranyloxyhexyl, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecanyl, palmitic acid, myristic acid, O3-(oleoyl) chalcedony). Acids, O3-(oleoyl)cholenic acid, dimethoxytriphenylmethyl or phenoxazine), peptide conjugates (e.g., tentacles, Tat peptides), alkylating agents, phosphates, amino groups, thiol groups, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption promoters (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, diimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraaza macrocyclic compounds), dinitrophenyl, HRP or AP.

[0091] In some embodiments, an additional chemical portion is a targeting portion. In some embodiments, the oligonucleotides disclosed herein further comprise one or more targeting portions. In some embodiments, one or more targeting portions comprise lipids, sterols, carbohydrates, vitamins, and / or peptides. In some embodiments, one or more targeting portions comprise thyroid-stimulating hormone, melanocyte-stimulating hormone, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polyvalent lactose, polyvalent galactose, ASGPR-binding portion, N-acetyl-galactosamine (GalNAc) portion, N-acetyl-glucosamine polymannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, polyglutamate, polyaspartate, cholesterol, bile acids, folic acid, vitamin B12, vitamin A, biotin, RGD peptide, or RGD peptide mimic. In some embodiments, the ASGPR-binding portion is N-acetyl-galactosamine (GalNAc) or N-acetyl-glucosamine polymannose. In some embodiments, one or more targeting portions comprise N-acetyl-galactosamine (GalNAc) portion. In some embodiments, one or more targeting portions are located at the 5' end, the 3' end, or both of the oligonucleotide. In some embodiments, one or more targeting portions are located at both the 5' end and the 3' end of the oligonucleotide. In some embodiments, one or more targeting portions are located at the 5' end of the oligonucleotide. In some embodiments, one or more targeting portions are located at the 3' end of the oligonucleotide. In some embodiments, one or more targeting portions are linked to the oligonucleotide via a linker portion. In some embodiments, the linker portion comprises an alkylene oxide, polyalkylene oxide, and / or peptide portion. In some embodiments, an additional chemical portion is or comprises a carbohydrate portion. In some embodiments, an additional chemical portion is or comprises a lipid portion. In some embodiments, an additional chemical portion is or comprises a ligand portion, such as a ligand portion of a cell receptor (e.g., a σ receptor) or a desialyl glycoprotein receptor. In some embodiments, the ligand portion is or comprises an anisamide portion, which may be a ligand portion of a σ receptor. In some embodiments, the ligand portion is or comprises a GalNAc portion, which may be a ligand portion of a desialyl glycoprotein receptor. In some embodiments, the additional chemical portion facilitates delivery to the liver. In some cases, the oligonucleotide comprises a GalNAc portion. In some cases, the GalNAc moiety is located at the 5' end of the oligonucleotide. In other cases, the GalNAc moiety is located at the 3' end of the oligonucleotide.

[0092] Ligands can be proteins (e.g., glycoproteins), peptides (e.g., molecules with specific affinity for the coligand), or antibodies (e.g., antibodies that bind to specific cell types, such as hepatocytes). Ligands can also include hormones and hormone receptors. Ligands can also include non-peptide substances such as lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, or polyvalent fucose.

[0093] Ligands can be substances that can increase the uptake of oligonucleotide agents into cells, for example, by disrupting the cell's cytoskeleton, such as by disrupting the cell's microtubules, microfilaments, and / or intermediate filaments. Drugs can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoseverin.

[0094] In some embodiments, ligands linked to oligonucleotides as described herein act as pharmacokinetic modulators (PK modulators). PK modulators include lipophiles, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEGs, vitamins, etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, bile acids, lithocholic acids, dialkyl glycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, etc. Oligonucleotides comprising numerous thiophosphate bonds are also known to bind to serum proteins; therefore, short oligonucleotides, such as those of about 5, 10, 15, or 20 bases, including thiophosphate bonds in their backbone as ligands (e.g., as PK-regulating ligands), are also suitable as the oligonucleotides disclosed herein. Additionally, aptamers that bind to serum components (e.g., serum proteins) are also suitable as PK-regulating ligands in the embodiments described herein.

[0095] In some embodiments, the additional chemical portion conjugated to the oligonucleotide can target the oligonucleotide to cells in the central nervous system.

[0096] In some embodiments, the additional chemical portion comprises or is a cell receptor ligand. In some embodiments, the additional chemical portion comprises or is a protein binder, such as a protein binder that binds to cell surface proteins. Such portions, etc., can be used to target the delivery of oligonucleotides to cells expressing corresponding receptors or proteins. In some embodiments, the additional chemical portion of the provided oligonucleotide comprises anisamide or a derivative or analogue thereof, and is capable of targeting the oligonucleotide to cells expressing a specific receptor (such as the σ1 receptor).

[0097] In some embodiments, the provided oligonucleotide is formulated for application to somatic cells and / or tissues expressing its target. In some embodiments, an additional chemical moiety conjugated to the oligonucleotide enables the oligonucleotide to be targeted to the cell.

[0098] In some embodiments, an additional chemical component is or comprises an asialic acid glycoprotein receptor (ASGPR) ligand. ASGPR1 expression in the hippocampus and / or cerebellum Purkinje cell layer of mice has been reported without wishing to be bound by any particular theory. http: / / mouse.brain-map.org / experiment / show / 2048. Various ASGPR ligands are known in the art and can be used according to this disclosure. In some embodiments, the ASGPR ligand is a carbohydrate. In some embodiments, the ASGPR ligand is GalNac or a derivative or analog thereof. In some embodiments, the ASGPR ligand is the ligand described by Sanhueza et al., *Journal of the American Chemical Society*, 2017, 139(9), pp. 3528–3536. In some embodiments, the ASGPR ligand is the ligand described by Mamidyala et al., *Journal of the American Chemical Society*, 2012, 134, pp. 1978–1981. In some embodiments, the ASGPR ligand is the ligand described in US 20160207953. In some embodiments, the ASGPR ligand is, for example, a substituted 6,8-dioxabicyclo[3.2.1]octane-2,3-diol derivative disclosed in US 20160207953. In some embodiments, the ASGPR ligand is, for example, the ligand described in US 20150329555. In some embodiments, the ASGPR ligand is a substituted 6,8-dioxabicyclo[3.2.1]octane-2,3-diol derivative disclosed in US 20150329555. In some embodiments, the ASGPR ligand is the ligand described in US 8877917, US 20160376585, US 10086081, or US 8106022. The ASGPR ligands described in these documents are incorporated herein by reference. Those skilled in the art will understand that various techniques are known in the art, including those described in the document for evaluating the binding of chemical moieties to ASGPR, and these techniques can be used according to this disclosure. In some embodiments, the provided oligonucleotide is conjugated to an ASGPR ligand. In some embodiments, the provided oligonucleotide comprises an ASGPR ligand.

[0099] In some embodiments, additional chemical moieties on oligonucleotides as disclosed herein are linked by adapters. Various adapters are available in the art and can be used according to this disclosure, for example, adapters for conjugating various moieties to proteins (e.g., to antibodies to form antibody-drug conjugates), nucleic acids, etc. Some available connectors are described in US9982257, US 20170037399, US 20180216108, US 20180216107, US 9598458, WO 2017 / 062862, WO 2018 / 067973, WO 2017 / 160741, WO 2017 / 192679、WO 2017 / 210647、WO 2018 / 098264、WO 2018 / 223056、WO 2018 / 237194、WO 2019 / 032607、WO 2019 / 055951、WO 2019 / 075357、WO 2019 / 200185、WO In WO 2019 / 217784, WO 2019 / 032612, WO 2020 / 191252 and / or WO2021 / 071858, the linker portion of each linker is independently incorporated herein by reference. In some embodiments, the linker may connect two or more additional chemical moieties to the oligonucleotide chain as described herein. For example, in some embodiments, one or two or three or more additional chemical moieties (e.g., GalNAc moieties) are connected to the oligonucleotide chain (e.g., at the 5' end) via a multivalent linker portion.

[0100] In some embodiments, additional chemical portions are cleaved from the remainder of the oligonucleotide (e.g., the oligonucleotide chain) after application to a system, cell, tissue, organ, subject, etc. In some embodiments, the additional chemical portions promote, increase, and / or accelerate delivery to certain cells, and are cleaved from the oligonucleotide after delivery to such cells. In some embodiments, the adapter portion comprises one or more cleavable portions that can be cleaved at a desired location (e.g., within certain types of cells, daughter cell compartments (e.g., lysosomes, etc.)) and / or at a desired time point. In some embodiments, the cleavable portions are selectively cleaved by peptides, such as enzymes (e.g., nucleases). Numerous useful cleavable portions and cleavable adapters have been reported, and these cleavable portions and adapters can be used according to this disclosure. In some embodiments, the cleavable portion is or contains one or more functional groups selected from amides, esters, ethers, phosphodiesters, disulfide bonds, carbamates, etc. In some embodiments, connectors are described in WO 2012 / 030683, WO 2021 / 030778, WO 2020 / 154344, WO 2020 / 154343, WO 2020 / 154342, WO 2020 / 165077, WO 2020 / 201406, WO 2020 / 216637 or WO 2020 / 252376.

[0101] lipid conjugates In some embodiments, the ligand or conjugate is a lipid or lipid-based molecule. Such lipid or lipid-based molecules preferably bind to a serum protein, such as human serum albumin (HSA). HSA-binding ligands allow the conjugate to distribute to target tissues, such as non-renal target tissues of the body. For example, the target tissue could be the liver, including hepatic parenchymal cells. Other molecules that can bind to HSA can also be used as ligands. For example, naproxen or aspirin can be used. Lipid or lipid-based ligands can (a) enhance resistance to conjugate degradation; (b) increase targeting or transport to target cells or cell membranes; and / or (c) can be used to modulate binding to serum proteins, such as HSA.

[0102] Lipid-based ligands can be used to inhibit (e.g., control) the binding of conjugates to target tissues. For example, lipids or lipid-based ligands that bind more strongly to HSA are less likely to be targeted to the kidney and therefore less likely to be cleared from the body. Lipids or lipid-based ligands that bind less strongly to HSA can be used to target conjugates to the kidney.

[0103] In some embodiments, the ligand is a portion taken up by target cells (e.g., proliferating cells), such as a vitamin. Exemplary vitamins include vitamins A, E, and K.

[0104] Cell permeabilizer In some embodiments, the ligand is a cell permeabilizer, preferably a helical cell permeabilizer. Preferably, the agent is amphiphilic. Exemplary agents are peptides, such as tat or tentacles. If the agent is a peptide, it can be modified, including by peptide analogs, inversions, non-peptide or pseudopeptide bonds, and the use of D-amino acids. The helical agent is preferably an α-helical agent having both a lipophilic and a lipophobic phase.

[0105] The ligand can be a peptide or a peptide mimic. A peptide mimic (also referred to herein as an oligopeptide mimic) is a molecule that, like a natural peptide, can fold into a defined three-dimensional structure. The conjugation of peptides and peptide mimics to oligonucleotide drugs can affect the pharmacokinetic distribution of oligonucleotides, such as by enhancing cellular recognition and uptake. The length of the peptide or peptide mimic moiety can be approximately 5–50 amino acids, for example, approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids.

[0106] The peptide or peptide mimic can be, for example, a cell-permeable peptide, a cationic peptide, an amphiphilic peptide, or a hydrophobic peptide (e.g., primarily composed of Tyr, Trp, or Phe). The peptide moiety can be a dendritic peptide, a bound peptide, or a cross-linked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary peptide containing a hydrophobic MTS is an RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 64). RFGF analogs containing hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 65)) can also be targeting moieties. The peptide moiety can be a "delivery" peptide that can carry highly polar molecules, including peptides, oligonucleotides, and proteins, across the cell membrane. For example, sequences from HIVTat protein (GRKKRRQRRRPPQ (SEQ ID NO: 66) and Drosophila tentacles (RQIKIWFQNRRMKWKK (SEQ ID NO: 67)) have been found to act as delivery peptides. Peptides or peptide mimics can be encoded by random sequences of DNA, such as peptides identified from phage display libraries or one-to-one-compound (OBOC) combinatorial libraries (Lam et al., Nature 354:82-84, 1991). Examples of peptides or peptide mimics tethered to oligonucleotide agents by incorporating monomeric units for cell-targeting purposes are arginine-glycine-aspartic acid (RGD)-peptides or RGD mimics. The length of the peptide moiety can range from about 5 amino acids to about 40 amino acids. The peptide moiety can have structural modifications, such as structural modifications to increase stability or direct conformational properties. Any structural modifications described below can be used.

[0107] RGD peptides used in the compositions and methods described herein can be linear or cyclic and can be modified, for example, by glycosylation or methylation, to facilitate targeting to specific tissues. RGD-containing peptides and peptide mimics may include D-amino acids as well as synthetic RGD mimics. In addition to RGD, other parts of the targeting integrin ligand may be used. Some conjugates of this ligand target PECAM-1 or VEGF.

[0108] "Cell-permeable peptides" are capable of penetrating cells, such as microbial cells (e.g., bacterial or fungal cells) or mammalian cells (e.g., human cells). Microbial cell-permeable peptides can be, for example, α-helical linear peptides (e.g., LL-37 or CeropinP1), disulfide-containing peptides (e.g., α-defensins, β-defensins, or bacteriocins), or peptides containing only one or two major amino acids (e.g., PR-39 or indolicidin). Cell-permeable peptides can also include nuclear localization signals (NLS). For example, cell-permeable peptides can be amphiphilic peptides derived from the NLS of the fusion peptide domain of HIV-1 gp41 and the SV40 large T antigen, such as MPG (Simeoni et al., Nucleic Acid Research 31:2717-2724, 2003).

[0109] carbohydrate conjugates In some embodiments, the oligonucleotides described herein further include carbohydrates. Carbohydrate-conjugated oligonucleotides are advantageous for in vivo delivery of nucleic acids and for compositions suitable for in vivo therapeutic use as described herein. As used herein, "carbohydrate" means a compound that is itself composed of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic) wherein oxygen, nitrogen, or sulfur atoms are bonded to each carbon atom; or a compound having as part of a carbohydrate portion composed of one or more monosaccharide units each having at least six carbon atoms (which may be linear, branched, or cyclic) wherein oxygen, nitrogen, or sulfur atoms are bonded to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Certain monosaccharides include sugars with 5 or more saccharides (e.g., C5, C6, C7, or C8); disaccharides and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

[0110] In some embodiments, the carbohydrate conjugate is a monosaccharide.

[0111] In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, PK regulators and / or cell-penetrating peptides.

[0112] Additional carbohydrate conjugates (and linkers) applicable to the oligonucleotides described herein include the carbohydrates described in PCT Publications WO 2014 / 179620 and WO 2014 / 179627, the entire contents of each of which are incorporated herein by reference.

[0113] connector In some embodiments, the conjugates or ligands described herein can be linked to oligonucleotides via various linkers, which can be cleavable or non-cleavable.

