Compositions and methods for inhibiting gene expression in the central nervous system - Patents.com

Abstract

Provided herein are oligonucleotide conjugates that inhibit or reduce expression of a target gene in the CNS. Also provided are compositions containing the same and uses thereof, particularly for treating diseases, disorders and / or conditions associated with target gene expression in the CNS.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 142,877, filed January 28, 2021, the contents of which are incorporated herein by reference.

[0002] The present disclosure relates to nucleic acid-hydrophobic ligand conjugates and oligonucleotide-hydrophobic ligand conjugates.Specifically, the present disclosure relates to nucleic acid-lipid conjugates and oligonucleotide-lipid conjugates, their preparation methods, their chemical composition, and the method of using the nucleic acid and oligonucleotide conjugated according to the description provided herein to regulate (e.g., inhibit or reduce) the expression of target genes in the central nervous system (abbreviated as "CNS" herein) (e.g., cells, tissues, or regions of CNS).The present disclosure also provides pharma-ceutically acceptable compositions comprising the conjugates described herein, and the method of using the aforementioned compositions in the treatment of various diseases or disorders. [Background technology]

[0003] Modulation of gene expression by modified nucleic acids has shown great potential both as a research tool in the laboratory and as a therapeutic approach in the clinic. Several classes of oligonucleotide or nucleic acid-based therapeutic agents are in clinical trials, including antisense oligonucleotides (ASOs), short interfering RNA (siRNA), double-stranded nucleic acids (dsNAs), aptamers, ribozymes, exon skipping and splice-altering oligonucleotides, immune-modulating oligonucleotides, mRNA, and CRISPR. Chemical modifications in the relevant molecules play an important role in overcoming the challenges of oligonucleotide therapeutic agents, including improving nuclease stability, RNA binding affinity, and pharmacokinetics. Various chemical modification strategies for oligonucleotides have been developed in the past 30 years, including modifications of sugars, nucleobases, and phosphodiester backbones to improve and optimize performance and therapeutic efficacy ([Non-Patent Document 1], [Non-Patent Document 2], and [Non-Patent Document 3]).

[0004] Therapeutic gene silencing mediated by RNAi oligonucleotide-based therapeutics, including siRNA or double-stranded nucleic acid (dsNA), offers the possibility of a substantial expansion of the druggable target space and the possibility of treating rare diseases that are inaccessible to treatment by other drug modalities (e.g., antibodies and / or small molecules). RNAi oligonucleotide-based therapeutics that inhibit or reduce the expression of specific target genes in the liver have been developed and are currently in clinical use ([Non-Patent Document 4]). Technical hurdles remain for the development and clinical use of RNAi oligonucleotides in extrahepatic cells, tissues, and organs (e.g., the central nervous system or CNS). Therapeutic gene silencing mediated by RNAi oligonucleotide-based therapeutics in the CNS is of particular interest for treating neurological diseases ([Non-Patent Document 5]). Thus, there is a continuing need in the art for the successful development of novel and effective RNAi oligonucleotides to regulate the expression of target genes in extrahepatic cells, tissues, and / or organs (e.g., the CNS). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Deleavey and Darma,CHEM.BIOL.2012,19(8):937-54 [Non-Patent Document 2] Wan and Seth,J.MED.CHEM.2016,59(21):9645-67 [Non-Patent Document 3] Egli and Manoharan,ACC.CHEM RES.2019,54(4):1036-47 [Non-Patent Document 4] Sehgal et al.,(2013)JOURNAL OF HEPATOLOGY59:1354-1359 [Non-Patent Document 5] Boudreau&Davidson(2010)BRAIN RESEARCH1338:112-21 Summary of the Invention

[0006] The mammalian CNS is a complex system of tissues, including cells, fluids, and chemicals, which work together to interact to enable a wide range of functions, including movement, navigation, cognition, speech, vision, and emotion. Unfortunately, a variety of diseases and disorders of the CNS (e.g., neurological disorders) are known that affect or disrupt some or all of these functions. Typically, treatments for diseases and disorders of the CNS are limited to small molecule drugs, antibodies, and / or adaptive or behavioral therapies. There is a continuing need to develop treatments for diseases and disorders of the CNS associated with inappropriate gene expression. Thus, the present disclosure is directed to compositions comprising oligonucleotide-ligand conjugates (e.g., RNAi oligonucleotide conjugates) that include one or more hydrophobic moieties that modulate (e.g., reduce or inhibit) target gene expression in the CNS. The present disclosure is also directed to methods of preparation and methods of use and treatment of the aforementioned oligonucleotide conjugates.

[0007] The present application relates to novel nucleic acids, oligonucleotides, or analogs thereof, containing hydrophobic ligands, including but not limited to adamantyl and lipid conjugates. The present disclosure relates to nucleic acid-lipid conjugates and oligonucleotide-lipid conjugates that function to regulate the expression of target genes in cells, as well as methods of preparation and use thereof. Without being bound by theory, lipophilic / hydrophobic moieties, such as fatty acids and adamantyl groups, when attached to these highly hydrophilic nucleic acids / oligonucleotides, substantially enhance plasma protein binding, resulting in increased circulatory half-life. The conjugated nucleic acids, oligonucleotides, and analogs thereof provided herein are stable and bind to RNA targets to trigger extensive extrahepatic RNase H activity, and are also useful for splice switching and RNAi. Without intending to be bound by theory, the incorporation of hydrophobic moieties, such as lipids, facilitates systemic delivery of novel nucleic acids, oligonucleotides, or analogs thereof to several tissues, including but not limited to the CNS, muscle, fat, and adrenal gland.

[0008] Suitable nucleic acid-hydrophobic ligand conjugates and oligonucleotide-hydrophobic ligand conjugates include nucleic acid inhibitor molecules such as dsRNA inhibitor molecules, dsRNAi inhibitor molecules, antisense oligonucleotides, miRNA, ribozymes, antagomirs, aptamers, and single-stranded RNAi inhibitor molecules. In particular, the present disclosure provides nucleic acid-lipid conjugates, oligonucleotide-lipid conjugates, and analogs thereof that find utility as regulators of intracellular RNA levels. The nucleic acid inhibitor molecules of the present disclosure regulate RNA expression through a diverse array of mechanisms, for example, by RNA interference (RNAi). An advantage of the nucleic acid-hydrophobic ligand conjugates, oligonucleotide-hydrophobic ligand conjugates, and analogs thereof provided herein is that they are capable of a wide range of pharmacological activities consistent with regulating intracellular RNA levels. In addition, the present disclosure provides methods of using an effective amount of the conjugates described herein for the treatment or amelioration of disease conditions by regulating intracellular RNA levels.

[0009] The present disclosure relates to the RNAi oligonucleotide conjugate, which comprises one or more nucleic acid-ligand conjugate units that regulate target gene expression in CNS through RNA interference (RNAi).In particular, the present disclosure relates to the novel RNAi oligonucleotide conjugate, which comprises one or more hydrophobic moiety ligands, including but not limited to lipid moieties, that regulate (e.g., reduce or inhibit) target gene expression in CNS, the composition of the aforementioned RNAi oligonucleotide conjugate, and the preparation method and use thereof.

[0010] The present disclosure is based, at least in part, on the discovery of RNAi oligonucleotide-lipid conjugates that effectively reduce target gene expression in the CNS for extended periods of time. The exemplary RNAi oligonucleotide-lipid conjugates provided herein have demonstrated sustained reduction of target gene expression in the CNS for multiple months after a single administration. Furthermore, the exemplary RNAi oligonucleotide-lipid conjugates provided herein have demonstrated pharmacological activity in multiple regions throughout the CNS. Without being bound by theory, the hydrophobic moiety (e.g., lipid) facilitates the delivery and distribution of the RNAi oligonucleotide-lipid conjugate to the CNS, thereby increasing the efficacy and persistence of gene knockdown. Furthermore, the present disclosure provides RNAi oligonucleotide-lipid conjugates that effectively reduce target gene expression in the CNS without reducing target gene expression in the liver. Thus, the present disclosure provides a method of treating a disease or disorder by regulating target gene expression in the CNS using the RNAi oligonucleotide-lipid conjugates described herein and their pharma- ceutical acceptable compositions. The disclosure further provides methods of using the RNAi oligonucleotide-lipid conjugates in the manufacture of a medicament for treating a disease or disorder by modulating target gene expression in the CNS.

[0011] RNAi has rapidly evolved as a tool for directional gene silencing. The RNAi machinery present in cells can be co-opted in many ways to achieve gene expression knockdown of selected targets, even in the CNS. According to the present disclosure, without being bound by theory, synthetic siRNAs are introduced into cells and loaded directly into the RISC, or, in the case of longer dsRNAs (25-27 nucleotides), are first processed by Dicer and then loaded into the RISC to achieve gene silencing. RNAi therapy provided by various embodiments of the present disclosure is suitable for diseases and disorders in which disease-causing genes acquire negative or destructive "gain-of-function" effects. Identification of such disease-causing genes in the literature along with the present disclosure allows for the design of RNAi oligonucleotide molecules that target disease-causing alleles and provide therapeutic relief.The types of diseases / disorders targeted by the disclosure provided herein include, but are not limited to, the following: Progressive Supranuclear Palsy (PSP), Corticobasal Degeneration (CBD), Argyrophilic Granulopathy (AGD), Globular Glial Tauopathy (GGT), Age-Related Tau Astrogliopathy (ARTAG), Familial Frontotemporal Dementia 17 (FTD-17), Tauopathy with Respiratory Failure, Dementia with Stroke, Pick's Disease, and Neuropsychiatric Disorders. Myotonic dystrophy 1 or 2 (MD1 or MD2), Down syndrome, Spastic paraplegia (SP), Niemann-Pick disease type C, Dementia with Lewy bodies (DLB), Lewy body dysphagia, Lewy body disease, Olivopontocerebellar atrophy, Striatonigral degeneration, Shy-Drager syndrome, Spinal muscular atrophy V (SMAV), Huntington's disease (HD), Alzheimer's disease, SCA1, SCA2, SCA3, SCA7, SCA10 (Spinocerebellar Ataxia Types 1, 2, 3, 7, or 10), Multiple System Atrophy (MSA), Spinal-bulbar Muscular Atrophy (SBMA, Kennedy Disease), Friedreich's Ataxia, Fragile X-associated Tremor / Ataxia Syndrome (FXTAS), Fragile X Syndrome (FRAXA), X-linked Mental Retardation (XLMR), Parkinson's Disease, Dystonia, SBMA (Spinal-bulbar Muscular Atrophy), Neuropathic Pain Disorders, Spinal Cord Injury, Dentatorubral Nucleus Pallidoluysian atrophy (DRPLA), recessive CNS disorder, ALS (amyotrophic lateral sclerosis), M2DS (MECP2 duplication syndrome), FTD (frontotemporal dementia), prion disease, adult-onset leukodystrophy, Alexander disease, Krabbe disease, chronic traumatic encephalopathy, Pelizaeus-Merzbach disease (PMD), Lafora disease, stroke, cerebral amyloid angiopathy (CAA), and metachromatic leukodystrophy (MLD). In some embodiments, the disease or disorder is PMD, spinal cord injury, stroke, Krabbe disease, MLD, SCA3, prion disease, Alzheimer's disease, Alexander disease, adult-onset leukodystrophy, MECP2 duplication syndrome, Charcot-Marie-Tooth disease, multiple sclerosis, Kennedy disease, Huntington's disease, X-linked adrenoleukodystrophy, or SCA1. In some embodiments, the disease or disorder is PMD. In some embodiments, the disease or disorder is spinal cord injury, hi some embodiments, the disease or disorder is stroke.In some embodiments, the disease or disorder is Krabbe disease. In some embodiments, the disease or disorder is MLD. In some embodiments, the disease or disorder is SCA3. In some embodiments, the disease or disorder is a prion disease. In some embodiments, the disease or disorder is Alzheimer's disease. In some embodiments, the disease or disorder is Alexander disease. In some embodiments, the disease or disorder is adult-onset leukodystrophy. In some embodiments, the disease or disorder is MECP2 duplication syndrome. In some embodiments, the disease or disorder is Charcot-Marie-Tooth disease. In some embodiments, the disease or disorder is multiple sclerosis. In some embodiments, the disease or disorder is Kennedy disease. In some embodiments, the disease or disorder is Huntington's disease. In some embodiments, the disease or disorder is X-linked adrenoleukodystrophy. In some embodiments, the disease or disorder is SCA1.