[0114] Linkages typically include direct bonds or atoms (such as oxygen or sulfur), units (such as NR8, C(O), C(O)NH, SO, SO2, SO2NH), or atomic chains, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, aralkyl, areneyl, arynyl, heteroaralkyl, heteroareneyl, heteroarynyl, heterocyclic alkyl, heterocyclic alkenyl, heterocyclic alynyl, aryl, heteroaryl, heterocyclic, cycloalkyl, cycloalkenyl, alkylaralkyl, alkylareneyl, alkylarynyl, alkenylaralkyl, alkenylareneyl, alkenylarynyl, alynylaralkyl, alynylareneyl, alynylarynyl, alkylheteroalkyl, alkylheteroalkenyl, alkylheteroalynyl, alkenylheteroalkyl, alkylheteroalynyl, alkenylheteroalkyl, alkylheteroalkyl, alkylheteroalynyl, alkenylheteroalkylane Alkyl, alkenyl heteroaryl, alkenyl heteroarylynyl, alkyneyl heteroarylalkyl, alkyneyl heteroaryl, alkyneyl heteroarylynyl, alkyl heterocyclic alkyl, alkyl heterocyclic alkenyl, alkyl heterocyclic alkyne, alkenyl heterocyclic alkenyl, alkenyl heterocyclic alkyne, alkyneyl heterocyclic alkyl, alkyneyl heterocyclic alkenyl, alkyneyl heterocyclic alkyne, alkyl aryl, alkenyl aryl, alkyneyl aryl, alkyl heteroaryl, alkenyl heteroaryl, alkyneyl heteroaryl, alkyl aryl, alkenyl aryl, alkyneyl aryl, alkyl heteroaryl, alkenyl heteroaryl, alkyneyl heteroaryl, alkyl aryl, alkenyl heteroaryl, alkyneyl heteroaryl, alkyl aryl, alkenyl heteroaryl, alkynyl heteroaryl, wherein one or more methylene groups may be interrupted or capped by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle; wherein R8 is hydrogen, acyl, aliphatic compound or substituted aliphatic compound. In some embodiments, the connector is about 1-24 atoms, 2-24 atoms, 3-24 atoms, 4-24 atoms, 5-24 atoms, 6-24 atoms, 6-18 atoms, 7-18 atoms, 8-18 atoms, 7-17 atoms, 8-17 atoms, 6-16 atoms, 7-17 atoms, or 8-16 atoms.

[0115] A cleavable linker is a linker that is sufficiently stable outside the cell but is cleaved upon entry into a target cell to release the two parts of the linker that remain together. In a preferred embodiment, the cleavage rate of the cleavable linker in the target cell or under a first reference condition (which may be selected, for example, to simulate or represent intracellular conditions) is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or more times, or at least about 100 times, the cleavage rate in the subject's blood or under a second reference condition (which may be selected, for example, to simulate or represent conditions present in blood or serum).

[0116] Cleavable linker groups are susceptible to the effects of cleaving agents (e.g., pH, redox potential, or the presence of degrading molecules). Generally, cleaving agents are more prevalent or present at higher levels or with higher activity inside cells than in serum or blood. Examples of such degrading agents include: redox agents that are selective or non-substrate-specific to a particular substrate, including, for example, oxidases or reductases (e.g., thiols) present in cells that can degrade redox-cleavable linker groups by reduction; esterases; endosomes or agents that can produce an acidic environment, for example, endosomes or agents that produce a pH of five or lower; and enzymes that can hydrolyze or degrade acid-cleavable linker groups by acting as universal acids, peptidases (which may be substrate-specific), and phosphatases.

[0117] Cleavable linker groups (such as disulfide bonds) may be susceptible to pH influence. Human serum has a pH of 7.4, while the average intracellular pH is slightly lower, ranging from approximately 7.1 to 7.3. Endosomes have a more acidic pH, in the range of 5.5 to 6.0, and lysosomes have a pH of approximately 5.0 or even more acidic. Some linkers will have cleavable linker groups that are cleaved at a preferred pH, thereby releasing cationic lipids from intracellular ligands or into desired compartments of the cell.

[0118] Linkers can include cleavable linker groups that can be cleaved by specific enzymes. The type of cleavable linker group incorporated into the linker can depend on the cell type to be targeted. For example, liver-targeting ligands can be linked to cationic lipids via linkers that include ester groups. Hepatocytes are rich in esterases, and therefore the linker will cleave more efficiently in hepatocytes compared to non-esterase-rich cell types. Other esterase-rich cell types include lung, renal cortex, and testicular cells.

[0119] When targeting cell types rich in peptidase, such as hepatocytes and synovial cells, a linker containing peptide bonds can be used.

[0120] Generally, the suitability of a candidate cleavable linker can be evaluated by testing its ability to cleave the candidate linker group by a degrading agent (or condition). It will also be necessary to test the ability of the candidate cleavable linker group to resist cleavage in blood or upon contact with other non-target tissues. Thus, a relative sensitivity to cleavage can be determined between a first condition and a second condition, wherein the first condition is selected to indicate cleavage in target cells, and the second condition is selected to indicate cleavage in other tissues or biological fluids (e.g., blood or serum). Evaluation can be performed in cell-free systems, cells, cell cultures, organ or tissue cultures, or in whole animals. Initial evaluation under cell-free or culture conditions, followed by confirmation through further evaluation in whole animals, may be useful. In some embodiments, the cleavage rate of a useful candidate compound in cells (or under in vitro conditions selected to simulate intracellular conditions) is at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times that in blood or serum (or under in vitro conditions selected to simulate extracellular conditions).

[0121] Oxidative-reduction cleavage linker In some embodiments, the cleavable linker is an oxidoreductile linker that is cleaved upon reduction or oxidation. An example of an oxidoreductile linker is a disulfide linker (--S--S--). To determine whether a candidate cleavable linker is a suitable “oxidoreductile linker,” or, for example, whether it is suitable for use with a specific oligonucleotide moiety and a specific target, reference can be made to the methods described herein. For example, candidates can be evaluated using reagents known in the art by incubation with dithiothreitol (DTT) or other reducing agents that simulate the cleavage rate to be observed in cells (e.g., target cells). Candidates can also be evaluated under conditions selected to simulate blood or serum conditions. In one embodiment, the candidate compound is cleaved by up to about 10% in blood. In other embodiments, the degradation rate of the useful candidate compound in cells (or under in vitro conditions selected to simulate intracellular conditions) is at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times that in blood (or under in vitro conditions selected to simulate extracellular conditions). The cleavage rate of the candidate compound can be determined using standard enzyme kinetic assays under conditions selected to simulate intracellular media and compared to conditions selected to simulate extracellular media.

[0122] Phosphate-based cleavable linker groups In another embodiment, the cleavable connector comprises a phosphate-based cleavable linker group. The phosphate-based cleavable linker group is cleaved by an agent that degrades or hydrolyzes the phosphate group. Examples of agents in cells that cleave phosphate groups are cellular enzymes, such as phosphatases. Examples of phosphate-based linker groups are -OP(O)(ORk)-O-, OP(S)(ORk) O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, OP(S)(ORk) S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, SP(O)(Rk)-S-, and -OP(S)(Rk)-S-, wherein R k For H or C 1-6 Alkyl groups. These candidates can be evaluated using methods similar to those described above.

[0123] Acid-cutting linker In another embodiment, the cleavable connector includes an acid-cleavable linker group. An acid-cleavable linker group is a linker group that is cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable linker group is cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0 or lower) or by an agent (such as an enzyme) that can act as a generalized acid. In cells, specific low-pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for the acid-cleavable linker group. Examples of acid-cleavable linker groups include, but are not limited to, hydrazones, esters, and esters of amino acids. The acid-cleavable group can have the general formula –C=NN--, C(O)O, or --OC(O). A preferred embodiment is when the carbon (alkoxy group) attached to the oxygen of the ester is aryl, substituted alkyl, or tertiary alkyl (such as dimethylpentyl or tert-butyl). These candidates can be evaluated using methods similar to those described above.

[0124] Based on ester linking groups In some embodiments, the cleavable linker comprises an ester-based cleavable linker group. Ester-based cleavable linker groups are cleaved by cellular enzymes such as esterases and amidases. Examples of ester-based cleavable linker groups include, but are not limited to, alkylene, alkenene, and ynylene esters. Ester-cleavable linker groups have the general formula -C(O)O-- or --OC(O)--. These candidates can be evaluated using methods similar to those described above.

[0125] Based on peptide cleavage groups In yet another embodiment, the cleavable linker comprises a peptide-based cleavable linker group. The peptide-based cleavable linker group is cleaved by enzymes in the cell, such as peptidases and proteases. The peptide-based cleavable linker group is a peptide bond formed between amino acids to produce oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The peptide-based cleavable linker group does not include an amide group (--C(O)NH--). The amide group can be formed between any alkylene, alkenyl, or alkyne groups. A peptide bond is a specific type of amide bond formed between amino acids to produce peptides and proteins. The peptide-based cleavable linker group is generally limited to peptide bonds (i.e., amide bonds) formed between amino acids that produce peptides and proteins, and does not include the entire amide functional group. The peptide-based cleavable linker group has the general formula --NHCHRAC(O)NHCHRBC(O)--, where RA and RB are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0126] In one embodiment, the oligonucleotides described herein are conjugated to carbohydrates via linkers. Linkers comprise divalent and trivalent branched linker groups. Exemplary oligonucleotide-carbohydrate conjugates having linkers include, but are not limited to, the conjugates described in Formulas 24-35 of PCT Publication WO 2018 / 195165.

[0127] Representative U.S. patents teaching the preparation of oligonucleotide conjugates include, but are not limited to, U.S. Patent Nos. 4,828,979, 4,948,882, 5,218,105, 5,525,465, 5,541,313, 5,545,730, 5,552,538, 5,578,717, 5,580,731, 5,591,584, 5,109,124, 5,118,802, 5,138,045, 5,414,077, and 5,486,603. No. 5,512,439; No. 5,578,718; No. 5,608,046; No. 4,587,044; No. 4,605,735; No. 4,667,025; No. 4,762,779; No. 4,789,737; No. 4,824,941; No. 4,835,263; No. 4,876,335; No. 4,904,582; No. 4,958,013; No. 5,082,830; No. 5,112,963; No. 5,214,136; No. 5,082,830; No. 5,1 No. 12,963; No. 5,214,136; No. 5,245,022; No. 5,254,469; No. 5,258,506; No. 5,262,536; No. 5,272,250; No. 5,292,873; No. 5,317,098; No. 5,371,241; No. 5,391,723; No. 5,416,203; No. 5,451,463; No. 5,510,475; No. 5,512,667; No. 5,514,785; No. 5,565,552; No. 5,567,81 No. 0; No. 5,574,142; No. 5,585,481; No. 5,587,371; No. 5,595,726; No. 5,597,696; No. 5,599,923; No. 5,599,928; and No. 5,688,941; No. 6,294,664; No. 6,320,017; No. 6,576,752; No. 6,783,931; No. 6,900,297; No. 7,037,646; No. 8,106,022, the entire contents of each of these documents are hereby incorporated herein by reference.

[0128] In some cases, the oligonucleotides described herein can be modified with non-ligand groups. Many non-ligand molecules have been conjugated to oligonucleotides to enhance their activity, cellular distribution, or cellular uptake, and procedures for such conjugation are available in the scientific literature. Such non-ligand moieties already include lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proceedings of the National Academy of Sciences, 1989, 86:6553); bile acids (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053); thioethers, for example, hexyl-S-triphenylmethylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3:2765); mercaptocholesterol (Oberhauser et al., Nucleic Acid Research, 1992, 20:533); aliphatic chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., Journal of the European Society for Molecular Biology, 1991, 10:111; Kabanov et al., FEBS Letters, 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49); phospholipids, such as di-hexadecyl-rac-glycerol or 1,2-di-O-hexadecyl-rac-glycerol-3-H-phosphonate triethylammonium (Manoharan et al., Tetrahedral Letters, 1995, 36:3651; Shea et al., Nucleic Acid Research, 1990, 18:3777); polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides and Nucleotides, 1995, 14:969); or adamantaneacetic acid (Manoharan et al., Tetrahedral Letters, 1995, 36:3651); palmityl moiety (Mishra et al., Chinese Journal of Biochemistry and Biophysics, 1995, 1264229); or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., Journal of Pharmacology and Experimental Therapeutics, 1996, 277:923). The above lists representative U.S. patents teaching the preparation of such oligonucleotide conjugates. Typical conjugation schemes involve the synthesis of oligonucleotides with amino linkers at one or more positions in the sequence.The amino group is then reacted with the conjugated molecule using a suitable coupling or activating reagent. The conjugation reaction can be carried out in solution while the oligonucleotide is still bound to the solid support or after oligonucleotide cleavage. Purification of the oligonucleotide conjugate by HPLC typically yields a pure conjugate.

[0129] Methods for preparing oligonucleotides The ligand-conjugated oligonucleotides described herein can be synthesized using oligonucleotides with side-group reactive capabilities, such as oligonucleotides derived from linkers attached to oligonucleotides (as described herein). Such reactive oligonucleotides can react directly with commercially available ligands, synthetic ligands with any of a variety of protecting groups, or ligands having a linker motif attached to them.

[0130] The oligonucleotides used in the conjugates described herein can be conveniently and routinely prepared using well-known solid-phase synthesis techniques. Equipment for such synthesis is sold by several vendors, including, for example, Applied Biosystems, Inc. (Foster City, California). The oligonucleotides described herein can be synthesized using standard methods known in the art, as discussed further below, for example, by using an automated DNA synthesizer, such as those commercially available from, for example, Biosearch, Applied Biosystems, Inc. Any other methods known in the art for such synthesis may be used, either alternatively or as a substitute. The preparation of other oligonucleotides, such as phosphate thioesters and alkylated derivatives, using similar techniques is also known.

[0131] In the ligand-conjugated oligonucleotides described herein, such as ligand molecules containing sequence-specific links to nucleosides of the oligonucleotides described herein, the oligonucleotides and oligonucleotides can be assembled onto a suitable DNA synthesizer using standard nucleotides or nucleoside precursors, or nucleotides or nucleoside conjugate precursors already containing linking portions, ligand-nucleotide or nucleoside conjugate precursors already containing ligand molecules, or building blocks containing non-nucleoside ligands.

[0132] When using a nucleotide conjugate precursor that already has a linker moiety, the synthesis of a sequence-specific linked nucleoside is typically performed, and then the ligand molecule reacts with the linker moiety to form a ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides described herein are synthesized via an automated synthesizer using phosphorusamide derived from the ligand-nucleoside conjugate, along with commercially available standard and non-standard phosphorusamides commonly used in oligonucleotide synthesis.

[0133] The oligonucleotides described herein were synthesized and / or modified using methods recognized in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, SL et al. (eds.), John Wiley & Sons, Inc., New York, NY, USA, which are hereby incorporated by reference. Representative U.S. patents teaching the preparation of oligonucleotides include, but are not limited to, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; and 5,489,677. Nos. 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, the entire contents of each of these documents are hereby incorporated herein by reference.

[0134] Oligonucleotides can be prepared using solution-phase synthesis, solid-phase organic synthesis, or both. Organic synthesis offers the advantage of readily preparing oligonucleotides that include non-natural or alternative nucleotides. The single-stranded oligonucleotides described herein can be prepared using solution-phase synthesis, solid-phase organic synthesis, or both.

[0135] It is envisioned that for any sequence identified herein, further optimization could be achieved by systematically adding or removing linking nucleotides to produce longer or shorter sequences. Such optimized sequences could be modified, for example, by introducing alternative nucleobases, alternative sugar moieties, and / or alternative internucleotide bonds as described herein or known in the art, including alternative nucleobases, alternative sugar moieties, and / or alternative internucleotide bonds as known in the art and / or discussed herein, to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting specific locations or cell types, and / or increasing interaction with RNA editing enzymes (e.g., ADAR).

[0136] How to use This document provides the use of oligonucleotides as therapeutic agents as described herein. In various embodiments, the oligonucleotides are structural genes, genes including control and termination regions, self-replicating systems such as viral DNA or plasmid DNA, single-stranded or double-stranded RNAi agents, shRNA, antisense oligonucleotides, ribozymes, microRNAs, microRNA mimics, supermir, aptamers, antimir, antagomir, adaptors, triplet-forming oligonucleotides, G-quadruplex oligonucleotides, RNA activators, immunostimulatory oligonucleotides, or decoy oligonucleotides.