[0012] In some embodiments, the nucleic acid-hydrophobic ligand conjugates and pharma- ceutically acceptable compositions thereof of the present disclosure are effective as regulators of intracellular RNA levels and / or RNA function. Such nucleic acid-lipid conjugates, including one or more lipid conjugates, are represented by Formula Ia or: [ka] or a pharma- ceutically acceptable salt thereof, in which each variable is as defined and described herein.

[0013] In certain embodiments, the nucleic acid-ligand conjugate is represented by formula Ib, Ic, I-Ib, or I-Ic, [ka] or a pharma- ceutically acceptable salt thereof, in which each variable is as defined and described herein.

[0014] In another aspect, the present disclosure provides an oligonucleotide-ligand conjugate represented by formula II-a: [ka] or a pharma- ceutically acceptable salt thereof, wherein each variable is as defined and described herein.

[0015] In certain embodiments, the oligonucleotide-lipid conjugate is represented by formula II-b, II-c, II-Ib, or II-Ic, [ka] or a pharma- ceutically acceptable salt thereof.

[0016] In certain embodiments, the oligonucleotide-ligand conjugate of any of the above disclosed embodiments comprises 1 to 10 nucleic acid-ligand or nucleic acid analog-ligand conjugate units, hi some embodiments, the oligonucleotide-ligand conjugate comprises 1, 2, or 3 nucleic acid-ligand conjugate units.

[0017] In some embodiments, the oligonucleotide-ligand conjugate comprises a sense strand 10-53 nucleotides in length and an antisense strand 15-53 nucleotides in length, the antisense oligonucleotide strand having a sequence complementary to at least 15 consecutive nucleotides of a target gene sequence, and when the oligonucleotide-conjugate is introduced into a mammalian cell, expression of the target gene is reduced. In some embodiments, the oligonucleotide-ligand conjugate comprises a nucleotide strand 9-30 nucleotides in length. In some embodiments, the oligonucleotide-ligand conjugate is single-stranded. In some embodiments, the oligonucleotide-ligand conjugate is double-stranded.

[0018] In certain embodiments of the oligonucleotide-ligand conjugates, the antisense strand is 19-27 nucleotides in length. In certain embodiments of the oligonucleotide-ligand conjugates, the sense strand is 12-40 nucleotides in length. In certain embodiments of the oligonucleotide-ligand conjugates, the sense strand forms a double-stranded region with the antisense strand. In certain embodiments of the oligonucleotide-ligand conjugates, the region of complementarity is fully complementary to the target sequence.

[0019] In certain embodiments of the oligonucleotide-ligand conjugates, the sense strand comprises a stem-loop at its 3' end designated as S1-L-S2, where S1 is complementary to S2 and L forms a loop 3-5 nucleotides long between S1 and S2. In certain embodiments of the oligonucleotide-ligand conjugates, L is a tetraloop and L comprises a sequence designated as GAAA. In some embodiments, L is a tetraloop having a nucleotide sequence designated as 5'-GAAA-3'.

[0020] In certain embodiments, the oligonucleotide-ligand conjugate further comprises a 3' overhang sequence on the antisense strand that is 2 nucleotides in length. In certain embodiments, the oligonucleotide-ligand conjugate further comprises a 3' overhang sequence that is 1 or more nucleotides in length, the 3' overhang sequence being present on the antisense strand, the sense strand, or the antisense strand and the sense strand.

[0021] In certain embodiments, the oligonucleotide-ligand conjugate comprises at least one modified nucleotide, the modified nucleotide comprising a 2'-modification. In some embodiments, the 2'-modification is a modification selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, 2'-deoxy-2'-fluoro and 2'-deoxy-2'-fluoro-β-d-arabino.

[0022] In certain embodiments of the oligonucleotide-ligand conjugate, all nucleotides of the oligonucleotide are modified.

[0023] In certain embodiments of the oligonucleotide-ligand conjugate, the oligonucleotide comprises at least one modified internucleotide bond. In some embodiments of the oligonucleotide-ligand conjugate, at least one modified internucleotide bond is a phosphorothioate bond.

[0024] In some embodiments of the oligonucleotide-ligand conjugate, the 4'-carbon of the sugar of the 5'-nucleotide of the antisense strand comprises a phosphate analog, which is an oxymethylphosphonate, vinylphosphonate, or malonylphosphonate.

[0025] In some aspects, the present disclosure provides a nucleic acid-ligand conjugate represented by formula Ia: [ka] or a pharma- ceutically acceptable salt thereof, wherein: B is a nucleobase or hydrogen; R 1 and R 2 are independently hydrogen, halogen, or R A , -CN, -S(O)R, -S(O)2R, -Si(OR)2R, -Si(OR)R2, or -SiR3, or R on the same carbon 1 and R 2 together with their intervening atoms form a 3-membered saturated or partially unsaturated ring having 0 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; Each R A is independent, C 1-6an optionally substituted group selected from aliphatic; phenyl; a 4-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; Each R is independently hydrogen, a suitable protecting group, or C 1-6 an optionally substituted group selected from aliphatic; phenyl; a 4-7 membered saturated or partially unsaturated heterocycle having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or two R groups on the same atom together with their intervening atoms form a 4-7 membered saturated, partially unsaturated, or heteroaryl ring having 0-3 heteroatoms independently selected from nitrogen, oxygen, silicon, and sulfur; L A is independent, P.G. 1 or -L-ligand, PG 1 is hydrogen or a suitable hydroxyl protecting group, Each ligand is independently -(LC) n and / or an adamantyl group, Each LC can be independently saturated or unsaturated, linear or branched C 1-50 a lipid conjugate moiety comprising a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently replaced by -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR-, or -P(S)OR-; Each -Cy- is independently selected from phenylenyl; 8-10 membered bicyclic arylenyl; 4-7 membered saturated or partially unsaturated carbocyclylenyl; 4-11 membered saturated or partially unsaturated spirocarbocyclylenyl; 8-10 membered bicyclic saturated or partially unsaturated carbocyclylenyl; adamantanenyl; 4-7 membered saturated or partially unsaturated heterocyclylenyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and nitrogen, oxygen, and sulfur. an optionally substituted bivalent ring selected from: a 4-11 membered saturated or partially unsaturated spiroheterocyclylenyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; an 8-10 membered bicyclic saturated or partially unsaturated heterocyclylenyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; a 5-6 membered heteroarylenyl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or an 8-10 membered bicyclic heteroarylenyl having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; n is 1 to 10; L is a covalent bond or a divalent saturated or unsaturated, linear or branched C 1-50 a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently selected from the group consisting of -Cy-, -O-, -NR-, -N(R)-C(O)-, -S-, -C(O)-, -S(O)-, -S(O)2-, -P(O)OR-, -P(S)OR-, -V 1 CR 2 W 1 -or [ka] is replaced by m is 1 to 50; X 1 , V 1 , and W 1 are independently -C(R)2-, -OR, -O-, -S-, -Se-, or -NR-; Z is -O-, -S-, -NR-, or -CR2-; PG 2 is hydrogen, a phosphoramidite analog, or a suitable protecting group.

[0026] In some embodiments, the conjugate is represented by formula Ib or Ic: [ka] or a pharma- ceutically acceptable salt thereof, wherein L 1 is a covalent bond or a divalent saturated or unsaturated, linear or branched C 1-50 a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR-, -P(S)OR-, or [ka] is replaced by R 4 is hydrogen, R A or a suitable amine protecting group, R 5 is adamantyl or saturated or unsaturated, linear or branched C 1-50 a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently replaced by -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR-, or -P(S)OR-.

[0027] In some embodiments, the nucleic acid-ligand conjugate is represented by formula I-Ib or I-Ic: [ka] or a pharma- ceutically acceptable salt thereof, wherein B is a nucleobase or hydrogen; m is 1 to 50; PG1 and P.G. 2 are independently hydrogen, a phosphoramidite analog, or a suitable protecting group; R 5 is adamantyl or saturated or unsaturated, linear or branched C 1-50 a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently replaced by -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR-, or -P(S)OR-.

[0028] In any of the above or related aspects, R 5 is selected from the following: [ka]

[0029] In any of the above or related aspects, R 5 is selected from the following: [ka]

[0030] In some embodiments, the present disclosure provides an oligonucleotide-ligand conjugate comprising one or more of the nucleic acid-ligand conjugates described herein. In some embodiments, the oligonucleotide-ligand conjugate comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleic acid-ligand conjugates.

[0031] In some aspects, the present disclosure provides an oligonucleotide-ligand conjugate comprising one or more nucleic acid-ligand conjugates represented by formula II-a: [ka] or a pharma- ceutically acceptable salt thereof, wherein: B is a nucleobase or hydrogen; R 1and R 2 are independently hydrogen, halogen, or R A , -CN, -S(O)R, -S(O)2R, -Si(OR)2R, -Si(OR)R2, or -SiR3, or R on the same carbon 1 and R 2 together with their intervening atoms form a 3- to 7-membered saturated or partially unsaturated ring having 0-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; Each R A is independent, C 1-6 an optionally substituted group selected from aliphatic; phenyl; a 4-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; Each R is independently hydrogen, a suitable protecting group, or C 1-6 an optionally substituted group selected from aliphatic; phenyl; a 4-7 membered saturated or partially unsaturated heterocycle having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or two R groups on the same atom together with their intervening atoms form a 4-7 membered saturated, partially unsaturated, or heteroaryl ring having 0-3 heteroatoms independently selected from nitrogen, oxygen, silicon, and sulfur; Each LC can be independently saturated or unsaturated, linear or branched C 1-50 a lipid conjugate moiety comprising a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently replaced by -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)-OR-, -P(S)OR-; Each -Cy- is independently selected from phenylenyl; 8-10 membered bicyclic arylenyl; 4-7 membered saturated or partially unsaturated carbocyclylenyl; 4-11 membered saturated or partially unsaturated spirocarbocyclylenyl; 8-10 membered bicyclic saturated or partially unsaturated carbocyclylenyl; 4-7 membered saturated or partially unsaturated heterocyclylenyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; 4-11 membered saturated or partially unsaturated heterocyclylenyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. an optionally substituted bivalent ring selected from: a saturated or partially unsaturated spiroheterocyclylenyl; an 8-10 membered bicyclic saturated or partially unsaturated heterocyclylenyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; a 5-6 membered heteroarylenyl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or an 8-10 membered bicyclic heteroarylenyl having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; n is 1 to 10; L is a covalent bond or a divalent saturated or unsaturated, linear or branched C 1-50 a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently selected from the group consisting of -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, S(O)-, -S(O)2-, -P(O)OR-, -P(S)OR-, -V 1 CR 2 W 1 -or [ka] is replaced by m is 1 to 50; X 1 , V 1 , and W 1 are independently -C(R)2-, -OR, -O-, -S-, -Se-, or -NR-; Y is hydrogen, a suitable hydroxyl protecting group, [ka] and R 3 is hydrogen, a suitable protecting group, a suitable prodrug, or C 1-6 an optionally substituted group selected from aliphatic; phenyl; a 4-7 membered saturated or partially unsaturated heterocycle having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; X 2 is O, S, or NR; X 3 is -O-, -S-, -BH2-, or a covalent bond, Y 1 is a linking group attached to the 2' or 3' end of a nucleoside, nucleotide, or oligonucleotide, Y 2 is hydrogen, a suitable protecting group, a phosphoramidite analog; an internucleotide linkage attached to the 5' end of a nucleoside, nucleotide, or oligonucleotide, or a linkage attached to a solid support; Z is -O-, -S-, -NR-, or -CR2-.

[0032] In any of the foregoing or related embodiments, the oligonucleotide-ligand conjugate is represented by formula II-b or II-c: [ka] or a pharma- ceutically acceptable salt thereof, wherein L 1 is a covalent bond, monovalent or divalent saturated or unsaturated, linear or branched C 1-50 a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR-, -P(S)OR-, or [ka] is replaced by R 4 is hydrogen, R A or a suitable amine protecting group, R 5 is adamantyl or saturated or unsaturated, linear or branched C 1-50 a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently replaced by -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR-, or -P(S)OR.