[0137] In some instances, methods for treating a disease or condition in a patient in need are provided, the methods comprising administering to the patient a therapeutically effective amount of the oligonucleotide disclosed herein. The term "therapeuticly effective amount" refers to an amount that effectively treats and / or improves a patient's disease or condition. Further, methods for delivering the oligonucleotides disclosed herein to cells are provided, the methods comprising contacting the cells with the oligonucleotides as disclosed herein or pharmaceutical compositions thereof. In some embodiments, the cells may be contacted in vitro. In other embodiments, the cells are contacted in vivo. In some cases, the patient is a mammalian subject. Mammal subjects may include, but are not limited to, human or mouse subjects. In yet other embodiments in which cells are contacted in vitro, the cells are obtained from a human or mouse subject. In some cases, the cells are tumor cells. In some cases, the cells are muscle cells.

[0138] In some embodiments, oligonucleotides are administered to patients to treat diseases or conditions. Non-limiting examples of diseases or conditions that can be treated with the oligonucleotides disclosed herein include cancer, infectious diseases, autoimmune diseases, and neurological disorders. In some embodiments, the oligonucleotides disclosed herein are used as vaccines. Genetic vaccination or administration of nucleic acid molecules (e.g., RNA) to patients, followed by transcription and / or translation of the encoded genetic information, can be used to treat and / or prevent inherited genetic diseases, but also to treat autoimmune diseases, infectious diseases, cancerous or tumor-related diseases, and inflammatory diseases. Genetic vaccination can be particularly used to treat cancer because cancer cells express antigens, and tumors are often not easily recognized and eliminated by the host, as demonstrated by disease development.Non-limiting examples of specific diseases and conditions for which the oligonucleotides disclosed in this paper may be used for treatment include cystic fibrosis, albinism, α-1-antitrypsin deficiency, Alzheimer's disease, amyotrophic lateral sclerosis, asthma, 11-thalassemia, Cadasil syndrome, Charcot-Marie-Tooth disease, chronic obstructive pulmonary disease, distal spinal muscular atrophy, Duchenne / Becker muscular dystrophy, dystrophic epidermolysis bullosa, epidermolysis bullosa, Fabry disease, Factor V Leiden associated disorder, familial adenoma, polyposis, galactosemia, Gaucher's disease, glucose-6-phosphate dehydrogenase deficiency, hemophilia, hereditary hemochromatosis, and Hunter syndrome. Syndrome, Huntington's disease, Hurler syndrome, inflammatory bowel disease, hereditary polyagglutination syndrome, Leber congenital amaurosis, Lesch-Nyhan syndrome, Lynch syndrome, Marfan syndrome, mucopolysaccharidosis, muscular dystrophy, myotonic dystrophy type I and II, neurofibromatosis, Niemann-Pick disease type A, B and C, NY-ESO-1 related cancers, Parkinson's disease, Peutz-Jeghers syndrome, phenylketonuria, Pompe's disease, primary ciliary disease, prothrombin mutation-related diseases, pulmonary hypertension, retinitis pigmentosa, Sandhoff's disease. Diseases, severe combined immunodeficiency syndrome, sickle cell anemia, spinal muscular atrophy, Stargardt's disease, Tay-Sachs disease, Usher syndrome, X-linked immunodeficiency, Sturge-Weber syndrome, Rett syndrome, and cancer.

[0139] vaccine The oligonucleotides disclosed herein can be used as vaccines, wherein the oligonucleotides are DNA or RNA that can encode immunogens, antigens, or neoantigens. The host's immune system provides a means for rapidly and specifically generating a protective response against pathogenic microorganisms and also helps to reject malignant tumors. Immune responses are generally described as including humoral responses and cell-mediated responses, in which differentiated B lymphocytes produce antibodies specific to antigens, and in which various types of T lymphocytes eliminate antigens through multiple mechanisms. For example, CD4 (also known as CD4+) helper T cells capable of recognizing specific antigens can respond by releasing soluble mediators such as cytokines, thereby recruiting other cells of the immune system to participate in the immune response. CD8 (also known as CD8+) cytotoxic T cells are also capable of recognizing specific antigens and can bind to cells or particles carrying antigens, and destroy or damage said antigen-carrying cells or particles. In particular, cell-mediated immune responses, including cytotoxic T lymphocyte (CTL) responses, may be very important for the elimination of tumor cells and cells infected by microorganisms such as viruses, bacteria, or parasites.

[0140] Therefore, this disclosure includes methods for inducing an immune response in a patient in need, the methods comprising administering to the patient a therapeutically effective amount of the oligonucleotide of this disclosure (e.g., formulated as an antigen composition). In some embodiments, administration is performed via intramuscular, intratumoral, intravenous, intraperitoneal, or subcutaneous delivery.

[0141] In various embodiments, administration of the oligonucleotides of this disclosure (e.g., formulated as compositions, pharmaceutical formulations, or antigen compositions) to a subject can increase the amount of antibodies against the antigen (e.g., neutralizing antibodies) produced in the subject relative to the amount of antibodies against the antigen produced in a subject who has not been administered the oligonucleotides. In some embodiments, the increase is a 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 700-fold, or 1000-fold increase.

[0142] The immune response induced by the methods of this disclosure typically includes antibody responses, preferably neutralizing antibody responses, maturation and memory of T and B cells, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and T cell-mediated responses such as CD4+ and CD8+. Immune responses generated by oligonucleotides encoding antigens as disclosed herein produce immune responses that recognize and preferably alleviate and / or neutralize infections as described herein. Methods for assessing antibody responses following administration of the antigen composition (immunization or vaccination) are known in the art and / or described herein. In some embodiments, the immune response comprises a T cell-mediated response (e.g., a peptide-specific response, such as a proliferative response or a cytokine response). In some embodiments, the immune response comprises both B cell and T cell responses. The antigen composition can be administered in a variety of suitable manner, such as intramuscular injection, intratumoral injection, subcutaneous injection, intradermal application, and mucosal application, such as oral or intranasal application. Other modes of administration include, but are not limited to, intravenous, intraperitoneal, intranasal, intravaginal, rectal, and oral administration. This disclosure also envisions combinations of different routes of administration in immunized subjects, such as simultaneous intramuscular and intranasal administration.

[0143] cancer Various cancers (e.g., cervical cancer) can be treated with the oligonucleotides disclosed herein. As used herein, the term "cancer" refers to any of the various malignant growths characterized by the proliferation of anaplastic cells: these anaplastic cells tend to invade surrounding tissues and metastasize to new body sites, and also refers to the pathogenic condition characterized by the growth of such malignant growths. Cancer can be a tumor or a hematologic malignancy, and includes, but is not limited to, all types of lymphoma / leukemia; carcinomas and sarcomas, such as those found in the anus, bladder, bile ducts, bones, brain, breast, cervix, colon / rectum, endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lungs, mediastinum (thoracic cavity), oral cavity, ovary, pancreas, penis, prostate, skin, small intestine, stomach, spinal cord, coccyx, testis, thyroid, and uterus.

[0144] As a non-limiting example, treatable cancers include acute myeloid leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, adenocarcinoma, adenosarcoma, adrenal carcinoma, adrenocortical carcinoma, anal cancer, anaplastic astrocytoma, angiosarcoma, appendiceal cancer, astrocytoma, basal cell carcinoma, B-cell lymphoma, bile duct cancer, bladder cancer, bone cancer, colorectal cancer, brain cancer, brainstem glioma, brain tumor, breast cancer, carcinoid tumor, cervical cancer, bile duct cancer, chondrosarcoma, chronic lymphocytic leukemia, chronic myeloid leukemia, and colon cancer. Colorectal cancer, craniopharyngioma, cutaneous lymphoma, cutaneous melanoma, diffuse astrocytoma, ductal carcinoma in situ, endometrial cancer, ependymoma, epithelioid sarcoma, esophageal cancer, Ewing sarcoma, extrahepatic bile duct cancer, ocular cancer, fallopian tube cancer, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, general germ cell tumor, glioblastoma multiforme, glioma, hairy cell leukemia, head and neck cancer, hemangioendothelioma, Hodgkin lymphoma. Lymphoma, Hodgkin's disease, Hodgkin's lymphoma, hypopharyngeal cancer, invasive ductal carcinoma, invasive lobular carcinoma, inflammatory breast cancer, colorectal cancer, intrahepatic bile duct cancer, invasive / invasive breast cancer, islet cell carcinoma, mandibular cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, meningeal metastasis, leukemia, lip cancer, liposarcoma, liver cancer, lobular carcinoma in situ, low-grade astrocytoma, lung cancer, lymph node cancer, lymphoma, male breast cancer, medullary carcinoma, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma. Mesenchymal chondrosarcoma, mesenchymal tumor, mesothelioma, metastatic breast cancer, metastatic melanoma, metastatic squamous neck cancer, mixed glioma, oral cancer, mucinous carcinoma, mucosal melanoma, multiple myeloma, nasal cavity cancer, nasopharyngeal carcinoma, neck cancer, neuroblastoma, neuroendocrine tumor, non-Hodgkin lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oat cell carcinoma, ocular cancer, ocular melanoma, oligodendroglioma, oral cancer (oral cavity cancer), oropharyngeal cancer, osteoblastic sarcoma, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, primary ovarian peritoneal cancer, ovarian sex cord-stromal tumor, Paget's disease.Diseases, pancreatic cancer, papillary carcinoma, paranasal sinus cancer, parathyroid carcinoma, pelvic cancer, penile cancer, peripheral nerve cancer, peritoneal cancer, pharyngeal cancer, pheochromocytoma, pilocytic astrocytoma, pineal region tumors, pineal blastoma, pituitary adenocarcinoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, osteosarcoma, soft tissue sarcoma, uterine sarcoma, sinus cancer, skin cancer Small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cancer, spinal cord cancer, spinal tumor, squamous cell carcinoma, gastric cancer, synovial sarcoma, T-cell lymphoma, testicular cancer, laryngeal cancer, thymoma / thymic carcinoma, thyroid cancer, tongue cancer, tonsil cancer, transitional cell carcinoma, transitional cell carcinoma, triple-negative breast cancer, fallopian tube cancer, tubular carcinoma, ureteral cancer, ureteral cancer, uterine adenocarcinoma, uterine cancer, uterine sarcoma, vaginal cancer, and vulvar cancer.

[0145] infectious disease. This document also discloses a method for treating infectious diseases (bacterial, viral, fungal, or parasitic infections) in patients in need, the method comprising administering an effective amount of the disclosed oligonucleotide to the patient. Non-limiting examples of infectious diseases include hepatitis (such as HBV or HCV infection), RSV, influenza, adenovirus, rhinovirus, or other viral infections. In some embodiments, administration is performed via intramuscular, intratumoral, intravenous, intraperitoneal, or subcutaneous delivery.

[0146] Autoimmune diseases.Various autoimmune diseases and autoimmune-related diseases can be treated with the oligonucleotides disclosed herein. As used herein, the term "autoimmune disease" refers to a disease in which the body produces antibodies that attack its own tissues. As non-limiting examples, autoimmune diseases can include acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, hypogammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune familial autonomic dysfunction, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticaria, axonal or neuronal neuropathies, Balo disease, and Behcet's disease. Diseases including bullous pemphigoid, cardiomyopathy, giant lymphadenopathy, celiac disease, Chagas disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid / benign mucosal pemphigoid, Crohn's disease, Cogans syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST disease, and essential mixed cryoglobulinemia. Mixed cryoglobulinemia, demyelinating neuropathy, herpetic dermatitis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, Dressler's syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibromyalgia**, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndromeGastrointestinal syndrome, granulomatous polyangiitis (GPA) (formerly known as Wegener's granulomatosis), Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosis, immunomodulatory lipoproteins, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile diabetes (type 1 diabetes), juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome Syndrome, leukocytic clotting vasculitis, lichen planus, lichen sclerosus, woody conjunctivitis, linear IgA disease (LAD), lupus (SLE), Lyme disease, chronic Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devi's disease), neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing rheumatoid arthritis, PANDAS (streptococcal-associated childhood autoimmune neuropsychiatric disorder), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome. syndromes including Parsonnage-Turner syndrome, Pars planitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, type I, II, and III autoimmune polyglandular syndrome, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, and postpericardiotomy syndrome.Syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosa, pure red cell aplasia, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm and testicular autoimmunity, stiff-person syndrome, subacute bacterial endocarditis (SBE), Susac's syndrome, sympathetic ophthalmia, Takayasu'sarteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome Syndrome), transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vasculobulbar dermatitis, vitiligo, and Wegener's granulomatosis (now known as granulomatous polyangiitis (GPA)). In some implementations, administration is via intramuscular, intratumoral, intravenous, intraperitoneal, or subcutaneous delivery.

[0147] Nervous system diseases.Various neurological disorders can be treated with the oligonucleotides disclosed herein. As non-limiting examples, neurological disorders may include septum pellucida agenesis, acid lipase disorder, acid maltase deficiency, acquired epileptic aphasia, acute disseminated encephalomyelitis, attention deficit hyperactivity disorder (ADHD), Adie's Pupil, Adie's Syndrome, adrenoleukodystrophy, corpus callosum dysplasia, agnosia, Aicardi Syndrome, Aicardi-Goutieres Syndrome Disorder, AIDS-related neurological complications, Alexander disease, Alpers' disease, alternating hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), anencephaly, aneurysm, and Angelman syndrome. Syndrome, hemangioma, hypoxia, antiphospholipid syndrome, aphasia, apraxia, arachnoid cysts, arachnoiditis, Arnold-Chiari malformation, arteriovenous malformation, Asperger syndrome, ataxia, telangiectatic ataxia, ataxia and cerebellar or spinocerebellar degeneration, atrial fibrillation and stroke, attention deficit hyperactivity disorder, autism spectrum disorder, autonomic dysfunction, back pain, Barth syndrome, Batten disease, Becker's myotonia, Behcet's disease, Bell's palsy, benign primary blepharospasm, benign focal muscular atrophy, benign intracranial hypertension, Bernhardt-Roth syndrome. Syndrome, Binswanger's Disease, blepharospasm, Bloch-Sulzberger Syndrome, Brachial Plexus Birth Injuries, Brachial Plexus Injury, Bradbury-Eggleston Syndrome, Brain and Spinal Tumors, Cerebral Aneurysms, Brain Injury, Brown-Sequard Syndrome, Bulbospinal Muscular AtrophyMuscular Atrophy, autosomal dominant cerebral arteriosclerosis with subcortical infarction and leukoencephalopathy (CADASIL), Canavan Disease, Carpal Tunnel Syndrome, Burning Neuralgia, Cavernous Tumor, Cavernous Hemangioma, Cavernous Malformation, Central Cervical Spinal Cord Syndrome, Central Cord Syndrome, Central Pain Syndrome, Central Pontic Myelinolysis, Head Development Disorder, Ceramide Deficiency, Cerebellar Degeneration, Cerebellar Dysplasia, Cerebral Aneurysm, Cerebral Arteriosclerosis, Cerebral Atrophy, Cerebral Beriberi, Cerebral Cavernous Malformation, Cerebral Gigantism, Cerebral Hypoxia, Cerebral Palsy, Brain-Eye-Face-Skeleton Syndrome (COFS), Shaco-Mali-Duss Disease, Chiari Malformation, Cholesterol Lipid Deposition Disease, Chorea, Chorea-Acanthocytosis, Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), Chronic Orthostatic Intolerance, Chronic Pain, Cockayne Syndrome Type II, Coffin Lowry Syndrome Syndrome, Colpocephaly, Coma, Complex Regional Pain Syndrome, Congenital Bilateral Facial Paralysis, Congenital Myasthenia Gravis, Congenital Myopathy, Congenital Vascular Cavernous Malformation, Corticobasal Degeneration, Cranial Arteritis, Craniosynostosis, Cree encephalitis, Creutzfeldt-Jakob Disease, Cushing's Syndrome, Mast Inclusion Disease, Cytomegalovirus Infection, Dancey-Eye and Dancey Foot Syndrome, Dandy-Walker Syndrome, Dawson Disease, De Morsier's Syndrome, Dejerine-Klumpke Syndrome Palsy, dementia, multiple infarct dementia, semantic dementia, subcortical vascular dementia, Lewy body dementia, dentate nucleus-cerebellar ataxia, basalis muscle atrophy, dermatomyositis, developmental limb atrophy, Devic's syndrome, diabetic neuropathy, diffuse sclerosis, Dravet syndrome, familial autonomic dysfunction, writing disorders, reading disorders, dysphagia, motor disorders, myoclonic cerebellar coordination disorder, myoclonic cerebellar coordination disorder, dystonia, early infantile epileptic encephalopathy, empty sella syndrome, encephalitis, somnolencephalitis, encephalocele, encephalopathy, familial infantile encephalopathy, trigeminal neuralgia, epilepsy, epileptic hemiplegia, Erb's palsyPalsy, Erb-Duchenne and Dejerine-Klumpke Palsies, Essential Tremor, Extracerebral Myelinolysis, Fabry Disease, Fahr's Syndrome, Syncope, Familial Autonomic Dysfunction, Familial Hemangioma, Familial Idiopathic Basal Ganglion Calcification, Familial Periodic Paralysis, Familial Spastic Paralysis, Farber's Disease, Febrile Seizures, Fibromuscular Dysplasia, Fisher's Syndrome, Infantile Hypotonia, Foot Drop, Friedreich's Ataxia, Frontotemporal Dementia, Gaucher Disease, Extensive Ganglioside Depression, Gerstmann's Syndrome, Gerstmann-Straussler-Scheinker Disease Diseases including giant axonal neuropathy, giant cell arteritis, giant cell inclusion body disease, globoid leukoencephalopathy, glossopharyngeal neuralgia, glycogen storage disease, Guillain-Barré syndrome, Hallervorden-Spatz disease, head injury, headache, continuous hemiscarriage, hemifacial spasm, alternating hemiparesis, hereditary neuropathy, hereditary spastic paraplegia, polyneuritis-type hereditary ataxia, Herpes zoster, otitis herpes zoster, Hirayama syndrome, Holmes-Adie syndrome, holohemorrhagic forebrain malformation, HTLV-1 related myelopathy, and Hughes syndrome. Syndrome, Huntington's disease, hydrocephalus, hydrocephalus-normal pressure type, hydramnios, Cushing's syndrome, hypersomnia, hypertonia, hypotonia, hypoxia, immune-mediated encephalomyelitis, inclusion body myositis, pigmentary disorders, infantile hypotonia, infantile axonal dysplasia, infantile phytate storage disease, infantile Refsum disease, infantile spasms, inflammatory myopathy, occipital cleft with exposed brain malformation, enterogenic lipodystrophy, intracranial cysts, intracranial hypertension, Isaacs' syndrome, Jubert's syndrome, Kearns-Sayre syndrome, Kennedy's disease, Kinsbourne syndromeKleine-Levin Syndrome, Klippel-Feil Syndrome, Klippel-Trenaunay Syndrome (KTS), Kliiver-Bucy Syndrome, Korsakoff's Amnesic Syndrome, Krabbe Disease, Kugelberg-Welander Disease, Kuru, Lambert-Eaton Myasthenic Syndrome, Landau-Kleffner Syndrome, lateral femoral cutaneous nerve compression, lateral medullary syndrome, learning disability, Leigh's Disease, Lennox-Gastaut Syndrome, Lesch-Nair Syndrome, leukodystrophy, Levine-Critchley Syndrome Syndrome, Lewy body dementia, lipid storage disease, lipoprotein deposition disease, lissencephaly, locked-in syndrome, Lou Gehrig's disease, lupus-neurological sequelae, Lyme disease-neurological complications, Machado-Joseph disease, macrocephaly, megalencephaly, Melkersson-Rosenthal syndrome, meningitis, meningitis and encephalitis, Menkes disease, Menkes syndrome, paresthesia femoris, metachromatic leukodystrophy, microcephaly, migraine, Miller Fisher syndrome, mini-stroke, mitochondrial myopathy, Moebius syndrome, unilateral muscular atrophy, motor neuron disease, Moyamoya diseaseDiseases, mucolipid storage diseases, mucopolysaccharidosis, multiple infarct dementia, multifocal motor neuropathy, multiple sclerosis, multiple system atrophy, multiple system atrophy with orthostatic hypotension, muscular dystrophy, congenital myasthenia gravis, myasthenia gravis, myelosporotic diffuse sclerosis, infantile myoclonic encephalopathy, myoclonus, myopathy, congenital myopathy, thyroid myopathy, myotonia, congenital myotonia, narcolepsy, neuroacanthocytosis, neurodegeneration with iron accumulation in the brain, neurofibroma. Neurological diseases, antipsychotic malignant syndrome, neurological complications of AIDS, neurological complications of Lyme disease, neurological consequences of cytomegalovirus infection, neurological manifestations of Pompe disease, neurological sequelae of lupus, neuromyelitis optica, neurogenic myotonia, neuronal ceroid lipofuscin deposition, neuronal migration abnormalities, hereditary neuropathies, neuronal sarcoidosis, neurosyphilis, neurotoxicity, cavernous nevus, Niemann-Pick disease, O'Sullivan-McLeod syndrome Syndrome, occipital neuralgia, Ohtahara syndrome, olivopontocerebellar atrophy, myoclonic ataxia, orthostatic hypotension, overuse syndrome, chronic pain, pantothenic kinase-related neurodegeneration, paraneoplastic syndrome, paresthesia, Parkinson's disease, paroxysmal choreoathetosis, paroxysmal migraine, Parry-Romberg disease, Pelizaeus-Merzbacher disease, Pena-Shokeir II syndrome, perineural cysts, periodic paralysis, peripheral neuropathy, periventricular leukomalacia, persistent vegetative state, pervasive developmental disorders, phytate storage disease, Pick's disease Diseases, nerve pinching, piriformis syndrome, pituitary tumor, polymyositis, Pompe disease, hole-brain syndrome, post-poliomyelitis syndrome, postherpetic neuralgia, post-infectious encephalomyelitis, orthostatic hypotension, orthostatic orthostatic tachycardia syndrome, orthostatic tachycardia syndrome, primary dentin atrophy, primary lateral sclerosis, primary progressive aphasia, prion disease, progressive hemifacial atrophy, progressive motor ataxia, progressive multifocal leukoencephalopathy, progressive sclerotic poliomyelitis, progressive supranuclear palsy, prosopagnosia, pseudo-Torch syndrome, pseudotoxoplasmosis syndrome, pseudotumor cerebri, psychogenic movement disorder, Ramsay Hunt syndrome I, Ramsay Hunt syndrome II, Rasmussen's encephalitisEncephalitis, reflex sympathetic dystrophy syndrome, Refsum disease, Refsum disease-infantile form, repetitive motion disorder, repetitive stress injury, restless legs syndrome, retrovirus-associated myelopathy, Rett syndrome, Reye's syndrome, rheumatic encephalitis, Riley-Day syndrome, sacral nerve root cyst, Saint Vitus Dance, salivary gland disorders, Sandhof's disease, Schilder's disease, split brain, Seitelberger disease, seizures, semantic dementia, optic-septal dysplasia, severe myoclonic epilepsy of infancy (SMEI), shaken baby syndrome, shingles, Shy-Drager syndrome, Sjögren's syndrome, sleep apnea, sleep disorders, Sotos syndrome Syndrome, spasm, spina bifida, spinal cord infarction, spinal cord injury, spinal cord tumor, spinal muscular atrophy, spinocerebellar ataxia, spinocerebellar atrophy, spinocerebellar degeneration, Steele-Richardson-Olszewski syndrome, stiff person syndrome, striatal nigrostriatum degeneration, stroke, Sturge-Weber syndrome, subacute sclerosing panencephalitis, subcortical arteriosclerotic encephalopathy, short-term unilateral glial headache (SUNCT), dysphagia, Sydenham Chorea, syncope, syphilitic spinal sclerosis, syringomyelia, systemic lupus erythematosus, tabes dorsalis, tardive dyskinesia, Tarlov cysts, Tey-Sachs disease, temporal arteritis, tethered cord syndrome, Thomsen's myotonia. Myotonia, thoracic outlet syndrome, thyrotoxic myopathy, trigeminal neuralgia (TicDouloureux), Todd's paralysis, Tourette syndrome, transient ischemic attack, transmissible cavernous encephalopathy, transverse myelitis, traumatic brain injury, tremor, trigeminal neuralgia, tropical spastic paraplegia, Troyer syndrome, tuberous sclerosis, vascular erectile tumor, vasculitis syndrome of the central and peripheral nervous systems, von Econo's disease, von Hippel-Lindau disease.Diseases including VHL, von Recklinghausen's Disease, Wallenberg's Syndrome, Werdnig-Hoffman Disease, Wernicke-Korsakoff Syndrome, West Syndrome, Whiplash, Whipple's Disease, Williams Syndrome, Wilson's Disease, Wolman's Disease, and X-linked spinal and bulbar muscular atrophy. In some implementations, administration is via intramuscular, intratumoral, intravenous, intraperitoneal, or subcutaneous delivery.

[0148] In some cases, the oligonucleotides disclosed herein have sequences that are fully complementary to the target RNA. The target RNA may be associated with a disease or condition that is conceived to be treated with the oligonucleotides disclosed herein. In some cases, the target RNA is selected from Serpina1, LRRK2, NRF2, TDP-43, Nav1.7, and PCKS9.

[0149] ADAR Editor RNA-acting adenosine deaminases (ADARs) are enzymes that bind to double-stranded RNA (dsRNA) and convert adenosine to inosine via deamination. In RNA, inosine functions similarly to guanosine for translation and replication. Therefore, the conversion of adenosine to inosine in mRNA can cause codon changes, which may alter the encoded protein and its function. Three ADAR proteins are known to be expressed in humans: ADAR1, ADAR2, and ADAR3. ADAR1 and ADAR2 are expressed systemically, while ADAR3 is expressed only in the brain. ADAR1 and ADAR2 are catalytically active, while ADAR3 is considered inactive.

[0150] Synthetic single-stranded oligonucleotides have been shown to utilize ADAR proteins to edit target RNA by deaminating specific adenosines in the target RNA. The oligonucleotides are complementary to the target RNA, except for at least one mismatch with the adenosine to be deainated. However, previously disclosed methods have not yet demonstrated the selectivity and / or stability required for their therapeutic use. Therefore, there is a need for novel oligonucleotides capable of selectively editing target RNA using ADAR proteins in a therapeutically effective manner.

[0151] In some embodiments, the oligonucleotides described herein are complementary to the target RNA and are capable of recruiting an ADAR enzyme for editing a target nucleobase on the target RNA, for example, for deaminating a target adenosine on the target RNA. In some embodiments, only one nucleobase (e.g., one adenosine) is edited (e.g., deamination). In some embodiments, one, two, or three nucleobases are edited. In some embodiments, the oligonucleotide includes at least one mismatch, wobble, insertion, or deletion. In some cases, the oligonucleotide includes a mismatch opposite to the target nucleobase. The oligonucleotides described herein may further include modifications (e.g., alternative nucleotides) for increasing stability and / or improving deamination efficiency. In some embodiments, the oligonucleotides described herein contain one, two, three, four, or five mismatches, wobbles, insertions, or deletions (or any combination thereof).

[0152] Therefore, this disclosure contemplates the use of the disclosed oligonucleotides in methods described herein for altering target RNA sequences in mammalian cells (preferably human cells). Similarly, this disclosure provides the use of these oligonucleotides as described herein in the manufacture of medicaments for altering target RNA sequences in mammalian cells (preferably human cells). In some embodiments, the target RNA is mRNA.

[0153] This disclosure also relates to a method for deaminating at least one specific target adenosine present in a target RNA sequence in a cell, the method comprising the steps of: providing the cell with an oligonucleotide as described herein; allowing the cell to take up the oligonucleotide; allowing the oligonucleotide to anneal to the target RNA sequence; allowing a mammalian ADAR enzyme to deaminate the target adenosine in the target RNA sequence to inosine; and optionally identifying the presence of inosine in the RNA sequence.

[0154] In some embodiments, this document provides oligonucleotides, compositions, and methods in which two adjacent adenosines are co-deaminated by an RNA editing enzyme such as ADAR. In this particular case, the UAA stop codon is converted to the UII Trp encoding codon. Other examples of modifications resulting from the deamination of the target adenosine within the target codon are provided in Tables 2 and 3 below.

[0155] Table 2 Table 3. Base composition of the triplet and the resulting edited triplets Because the deamination of adenosine to inosine may result in the absence of the mutated A protein at the target site, the identification of the deamination to inosine can be a functional readout, such as an assessment of the presence of a functional protein or even an assessment of whether the disease caused by the presence of adenosine is (partially) reversed. Functional assessments for each disease mentioned herein will generally be performed according to methods known to those skilled in the art. When the presence of target adenosine leads to aberrant splicing, the readout can be an assessment of whether aberrant splicing still occurs, does not occur, or occurs less frequently. On the other hand, when it is desired that the deamination of target adenosine introduces a splicing site, a similar approach can be used to check whether the desired type of splicing is indeed occurring. Suitable methods for identifying the presence of inosine after the deamination of target adenosine are RT-PCR and sequencing performed using methods well known to those skilled in the art.

[0156] Typically, any mutation in target RNA that can be reversed using the oligonucleotides disclosed herein is a G-to-A mutation, and oligonucleotide constructs can be designed accordingly. Mutations that can be targeted using oligonucleotide constructs also include C-to-A and U-to-A (T-to-A at the DNA level) mutations that recruit adenosine deaminase. Although in the latter case, RNA editing may not necessarily restore the mutation to the wild type, the edited nucleotides may produce improvements superior to the original mutation. For example, a mutation that produces an in-frame stop codon resulting in a truncated protein during translation can be changed to a codon encoding an amino acid at said position, which may not be the original amino acid, but produces a (full-length) protein with at least some function (at least more than the function of the truncated protein).

[0157] The oligonucleotides described herein are particularly suitable for treating genetic diseases such as cystic fibrosis, albinism, alpha-1-antitrypsin (A1AT) deficiency, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), asthma, 11-thalassemia, Cadasil syndrome, Shaco-Malley-Duss disease, chronic obstructive pulmonary disease (COPD), distal spinal muscular atrophy (DSMA), Duchenne / Becker muscular dystrophy, dystrophic epidermolysis bullosa, epidermolysis bullosa, Fabry disease, factor V Leiden-related disorders, familial adenomas, polyposis, galactosemia, Gaucher's disease, glucose-6-phosphate dehydrogenase deficiency, hemophilia, hereditary hemochromatosis, Huntington's disease, Heller syndrome, inflammatory bowel disease (IBD), hereditary polyagglutination syndrome, and Leber congenital melanocytosis. Montgomery syndrome, Lesch-Nair syndrome, Lynch syndrome, Marfan syndrome, mucopolysaccharidosis, muscular dystrophy, myotonic dystrophy type I and II, neurofibromatosis, Niemann-Pick disease type A, B and C, NY-ESO-1 related cancers, Parkinson's disease, Boytz-Yage syndrome, phenylketonuria, Pompe disease, primary ciliary disease, prothrombin mutation-related diseases (e.g., prothrombin G20210A mutation), pulmonary hypertension, retinitis pigmentosa, Sandhof disease, severe combined immunodeficiency syndrome (SCID), sickle cell anemia, spinal muscular atrophy, Sturgeon's disease, Tay-Sachs II disease, Ussell's syndrome, X-linked immunodeficiency, Sturgeon-Weber syndrome, Rett syndrome, and various forms of cancer (e.g., BRCA1 and 2 linked breast and ovarian cancer).

[0158] The oligonucleotides described in this article can mutate and deaminate adenosine, thereby increasing protein activity.