[0033] In any of the above or related aspects, R 5 is selected from the following: [ka]

[0034] In some embodiments, R 5 is selected from the following: [ka]

[0035] In some aspects, the present disclosure provides an oligonucleotide-ligand conjugate represented by formula II-Ib or II-Ic: [ka] or a pharma- ceutically acceptable salt thereof, wherein: B is a nucleobase or hydrogen; m is 1 to 50; X 1 is -O- or -S-; Y is hydrogen, [ka] and R 3 is hydrogen or a suitable protecting group, X 2 is O or S, X 3 is -O-, -S-, or a covalent bond; Y 1 is a linking group attached to the 2' or 3' end of a nucleoside, nucleotide, or oligonucleotide, Y 2 is hydrogen, a phosphoramidite analog; an internucleotide linkage attached to the 5' end of a nucleoside, nucleotide, or oligonucleotide, or a linkage attached to a solid support; R 5 is adamantyl or saturated or unsaturated, linear or branched C 1-50 a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently replaced by -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR-, or -P(S)OR-; R is hydrogen, a suitable protecting group, or C 1-6 an optionally substituted group selected from aliphatic; phenyl; a 4-7 membered saturated or partially unsaturated heterocycle having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0036] In any of the above or related aspects, R 5 is selected from the following: [ka]

[0037] In any of the above or related aspects, R 5 is as follows: [ka]

[0038] In any of the above or related aspects, R 5 is as follows: [ka]

[0039] In any of the above or related aspects, R 5 is as follows: [ka]

[0040] In any of the above or related aspects, R 5 is as follows: [ka]

[0041] In any of the above or related aspects, R 5 is as follows: [ka]

[0042] In any of the above or related aspects, R 5 is as follows: [ka]

[0043] In any of the above or related aspects, R 5 is as follows: [ka]

[0044] In any of the above or related aspects, R 5 is as follows: [ka]

[0045] In any of the above or related aspects, R 5 is as follows: [ka]

[0046] In any of the above or related aspects, R 5 is as follows: [ka]

[0047] In any of the foregoing or related embodiments, the oligonucleotide-ligand conjugate comprises between 1 and 10 nucleic acid-ligand conjugate units. In some embodiments, the conjugate comprises 1, 2, or 3 nucleic acid-ligand conjugate units.

[0048] In any of the foregoing or related embodiments, the oligonucleotide comprises a sense strand 10-53 nucleotides in length and an antisense strand 15-53 nucleotides in length, the antisense oligonucleotide strand having a sequence complementary to at least 15 contiguous nucleotides of a target gene sequence, wherein the oligonucleotide conjugate reduces target gene expression when introduced into a mammalian cell.

[0049] In any of the foregoing or related embodiments, the nucleic acid-ligand conjugate unit is in the sense strand. In some embodiments, the antisense strand is 19-27 nucleotides in length. In some embodiments, the sense strand is 12-40 nucleotides in length.

[0050] In any of the above or related embodiments, the sense strand forms a duplex region with the antisense strand.

[0051] In any of the foregoing or related aspects, the region of complementarity is perfectly complementary to the target sequence.

[0052] In any of the foregoing or related embodiments, the sense strand comprises at its 3' end a stem-loop designated as S1-L-S2, where S1 is complementary to S2 and L forms a loop 3-5 nucleotides long between S1 and S2. In some embodiments, L is a tetraloop. In some embodiments, L comprises a sequence designated as GAAA. In some embodiments, L comprises a nucleotide sequence designated as 5'-GAAA-3'. In some embodiments, L consists of a nucleotide sequence designated as 5'-GAAA-3'.

[0053] In some aspects, the disclosure provides an oligonucleotide-ligand conjugate for reducing expression of a target mRNA in the central nervous system (CNS), the oligonucleotide-ligand conjugate comprising: (i) a double-stranded oligonucleotide comprising an antisense strand 15-30 nucleotides in length and a sense strand 15-40 nucleotides in length, wherein the antisense strand and the sense strand each comprise a 5' end and a 3' end, and wherein the antisense strand and the sense strand form a double-stranded region, wherein the antisense strand has a region of complementarity to a target sequence of a target mRNA in the CNS, the region of complementarity being at least 15 contiguous nucleotides in length, and wherein the oligonucleotide comprises a stem loop; and (ii) one or more lipid moieties conjugated to the stem loop.

[0054] In any of the foregoing or related aspects, the lipid moiety comprises a saturated C16, C17, C18, C19, C20, C21, or C22 hydrocarbon chain. In some aspects, the lipid moiety comprises an unsaturated C16, C17, C18, C19, C20, C21, or C22 hydrocarbon chain, and optionally, the lipid moiety comprises C22:6. In some embodiments, the unsaturated hydrocarbon chain comprises at least one double bond. For example, a C18 hydrocarbon chain with one double bond is referred to as C18:1, while a C18 hydrocarbon chain with two double bonds is referred to as C18:2. In some aspects, the lipid moiety comprises an unsaturated C18 hydrocarbon chain (e.g., C18:1). In some aspects, the lipid moiety comprises an unsaturated C22 hydrocarbon chain (e.g., C22:6).

[0055] In any of the above or related embodiments, one or more lipid moieties comprise a saturated or unsaturated C1-C50 hydrocarbon chain. In some embodiments, one or more lipid moieties comprise a saturated or unsaturated C5-C25 hydrocarbon chain. In some embodiments, one or more lipid moieties comprise a saturated C8, C9, C10, C11, C12, C13, or C14 hydrocarbon chain. In some embodiments, the oligonucleotide-ligand conjugate reduces expression of the target mRNA in the CNS without reducing expression of the target mRNA outside the CNS, and optionally without reducing expression of the target mRNA in the liver. In some embodiments, the target mRNA is expressed in liver cells, and the oligonucleotide-ligand conjugate does not substantially reduce expression of the target mRNA in liver cells compared to expression of the target mRNA in cells of the CNS. In some embodiments, the target mRNA is expressed in liver cells, and the oligonucleotide-ligand conjugate does not reduce expression of the target mRNA in liver cells to a similar level as in cells of the CNS.

[0056] In some embodiments, the present disclosure provides an oligonucleotide-ligand conjugate for reducing expression of a target mRNA in the CNS, comprising: (i) a double-stranded oligonucleotide comprising an antisense strand 15-30 nucleotides long and a sense strand 15-40 nucleotides long, the antisense strand and the sense strand each comprising a 5'-end and a 3'-end, the antisense strand and the sense strand form a double-stranded region, the antisense strand has a complementary region to a target sequence of a target mRNA in the CNS, the complementary region is at least 15 consecutive nucleotides long, and the oligonucleotide comprises a stem-loop; and (ii) one or more lipid moieties conjugated to the stem-loop, the one or more lipid moieties being selected from a saturated or unsaturated C8-C14 hydrocarbon chain, the oligonucleotide-ligand conjugate not reducing expression of the target mRNA in the liver to a level similar or similar to that in the CNS. In some embodiments, the one or more lipid moieties are saturated or unsaturated C8 hydrocarbon chains. In some embodiments, one or more lipid moieties are saturated or unsaturated C9 hydrocarbon chains. In some embodiments, one or more lipid moieties are saturated or unsaturated C10 hydrocarbon chains. In some embodiments, one or more lipid moieties are saturated or unsaturated C11 hydrocarbon chains. In some embodiments, one or more lipid moieties are saturated or unsaturated C12 hydrocarbon chains. In some embodiments, one or more lipid moieties are saturated or unsaturated C13 hydrocarbon chains. In some embodiments, one or more lipid moieties are saturated or unsaturated C14 hydrocarbon chains.

[0057] In some embodiments, the sense strand comprises a stem loop at its 3' end. In some embodiments, the stem loop comprises a nucleotide sequence represented by the following formula: 5'-S1-L-S2-3', where S1 is complementary to S2 and L forms a loop between S1 and S2. In some embodiments, S1 and S2 are 1-8 nucleotides in length. In some embodiments, one or more lipid moieties are conjugated to the nucleotides of S1. In some embodiments, one or more lipid moieties are conjugated to the nucleotides of S2.

[0058] In any of the foregoing or related embodiments, L is 3-5 nucleotides in length. In some embodiments, L is a tetraloop, and optionally L is 4 nucleotides in length. In some embodiments, L comprises a nucleotide sequence depicted as 5'-GAAA-3'. In some embodiments, L consists of a nucleotide sequence depicted as 5'-GAAA-3'. In some embodiments, one or more lipid moieties are conjugated to the nucleotides of L.

[0059] In any of the foregoing or related embodiments, L is 3 nucleotides long and has a first, second, and third nucleotide from 5' to 3', the oligonucleotide-ligand conjugate comprises one lipid moiety, and the lipid moiety is conjugated to the first, second, or third nucleotide of L. In some embodiments, L is 4 nucleotides long and has a first, second, third, and fourth nucleotide from 5' to 3', the oligonucleotide-ligand conjugate comprises one lipid moiety, and the lipid moiety is conjugated to the first, second, third, or fourth nucleotide of L. In some embodiments, the lipid moiety is conjugated to the second nucleotide of L. In some embodiments, L consists of 5'-GAAA-3'. In some embodiments, the lipid moiety is conjugated to the second nucleotide of L. In some embodiments, L is 5 nucleotides in length and has, from 5' to 3', a first, second, third, fourth, and fifth nucleotide, and the oligonucleotide-ligand conjugate comprises one lipid moiety, wherein the lipid moiety is conjugated to the first, second, third, fourth, or fifth nucleotide of L.

[0060] In any of the foregoing or related embodiments, the antisense strand is 19-27 nucleotides in length. In some embodiments, the antisense strand is 21-27 nucleotides in length. In some embodiments, the antisense strand is 22 nucleotides in length.

[0061] In any of the above or related embodiments, the sense strand is 19-40 nucleotides in length, hi some embodiments, the sense strand is 36 nucleotides in length.

[0062] In any of the foregoing or related embodiments, the double-stranded region is at least 19 nucleotides in length. In some embodiments, the double-stranded region is at least 20 nucleotides in length. In some embodiments, the double-stranded region is 21 nucleotides in length.

[0063] In any of the above or related embodiments, the oligonucleotide-ligand conjugate comprises a 3' overhang sequence on the antisense strand that is 2 nucleotides long.In some embodiments, the oligonucleotide comprises a 3' overhang sequence that is 1 or more nucleotides long, and the 3' overhang sequence is on the antisense strand, the sense strand, or the antisense strand and the sense strand.In some embodiments, the 3' overhang sequence is on the antisense strand.In some embodiments, the 3' overhang sequence is 2 nucleotides long.

[0064] In any of the above or related embodiments, the oligonucleotide comprises at least one modified nucleotide. In some embodiments, the modified nucleotide comprises a 2'-modification. In some embodiments, the 2'-modification is a modification selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, 2'-deoxy-2'-fluoro and 2'-deoxy-2'-fluoro-β-d-arabino. In some embodiments, all nucleotides of the oligonucleotide are modified.

[0065] In any of the above or related embodiments, the oligonucleotide comprises at least one modified internucleotidic bond. In some embodiments, at least one modified internucleotidic bond is a phosphorothioate bond. In any of the above or related embodiments, the oligonucleotide comprises at least one modified internucleoside bond. In some embodiments, at least one modified internucleoside bond is a phosphorothioate bond.

[0066] In any of the above or related embodiments, the 4'-carbon of the sugar of the 5'-nucleotide of the antisense strand comprises a phosphate analog. In some embodiments, the phosphate analog is an oxymethylphosphonate, a vinylphosphonate, or a malonylphosphonate.

[0067] In some aspects, the present disclosure provides a composition comprising an oligonucleotide-ligand conjugate described herein and an excipient.

[0068] In some aspects, the present disclosure provides a pharmaceutical composition comprising an oligonucleotide-ligand conjugate described herein and a pharma- ceutically acceptable carrier.

[0069] In some aspects, the disclosure provides a method of delivering an oligonucleotide-ligand conjugate to a subject, the method comprising administering to the subject a composition described herein.