[0159] In some embodiments, the treatment is administered to subjects who have been diagnosed with a mutation in a gene but do not yet have disease symptoms (e.g., infants aged 1 to 12 months or subjects under 2 years of age). In other embodiments, the treatment is administered to individuals with at least one symptom.

[0160] Treatment can be administered to subjects of any age, from infancy to adulthood. Subjects may begin treatment, for example, at birth, six months, or at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, or 18 years of age.

[0161] In some embodiments, the oligonucleotide increases (e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more, or by more than 1.2, 1.4, 1.5, 1.8, 2.0, 3.0, 3.5, 4.5, 5.0, 10, 15, 20, 30, 40, 50, 100, 1000 or more times) the protein activity in vitro and / or in vivo.

[0162] In some embodiments, oligonucleotides increase (e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more, or by more than 1.2, 1.4, 1.5, 1.8, 2.0, 3.0, 3.5, 4.5, 5.0, 10, 15, 20, 30, 40, 50, 100, 1000 or more times) protein activity in the brain.

[0163] In jurisdictions where patentability is prohibited for methods practiced on humans, the meaning of "application" of a composition to a human subject or patient should be limited to any controlled substance that a human subject or patient would administer by themselves using any technique (e.g., oral, inhalation, topical application, injection, insertion, etc.). This is intended to be the broadest reasonable interpretation consistent with the laws or regulations defining registrable patentable subject matter. In jurisdictions where patentability is not prohibited for methods practiced on humans, "application" of a composition includes both the method practiced on humans and the aforementioned activities.

[0164] Pharmaceutical Compositions and Routes of Administration The oligonucleotides described herein are preferably formulated into pharmaceutical compositions for administration to patients (human patients) in a biocompatible form suitable for in vivo administration.

[0165] The oligonucleotides described herein can be administered, for example, orally, parenterally, intrathecally, intraventricularly, intra-organ parenchymally, buccally, sublingually, nasally, rectally, via patch, pump, intratumorally, or percutaneously, as well as in corresponding formulated pharmaceutical compositions. Parenterical administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, intraventricular, intra-organ parenchymal, rectal, and local administration. Parenterical administration can be achieved through continuous infusion over a selected time period.

[0166] The oligonucleotides described herein can be administered orally, for example, with an inert diluent or with an assimilated edible carrier, or the oligonucleotides can be encapsulated in hard or soft-shell capsules, or the oligonucleotides can be compressed into tablets, or the oligonucleotides can be directly incorporated into dietary foods. For oral therapeutic use, the oligonucleotides described herein can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, and flakes. The oligonucleotides described herein can also be administered parenterally. Solutions of the oligonucleotides described herein can be prepared in water with a suitable surfactant, such as hydroxypropyl cellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, DMSO, and mixtures thereof with or without alcohol, as well as in oils. Under normal storage and use conditions, these formulations may contain preservatives to prevent microbial growth. The routine procedures and ingredients for selecting and preparing suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2012, 22nd edition) and The United States Pharmacopeia: The National Formulary (USP 41 NF 36), published in mid-2018. Suitable drug forms for injectable use include sterile aqueous solutions or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid in a manner readily applicable by syringe. Compositions for nasal administration can routinely be formulated as aerosols, drops, gels, and powders. Aerosol formulations typically comprise solutions or fine suspensions of the active substance in physiologically acceptable aqueous or non-aqueous solvents and are typically presented sterilely in single- or multiple-dose amounts in sealed containers, which may be in the form of boxes or refillable for use with nebulizers. Alternatively, the sealed container can be a single dispensing device, such as a single-dose nasal inhaler or an aerosol dispenser equipped with a metering valve intended for post-use disposal. In dosage forms that include aerosol dispensers, the dispenser will contain a propellant, which can be a compressed gas, such as compressed air, or an organic propellant, such as chlorofluorocarbons. Aerosol dosage forms can also be in the form of a pump-nebulizer. Compositions suitable for buccal or sublingual administration include tablets, lozenges, and soft lozenges in which the active ingredient is formulated with a carrier (such as sugar, guar gum, gum, gelatin, and glycerin). Compositions for rectal administration are conveniently presented as suppositories containing a conventional suppository base, such as cocoa butter. The oligonucleotides described herein can also be administered intratumorally, for example, via intratumoral injection. Intratumoral injection involves direct injection into the tumor's blood vessels and is specifically envisioned for discrete, solid, and easily accessible tumors.Local, regional, or systemic application may also be appropriate.

[0167] The oligonucleotides described herein may be administered to animals, such as humans, alone or in combination with pharmaceutically acceptable carriers as mentioned herein, in proportions determined by the solubility and chemical properties of the oligonucleotides, the chosen route of administration, and standard pharmaceutical practice.

[0168] i. Membrane-like molecular assembly and delivery methods The oligonucleotides described herein can be delivered using a variety of membrane-based molecular assembly delivery methods, including polymers, biodegradable microparticles, or microcapsule delivery devices known in the art. For example, colloidal dispersion systems can be used to target and deliver the oligonucleotide agents described herein. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Liposomes are artificial membrane vesicles that can be used as delivery mediators in vitro and in vivo. Large monolayer vesicles (LUVs) ranging in size from 0.2 to 4.0 µm have been shown to encapsulate a considerable percentage of aqueous buffers containing large macromolecules. Liposomes can be used to transfer and deliver active ingredients to the site of action. Because the liposome membrane is structurally similar to a biological membrane, when liposomes are applied to tissues, the liposome bilayer fuses with the cell membrane bilayer. As the fusion of the liposome and cell progresses, the internal aqueous contents, including the oligonucleotides, are delivered into the cell, where the oligonucleotides can specifically bind to target RNA and mediate RNase H-mediated gene silencing. In some cases, liposomes are also specifically targeted, for example, to deliver oligonucleotides to specific cell types. Liposome compositions are typically combinations of phospholipids, often in combination with steroids, particularly cholesterol. Other phospholipids or other lipids may also be used. The physical properties of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0169] Liposomes containing oligonucleotides can be prepared by a variety of methods. In one example, the lipid component of the liposome is dissolved in a detergent, such that micelles are formed together with the lipid component. For example, the lipid component can be an amphiphilic cationic lipid or a lipid conjugate. The detergent can have a high critical micelle concentration and can be nonionic. Exemplary detergents include bile salts, CHAPS, octyl glucoside, deoxycholate, and lauroyl sarcosine. An oligonucleotide formulation is then added to the micelles comprising the lipid component. The cationic groups on the lipids interact with the oligonucleotide and condense around the oligonucleotide to form liposomes. After condensation, the detergent is removed, for example by dialysis, to produce a liposome formulation of the oligonucleotide.

[0170] If desired, a carrier compound that facilitates condensation can be added during the condensation reaction, for example, through controlled addition. For instance, the carrier compound could be a polymer other than nucleic acids (e.g., spermine or spermidine). The pH can also be adjusted to favor condensation.

[0171] Methods for generating stable oligonucleotide delivery mediators incorporating structural components of oligonucleotide / cationic lipid complexes as delivery mediators are further described, for example, in WO 96 / 37194, the entire contents of which are incorporated herein by reference. Liposome formation may also include one or more aspects of the exemplary methods described in the following: Feigner, PL et al., (1987) Proceedings of the National Academy of Sciences 8:7413-7417; U.S. Patent No. 4,897,355; U.S. Patent No. 5,171,678; Bangham et al., (1965) M. Molecular Biology 23:238; Olson et al., (1979) Acta Biochimica et Biophysica Sinica 557:9; Szoka et al., (1978) Proceedings of the National Academy of Sciences 75:4194; Mayhew et al., (1984) Acta Biochimica et Biophysica Sinica 775:169; Kim et al., (1983) Acta Biochimica et Biophysica Sinica 728:339; and Fukunaga et al., (1984) Endocrinol. 115:757. Common techniques for preparing lipid aggregates of appropriate size for use as delivery media include sonication and freeze-thaw extrusion (see, for example, Mayer et al., (1986) *Chinese Journal of Biochemistry and Biophysics* 858:161). When consistently small (50 nm to 200 nm) and relatively homogeneous aggregates are desired, microfluidics can be used (Mayhew et al., (1984) *Chinese Journal of Biochemistry and Biophysics* 775:169). These methods are readily applicable to packaging oligonucleotide formulations into liposomes.

[0172] Liposomes fall into two broad categories. Cationic liposomes are positively charged liposomes that interact with negatively charged nucleic acid molecules to form stable complexes. The positively charged nucleic acid / liposome complexes bind to the negatively charged cell surface and are internalized in endosomes. Due to the acidic pH in the endosomes, the liposomes rupture, thereby releasing their contents into the cytoplasm (Wang et al. (1987), Biochem. Biophys. Res. Commun., 147:980-985).

[0173] Liposomes trap nucleic acids rather than complexing with them. Since both nucleic acids and lipids carry similar charges, repulsion rather than complex formation occurs. However, some nucleic acids are trapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene into cultured cell layers. Expression of the exogenous gene was detected in the target cells (Zhou et al. (1992), *Journal of Controlled Release*, 19:269-274).

[0174] One major type of liposome composition comprises phospholipids other than naturally derived phosphatidylcholine. Neutral liposome compositions may be formed, for example, from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions are typically formed from dimyristoyl phosphatidylglycerol, while anionic fused liposomes are primarily formed from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), such as soybean PC and egg PC. Yet another type is formed from phospholipids and / or mixtures of phosphatidylcholine and / or cholesterol.

[0175] Examples of other methods for introducing liposomes into cells in vitro and in vivo include U.S. Patent Nos. 5,283,185; 5,171,678; WO 94 / 00569; WO 93 / 24640; WO 91 / 16024; Felgner, (1994) Journal of Biol. Chem. 269:2550; Nabel, (1993) Proceedings of the National Academy of Sciences 90:11307; Nabel, (1992) Human Gene Therapy 3:649; Gershon, (1993) Biochem. 32:7143; and Strauss, (1992) Journal of the European Society for Molecular Biology 11:417.

[0176] Nonionic liposome systems were also examined to determine their efficacy in drug delivery to cells, particularly systems involving nonionic surfactants and cholesterol. NOVASOME was used. TM I (Dilaurate / Cholesterol / Polyoxy-10-Stearyl Ether) and NOVASOME TMA nonionic liposome formulation of glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether was used to deliver cyclosporine A into the dermis of mouse skin. Results showed that this type of nonionic liposome system was effective in promoting the deposition of cyclosporine A into different layers of the skin (Hu et al., (1994) STP Pharma. Sci., 4(6):466).

[0177] Liposomes can also be stereostable liposomes comprising one or more specific lipids, which, compared to liposomes lacking such specific lipids, result in enhanced cycle life. An example of a stereostable liposome is one in which a portion of the vesicle-forming lipid portion of liposome (A) comprises one or more glycolipids, such as monosialotetrahexosylganglioside G. M1 (A) or (B) liposomes partially derived from one or more hydrophilic polymers, such as polyethylene glycol (PEG). While not wishing to be bound by any particular theory, it is believed in the art that, at least for spatially stable liposomes containing gangliosides, sphingomyelins, or PEG-derived lipids, the enhanced circulating half-life of these spatially stable liposomes is due to reduced uptake into cells entering the reticuloendothelial system (RES) (Allen et al., (1987) FEBS Letters, 223:42; Wu et al., (1993) Cancer Research, 53:3765).

[0178] Various liposomes, including one or more glycolipids, are known in the art. Papahadjopoulos et al. (Annals of the New York Academy of Sciences, (1987), 507:64) reported monosialotetrahexosylganglioside G M1 Galactocerebroside sulfate and phosphatidylinositol can improve the blood half-life of liposomes. These findings are described in Gabizon et al., Proceedings of the National Academy of Sciences, (1988), 85:6949. Both U.S. Patent Nos. 4,837,028 and WO 88 / 04924 to Allen et al. disclose liposomes comprising: (1) sphingomyelin and (2) ganglioside G. M1 Or galactocerebroside sulfate. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. WO 97 / 13499 (Lim et al.) discloses liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine.

[0179] In one embodiment, cationic liposomes are used. Cationic liposomes have the advantage of being able to fuse with cell membranes. Although non-cationic liposomes cannot fuse efficiently with the plasma membrane, they are absorbed by macrophages in vivo and can be used to deliver oligonucleotides to macrophages.

[0180] Other advantages of liposomes include: liposomes derived from natural phospholipids are biocompatible and biodegradable; liposomes can be incorporated into a variety of water-soluble and lipid-soluble drugs; and liposomes can protect oligonucleotides encapsulated in their internal compartments from metabolism and degradation (Rosoff, “Pharmaceutical Dosage Forms”, Lieberman, Rieger, and Banker (eds.), 1988, Vol. 1, p. 245). Important considerations in the preparation of liposome formulations are lipid surface charge, vesicle size, and the aqueous volume of the liposome.

[0181] The positively charged synthetic cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that spontaneously interact with nucleic acids to form lipid-nucleic acid complexes that can fuse with negatively charged lipids in the cell membrane of tissue cultured cells to deliver oligonucleotides (see, for example, Felgner, PL et al., (1987) Proceedings of the National Academy of Sciences 8:7413-7417; and U.S. Patent No. 4,897,355, which describes DOTMA and its use with DNA).

[0182] The DOTMA analog, 1,2-bis(oleoyloxy)-3-(trimethylamino)propane (DOTAP), can be used in combination with phospholipids to form DNA complex vesicles. LIPOFECTIN TM(Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for delivering highly anionic nucleic acids into living tissue culture cells comprising positively charged DOTMA liposomes that spontaneously interact with negatively charged polynucleotides to form a complex. When sufficiently positively charged liposomes are used, the resulting complex also has a positive net charge. The positively charged complex prepared in this way spontaneously attaches to the negatively charged cell surface, fuses with the plasma membrane, and efficiently delivers functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylamino)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Indiana) differs from DOTMA in that the oleoyl moiety is linked by an ester bond rather than an ether bond.

[0183] Other reported cationic lipid compounds include those conjugated with multiple portions, including, for example, those conjugated with one of two types of lipids and including, for example, 5-carboxy-arginine-glycine-octadecylamide (“DOGS”). TM The compounds such as Promega, Madison, Wisconsin (Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxy-arginyl-amide (“DPPES”) are carboxy-arginine compounds (see, for example, U.S. Patent No. 5,171,678).

[0184] Another type of cationic lipid conjugate includes lipid-cholesterol derivatives (“DC-Chol”), which have been formulated into liposomes in combination with DOPE (see, Gao, X. and Huang, L., (1991) Biochemical and Biophysical Research Communications 179:280). Lipopolylysine, prepared by conjugating polylysine with DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., (1991) Acta Biochimica and Biophysica Sinica 1065:8). For certain cell lines, these liposomes containing conjugated cationic lipids are said to exhibit lower toxicity and provide more efficient transfection compared to compositions containing DOTMA. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, California) and Lipofectamine (DOSPA) (LifeTechnology, Inc., Gaithersburg, Maryland). Other cationic lipids suitable for delivering oligonucleotides are described in WO 98 / 39359 and WO 96 / 37194.

[0185] Liposome formulations are particularly suitable for topical application, exhibiting several advantages over other formulations. These advantages include reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to deliver oligonucleotides to the skin. In some embodiments, liposomes are used to deliver oligonucleotides to epidermal cells and also to enhance the penetration of oligonucleotides into dermal tissues, such as the skin. For example, liposomes can be applied topically. Local delivery of drugs formulated as liposomes to the skin has been documented (see, for example, Weiner et al., (1992) *Journal of Drug Targeting*, Vol. 2, 405-410; du Plessis et al., (1992) *Antiviral Research*, 18:259-265; Mannino, RJ and Fould-Fogerite, S., (1998) *Biotechniques*, 6:682-690; Itani, T. et al., (1987) *Gene*, 56:267-276; Nicolau, C. et al. (1987) *Meth. Enzymol.*, 149:157-176; Straubinger, RM and Papahadjopoulos, D. (1983) *Enzymological Methods*). 101:512-527; Wang, CY and Huang, L., (1987) Proceedings of the National Academy of Sciences 84:7851-7855.