[0070] In some embodiments, the present disclosure provides a method for reducing or inhibiting the expression of target mRNA in a subject that is expressed by a cell population associated with the CNS of the subject, comprising administering to the subject the oligonucleotide-ligand conjugate described herein, the composition described herein, or the pharmaceutical composition described herein.In some embodiments, the expression level of target mRNA is reduced in a cell population associated with the CNS compared to a control cell population.In some embodiments, the expression level of target mRNA is not reduced in a cell population that exists outside the CNS compared to a control cell population.In some embodiments, the cell population that exists outside the CNS is in the liver.

[0071] In a further aspect, the disclosure provides an oligonucleotide-ligand conjugate for reducing expression of a target gene. In some aspects, the target gene is expressed in the CNS. In some aspects, the target gene is associated with a disease or disorder, optionally a neurological disease or disorder. In some aspects, the disease or disorder is progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), argyrophilic granulosis (AGD), globuloglial tauopathy (GGT), age-related tau astrogliopathy (ARTAG), familial frontotemporal dementia 17 (FTD-17), tauopathy with respiratory failure, dementia with seizures, Pick's disease, myotonic dystrophy 1 or 2 (MD1 or MD2), and / or MD3. ), Down syndrome, spastic paraplegia (SP), Niemann-Pick disease type C, Dementia with Lewy bodies (DLB), Lewy body dysphagia, Lewy body disease, Olivopontocerebellar atrophy, Striatonigral degeneration, Shy-Drager syndrome, Spinal muscular atrophy V (SMAV), Huntington's disease (HD), Alzheimer's disease, SCA1, SCA2, SCA3, SCA7, SCA10 (Spinocerebellar ataxia types 1, 2, 3, 7 type 103), multiple system atrophy (MSA), spinal-bulbar muscular atrophy (SBMA, Kennedy disease), Friedreich's ataxia, fragile X-associated tremor / ataxia syndrome (FXTAS), fragile X syndrome (FRAXA), X-linked mental retardation (XLMR), Parkinson's disease, dystonia, SBMA (spinal-bulbar muscular atrophy), neuropathic pain disorders, spinal cord injury, dentatorubral-pallidoluysian atrophy (DRPLA), recessive CNS disorders, and selected from ALS (amyotrophic lateral sclerosis), M2DS (MECP2 duplication syndrome), FTD (frontotemporal dementia), prion diseases, adult-onset leukodystrophy, Alexander disease, Krabbe disease, chronic traumatic encephalopathy, Pelizaeus-Merzbach disease (PMD), Lafora disease, stroke, cerebral amyloid angiopathy (CAA), and metachromatic leukodystrophy (MLD). [Brief description of the drawings]

[0072] [Figure 1]1 provides a graph showing the efficacy of RNAi oligonucleotides designed to inhibit Aldh2 mRNA expression in mice following administration to the mouse CNS (GalXC-ALDH2 RNAi oligonucleotides). As shown, the percent (%) of Aldh2 mRNA remaining in samples from various CNS tissues was measured in female CD-1 mice 5 days after intracerebroventricular (icv) administration of 100 μg of GalXC-ALDH2 RNAi oligonucleotides formulated in PBS, compared to the percent of Aldh2 mRNA in PBS-treated mice. [Diagram 2] Figure 2 provides a graph showing the dose response of GalXC-ALDH2 RNAi oligonucleotides after icv administration to the CNS of mice. As indicated, the percentage of Aldh2 mRNA remaining in various CNS tissues of mice was measured after icv administration of 250 μg or 500 μg of GalXC-ALDH2 RNAi oligonucleotides formulated in PBS at various time points as indicated, compared to the percentage of Aldh2 mRNA in PBS-treated mice. Aldh2 mRNA percentages were determined in the frontal cortex, hippocampus, hypothalamus, striatum, somatosensory cortex, and cerebellum as indicated. [Diagram 3] Figure 3 provides depictions of mouse brain and spinal cord regions, along with a graph showing the dose response of GalXC-ALDH2 RNAi oligonucleotides after icv administration to the CNS of mice. As indicated, the percentage of mouse Aldh2 mRNA remaining in various CNS tissues was measured in mice at various time points after icv administration of 250 μg or 500 μg of GalXC-ALDH2 RNAi oligonucleotides formulated in PBS, as compared to the percentage of Aldh2 mRNA in PBS-treated mice. Aldh2 mRNA percentages were determined in cervical, thoracic, and lumbar spinal cord, as indicated. [Figure 4]4 provides a graph showing the efficacy of RNAi oligonucleotides (GalXC-ALDH2 RNAi oligonucleotides) designed to inhibit Aldh2 mRNA expression in mice following administration to the CNS of the mice. As shown, the percent (%) of Aldh2 mRNA remaining in samples from various CNS tissues was measured in mice 7 days after intrathecal (it) administration of 500 μg of GalXC-ALDH2 RNAi oligonucleotides formulated in PBS, compared to the percent of Aldh2 mRNA in PBS-treated mice. [Diagram 5] Figure 5 provides a graph showing the dose response of GalXC-ALDH2 RNAi oligonucleotides following administration to the CNS of rats. As indicated, the percent (%) of rat Aldh2 mRNA remaining in various CNS tissues was measured following lumbar it administration of 1000 μg or 6000 μg of GalXC-ALDH2 RNAi oligonucleotides formulated in PBS in mice at days 7 and 28 as indicated, compared to the percent Aldh2 mRNA in PBS-treated rats. Aldh2 mRNA percent (%) was determined in the frontal cortex, striatum, hippocampus, brainstem, spinal cord regions SC1-SC8, C1-C7 dorsal root ganglia (DRG), T1-T12 DRG, L1-L6 DRG, and liver. [Figure 6] 6 provides a graph showing the efficacy of RNAi oligonucleotides (GalXC-ALDH2 RNAi oligonucleotides) designed to inhibit human / monkey ALDH2 mRNA expression following administration to the CNS of non-human primates (NHPs). As shown, the percent (%) of ALDH2 mRNA remaining in samples from various CNS tissues was measured in cynomolgus monkeys 28 days after it administration of 75 mg of GalXC-ALDH2 RNAi oligonucleotides formulated in PBS, compared to the percent of ALDH2 mRNA in PBS-treated NHPs. [Figure 7]Figure 7 provides a graph showing the dose response of GalXC-ALDH2 RNAi oligonucleotides after administration to the CNS of NHPs. As shown, the percent (%) of human / monkey ALDH2 mRNA remaining in various CNS tissues was measured in rhesus monkeys 28 days after it administration of 75 mg, 150 mg, or 300 mg of GalXC-ALDH2 RNAi oligonucleotides formulated in PBS, compared to the percent of ALDH2 mRNA in PBS-treated NHPs. The percent (%) of ALDH2 mRNA was determined in the frontal cortex, hippocampus, temporal cortex, cerebellum, brainstem, cervical spinal cord, thoracic spinal cord, lumbar spinal cord, dorsal root ganglion (DRG), and liver. [Figure 8] Figure 8 provides a graph showing the efficacy of GalXC-ALDH2 RNAi oligonucleotides after administration (infusion) into the CNS of NHPs via a surgically implanted lumbar port. As shown, the percent (%) of human / monkey ALDH2 mRNA remaining in various CNS tissues was measured in cynomolgus monkeys 28 days after it administration of 75 mg of GalXC-ALDH2 RNAi oligonucleotides formulated in PBS, compared to the percent of ALDH2 mRNA in PBS-treated NHPs. ALDH2 mRNA percent (%) was determined in the frontal cortex, caudate nucleus, hippocampus, midbrain, parietal cortex, occipital cortex, thalamus, temporal cortex, cerebellum, brainstem, cervical spinal cord, thoracic spinal cord, lumbar spinal cord, dorsal root ganglion (DRG), and liver. [Figure 9] Figure 9 provides a graph showing the efficacy of GalXC-ALDH2 RNAi oligonucleotides after administration to the CNS of NHPs.As shown, the percentage (%) of human / monkey ALDH2 mRNA remaining in various CNS tissues was measured in cynomolgus monkeys 28 days after intracisternal (icm) administration of 50 mg of GalXC-ALDH2 RNAi oligonucleotides formulated in PBS, compared to the percentage of ALDH2 mRNA in PBS-treated NHPs.The percentage (%) of ALDH2 mRNA was determined in the frontal cortex, caudate nucleus, hippocampus, midbrain, parietal cortex, occipital cortex, thalamus, temporal cortex, cerebellum, brainstem, cervical spinal cord, thoracic spinal cord, lumbar spinal cord, dorsal root ganglion (DRG), and liver. [Figure 10A] FIG. 10A provides a graph showing the efficacy of a series of GalXC-ALDH2 RNAi oligonucleotide-lipid conjugates (C8:0 to C22:6) following administration to the CNS of mice. As shown, the percent (%) of mouse Aldh2 mRNA remaining in various CNS tissues was measured in female CD-1 mice 7 days after icv administration of 250 μg of GalXC-ALDH2 RNAi oligonucleotide-lipid conjugates formulated in PBS, compared to the percent of Aldh2 mRNA in PBS-treated mice. Aldh2 mRNA percent (%) was determined in the somatosensory cortex (SS), hippocampus (HP), hypothalamus (HY), cervical spinal cord (CSC), thoracic spinal cord (TSC), and lumbar spinal cord (LSC). [Figure 10B] Figure 10B provides a graph showing the percent expression of Aldh2 mRNA in the livers of the same mice that received GalXC-ALDH2 RNAi oligonucleotide-lipid conjugates (C8:0 to C18:0) by icv administration as described in Figure 10A. Shown is the percent Aldh2 mRNA of mice remaining in liver tissue harvested 7 days after icv administration relative to the percent Aldh2 mRNA in liver tissue harvested from mice that received PBS only. [Figure 11] FIG. 11 provides a graph showing the efficacy of a series of GalXC-ALDH2 RNAi oligonucleotide-lipid conjugates (C10:0 to C18:2) following administration to the CNS of mice. As shown, the percent (%) of mouse Aldh2 mRNA remaining in various CNS tissues was measured in female C57BL / 6 mice 7 days after it administration of 250 μg of GalXC-ALDH2 RNAi oligonucleotide-lipid conjugates formulated in PBS, compared to the percent of Aldh2 mRNA in PBS-treated mice. Aldh2 mRNA percent (%) was determined in the frontal cortex, hippocampus (HP), brainstem (BS), cervical spinal cord (CSC), thoracic spinal cord (TSC), and lumbar spinal cord (LSC). [Figure 12]FIG. 12 provides a schematic diagram of a modified GalXC oligonucleotide. [Figure 13] FIG. 13 provides a schematic diagram of a modified lipid-conjugated GalXC oligonucleotide. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0073] In some aspects, the present disclosure provides an RNAi oligonucleotide conjugate (e.g., an RNAi oligonucleotide-lipid conjugate) that reduces the expression of a target gene in the CNS. In other aspects, the present disclosure provides a method of treating a disease or disorder (e.g., a neurological disease and / or inappropriate gene expression) using the RNAi oligonucleotide conjugate described herein or a pharma- ceutically acceptable composition thereof. In other aspects, the present disclosure provides a method of using the RNAi oligonucleotide-conjugate described herein in the manufacture of a medicament for treating a disease or disorder. In other aspects, the RNAi oligonucleotide conjugate provided herein is used to treat a neurological disease or disorder by regulating (e.g., inhibiting or reducing) the expression of a target gene associated with a neurological disease or disorder in the CNS. In some aspects, the present disclosure provides a method of treating a neurological disease or disorder by reducing the expression of a target gene associated with a neurological disease or disorder in the CNS (e.g., in a cell, tissue, or region of the CNS).

[0074] RNAi Oligonucleotide Conjugates The present disclosure provides, in particular, RNAi oligonucleotide conjugates (e.g., RNAi oligonucleotide-lipid conjugates) that reduce target gene expression in the CNS. In some embodiments, the RNAi oligonucleotide conjugates provided by the present disclosure target the mRNA encoding the target gene. The messenger RNA (mRNA) that encodes the target gene and is targeted by the RNAi oligonucleotide conjugates of the present disclosure is referred to herein as the "target mRNA". In some embodiments, the RNAi oligonucleotide conjugates reduce target gene expression in the CNS (e.g., somatosensory cortex (SS cortex), hippocampus (HP), striatum, frontal cortex, cerebellum, hypothalamus (HY), cervical spinal cord (CSC), thoracic spinal cord (TSC), and / or lumbar spinal cord (LSC)). In some embodiments, the RNAi oligonucleotide conjugates reduce target gene expression in the CNS (e.g., in the SS cortex, HP, HY, CSC, TSC, and / or LSC) without reducing the expression of the target mRNA outside the CNS. In some embodiments, the RNAi oligonucleotide conjugate reduces target gene expression in the CNS (e.g., SS cortex, HP, HY, CSC, TSC, and / or LSC) without reducing expression of the target mRNA in the liver. In some embodiments, the RNAi oligonucleotide conjugate does not reduce expression of the target mRNA in the liver to the same or similar level as in the CNS.

[0075] mRNA target sequence In some embodiments, the RNAi oligonucleotide conjugate targets a target sequence comprising a target mRNA. In some embodiments, the RNAi oligonucleotide conjugate targets a target sequence within a target mRNA. In some embodiments, the target mRNA is expressed in the CNS. In some embodiments, the target mRNA expressed in the CNS is referred to herein as a "CNS target mRNA". In some embodiments, the RNAi oligonucleotide conjugate, or a part, fragment, or strand thereof (e.g., the antisense strand or guide strand of a double-stranded oligonucleotide) binds or anneals to a target sequence comprising a target mRNA, thereby reducing target gene expression. In some embodiments, the RNAi oligonucleotide conjugate targets a target sequence comprising a target mRNA for the purpose of reducing target gene expression in vivo. In some embodiments, the amount or extent of reduction of target gene expression by an RNAi oligonucleotide conjugate targeting a particular target sequence correlates with the efficacy of the RNAi oligonucleotide conjugate. In some embodiments, the amount or degree of reduction in target gene expression by an RNAi oligonucleotide conjugate targeted to a particular target sequence correlates with the amount or degree of therapeutic effect in a subject or patient having a disease, disorder, or condition associated with target gene expression that is treated with the RNAi oligonucleotide conjugate.

[0076] Through the examination of the nucleotide sequence of the mRNA encoding the target gene, including the mRNA of several different species (e.g., human, cynomolgus monkey, mouse, and rat), and as a result of in vitro and in vivo testing, it has been discovered that certain nucleotide sequences that comprise the target mRNA are more susceptible to RNAi oligonucleotide conjugate-mediated reduction than other nucleotide sequences, and are therefore useful as target sequences for the RNAi oligonucleotide conjugates herein.In some embodiments, the sense strand of the RNAi oligonucleotide conjugates (e.g., RNAi oligonucleotide-lipid conjugates) described herein, or a portion or fragment thereof, comprises a nucleotide sequence that is similar (e.g., has no more than four mismatches) or identical to the target sequence that comprises the target mRNA.In some embodiments, a portion or region of the sense strand of the double-stranded oligonucleotide described herein comprises a target sequence that comprises the target mRNA.

[0077] RNAi oligonucleotide targeting sequences In some embodiments, the RNAi oligonucleotide conjugates provided by the present disclosure include a targeting sequence. As used herein, the term "targeting sequence" refers to a nucleotide sequence having a region of complementarity to a nucleotide sequence comprising an mRNA (e.g., a target mRNA). In some embodiments, the RNAi oligonucleotide conjugates provided by the present disclosure include a targeting sequence having a region of complementarity to a nucleotide sequence comprising a target sequence of a target mRNA. In some embodiments, the RNAi oligonucleotide conjugates provided by the present disclosure include a targeting sequence having a region of complementarity to a nucleotide sequence comprising a target sequence of a CNS target mRNA. The targeting sequence confers the RNAi oligonucleotide conjugate with the ability to specifically target an mRNA by binding or annealing to a target sequence comprising a target mRNA by complementary (Watson-Crick) base pairing. In some embodiments, the RNAi oligonucleotide conjugates herein (or a strand thereof, e.g., the antisense strand or guide strand of a double-stranded oligonucleotide) include a targeting sequence having a region of complementarity that binds or anneals to a target sequence comprising a target mRNA by complementary (Watson-Crick) base pairing. Targeting sequence is generally suitable length and base content to allow the RNAi oligonucleotide conjugate (or its strand) to bind or anneal to specific target mRNA for the purpose of inhibiting target gene expression.In some embodiments, targeting sequence is at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, or at least about 30 nucleotides in length.In some embodiments, targeting sequence is at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, or at least about 20 nucleotides in length.In some embodiments, the targeting sequence is about 12 to about 30 (e.g., 12 to 30, 12 to 22, 15 to 25, 17 to 21, 18 to 27, 19 to 27, or 15 to 30) nucleotides in length. In some embodiments, the targeting sequence is about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the targeting sequence is 18 nucleotides in length. In some embodiments, the targeting sequence is 19 nucleotides in length. In some embodiments, the targeting sequence is 20 nucleotides in length. In some embodiments, the targeting sequence is 21 nucleotides in length. In some embodiments, the targeting sequence is 22 nucleotides in length. In some embodiments, the targeting sequence is 23 nucleotides in length. In some embodiments, the targeting sequence is 24 nucleotides in length.

[0078] In some embodiments, the RNAi oligonucleotide conjugate herein comprises a targeting sequence that is completely complementary to the target sequence that comprises the target mRNA.In some embodiments, the targeting sequence is partially complementary to the target sequence that comprises the target mRNA.In some embodiments, the targeting sequence comprises a region of consecutive nucleotides that comprises the antisense strand.

[0079] In some embodiments, the RNAi oligonucleotide conjugates herein comprise a targeting sequence complementary to a contiguous sequence of nucleotides comprising a target mRNA, the contiguous sequence of nucleotides being about 12 to about 30 nucleotides in length (e.g., 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 20, 12 to 18, 12 to 16, 14 to 22, 16 to 20, 18 to 20, or 18 to 19 nucleotides in length). In some embodiments, the RNAi oligonucleotide conjugates comprise a targeting sequence complementary to a contiguous sequence of nucleotides comprising a target mRNA, the contiguous sequence of nucleotides being 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the RNAi oligonucleotide conjugates comprise a targeting sequence complementary to a contiguous sequence of nucleotides comprising a target mRNA, the contiguous sequence of nucleotides being 19 nucleotides in length. In some embodiments, the RNAi oligonucleotide conjugate comprises a targeting sequence that is complementary to a contiguous sequence of nucleotides that comprises a target mRNA, wherein the contiguous sequence of nucleotides is 20 nucleotides in length.

[0080] In some embodiments, the RNAi oligonucleotide conjugate comprises a targeting sequence that is complementary to a contiguous sequence of nucleotides that comprises a target mRNA (e.g., a CNS target mRNA), and the contiguous sequence of nucleotides is 15 nucleotides in length. In some embodiments, the RNAi oligonucleotide conjugate comprises a targeting sequence that is complementary to a contiguous sequence of nucleotides that comprises a target mRNA (e.g., a CNS target mRNA), and the contiguous sequence of nucleotides is 19 nucleotides in length.

[0081] In some embodiments, the targeting sequence of the RNAi oligonucleotide conjugates herein is fully complementary (e.g., has no mismatches) to a target sequence including a target mRNA and includes the entire length of the antisense strand. In some embodiments, the targeting sequence of the RNAi oligonucleotide conjugates herein is fully complementary (e.g., has no mismatches) to a target sequence including a target mRNA and includes a portion of the entire length of the antisense strand. In some embodiments, the targeting sequence of the RNAi oligonucleotide conjugates herein is fully complementary (e.g., has no mismatches) to a target sequence including a target mRNA and includes an antisense strand of 10 to 20 nucleotides. In some embodiments, the targeting sequence of the RNAi oligonucleotide conjugates herein is fully complementary (e.g., has no mismatches) to a target sequence including a target mRNA and includes an antisense strand of 15 to 19 nucleotides. In some embodiments, the targeting sequence of the RNAi oligonucleotide conjugate herein is fully complementary (e.g., has no mismatches) to the target sequence comprising the target mRNA, and comprises an antisense strand of 15, 16, 17, 18, 19, 20, 21, or 22 nucleotides. In some embodiments, the targeting sequence of the RNAi oligonucleotide conjugate herein is fully complementary (e.g., has no mismatches) to the target sequence comprising the target mRNA, and comprises an antisense strand of 19 nucleotides. In some embodiments, the targeting sequence of the RNAi oligonucleotide conjugate herein is fully complementary (e.g., has no mismatches) to the target sequence comprising the target mRNA, and comprises an antisense strand of 20 nucleotides.

[0082] In some embodiments, the targeting sequence of the RNAi oligonucleotide conjugates herein is partially complementary (e.g., has no more than four mismatches) to a target sequence including a target mRNA and includes the entire length of the antisense strand. In some embodiments, the targeting sequence of the RNAi oligonucleotide conjugates herein is partially complementary (e.g., has no more than four mismatches) to a target sequence including a target mRNA and includes a portion of the entire length of the antisense strand. In some embodiments, the targeting sequence of the RNAi oligonucleotide conjugates herein is partially complementary (e.g., has no more than four mismatches) to a target sequence including a target mRNA and includes an antisense strand of 10-20 nucleotides. In some embodiments, the targeting sequence of the RNAi oligonucleotide conjugates herein is partially complementary (e.g., has no more than four mismatches) to a target sequence including a target mRNA and includes an antisense strand of 15-19 nucleotides. In some embodiments, the targeting sequence of the RNAi oligonucleotide conjugate herein is partially complementary (e.g., has no more than four mismatches) to the target sequence comprising the target mRNA, and comprises an antisense strand of 15, 16, 17, 18, 19, 20, 21, or 22 nucleotides. In some embodiments, the targeting sequence of the RNAi oligonucleotide conjugate herein is partially complementary (e.g., has no more than four mismatches) to the target sequence comprising the target mRNA, and comprises an antisense strand of 19 nucleotides. In some embodiments, the targeting sequence of the RNAi oligonucleotide conjugate herein is partially complementary (e.g., has no more than four mismatches) to the target sequence comprising the target mRNA, and comprises an antisense strand of 20 nucleotides.

[0083] In some embodiments, the RNAi oligonucleotide conjugates herein comprise a targeting sequence that has one or more base pair (bp) mismatches with the corresponding target sequence comprising the target mRNA. In some embodiments, the targeting sequence has 1 bp mismatch, 2 bp mismatch, 3 bp mismatch, 4 bp mismatch, or 5 bp mismatch with the corresponding target sequence comprising the target mRNA, as long as the ability of the targeting sequence to bind or anneal to the target sequence under suitable hybridization conditions and / or the ability of the RNAi oligonucleotide conjugate to inhibit or reduce target gene expression is maintained (e.g., under physiological conditions). Alternatively, in some embodiments, the targeting sequence comprises no more than 1 bp mismatch, no more than 2 bp mismatch, no more than 3 bp mismatch, no more than 4 bp mismatch, or no more than 5 bp mismatch with the corresponding target sequence comprising the target mRNA, as long as the ability of the targeting sequence to bind or anneal to the target sequence under suitable hybridization conditions and / or the ability of the RNAi oligonucleotide conjugate to inhibit or reduce target gene expression is maintained. In some embodiments, the RNAi oligonucleotide conjugate comprises a targeting sequence having one mismatch with the corresponding target sequence. In some embodiments, the RNAi oligonucleotide conjugate comprises a targeting sequence having two mismatches with the corresponding target sequence. In some embodiments, the RNAi oligonucleotide conjugate comprises a targeting sequence having three mismatches with the corresponding target sequence. In some embodiments, the RNAi oligonucleotide conjugate comprises a targeting sequence having four mismatches with the corresponding target sequence. In some embodiments, the RNAi oligonucleotide conjugate comprises a targeting sequence having five mismatches with the corresponding target sequence.In some embodiments, the RNAi oligonucleotide conjugate comprises a targeting sequence having multiple mismatches (e.g., 2, 3, 4, 5 or more mismatches) with the corresponding target sequence, where at least two (e.g., all) of the mismatches are located consecutively (e.g., 2, 3, 4, 5 or more mismatches in a row), or where the mismatches are interspersed anywhere throughout the targeting sequence. In some embodiments, the RNAi oligonucleotide conjugate comprises a targeting sequence having multiple mismatches (e.g., 2, 3, 4, 5 or more mismatches) with the corresponding target sequence, where at least two (e.g., all) of the mismatches are located consecutively (e.g., 2, 3, 4, 5 or more mismatches in a row), or where at least one or more non-mismatched base pairs are located between the mismatches, or a combination thereof.