[0186] Nonionic liposome systems were also examined to determine their efficacy in drug delivery to cells, particularly systems comprising nonionic surfactants and cholesterol. Nonionic liposome formulations comprising Novasome I (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver drugs to the dermis of mouse skin. Such formulations containing oligonucleotides may be used to treat dermatological conditions.

[0187] The targeting of liposomes can also be achieved based on factors such as organ specificity, cell specificity, and organelle specificity, and is known in the art. In the case of a liposome-targeted delivery system, lipid groups can be incorporated into the lipid bilayer of the liposome to maintain stable association between the targeting ligand and the liposome bilayer. Various linker groups can be used to bind the lipid chain to the targeting ligand. Other methods are known in the art and described, for example, in U.S. Application Publication No. 20060058255, whose linker groups are incorporated herein by reference.

[0188] Liposomes containing oligonucleotides can be made highly deformable. This deformability allows the liposomes to penetrate through pores smaller than the average radius of the liposome. For example, delivery bodies are another type of liposome and are highly deformable lipid aggregates, which are attractive candidates for drug delivery mediators. Delivery bodies can be described as lipid droplets that are highly deformable, allowing them to easily penetrate through pores smaller than the droplets. Delivery bodies can be prepared by adding a surface-edge surfactant (typically a surfactant) to a standard liposome composition. Delivery bodies containing oligonucleotides can be delivered, for example, via subcutaneous infection to deliver the oligonucleotides to keratinocytes in the skin. To penetrate intact mammalian skin, lipid vesicles must pass through a series of pores less than 50 nm in diameter under the influence of a suitable transdermal gradient. Furthermore, due to their lipid properties, these delivery bodies can self-optimize (adapt to the shape of the pores, such as those in the skin), self-repair, and often reach their targets without fragmentation, and often self-load. Delivery bodies have been used to deliver serum albumin into the skin. Serum albumin delivery mediated by a serum albumin translocator has been shown to be as effective as subcutaneous injection of a serum albumin-containing solution.

[0189] Other formulations suitable for the disclosed oligonucleotides and methods are described in WO 2009 / 086558 and WO2009 / 088891. WO 2008 / 042973 also describes formulations suitable for the oligonucleotides and methods disclosed herein.

[0190] Surfactants are widely used in formulations such as emulsions (including microemulsions) and liposomes. The most common way to classify and rank the properties of many different types of surfactants (both natural and synthetic) is by using the hydrophilic / lipophilic balance (HLB). The properties of the hydrophilic group (also known as the “head”) provide the most useful way to classify the different surfactants used in formulations (Rieger, Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0191] If a surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants are widely used in pharmaceutical and cosmetic products and are available in a wide range of pH values. Typically, their HLB values ​​range from 2 to approximately 18, depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglycerol esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers, such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers, are also included in this category. Polyoxyethylene surfactants are the most popular members of the nonionic surfactant category.

[0192] Surfactants are classified as anionic if their molecules carry a negative charge when dissolved or dispersed in water. Anionic surfactants include carboxylic acid esters, such as soaps, lactic acid esters, acylamides of amino acids, esters of sulfuric acid (such as alkyl sulfates and ethoxylated alkyl sulfates), sulfonates (such as alkyl benzenesulfonates, isothioctanoic acid acyl esters, taurine acyl esters, and succinate sulfonates), and phosphate esters. The most important members of the anionic surfactant category are alkyl sulfates and soaps.

[0193] Surfactants are classified as cationic if their molecules carry a positive charge when dissolved or dispersed in water. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most commonly used members of this category of cationic surfactants.

[0194] Surfactants are classified as amphoteric if their molecules can carry either a positive or negative charge. Amphoteric surfactants include acrylic acid derivatives, substituted alkyl amides, N-alkyl betaines, and phospholipids.

[0195] The use of surfactants in pharmaceutical products, formulations and emulsions is reviewed (Rieger, Pharmaceutical Dosage Forms, Marcel Decker, NY, 1988, p. 285).

[0196] The oligonucleotides used in the methods described herein can also be provided in the form of micelle formulations. Micelles are a specific type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure such that all hydrophobic portions of the molecules face inward, thereby allowing the hydrophilic portions to come into contact with the surrounding water. If the environment is hydrophobic, the opposite arrangement exists.

[0197] In some embodiments, formulations are provided herein comprising the oligonucleotides disclosed herein and pharmaceutically acceptable excipients. In some embodiments, the pharmaceutically acceptable excipients comprise lipid nanoparticles. In some embodiments, the formulations are suitable for use as parenteral formulations.

[0198] In some embodiments, when an amount of the oligonucleotides disclosed herein or a formulation containing such an amount is administered parenterally to a patient, the concentration of the oligonucleotides measured in the patient's tissue is at least a specified percentage of the amount of oligonucleotides initially administered to the patient. The measurement can be performed in any suitable manner known to those skilled in the art, such as by assays or mass spectrometry techniques (e.g., quantitative mass spectrometry) used in the art. In some embodiments, when the oligonucleotides or formulations disclosed herein are administered parenterally to a patient, the concentration of the oligonucleotides in the patient's tissue is at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% of the amount of oligonucleotides administered to the patient. In some embodiments, the concentration of the oligonucleotides in the patient's tissue is at least 40% of the amount of oligonucleotides administered to the patient. In some embodiments, the concentration of the oligonucleotides in the patient's tissue is at least 45% of the amount of oligonucleotides administered to the patient. In some embodiments, the concentration of the oligonucleotides in the patient's tissue is at least 50% of the amount of oligonucleotides administered to the patient. In some embodiments, the concentration of the oligonucleotides in the patient's tissue is at least 55% of the amount of oligonucleotides administered to the patient. In some embodiments, the concentration of the oligonucleotides in the patient's tissue is at least 60% of the amount of oligonucleotides administered to the patient.

[0199] In some embodiments, the amount of oligonucleotides present in the tissue is measured at some time after application of the oligonucleotides disclosed herein or formulations containing said oligonucleotides. In some embodiments, the amount of oligonucleotides is measured at least 24 hours, at least 48 hours, or at least 72 hours after application. In some embodiments, the amount of oligonucleotides is measured at least 24 hours after application. In some embodiments, the amount of oligonucleotides is measured at least 48 hours after application. In some embodiments, the amount of oligonucleotides is measured at least 72 hours after application.

[0200] In some embodiments, when measured in tissue at least 24 hours, at least 48 hours, or at least 72 hours after application, the oligonucleotide concentration in the patient's tissue is at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% of the amount of oligonucleotides administered to the patient. In some embodiments, when measured in tissue at least 24 hours, at least 48 hours, or at least 72 hours after application, the oligonucleotide concentration in the patient's tissue is at least 40% of the amount of oligonucleotides administered to the patient. In some embodiments, when measured in tissue at least 24 hours, at least 48 hours, or at least 72 hours after application, the oligonucleotide concentration in the patient's tissue is at least 45% of the amount of oligonucleotides administered to the patient. In some embodiments, when measured in tissue at least 24 hours, at least 48 hours, or at least 72 hours after application, the oligonucleotide concentration in the patient's tissue is at least 50% of the amount of oligonucleotides administered to the patient. In some embodiments, when measured in tissue at least 24 hours, at least 48 hours, or at least 72 hours after application, the oligonucleotide concentration in the patient's tissue is at least 55% of the amount of oligonucleotides administered to the patient. In some embodiments, when measured in tissue at least 24 hours, at least 48 hours, or at least 72 hours after application, the oligonucleotide concentration in the patient's tissue is at least 60% of the amount of oligonucleotides applied to the patient. In some embodiments, when measured in tissue at least 24 hours after application, the oligonucleotide concentration in the patient's tissue is at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% of the amount of oligonucleotides applied to the patient. In some embodiments, when measured in tissue at least 24 hours after application, the oligonucleotide concentration in the patient's tissue is at least 40% of the amount of oligonucleotides applied to the patient. In some embodiments, when measured in tissue at least 24 hours after application, the oligonucleotide concentration in the patient's tissue is at least 45% of the amount of oligonucleotides applied to the patient. In some embodiments, when measured in tissue at least 24 hours after application, the oligonucleotide concentration in the patient's tissue is at least 50% of the amount of oligonucleotides applied to the patient. In some embodiments, when measured in tissue at least 24 hours after application, the oligonucleotide concentration in the patient's tissue is at least 55% of the amount of oligonucleotides applied to the patient. In some embodiments, when measured in tissue at least 24 hours after administration, the oligonucleotide concentration in the patient's tissue is at least 60% of the amount of oligonucleotide administered to the patient. In some embodiments, when measured in tissue at least 48 hours after administration, the oligonucleotide concentration in the patient's tissue is at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% of the amount of oligonucleotide administered to the patient.In some embodiments, when measured in tissue at least 48 hours after administration, the oligonucleotide concentration in the patient's tissue is at least 40% of the amount of oligonucleotides administered to the patient. In some embodiments, when measured in tissue at least 48 hours after administration, the oligonucleotide concentration in the patient's tissue is at least 45% of the amount of oligonucleotides administered to the patient. In some embodiments, when measured in tissue at least 48 hours after administration, the oligonucleotide concentration in the patient's tissue is at least 50% of the amount of oligonucleotides administered to the patient. In some embodiments, when measured in tissue at least 48 hours after administration, the oligonucleotide concentration in the patient's tissue is at least 55% of the amount of oligonucleotides administered to the patient. In some embodiments, when measured in tissue at least 48 hours after administration, the oligonucleotide concentration in the patient's tissue is at least 60% of the amount of oligonucleotides administered to the patient. In some embodiments, when measured in tissue at least 72 hours after administration, the oligonucleotide concentration in the patient's tissue is at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% of the amount of oligonucleotides administered to the patient. In some embodiments, when measured in tissue at least 72 hours after administration, the oligonucleotide concentration in the patient's tissue is at least 40% of the amount of oligonucleotides administered to the patient. In some embodiments, when measured in tissue at least 72 hours after administration, the oligonucleotide concentration in the patient's tissue is at least 45% of the amount of oligonucleotides administered to the patient. In some embodiments, when measured in tissue at least 72 hours after administration, the oligonucleotide concentration in the patient's tissue is at least 50% of the amount of oligonucleotides administered to the patient. In some embodiments, when measured in tissue at least 72 hours after administration, the oligonucleotide concentration in the patient's tissue is at least 55% of the amount of oligonucleotides administered to the patient. In some embodiments, when measured in tissue at least 72 hours after administration, the oligonucleotide concentration in the patient's tissue is at least 60% of the amount of oligonucleotides administered to the patient.

[0201] ii. Lipid nanoparticle-based delivery methods The oligonucleotides described herein can be completely encapsulated in lipid formulations, such as lipid nanoparticles (LNPs) or other nucleic acid-lipid particles. LNPs are particularly useful for systemic application because they exhibit prolonged circulation life after intravenous (iv) injection and accumulate at distant sites (e.g., sites physically separate from the application site). LNPs include “pSPLP”, which comprises an encapsulated condenser-nucleic acid complex as shown in PCT Publication WO 00 / 03683. The particles of this disclosure typically have an average diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, and most typically about 70 nm to about 90 nm, and are substantially non-toxic. Furthermore, the nucleic acids, when present in the nucleic acid-lipid particles, are resistant to degradation by nucleases in aqueous solutions. Nucleic acid-lipid particles and methods for their preparation are disclosed, for example, in U.S. Patent Nos. 5,976,567, 5,981,501, 6,534,484, 6,586,410, and 6,815,432; U.S. Publication No. 2010 / 0324120; and PCT Publication No. WO 96 / 40964.

[0202] In one embodiment, the lipid to drug ratio (mass / mass ratio) (e.g., lipid to oligonucleotide ratio) will be in the range of about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges between these ranges are also contemplated as part of this description.

[0203] Non-limiting examples of cationic lipids include N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), N,N-distearate-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-(dioleoyloxy)propylamine (DODMA), and 1,2-dilinoleoyloxy-N,N-dimethylamine. 1,2-Dilinoleoyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinoleoylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleoylcarbamoyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dialkylcarbamoyloxy-3-morpholinopropane (DLin-MA), 1,2-dihydrooleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleoylthio-3 -Dimethylaminopropane (DLin-S-DMA), 1-linoleyl-2-linoleyl-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-dihydrooleoyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazine)propane (DLin-MPZ) or 3-(N,N-dioleylamino)-1,2-propanediol (DLinAP), 3-(N,N- Dioleoylamino)-1,2-propanediol (DOAP), 1,2-dioleo-oxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleo-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-DMA) or analogues thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-bis((9Z,12Z)-octadec-9,12-dienyl)tetrahydro- 3aH-cyclopenteno[d][1,3]-dioxacyclopenten-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptadecane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazadiyl)docosahexadecane-2-ol (Tech G1), or mixtures thereof.Cationic lipids may comprise, for example, about 20 mol% to about 50 mol% or about 40 mol% of the total lipids present in the particles.

[0204] Ionizable / non-cationic lipids can be anionic or neutral lipids, including but not limited to distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoylphosphatidylcholine (POPC), and palmitoylphosphatidylethanolamine (P... OPE, dioleoyl-phosphatidylethanolamine 4-(N-maleimidemethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearate-phosphatidyl-ethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol, or mixtures thereof. Non-cationic lipids may constitute, for example, about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% of the total lipids present in the particles (if cholesterol is included).

[0205] The conjugated lipids that inhibit particle aggregation can be, for example, polyethylene glycol (PEG) lipids, including but not limited to PEG-diacylglycerol (DAG), PEG-dialkoxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), or mixtures thereof. PEG-DAA conjugates can be, for example, PEG-1-dilauryloxypropyl (Ci2), PEG-dimyristyloxypropyl (Ci4), PEG-dispalmityloxypropyl (Ci6), or PEG-distearateloxypropyl (Ci8). The conjugated lipids that prevent particle aggregation can comprise, for example, from 0 mol% to about 20 mol% or about 2 mol% of the total lipids present in the particles.

[0206] In some embodiments, the nucleic acid-lipid particles further include cholesterol, for example, cholesterol comprising about 10 mol% to about 60 mol% or about 50 mol% of the total lipids present in the particles.

[0207] dose The dosage of the compositions described herein (e.g., compositions comprising oligonucleotides) can vary depending on many factors, such as the pharmacokinetic properties of the compound; the administration method; the recipient's age, health, and weight; the nature and severity of symptoms; the frequency of treatment and the type of concurrent treatment (if any); and the clearance of the compound in the animal to be treated. Those skilled in the art can determine an appropriate dosage based on these factors. The compositions described herein can be initially administered at a suitable dosage, which can be adjusted as needed based on clinical response. In some embodiments, the dosage of the composition (e.g., a composition comprising oligonucleotides) is a preventative or therapeutically effective amount.

[0208] Reagent test kit This document provides a kit comprising (a) a pharmaceutical composition comprising an oligonucleotide that deamination adenosine in mRNA in cells or a subject as described herein, and (b) a packaging insert having instructions for performing any of the methods described herein. In some embodiments, the kit comprises (a) a pharmaceutical composition comprising an oligonucleotide that deamination adenosine in mRNA in cells or a subject as described herein, (b) an additional therapeutic agent, and (c) a packaging insert having instructions for performing any of the methods described herein. In some embodiments, a kit is provided comprising an oligonucleotide disclosed herein or a formulation comprising an oligonucleotide disclosed herein, and optionally: one or more containers, one or more additional therapeutic agents, a packaging insert having instructions for performing any of the methods described herein, one or more syringes, one or more filter needles, and / or one or more needles for parenteral injection. In some embodiments, the kit comprises an oligonucleotide disclosed herein or a formulation comprising an oligonucleotide disclosed herein, one or more containers, and a packaging insert having instructions for performing any of the methods described herein. In some embodiments, the kit further comprises one or more additional therapeutic agents. In some embodiments, the kit further comprises one or more syringes, one or more filter needles, and / or one or more needles for parenteral injection.