[0084] Oligonucleotide types Various RNAi oligonucleotide types and / or structures are useful for reducing target gene expression in the methods herein.Any of the RNAi oligonucleotide types described herein or elsewhere are contemplated to be used as the framework for incorporating targeting sequence herein for the purpose of inhibiting or reducing the corresponding target gene expression in CNS.

[0085] In some embodiments, the RNAi oligonucleotide conjugates herein inhibit target gene expression by participating in the RNA interference (RNAi) pathway upstream or downstream of Dicer participation. For example, RNAi oligonucleotides have been developed with each strand having a size of about 19-25 nucleotides with at least one 3' overhang of 1-5 nucleotides (see, e.g., U.S. Pat. No. 8,372,968). Longer oligonucleotides have also been developed that are processed by Dicer to generate active RNAi products (see, e.g., U.S. Pat. No. 8,883,996). Further research has produced extended double-stranded oligonucleotides in which at least one end of at least one strand is extended beyond the double-stranded targeting region, with one of the strands including a structure that includes a thermodynamically stabilizing tetraloop structure (see, e.g., U.S. Pat. Nos. 8,513,207 and 8,927,705, and International Patent Application Publication No. WO 2010 / 033225). Such structures may contain single-stranded extensions (on either or both sides of the molecule) as well as double-stranded extensions.

[0086] In some embodiments, the RNAi oligonucleotide conjugates herein participate in the RNAi pathway downstream of Dicer intervention (e.g., Dicer cleavage). In some embodiments, the RNAi oligonucleotide conjugates described herein are Dicer substrates. In some embodiments, endogenous Dicer processing produces a 19-23 nucleotide long double-stranded nucleic acid that can reduce expression of a target mRNA (e.g., a CNS target mRNA). In some embodiments, the RNAi oligonucleotide conjugate has an overhang (e.g., 1, 2, or 3 nucleotides long) at the 3' end of the sense strand. In some embodiments, the RNAi oligonucleotide conjugate (e.g., siRNA conjugate) comprises a 21 nucleotide guide strand that is antisense to the target mRNA and a complementary passenger strand, both strands annealing to form a 19 bp duplex and a 2 nucleotide overhang at either or both 3' ends. Longer oligonucleotide designs are also contemplated, including oligonucleotides with 23 nucleotides of guide strand and 21 nucleotides of passenger strand, with blunt end (3' end of passenger strand / 5' end of guide strand) on the right side of the molecule, and 2 nucleotides of 3'-guide strand overhang (5' end of passenger strand / 3' end of guide strand) on the left side of the molecule.In such molecules, there is a 21bp double-stranded region.See, for example, U.S. Patent Nos. 9,012,138, 9,012,621, and 9,193,753.

[0087] In some embodiments, the RNAi oligonucleotide conjugates disclosed herein include a sense strand and an antisense strand that are both in the range of about 17-26 (e.g., 17-26, 20-25, or 21-23) nucleotides in length. In some embodiments, the RNAi oligonucleotide conjugates disclosed herein include a sense strand and an antisense strand that are both in the range of about 19-22 nucleotides in length. In some embodiments, the sense strand and the antisense strand are of equal length. In some embodiments, the RNAi oligonucleotide conjugates disclosed herein include a sense strand and an antisense strand such that there is a 3' overhang on either the sense strand or the antisense strand, or on both the sense strand and the antisense strand. In some embodiments, for RNAi oligonucleotide conjugates having a sense strand and an antisense strand that are both in the range of about 21-23 nucleotides in length, the 3' overhang on the sense strand, on the antisense strand, or on both the sense strand and the antisense strand is 1 or 2 nucleotides in length. In some embodiments, the RNAi oligonucleotide conjugate has a 22 nucleotide guide strand and a 20 nucleotide passenger strand, with a blunt end on the right side of the molecule (3' end of passenger strand / 5' end of guide strand) and a 2 nucleotide 3'-guide strand overhang on the left side of the molecule (5' end of passenger strand / 3' end of guide strand).In such molecules, there is a 20bp double-stranded region.

[0088] Other oligonucleotide designs for use with the compositions and methods herein include 16-mer siRNAs (see, e.g., Nucleic Acids in Chemistry and Biology, Blackburn (ed.), ROYAL SOCIETY OF CHEMISTRY, 2006), shRNAs (e.g., with stems of 19 bp or shorter; see, e.g., Moore et al. (2010) METHODS MOL. BIOL. 629:141-58), blunt siRNAs (e.g., 19 bp in length; see, e.g., Kraynack & Baker (2006) RNA 12:163-76), asymmetric siRNAs (aiRNAs; see, e.g., Sun et al. (2008) NAT. BIOTECHNOL. 26:1379-82), asymmetric short double-stranded siRNAs (see, e.g., Chang et al. (2008) NAT. BIOTECHNOL. 26:1379-82), and the like. al. (2009) Mol. Ther. 17:725-732), forked siRNAs (see, e.g., Hohjoh (2004) FEBS Lett. 557:193-98), single-stranded siRNAs (Elsner (2012) NAT. BIOTECHNOL. 30:1063), dumbbell-shaped circular siRNAs (see, e.g., Abe et al. (2007) J. AM. CHEM. SOC. 129:15108-09), and small internal segmented interfering RNAs (siRNAs; see, e.g., Bramsen et al. (2007) Nucleic Acids Res. 35:5886-97). Further non-limiting examples of oligonucleotide structures that may be used in some embodiments to reduce or inhibit expression of target genes are microRNAs (miRNAs), short hairpin RNAs (shRNAs), and short siRNAs (see, e.g., Hamilton et al. (2002) EMBO J. 21:4671-79; see also U.S. Patent Application Publication No. 2009 / 0099115).

[0089] Furthermore, in some embodiments, the RNAi oligonucleotide conjugate for reducing or inhibiting target gene expression herein is single-stranded (ss). Such structures include, but are not limited to, single-stranded RNAi molecules. Recent studies have demonstrated the activity of single-stranded RNAi molecules (see, for example, Matsui et al. (2016) MOL.THER.24:946-955). However, in some embodiments, the oligonucleotide-ligand conjugate described herein comprises an antisense oligonucleotide (ASO). An antisense oligonucleotide is a single-stranded oligonucleotide having a nucleobase sequence that, when described or shown in the 5' to 3' direction, comprises the reverse complement of the targeted segment of a particular nucleic acid and is suitably modified to induce RNaseH-mediated cleavage of its target RNA in cells (e.g., as a gapmer) or inhibit translation of a target mRNA in cells (e.g., as a mixmer). ASOs for use herein may be modified in any suitable manner known in the art, including, for example, those set forth in U.S. Patent No. 9,567,587 (including, for example, modifications of length, sugar moieties of nucleobases (pyrimidines, purines), and heterocyclic moieties of nucleobases). Additionally, ASOs have been used for decades to reduce the expression of specific target genes (see, for example, Bennett et al. (2017) ANNU. REV. PHARMACOL. 57:81-105).

[0090] Structure of the nucleic acid ligand conjugate In certain aspects, the present disclosure provides a nucleic acid-ligand conjugate represented by formula Ia: [ka] or a pharma- ceutically acceptable salt thereof, wherein: B is a nucleobase or hydrogen; R 1 and R 2 are independently hydrogen, halogen, or R A, -CN, -S(O)R, -S(O)2R, -Si(OR)2R, -Si(OR)R2, or -SiR3, or R on the same carbon 1 and R 2 together with their intervening atoms form a 3-membered saturated or partially unsaturated ring having 0 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; Each R A is independent, C 1-6 an optionally substituted group selected from aliphatic; phenyl; a 4-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; Each R is independently hydrogen, a suitable protecting group, or C 1-6 an optionally substituted group selected from aliphatic; phenyl; a 4-7 membered saturated or partially unsaturated heterocycle having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or two R groups on the same atom together with their intervening atoms form a 4-7 membered saturated, partially unsaturated, or heteroaryl ring having 0-3 heteroatoms independently selected from nitrogen, oxygen, silicon, and sulfur; L A is independent, P.G. 1 or -L-ligand, PG 1 is hydrogen or a suitable hydroxyl protecting group, Each ligand is independently -(LC) n and / or an adamantyl group, Each LC can be independently saturated or unsaturated, linear or branched C 1-50a lipid conjugate moiety comprising a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently replaced by -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR-, or -P(S)OR-; Each -Cy- is independently selected from phenylenyl; 8-10 membered bicyclic arylenyl; 4-7 membered saturated or partially unsaturated carbocyclylenyl; 4-11 membered saturated or partially unsaturated spirocarbocyclylenyl; 8-10 membered bicyclic saturated or partially unsaturated carbocyclylenyl; adamantanenyl; 4-7 membered saturated or partially unsaturated heterocyclylenyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; 4-7 membered saturated or partially unsaturated heterocyclylenyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; an optionally substituted bivalent ring selected from: an 8- to 11-membered saturated or partially unsaturated spiroheterocyclylenyl; an 8- to 10-membered bicyclic saturated or partially unsaturated heterocyclylenyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; a 5- to 6-membered heteroarylenyl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or an 8- to 10-membered bicyclic heteroarylenyl having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; n is 1 to 10; L is a covalent bond or a divalent saturated or unsaturated, linear or branched C 1-50 a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently selected from the group consisting of -Cy-, -O-, -NR-, -N(R)-C(O)-, -S-, -C(O)-, -S(O)-, -S(O)2-, -P(O)OR-, -P(S)OR-, -V 1 CR 2 W 1 -or [ka] is replaced by m is 1 to 50; X 1 , V 1 , and W1 are independently -C(R)2-, -OR, -O-, -S-, -Se-, or -NR-; Z is -O-, -S-, -NR-, or -CR2-; PG 2 is hydrogen, a phosphoramidite analog, or a suitable protecting group.

[0091] In certain embodiments, the nucleic acid-ligand conjugate is represented by formula Ib or Ic: [ka] or a pharma- ceutically acceptable salt thereof, wherein L 1 is a covalent bond or a divalent saturated or unsaturated, linear or branched C 1-50 a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR-, -P(S)OR-, or [ka] is replaced by R 4 is hydrogen, R A or a suitable amine protecting group, R 5 is adamantyl or saturated or unsaturated, linear or branched C 1-50 a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently replaced by -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR-, or -P(S)OR-.

[0092] In some embodiments, the nucleic acid-ligand conjugate is represented by formula I-Ib or I-Ic: [ka] or a pharma- ceutically acceptable salt thereof, wherein B is a nucleobase or hydrogen; m is 1 to 50; PG 1 and P.G. 2 are independently hydrogen, a phosphoramidite analog, or a suitable protecting group; R 5 is adamantyl or saturated or unsaturated, linear or branched C 1-50 a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently replaced by -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR-, or -P(S)OR-.