[0209] Example General methods All guide oligonucleotides were chemically synthesized using standard β-cyanoethyl phosphoramide chemistry and universal solid supports, such as controlled-porosity glass (CPG), on an automated RNA / DNA synthesizer. N-protected β-homo-DNA phosphoramide was synthesized using a reported procedure. See Matheus Froeyen et al., (2001) *Chem. Eur. J.*, 7: 5183-5794; Herdewijn, (2010) *Chem. Biodivers.*, 7: 1-59; Jabgunde et al., (2019) *Tetrahedron*, 75: 1107-1114. Other 5'-O-DMT-3'-phosphoramide RNA, 2'-O-methyl RNA, and DNA monomers, namely A, C, G, U, and T, were purchased from commercial sources. When introducing alkylsulfonyl phosphoramidide linkers L1-L15, the corresponding alkylsulfonyl azides were used as oxidants as described in Table 4. After synthesis, the oligonucleotides were cleaved from the solid support, deprotected, and purified using standard protocols via HPLC. The oligonucleotides were desalted, dialyzed, and lyophilized. The purity of each lyophilized oligonucleotide was ≥ 80%, as determined by analytical reversed-phase HPLC. The sequence integrity of the oligonucleotides was determined by ESI-MS. (Sequences of the various oligonucleotides are provided in Tables 5 and 6).

[0210] The human ADAR2 sequence (NM_001112.4) was cloned into the pcDNA3.1 plasmid using BamHI and XbaI restriction sites (Quintara Bio, Berkeley, CA) under the control of the CMV promoter, and the correct insert was validated. This plasmid will therefore be designated ADAR2 / pcDNA3.1. For editing experiments, 25 μL of culture medium was used per 10 cm² culture dish. L of Lipofectamine 3000 and 24 L's P3000 (Life Technologies) will 2 g of ADAR2 / pcDNA3.1 plasmid was transfected into 5 x 10⁻⁶ cells. 6HEK293T cells (American Type Culture Collection (ATCC)). Four hours later, the medium was replenished with freshly heated DMEM high-glucose medium (Lifetechnologies, Inc.). 12–16 hours post-transfection, the transfected HEK293T cells were transfected with guide oligonucleotides to achieve a final concentration of 100 nM in each well. All transfections were performed using Lipofectamine 3000 (0.4 g / L). Cells were transfected in 96-well batches (L / well) according to the manufacturer's instructions. 12–16 hours after the second transfection, cells were washed once with ice-cold PBS, and total mRNA isolation was performed using the DynaBeads mRNA Direct Kit for KingFisher Flex Purification (Lifetech Corporation) according to the manufacturer's instructions. Samples were treated with TURBO DNase (Lifetech Corporation) prior to elution. The resulting isolated mRNA was used for cDNA synthesis using SuperScript IV Vilo according to the manufacturer's instructions (Lifetech Corporation). 1 µl of cDNA was used as a template for PCR using gene-specific primers (Platinum II Hot-Start PCR Master Mixture; Lifetech Corporation) to generate amplicons for Sanger sequencing. Sanger sequencing was performed by Quintiles Biosciences (Berkeley, CA). The editing yield of adenosine to guanosine was quantified by measuring the peak heights of adenosine and guanosine and dividing the guanosine peak height by the combined peak height of adenosine and guanosine.

[0211] Example 1: Synthesis of oligonucleotides The disclosed oligonucleotides were synthesized using standard solid-phase phosphoramidite chemistry at a scale of 200 nM – 25 µM as described below. The monomeric phosphoramidite (amidite) was dissolved in anhydrous acetonitrile at 0.05 M. It was then delivered to a solid-phase synthesis column containing 0.25 M of acetonitrile with 5-(ethylthio)-1H-tetrazole (ETT). Two couplings were performed, each lasting 6 minutes.

[0212] According to Luo L, Tang J, Sun R, et al., (2022) Organic Chemistry Communications ( Org LettAlkylsulfonyl azides were prepared according to procedures described in references such as (15):2821-2825; and the alkylsulfonyl azides were separated after washing with 0.1 M sodium bicarbonate, followed by an aqueous post-treatment of extraction evaporation. The reagent thus prepared was dissolved in dry acetonitrile to produce a final concentration of 0.5 M. During the synthesis cycle, the corresponding alkylsulfonyl azide solution was used as an oxidant when introducing the alkylsulfonyl phosphoramidite (PAX) linker of this disclosure into the continuously growing oligonucleotide sequence (the optimized solid-phase synthesis procedure is shown in Table 4).

[0213] Table 4. Solid-phase synthesis conditions. After synthesis, the solid support was dried under vacuum, and the bound oligonucleotides were cleaved from the support at 55°C with concentrated ammonia for 16 hours. Ammonia was removed under reduced pressure. The product absorbed on the support was reduced in water and separated from the solid support by filtration, and analyzed by LC / MS. The crude solution was purified and desalted by preparative IEx-HPLC. The HELM sequences of the synthesized oligomers are shown in Table 5, and the nucleotide sequences are shown in Table 6.

[0214] Table 5. Synthesized sequences Table 6. Nucleotide Sequences Example 2 - In vitro editing efficiency of 42-meric oligonucleotides with PA1 bonds relative to those without PA1 bonds The efficiency of oligonucleotide editing in Piz mouse hepatocytes was tested in the absence of interferon-alpha. These were tested using RNAiMAX and at desired concentrations without lipofectamine. Piz mouse hepatocytes were thawed in 50 ml tubes containing cryopreserved hepatocyte recovery medium (CHRM-Life Technologies) at 37°C. After centrifugation at 80 xg for 6 min, the supernatant was aspirated, and the cell pellet was resuspended in hepatocyte plating medium (MB Bioscience). Cells were plated at 20,000 cells / well onto 96-well collagen-coated tissue culture plates. Cells were transferred to an incubator (37°C) and, after 4 to 6 hours, the medium was replaced with hepatocyte maintenance medium (MB Bioscience), and cells were transfected with ASO at the desired concentration with or without RNAiMAX (Life Technologies, California), according to the manufacturer's protocol, and then returned to the incubator.

[0215] Forty-eight hours after the addition of oligonucleotides, mRNA was isolated from PiZ hepatocytes using Oligo(dT)25 magnetic beads and relevant buffer from New England Biolabs. Following elution, the samples were processed with EZ DNase (Lifetech Corporation). The resulting isolated mRNA was used for cDNA synthesis using SuperScript IV VILO™ according to the manufacturer's instructions (Lifetech Corporation). 10 µl of cDNA was used for next-generation sequencing (NGS), i.e., amplicon sequencing, by Quintiles Biotechnology.

[0216] DNA amplicon was used directly for amplicon next-generation sequencing (NGS). The percentage of editing at the site of interest was quantified based on NGS counts, as a percentage of the number of edited nucleotides. Each oligonucleotide was measured in at least three replicates. Primers used for sequencing are described in Table 7, and the data used for the measurements are presented in… Figure 1A and 1B middle.

[0217] The results of free uptake comparisons using sequences (42-mer A1 and 30-mer A12) showed that editing activity remained unchanged when PA-1 was replaced with the L1 or L2 backbone. Figure 1A The transfection results showed that the new L1 and L2 backbones exhibited similar editing activity. Figure 1B ).

[0218] Table 7. PCR and sequencing primers Oligonucleotides containing L4 and L15 bonds were studied in the same manner, and the editing percentage was compared with that of two controls (oligonucleotides A1 and A1a) containing only PS bonds. In both cases where the oligonucleotides contained one L4 bond at the 3' end and two L4 bonds at the 5' end, at a dose of 100 nM, oligonucleotides containing L4 bonds showed significantly higher editing than A1. Oligonucleotides containing L15 bonds were edited in a similar manner to the control oligonucleotide A1, and oligonucleotides containing either L4 or L15 bonds were edited in a similar manner to the control oligonucleotide A1a. Data are shown in... Figure 5 and 6 middle.

[0219] These data indicate that replacing PA1 with L4 or L15 produces comparable editing efficiency, and in some cases, editing efficiency is improved with L4.

[0220] Additional data on oligonucleotides that use L1 instead of PA1 are given in Table 8 below.

[0221] Table 8 Further studies were conducted in DefiniGEN ZZ HLC hepatocytes.

[0222] Hepatocyte-like cells (HLCs) derived from ZZ genotype patients (DefiniGEN) were thawed at 37°C using a thawing medium consisting of minimum essential medium (MEM, Giboc 51200-046), MEM non-essential amino acid solution (11140-050), a chemically defined lipid concentrate (Giboc 11905-031), insulin (Sigma-Aldrich 11376497001), and Def-HEP components A, B, C, D, and H (DefiniGEN). Cells were seeded using Def-HEP recovery and maintenance medium, which consisted of HepatoZYME SFM (Gibco 17705-021), MEM non-essential amino acid solution, chemically defined lipid concentrate, insulin, and Def-HEP components A, B, C, E, F, and G (DefiniGEN) with a Rock inhibitor (Stemcell 72304). The Rock inhibitor was used only for seeding and not for refeeding. Cells were seeded onto 384-well collagen I-coated plates and incubated at 37°C in a hypoxic incubator. The medium was changed every 48 hours after seeding until the cells matured in 12–14 days. For transfection, HLCs were treated with oligonucleotides and a transfection reagent consisting of OPTI-MEM (Gibco 31985-0662) and Lipofectamine RNAiMAX (Invitrogen 13778-150) at a 1:25 ratio.

[0223] The results are shown in Table 9 below.

[0224] Table 9 Example 3 - In vitro editing efficiency of 30-meric oligonucleotides with PA1 bonds relative to those without PA1 bonds The editing efficiency of oligonucleotides in primary normal mouse hepatocytes was tested in the absence of interferon-alpha. These were tested using RNAiMAX and at desired concentrations without the presence of lipofectamine. Primary mouse hepatocytes were thawed in 50 ml tubes containing cryopreserved hepatocyte recovery medium (CHRM-Life Sciences) at 37°C. After centrifugation at 80 xg for 6 min, the supernatant was aspirated, and the cell pellet was resuspended in hepatocyte plating medium (MB Biosciences). Cells were plated at 20,000 cells / well on 96-well collagen-coated tissue culture plates or at 5,000 cells / well on 384-well collagen-coated tissue culture plates. The cells were transferred to an incubator (37°C) and after 4 to 6 hours, the culture medium was replaced with hepatocyte maintenance medium (MB Biosciences). The cells were then transfected with ASO at the desired concentration with or without RNAiMax (Life Technologies, California) according to the manufacturer’s protocol, and the cells were returned to the incubator.

[0225] The results are shown in Figure 7 The transfection data are shown in (A), and the free uptake data are shown in (B).

[0226] Compared to editing via free uptake, editing of the test oligonucleotides via transfection was more comparable to editing performed on controls. In vivo data from this study indicate that almost all editing performed on the test oligonucleotides was comparable to editing performed on controls, with some test oligonucleotides being superior to controls. Given that L1 and L2 were performed with the aim of enhancing stability and improving nuclease resistance, they are expected to have the greatest effect in vivo. Figure 7 and Figure 8 The combination of in vitro and in vivo results shown in the figure demonstrates that L1 and L2 enhance stability and are viable alternatives to PA-1, with improvements in certain scenarios.

[0227] Example 4 - In vivo editing efficiency of oligonucleotides with aminophosphate linkers disclosed herein In vivo delivery – All procedures used in animal experiments were approved by the Institutional Animal Care and Use Committee and performed according to its guidelines. PiZ male mice (8–10 weeks old) were weighed and randomly assigned to groups of three mice each. The LNP formulation in MC3 buffer was administered via the lateral tail vein at a dose of 2 mg / kg, in a volume of 0.2 mL per animal. Animals were euthanized by exsanguination, and blood was collected via cardiac puncture using a 1 mL syringe with a 25G needle. At least 500 µL of whole blood was collected and transferred to a serum micro-blood collection tube. Liver tissue was also extracted from the mice.

[0228] Mulch mouse liver tissue samples using the 2010 Geno / Grinder® following the supplier's manual. Transfer 5–10 mg of pulverized tissue to a Fisherbrand™ 24-bead homogenizer tube and homogenize with Trizol. Add chloroform to each sample. After centrifugation, transfer the aqueous layer to a silica membrane RNeasy 96-well plate. Perform RNA extraction in a Qiacube HT following the supplier's standard miRNeasy protocol. Measure the isolated mRNA in a nanodrop and use up to 1 µg of RNA from each sample to generate cDNA. Add 4 µL of SupersciptVILO IV master mixture, 14 µL of RNase-free water, and 2 µL of diluted RNA (up to 1 µg of RNA) to each reaction mixture in a 96-well plate. Seal the plate and centrifuge at 1000 rpm for 1 min, allowing incubation in a thermal cycler at 25°C for 10 min, 50°C for 10 min, and 85°C for 5 min. On the QIAcuity eight dPCR system, dPCR was performed using the QIAcuity Probe PCR Kit following the vendor's protocol on 8.5 kb 96-well QIAcuity nanoplates. Primers and probes for WT and mutant SerpinA1 are listed in Table 10. Raw data were analyzed in the Qiagen Plate Configurator, and the percentage of edits was calculated based on the concentration of each target.

[0229] Table 10. Primers used for in vivo editing assays The percentage of edits in PiZ mouse liver tissue was assessed at days 4, 7, and 14 following a single dose of oligonucleotide A3 encapsulated in MC3 LNP. PiZ mice (n = 3) were administered a single intravenous dose of either the carrier (DPBS) or ASO as indicated at 2 mg / kg. Liver tissue was collected at days 4, 7, and 14 post-administration. The percentage of edits was assessed at each time point by performing NGS (next-generation sequencing) on ​​mRNA collected from the fragmented liver tissue. The percentage of edits data at each time point (e.g., measured via E342K) are presented in [data missing]. Figure 2 In vivo administration of oligonucleotide A3 using MC3 resulted in high levels of M-A1AT concentrations in serum.

[0230] At all time points, similar or equivalent editing activity was observed when PA1 was replaced by L2, and this activity was also maintained when PA1 was replaced by L1. Regarding both wild-type A1AT concentration and the percentage of wild-type A1AT in serum, oligonucleotide A1 and the modified oligonucleotide showed similarities at day 4 (peak), day 7, and day 14 (final time point). Regarding wild-type A1AT production and the percentage of wild-type A1AT in serum, oligonucleotide A12 tended to be higher than the modified oligonucleotide for both editing activity and wild-type A1AT concentration.

[0231] The results are shown in Figure 3A (Compare oligonucleotides A1, A10, and A11) and Figure 3B (Compare oligonucleotides A12, A13 and A14).

[0232] Example 5: In vitro stability determination of mouse liver homogenate The stability of different oligonucleotides in mouse liver homogenate was evaluated.

[0233] Approximately 100 mg of PiZ mouse liver powder was added to a 2 mL homogenization vial containing beads, using approximately 6 beads. For every 1 g of liver powder, 7 mL of stability buffer was added. The sample was briefly homogenized, and all homogenates were combined into 15 mL centrifuge tubes. 10 μL of MgCl2 and 10 μL of Anti-Anti were added to each 1 mL of homogenate. The homogenate was aliquoted into 1.5 mL Eppendorf tubes, triplicate for each time point and each oligonucleotide. The homogenate was pre-incubated at 37°C for 0.5 h, after which the oligonucleotide test product was added. 2 μL of 100 μM oligonucleotide stock solution was added to each 100 μL of homogenate. The sample was incubated at 37°C until the desired time point was reached. To quench at each time point, 100 μL of stop buffer was added, the sample was vortexed, and the sample was frozen at -80°C.

[0234] Sample extraction (LLE) Remove the samples from the freezer and thaw them in water for 5 minutes, followed by a brief vortex. Add 10 µL of KB6400 (25 µg / mL) to each sample and vortex again. Add 400 µL of 10% ammonium hydroxide to each sample. Next, add 400 µL of phenol / chloroform / isoamyl alcohol to each sample. Cap the samples and then mix thoroughly using a multi-tube vortex mixer (2 minutes), followed by individual vortexing. Centrifuge the samples at 8,000 g (5194 rpm) at room temperature for 30 minutes. Next, remove approximately 600 µL of the top aqueous solution from each sample and transfer it to a clean, labeled 1.4 mL Matrix vial. Discard the bottom organic layer and add 300 µL of chloroform to each sample.

[0235] Cap the samples and then mix thoroughly using a multi-tube vortex mixer (2 minutes), followed by individual vortexing. Next, centrifuge the samples at 8,000 g (5194 rpm) at room temperature for 30 minutes. Remove approximately 500 µL of the top aqueous layer from each sample and transfer it to a clean, labeled 1.4 mL vial. Discard the bottom organic layer.

[0236] Sample evaporation and concentration Uncap the sample and place it in a commercial concentrator system with the following settings: top flow: 40 L / min; top temperature: 40 °C; bottom flow: 40 L / min; bottom temperature: 40 °C. Allow the sample to dry to approximately 200 µL. Then, transfer 70 µL of the sample extract to a 96-well plate for LC / HRAM-MS analysis. Freeze any remaining sample extract at -80 °C. Then subject the extracted sample to LC-MS analysis.

[0237] At two edit percentages and wild-type A1AT concentrations ( Figure 4A Regarding edit percentage and wild-type A1AT% in serum, the 42-meric oligonucleotide A1 containing a PA1 bond and the modified oligonucleotides containing L2 and L3 bonds showed similar performance on days 4 (peak), 7, and 14 (final time point). In terms of edit percentage and wild-type A1AT yield and wild-type A1AT% in serum, the 30-meric oligonucleotide A12 containing a PA1 bond tended to be higher than the modified oligonucleotides. The L2 and L3 modified 30-meric oligonucleotide analogs A13 and A14 maintained good edit and wild-type A1AT concentrations until day 14. Figure 4B ).

[0238] The results are shown in Table 11, and the trends of oligonucleotides A1-A4 are shown in... Figure 4A In, and the trend of oligonucleotides A5-A7 is shown in Figure 4B middle.

[0239] L1 and L2 oligonucleotides exhibited improved metabolic stability compared to the PS-containing control. The percentage of the remaining portion of the 3' and 5' oligonucleotide backbone replaced by L1 (A3) and L2 (A4) in A1 (PA-1) was increased (43% and 47% respectively compared to 30% at day 3). The percentage of the remaining portion of the 3' edge replaced by L1 (A5) and L2 (A6) in oligonucleotide A9 (PA-1) was increased on days 2 and 3. The percentage of the remaining portion of both the 3' and 5' edges replaced by L1 (A7) and L2 (A8) in oligonucleotide A9 (PA1) was increased on days 2 and 3. In early stability data for the 42-mer analogues of oligonucleotide A1, none showed improvement, including designs with additional PS bonds. Taken together, the above data suggest that the L1 and L2 bonds provide superior stability compared to the PS and PA-1 moieties.

[0240] Table 11. In vitro metabolic stability over time Example 6 - Editing of ACTb oligonucleotides in the liver The following example evaluates editing in the WT C57BL / 6 mouse model after administration of 5 mg / kg once daily for 5 days.

[0241] Female or male C57BL / 6 mice (6–10 weeks old) were weighed and randomly assigned to groups of three mice each. GalNAc formulation was administered subcutaneously at a volume of 0.2 mL per animal. Animals were euthanized by exsanguination 7 days after the last administration or on study day 11. In vivo editing was measured in liver tissue collected from the median or left lateral lobe of the euthanized mice. Tissue was collected in a cryogenic grinding jar and rapidly frozen on dry ice. The liver was ground into a fine powder to isolate RNA, thereby assessing editing activity.

[0242] Editing data in the liver is shown in Figure 8 middle.

Claims

1. An oligonucleotide comprising 10 to 300 nucleotides, each nucleotide comprising a sugar moiety, a nucleobase, and an internucleotide bond, wherein at least one internucleotide bond is a PAX internucleotide bond having the structure of formula (I): （I）， in R 1 It is isopropyl, isobutyl, sec-butyl, C 1-6 Haloalkyl, C 2-6 Hydroxyalkyl, C 2-8 Alkylene-N(R) N 2. C 0-2 Alkylene-C 3-8 The heterocyclic alkyl group, a 4-10 membered heterocyclic alkyl group having 1-3 cyclic heteroatoms selected from O, N, and S, or a 5-10 membered heteroaryl group having 1-3 cyclic heteroatoms selected from O, N, and S, provided that the heterocyclic alkyl group or the heteroaryl group is attached to sulfur via a carbocyclic atom, and the heterocyclic alkyl group, the heterocyclic alkyl group, or the heteroaryl group is surrounded by 0, 1, 2, or 3 R atoms. 2 Group substitution; Each R 2 Independently halogenated, CN, N(R) N 2. C 1-3 Alkyl, C 1-3 Haloalkyl, C 1-3 Alkoxy, oxo, CO2R N or C(O)C 1-3 Alkyl; and Each R N Independently H or C 1-3 alkyl.

2. The oligonucleotide according to claim 1, wherein R 1 It is isopropyl.

3. The oligonucleotide according to claim 1, wherein R 1 C 3-8 Cycloalkyl.

4. The oligonucleotide according to claim 3, wherein R 1 It is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

5. The oligonucleotide according to claim 3 or 4, wherein R 1 It is cyclopropyl.

6. The oligonucleotide according to claim 3, wherein R 1 It is spiro[3.3]heptyl.

7. The oligonucleotide of claim 1, wherein R 1 C 1-6 Halogenated alkyl or C 1-6 Hydroxyalkyl.

8. The oligonucleotide of claim 7, wherein R 1 C 1-6 Fluoroalkyl groups.

9. The oligonucleotide according to claim 7 or 8, wherein R 1 It is CH2F or CHF2.

10. The oligonucleotide of claim 1, wherein R 1 It is a 4-8 membered heterocyclic alkyl group.

11. The oligonucleotide of claim 10, wherein R 1 It is azacyclobutane, pyrrolidine, piperidine, oxacyclobutane, tetrahydrofuran, or tetrahydropyran.

12. The oligonucleotide of claim 1, wherein R 1 It consists of 5-10 heteroaryl groups.

13. The oligonucleotide of claim 12, wherein R 1 It can be furan, thiophene, thiazole, isoxazole, imidazole, pyridine, or pyrazine.

14. The oligonucleotide according to any one of claims 5 and 10 to 13, wherein R 1 It is not replaced.

15. The oligonucleotide according to any one of claims 5 and 10 to 13, wherein R 1 By 1, 2 or 3 R 2 replace.

16. The oligonucleotide of claim 15, wherein R 1 By 1 R 2 replace.

17. The oligonucleotide of claim 15, wherein R 1 By 2 R 2 replace.

18. The oligonucleotide according to any one of claims 15 to 17, wherein at least one R 2 It can be oxidized, CO2H-substituted, or halogenated.

19. The oligonucleotide according to any one of claims 15 to 18, wherein at least one R 2 It is fluorinated.

20. The oligonucleotide of claim 1, having at least one PAX nucleotide inter-bond as shown in Table 1.

21. The oligonucleotide according to any one of claims 1 to 20, having 30-100% phosphate thioester, aminophosphate ester and PAX nucleotide internucleotide bonds.

22. The oligonucleotide according to claim 21, wherein it has 30-70% phosphate thioester, aminophosphate and PAX nucleotide internucleotide bonds.

23. The oligonucleotide according to claim 21 or 22, having 40-60% phosphate thioester, aminophosphate ester and PAX nucleotide internucleotide bonds.

24. The oligonucleotide according to any one of claims 1 to 23, comprising a PAX internucleotide bond.

25. The oligonucleotide according to any one of claims 1 to 23, comprising 2 to 10 PAX nucleotide inter-bonds.

26. The oligonucleotide according to any one of claims 1 to 25, comprising a PAX nucleotide internucleotide bond located between the 3' terminal nucleotide and the 3'-1 nucleotide of the oligonucleotide.

27. The oligonucleotide according to any one of claims 1 to 26, comprising a PAX nucleotide internucleotide bond located between the 5' terminal nucleotide and the 5'-1 nucleotide of the oligonucleotide.

28. The oligonucleotide of claim 27, comprising a PAX nucleotide bond between the 3' terminal nucleotide and the 3'-1 nucleotide of the oligonucleotide and a PAX nucleotide bond between the 5' terminal nucleotide and the 5'-1 nucleotide of the oligonucleotide.

29. The oligonucleotide according to any one of claims 1 to 28, wherein at least one nucleotide bond is a phosphate thioester.

30. The oligonucleotide according to any one of claims 1 to 29, wherein at least one nucleotide internucleotide bond is a methanesulfonylaminophosphate.

31. The oligonucleotide according to any one of claims 1 to 30, comprising 25 to 100 nucleotides.

32. The oligonucleotide according to claim 31, comprising 25-60 nucleotides.

33. The oligonucleotide according to claim 30 or 31, comprising 30-52 nucleotides.

34. The oligonucleotide according to any one of claims 1 to 33, wherein at least one sugar moiety is ribose or modified ribose.

35. The oligonucleotide according to any one of claims 1 to 34, wherein each sugar moiety is independently 2'-methoxy-ribose, 2'-MOE-ribose, 5'-methyl-2'-deoxyribose, 2'-deoxy-2'-fluororibose, 2'-fluoro-arabinose, 2-methoxy-arabinose, 2'-deoxyribose, locked nucleic acid (LNA), or deoxyhexose.

36. The oligonucleotide according to any one of claims 1 to 35, further comprising one or more targeting moieties.

37. The oligonucleotide of claim 36, wherein the one or more targeting portions comprise lipids, sterols, carbohydrates, vitamins and / or peptides.

38. The oligonucleotide of claim 36 or 37, wherein the one or more targeting moieties comprise thyroid-stimulating hormone, melanocyte-stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, ASGPR binding moiety, N-acetyl-galactosamine (GalNAc) moiety, N-acetyl-glucosamine polymannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, polyglutamate, polyaspartate, cholesterol, bile acids, folic acid, vitamin B12, vitamin A, biotin, RGD peptide, or RGD peptide mimicry.

39. The oligonucleotide of claim 38, wherein the ASGPR binding moiety is N-acetyl-galactosamine (GalNAc) or N-acetyl-glucosamine polymannose.

40. The oligonucleotide according to any one of claims 35 to 39, wherein the one or more targeting moieties comprise an N-acetyl-galactosamine (GalNAc) moiety.

41. The oligonucleotide according to any one of claims 36 to 40, wherein the one or more targeting portions are located at the 5' end, the 3' end, or both of the oligonucleotide.

42. The oligonucleotide of claim 41, wherein the one or more targeting portions are located at the 5' end of the oligonucleotide.

43. The oligonucleotide of claim 41 or 42, wherein the one or more targeting portions are located at the 3' end of the oligonucleotide.

44. The oligonucleotide according to any one of claims 36 to 43, wherein one or more targeting portions are connected to the oligonucleotide via a linker portion.

45. The oligonucleotide of claim 44, wherein the linker portion comprises an alkylene oxide, a polyalkylene oxide, and / or a peptide portion.

46. ​​The oligonucleotide according to any one of claims 1 to 45, wherein the oligonucleotide is a structural gene, a gene including a control region and a termination region, a self-replicating system such as viral DNA or plasmid DNA, a single-stranded or double-stranded RNAi agent, shRNA, antisense oligonucleotide, ribozyme, microRNA, microRNA mimic, supermir, aptamer, antimir, antagomir, adaptor, triplet-forming oligonucleotide, tRNA (transfer RNA), G-quadruplex oligonucleotide, RNA activator, immunostimulatory oligonucleotide, or decoy oligonucleotide.

47. The oligonucleotide according to any one of claims 1 to 46, having a sequence that is fully complementary to the target RNA.

48. The oligonucleotide of claim 47, wherein the target RNA is selected from Serpina1, LRRK2, NRF2, TDP-43, Nav1.7 and PCKS9.

49. A formulation comprising an oligonucleotide according to any one of claims 1 to 48 and a pharmaceutically acceptable excipient.

50. The formulation of claim 49, wherein the pharmaceutically acceptable excipient comprises lipid nanoparticles.

51. The formulation according to claim 49 or 50, which is suitable for use as a parenteral formulation.

52. The formulation according to claim 51, wherein when administered parenterally to a patient, the concentration of oligonucleotides in the patient's tissues is at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% of the amount of oligonucleotides administered to the patient.

53. The formulation of claim 52, wherein when measured in tissue at least 24 hours, at least 48 hours, or at least 72 hours after administration, the concentration of oligonucleotides in the patient's tissue is at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% of the amount of oligonucleotides administered to the patient.

54. The formulation according to claim 52 or 53, wherein when measured in tissue at least 72 hours after parenteral administration, the concentration of oligonucleotides in the patient's tissue is at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% of the amount of oligonucleotides administered to the patient.

55. A method of treating a disease or condition in a patient in need, the method comprising administering to the patient a therapeutically effective amount of an oligonucleotide according to any one of claims 1 to 48 or a formulation according to any one of claims 49 to 54.

56. The method of claim 55, wherein the disease or condition is cystic fibrosis, albinism, α-1-antitrypsin deficiency, Alzheimer's disease, amyotrophic lateral sclerosis, asthma, 11-thalassemia, Cadasil syndrome, Charcot-Marie-Tooth disease, chronic obstructive pulmonary disease, distal spinal muscular atrophy, Duchenne / Becker muscular dystrophy, dystrophic epidermolysis bullosa, epidermolysis bullosa, Fabry disease, Factor V Leiden associated disorder, familial adenoma, polyposis, galactosemia, Gaucher's disease, glucose-6-phosphate dehydrogenase deficiency, hemophilia, hereditary hemochromatosis, Hunter syndrome. Syndrome, Huntington's disease, Hurler syndrome, inflammatory bowel disease, hereditary polyagglutination syndrome, Leber congenital amaurosis, Lesch-Nyhan syndrome, Lynch syndrome, Marfan syndrome, mucopolysaccharidosis, muscular dystrophy, myotonic dystrophy type I and II, neurofibromatosis, Niemann-Pick disease type A, B and C, NY-NY-ESO-1 related cancers, Parkinson's disease, Peutz-Jeghers syndrome, phenylketonuria, Pompe's disease Diseases, primary ciliary disease, prothrombin mutation-related diseases, pulmonary hypertension, retinitis pigmentosa, Sandhoff's disease, severe combined immunodeficiency syndrome, sickle cell anemia, spinal muscular atrophy, Stargardt's disease, Tay-Sachs disease, Usher syndrome, X-linked immunodeficiency, Sturge-Weber syndrome, Rett syndrome, or cancer.

57. A kit comprising an oligonucleotide according to any one of claims 1 to 48 or a formulation according to any one of claims 49 to 54.

58. The kit according to claim 57, further comprising one or more containers.

59. The kit according to claim 57 or 58, further comprising one or more additional therapeutic agents.

60. The kit according to any one of claims 57 to 59, further comprising a packaging insert having instructions for performing the method according to claim 55 or 56.

61. The kit according to any one of claims 57 to 60, further comprising one or more syringes, one or more filter needles and / or one or more needles for parenteral injection.

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