[0093] In certain embodiments of the nucleic acid-ligand conjugate, R 5 is selected from the following: [ka]

[0094] In certain embodiments of the nucleic acid-ligand conjugate, R 5 is selected from the following: [ka]

[0095] Double-stranded RNAi oligonucleotide conjugates structure In some aspects, the disclosure provides an oligonucleotide-ligand conjugate or oligonucleotide conjugate comprising one or more nucleic acid-ligand conjugates represented by formula II-a: [ka] or a pharma- ceutically acceptable salt thereof, wherein: B is a nucleobase or hydrogen; R1 and R 2 are independently hydrogen, halogen, or R A , -CN, -S(O)R, -S(O)2R, -Si(OR)2R, -Si(OR)R2, or -SiR3, or R on the same carbon 1 and R 2 together with their intervening atoms form a 3- to 7-membered saturated or partially unsaturated ring having 0-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; Each R A is independent, C 1-6 an optionally substituted group selected from aliphatic; phenyl; a 4-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; Each R is independently hydrogen, a suitable protecting group, or C 1-6 an optionally substituted group selected from aliphatic; phenyl; a 4-7 membered saturated or partially unsaturated heterocycle having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or two R groups on the same atom together with their intervening atoms form a 4-7 membered saturated, partially unsaturated, or heteroaryl ring having 0-3 heteroatoms independently selected from nitrogen, oxygen, silicon, and sulfur; Each LC can be independently saturated or unsaturated, linear or branched C 1-50 a lipid conjugate moiety comprising a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently replaced by -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)-OR-, -P(S)OR-; Each -Cy- is independently selected from phenylenyl; 8-10 membered bicyclic arylenyl; 4-7 membered saturated or partially unsaturated carbocyclylenyl; 4-11 membered saturated or partially unsaturated spirocarbocyclylenyl; 8-10 membered bicyclic saturated or partially unsaturated carbocyclylenyl; 4-7 membered saturated or partially unsaturated heterocyclylenyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; 4-11 membered saturated or partially unsaturated heterocyclylenyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. an optionally substituted bivalent ring selected from: a saturated or partially unsaturated spiroheterocyclylenyl; an 8-10 membered bicyclic saturated or partially unsaturated heterocyclylenyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; a 5-6 membered heteroarylenyl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or an 8-10 membered bicyclic heteroarylenyl having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; n is 1 to 10; L is a covalent bond or a divalent saturated or unsaturated, linear or branched C 1-50 a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently selected from the group consisting of -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, S(O)-, -S(O)2-, -P(O)OR-, -P(S)OR-, -V 1 CR 2 W 1 -or [ka] is replaced by m is 1 to 50; X 1 , V 1 , and W 1 are independently -C(R)2-, -OR, -O-, -S-, -Se-, or -NR-; Y is hydrogen, a suitable hydroxyl protecting group, [ka] and R 3 is hydrogen, a suitable protecting group, a suitable prodrug, or C 1-6 an optionally substituted group selected from aliphatic, phenyl, 4-7 membered saturated or partially unsaturated heterocycle having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; X 2 is O, S, or NR; X 3 is -O-, -S-, -BH2-, or a covalent bond, Y 1 is a linking group attached to the 2' or 3' end of a nucleoside, nucleotide, or oligonucleotide, Y 2 is hydrogen, a suitable protecting group, a phosphoramidite analog; an internucleotide linkage attached to the 5' end of a nucleoside, nucleotide, or oligonucleotide, or a linkage attached to a solid support; Z is -O-, -S-, -NR-, or -CR2-.

[0096] In certain embodiments, the oligonucleotide-ligand conjugate (oligonucleotide conjugate) is represented by formula II-b or II-c: [ka] or a pharma- ceutically acceptable salt thereof, wherein L 1 is a covalent bond, monovalent or divalent saturated or unsaturated, linear or branched C 1-50 a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR-, -P(S)OR-, or [ka] is replaced by R 4 is hydrogen, R A or a suitable amine protecting group, R 5 is adamantyl or saturated or unsaturated, linear or branched C 1-50 a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently replaced by -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR-, or -P(S)OR.

[0097] In some embodiments of the oligonucleotide-ligand conjugate, R 5 is selected from the following: [ka]

[0098] In certain embodiments of the oligonucleotide-ligand conjugate, R 5 is selected from the following: [ka]

[0099] In some embodiments, R 5 teeth [ka] It is.

[0100] In some embodiments, R 5 teeth [ka] It is.

[0101] In some embodiments, R 5 teeth [ka] It is.

[0102] In some embodiments, R 5 teeth [ka] It is.

[0103] In some embodiments, R 5 teeth [ka] It is.

[0104] In some embodiments, R 5 teeth [ka] It is.

[0105] In some embodiments, R 5 teeth [ka] It is.

[0106] In some embodiments, R 5 teeth [ka] It is.

[0107] In some embodiments, R 5 teeth [ka] It is.

[0108] In some embodiments, R 5 teeth [ka] It is.

[0109] In some embodiments, R 5 teeth, [ka] It is.

[0110] In some embodiments, R 5 teeth [ka] It is.

[0111] In some embodiments, R 5 teeth [ka] It is.

[0112] In some embodiments, R 5 teeth [ka] It is.

[0113] In some embodiments, the oligonucleotide-ligand conjugate is represented by formula II-Ib or II-Ic: [ka] or a pharma- ceutically acceptable salt thereof, wherein B is a nucleobase or hydrogen; m is 1 to 50; X 1 is -O- or -S-; Y is hydrogen, [ka] and R 3 is hydrogen or a suitable protecting group, X 2is O or S, X 3 is -O-, -S-, or a covalent bond; Y 1 is a linking group attached to the 2' or 3' end of a nucleoside, nucleotide, or oligonucleotide, Y 2 is hydrogen, a phosphoramidite analog; an internucleotide linkage attached to the 5' end of a nucleoside, nucleotide, or oligonucleotide, or a linkage attached to a solid support; R 5 is adamantyl or saturated or unsaturated, linear or branched C 1-50 a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently replaced by -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR-, or -P(S)OR-; R is hydrogen, a suitable protecting group, or C 1-6 an optionally substituted group selected from aliphatic; phenyl; a 4-7 membered saturated or partially unsaturated heterocycle having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0114] In certain embodiments of the oligonucleotide-ligand conjugate, R 5 is selected from the following: [ka]

[0115] In certain embodiments, the oligonucleotide-ligand conjugate comprises one or more nucleic acid-ligand conjugate configurations of any one of the above disclosed embodiments.

[0116] In certain embodiments, the oligonucleotide-ligand conjugate comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleic acid-ligand conjugate units.

[0117] In some aspects, the present disclosure provides RNAi oligonucleotide conjugates for targeting target mRNAs (e.g., target mRNAs expressed in the CNS) and inhibiting or reducing target gene expression (e.g., via the RNAi pathway), wherein the RNAi oligonucleotide conjugates are double-stranded (ds) nucleic acid molecules comprising a sense strand (e.g., also referred to herein as a passenger strand) and an antisense strand (e.g., also referred to herein as a guide strand). In some embodiments, the sense strand and the antisense strand are separate strands and are not covalently linked. In some embodiments, the sense strand and the antisense strand are covalently linked. In some embodiments, the sense strand and the antisense strand form a double-stranded region, and the sense strand and the antisense strand, or portions thereof, bind or anneal to each other in a complementary manner (e.g., by Watson-Crick base pairing).

[0118] In some embodiments, the sense strand has a first region (R1) and a second region (R2), R2 includes a first subregion (S1), a tetraloop (L) or a triloop (triL), and a second subregion (S2), L or triL is located between S1 and S2, and S1 and S2 form a second duplex (D2). D2 can have a variety of lengths. In some embodiments, D2 is about 1-6 bp in length. In some embodiments, D2 is 2-6, 3-6, 4-6, 5-6, 1-5, 2-5, 3-5, or 4-5 bp in length. In some embodiments, D2 is 1, 2, 3, 4, 5, or 6 bp in length. In some embodiments, D2 is 6 bp in length.

[0119] In some embodiments, R1 of the sense strand and the antisense strand form a first duplex (D1). In some embodiments, D1 is at least about 15 (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21) nucleotides in length. In some embodiments, D1 is in the range of about 12-30 nucleotides in length (e.g., 12-30, 12-27, 15-22, 18-22, 18-25, 18-27, 18-30, or 21-30 nucleotides in length). In some embodiments, D1 is at least 12 nucleotides in length (e.g., at least 12, at least 15, at least 20, at least 25, or at least 30 nucleotides in length). In some embodiments, D1 is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long. In some embodiments, D1 is 19 nucleotides long. In some embodiments, D1 is 20 nucleotides long. In some embodiments, D1 comprising a sense strand and an antisense strand does not span the entire length of the sense strand and / or the antisense strand. In some embodiments, D1 comprising a sense strand and an antisense strand spans the entire length of either the sense strand or the antisense strand or both. In certain embodiments, D1 comprising a sense strand and an antisense strand spans the entire length of both the sense strand and the antisense strand.

[0120] It should be understood that in some embodiments, when describing the structure of an oligonucleotide (e.g., an RNAi oligonucleotide conjugate) or other nucleic acid, reference may be made to the sequences presented in the sequence listing. In such embodiments, the actual oligonucleotide or other nucleic acid may have one or more alternative nucleotides (e.g., the RNA counterpart of a DNA nucleotide or the DNA counterpart of an RNA nucleotide) and / or one or more modified nucleotides and / or one or more modified internucleotide bonds and / or one or more other modifications when compared to the designated sequence, while retaining essentially the same or similar complementary properties as the designated sequence.

[0121] In some embodiments, the RNAi oligonucleotide conjugate herein comprises a sense strand of 25 nucleotides and an antisense strand of 27 nucleotides, and when acted by dicer enzyme, the antisense strand is incorporated into mature RISC.In some embodiments, the sense strand of the RNAi oligonucleotide conjugate is longer than 27 nucleotides (e.g., 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides).In some embodiments, the sense strand of the RNAi oligonucleotide conjugate is longer than 25 nucleotides (e.g., 26, 27, 28, 29 or 30 nucleotides).

[0122] In some embodiments, the RNAi oligonucleotide conjugates herein have one 5' end that is thermodynamically less stable compared to the other 5' end. In some embodiments, asymmetric RNAi oligonucleotide conjugates are provided that include a blunt end at the 3' end of the sense strand and a 3' overhang at the 3' end of the antisense strand. In some embodiments, the 3' overhang on the antisense strand is about 1-8 nucleotides long (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides long). Typically, the RNAi oligonucleotide conjugates have a 2-nucleotide overhang at the 3' end of the antisense (guide) strand. However, other overhangs are possible. In some embodiments, the overhang is a 3' overhang comprising 1-6 nucleotides, optionally 1-5, 1-4, 1-3, 1-2, 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-5, 5-6 nucleotides, or 1, 2, 3, 4, 5, or 6 nucleotides in length. However, in some embodiments, the overhang is a 5' overhang comprising 1-6 nucleotides, optionally 1-5, 1-4, 1-3, 1-2, 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-5, 5-6 nucleotides, or 1, 2, 3, 4, 5, or 6 nucleotides in length.

[0123] In some embodiments, the two terminal nucleotides on the 3' end of the antisense strand are modified. In some embodiments, the two terminal nucleotides on the 3' end of the antisense strand are complementary to the target mRNA (e.g., the target mRNA expressed in the CNS). In some embodiments, the two terminal nucleotides on the 3' end of the antisense strand are not complementary to the target mRNA. In some embodiments, the two terminal nucleotides on the 3' end of the antisense strand of the RNAi oligonucleotide conjugate herein are unpaired. In some embodiments, the two terminal nucleotides on the 3' end of the antisense strand of the RNAi oligonucleotide conjugate herein comprise unpaired GG. In some embodiments, the two terminal nucleotides on the 3' end of the antisense strand of the RNAi oligonucleotide conjugate herein are not complementary to the target mRNA. In some embodiments, the two terminal nucleotides on each 3' end of the RNAi oligonucleotide conjugate are GG. Typically, one or both of the two terminal GG nucleotides on each 3' end of the double-stranded oligonucleotide (e.g., the RNAi oligonucleotide conjugate) are not complementary to the target mRNA.

[0124] In some embodiments, there is one or more (e.g., 1, 2, 3, 4, or 5) mismatches between the sense strand and the antisense strand. When there are multiple mismatches between the sense strand and the antisense strand, they can be located consecutively (e.g., 2, 3, or more consecutively) or can be interspersed throughout the complementary region. In some embodiments, the 3' end of the sense strand contains one or more mismatches. In some embodiments, two mismatches are incorporated into the 3' end of the sense strand. In some embodiments, base mismatches or destabilization of the segment at the 3' end of the sense strand of the RNAi oligonucleotide conjugate herein improves or increases the potency and / or efficacy of the RNAi oligonucleotide conjugate.

[0125] In some embodiments, the present disclosure provides an RNAi oligonucleotide conjugate comprising an RNAi oligonucleotide and a lipid moiety. In some embodiments, the lipid moiety is conjugated to the sense strand of the RNAi oligonucleotide. In some embodiments, the RNAi oligonucleotide comprises a stem loop. In some embodiments, the ligand is conjugated to any of the nucleotides in the stem loop. In some embodiments, the ligand is conjugated to the first nucleotide 5' to 3' in the stem loop. In some embodiments, the ligand is conjugated to the second nucleotide 5' to 3' in the stem loop. In some embodiments, the ligand is conjugated to the third nucleotide 5' to 3' in the stem loop. In some embodiments, the ligand is conjugated to the fourth nucleotide 5' to 3' in the stem loop. In some embodiments, the ligand is conjugated to one, two, three, or four of the nucleotides in the stem loop. In some embodiments, the ligand is conjugated to three of the nucleotides in the stem loop.

[0126] In some embodiments, the oligonucleotide-ligand conjugate comprises a 36 nucleotide sense strand numbered 1-36 from the 5' to 3' positions. In some embodiments, the oligonucleotide-ligand conjugate comprises a lipid conjugated to position 27 of the 36 nucleotide sense strand. In some embodiments, the oligonucleotide-ligand conjugate comprises a lipid conjugated to position 28 of the 36 nucleotide sense strand. In some embodiments, the oligonucleotide conjugate comprises a lipid conjugated to position 29 of the 36 nucleotide sense strand. In some embodiments, the oligonucleotide conjugate comprises a lipid conjugated to position 30 of the 36 nucleotide sense strand.

[0127] In some embodiments, the oligonucleotide-ligand conjugate comprises an antisense strand of 15-30 nucleotides and a sense strand of 15-40 nucleotides, wherein the sense and antisense strands form a double-stranded region, wherein the antisense strand comprises a region of complementarity to a target sequence expressed in a tissue or cell of the CNS, and wherein the sense strand comprises at its 3' end a stem loop comprising a tetraloop comprising 4 nucleosides, wherein one or more of the 4 nucleosides are represented by formula II-Ib: [ka] where B is selected from adenine and guanine nucleobases; R 5 is a hydrocarbon chain. In some embodiments, m is 1, X1 is O, and Y2 is an internucleotide linkage group attached to the 5' end of the nucleoside; Y is [ka] where Y1 is a linking group attached to the 2' or 3' end of the nucleotide, X2 is O, X3 is O, and R3 is H. In some embodiments, the hydrocarbon chain is a C8-C30 hydrocarbon chain. In some embodiments, the hydrocarbon chain is a C16 hydrocarbon chain. In some embodiments, the C16 hydrocarbon chain is represented by: [ka] In some embodiments, the four nucleosides of the tetraloop are numbered 1-4 from 5' to 3', with position 1 being represented by formula II-Ib. In some embodiments, position 2 is represented by formula II-Ib. In some embodiments, position 3 is represented by formula II-Ib. In some embodiments, position 4 is represented by formula II-Ib. In some embodiments, the sense strand is 36 nucleotides with position numbering 1-36 from 5' to 3', the stem loop comprises nucleotides at positions 21-36, with one or more nucleosides at positions 27-30 being represented by formula II-Ib. In some embodiments, the antisense strand is 22 nucleotides.

[0128] Antisense strand In some embodiments, the antisense strand of RNAi oligonucleotide conjugate is called "guide strand".For example, antisense strand participates in RNA-induced silencing complex (RISC), binds to Argonaute protein such as Ago2, or participates in or binds to one or more similar factors, and directs the silencing of target gene, and antisense strand is called guide strand.In some embodiments, the sense strand that is complementary to guide strand is called "passenger strand".

[0129] In some embodiments, the RNAi oligonucleotide conjugates herein comprise an antisense strand up to about 50 nucleotides in length (e.g., up to 50, up to 40, up to 35, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, up to 15, or up to 12 nucleotides in length). In some embodiments, the RNAi oligonucleotide conjugates herein comprise an antisense strand at least about 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 22, at least 25, at least 27, at least 30, at least 35, or at least 38 nucleotides in length). In some embodiments, the RNAi oligonucleotide conjugates herein comprise an antisense strand ranging from about 12 to about 40 (e.g., 12-40, 12-36, 12-32, 12-28, 15-40, 15-36, 15-32, 15-30, 15-28, 17-22, 17-25, 19-27, 19-30, 20-40, 22-40, 25-40, or 32-40) nucleotides in length. In some embodiments, the RNAi oligonucleotide conjugates herein comprise an antisense strand ranging from 15-30 nucleotides in length. In some embodiments, the antisense strand of any one of the RNAi oligonucleotide conjugates disclosed herein is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length. In some embodiments, the RNAi oligonucleotide conjugate comprises an antisense strand that is 22 nucleotides in length.

[0130] Sense strand In some embodiments, the RNAi oligonucleotide conjugates disclosed herein comprise a sense strand (or passenger strand) of up to about 50 nucleotides in length (e.g., up to 50, up to 40, up to 36, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, or up to 12 nucleotides in length). In some embodiments, the RNAi oligonucleotide conjugates herein comprise a sense strand of at least about 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 25, at least 27, at least 30, at least 36, or at least 38 nucleotides in length). In some embodiments, the RNAi oligonucleotide conjugates herein comprise a sense strand ranging from about 12 to about 50 (e.g., 12-50, 12-40, 12-36, 12-32, 12-28, 15-40, 15-36, 15-32, 15-28, 17-21, 17-25, 19-27, 19-30, 20-40, 22-40, 25-40, or 32-40) nucleotides in length. In some embodiments, the RNAi oligonucleotide conjugates herein comprise a sense strand ranging from 15-50 nucleotides in length. In some embodiments, the RNAi oligonucleotide conjugates herein comprise a sense strand ranging from 18-36 nucleotides in length. In some embodiments, the RNAi oligonucleotide conjugates herein comprise a sense strand that is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the RNAi oligonucleotide conjugates herein comprise a sense strand that is 36 nucleotides in length.

[0131] In some embodiments, the sense strand comprises a stem-loop structure at its 3' end. In some embodiments, the stem-loop is formed by intrastrand base pairing. In some embodiments, the sense strand comprises a stem-loop structure at its 5' end. In some embodiments, the stem is double stranded, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleotides in length. In some embodiments, the stem-loop provides RNAi oligonucleotide conjugate protection against degradation (e.g., enzymatic degradation), promotes or improves targeting and / or delivery to target cells, tissues, or organs (e.g., liver), or both. For example, in some embodiments, the loop of the stem-loop provides nucleotides that include one or more modifications that promote, improve, or increase targeting to target mRNA (e.g., target mRNA expressed in the CNS), inhibition of target gene expression, and / or delivery to target cells, tissues, or organs (e.g., CNS), or a combination thereof. In some embodiments, the stem-loop itself or modifications to the stem-loop do not substantially affect the intrinsic gene expression inhibitory activity of the RNAi oligonucleotide conjugate, but promote, improve, or increase the stability (e.g., provide protection against degradation) and / or delivery of the RNAi oligonucleotide conjugate to a target cell, tissue, or organ (e.g., CNS). In certain embodiments, the RNAi oligonucleotide conjugate herein comprises a sense strand comprising a stem-loop (e.g., at its 3' end) shown as S1-L-S2, where S1 is complementary to S2, and L forms a single-stranded loop between S1 and S2 up to about 10 nucleotides long (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides long). In some embodiments, the loop (L) is 3 nucleotides long. In some embodiments, the loop (L) is 4 nucleotides long.

[0132] In some embodiments, the tetraloop comprises the sequence 5'-GAAA-3'. In some embodiments, the stem loop comprises the sequence 5'-GCAGCCGAAAGGCUGC-3' (SEQ ID NO: 15).

[0133] In some embodiments, the loop (L) of the stem-loop having the structure S1-L-S2 described above is a triloop. In some embodiments, the triloop comprises ribonucleotides, deoxyribonucleotides, modified nucleotides, delivery ligands, and combinations thereof.

[0134] In some embodiments, the loop (L) of the stem-loop having the structure S1-L-S2 described above is a tetraloop (e.g., in a nicked tetraloop structure). In some embodiments, the tetraloop comprises ribonucleotides, deoxyribonucleotides, modified nucleotides, delivery ligands, and combinations thereof.

[0135] In some embodiments, the loop (L) of the stem-loop having the structure S1-L-S2 described above is a tetraloop (e.g., in a nicked tetraloop structure) as described in U.S. Pat. No. 10,131,912, which is incorporated herein by reference.

[0136] Length of the double strand In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 12 (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21) nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is in the range of 12-30 nucleotides in length (e.g., 12-30, 12-27, 12-22, 15-25, 18-30, 18-22, 18-25, 18-27, 18-30, 19-30, or 21-30 nucleotides in length). In some embodiments, the duplex formed between the sense strand and the antisense strand is 12, 13, 14, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the duplex formed between sense strand and antisense strand does not cover the entire length of sense strand and / or antisense strand.In some embodiments, the duplex formed between sense strand and antisense strand covers the entire length of either sense strand or antisense strand.In some embodiments, the duplex formed between sense strand and antisense strand covers the entire length of both sense strand and antisense strand.

[0137] Oligonucleotide End In some embodiments, the RNAi oligonucleotide conjugates disclosed herein comprise a sense strand and an antisense strand, such that there is a 3' overhang on either the sense strand or the antisense strand, or on both the sense strand and the antisense strand. In some embodiments, the RNAi oligonucleotide conjugates herein have one 5' end that is thermodynamically less stable than the other 5' end. In some embodiments, asymmetric RNAi oligonucleotide conjugates are provided that comprise a blunt end on the 3' end of the sense strand and an overhang on the 3' end of the antisense strand. In some embodiments, the 3' overhang on the antisense strand is about 1-8 nucleotides in length (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides in length).

[0138] Typically, RNAi oligonucleotides have an overhang of two nucleotides on the 3' end of the antisense (guide) strand. However, other overhangs are possible. In some embodiments, the overhang is a 3' overhang that includes 1-6 nucleotides, optionally 1-5, 1-4, 1-3, 1-2, 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-5, 5-6 nucleotides, or 1, 2, 3, 4, 5, or 6 nucleotides in length. In some embodiments, the overhang is a 5' overhang that includes 1-6 nucleotides, optionally 1-5, 1-4, 1-3, 1-2, 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-5, 5-6 nucleotides, or 1, 2, 3, 4, 5, or 6 nucleotides in length.

[0139] In some embodiments, one or more (e.g., 2, 3, or 4) terminal nucleotides at the 3'-end or 5'-end of the sense strand and / or antisense strand are modified. For example, in some embodiments, one or two terminal nucleotides at the 3'-end of the antisense strand are modified. In some embodiments, the last nucleotide at the 3'-end of the antisense strand is modified, e.g., includes a 2' modification, e.g., 2'-O-methoxyethyl. In some embodiments, the last one or two terminal nucleotides at the 3'-end of the antisense strand are complementary to the target. In some embodiments, the last one or two nucleotides at the 3'-end of the antisense strand are not complementary to the target.

[0140] In some embodiments, the RNAi oligonucleotide conjugate disclosed herein comprises a stem-loop structure at the 3' end of the sense strand, and comprises two terminal overhang nucleotides at the 3' end of the antisense strand.In some embodiments, the RNAi oligonucleotide conjugate disclosed herein comprises a nicked tetraloop structure, the 3' end of the sense strand comprises a stem-tetraloop structure, and comprises two terminal overhang nucleotides at the 3' end of the antisense strand.In some embodiments, the two terminal overhang nucleotides are GG.Typically, one or both of the two terminal GG nucleotides of the antisense strand are not complementary to the target.

[0141] In some embodiments, the 5' and / or 3' end of the sense or antisense strand has an inverted cap nucleotide.

[0142] In some embodiments, one or more (e.g., 2, 3, 4, 5, 6) modified internucleotide linkages are provided between the terminal nucleotides at the 3' or 5' ends of the sense and / or antisense strands. In some embodiments, modified internucleotide linkages are provided between the overhanging nucleotides at the 3' or 5' ends of the sense and / or antisense strands.

[0143] Oligonucleotide Modification In some embodiments, the RNAi oligonucleotide conjugate disclosed herein comprises one or more modifications.Oligonucleotide (e.g., RNAi oligonucleotide) can be modified in various ways to improve or control specificity, stability, delivery, bioavailability, resistance to nuclease degradation, immunogenicity, base pairing properties, RNA distribution and cellular uptake, and other characteristics related to therapeutic research applications.

[0144] In some embodiments, the modification is a modified sugar. In some embodiments, the modification is a 5'-terminal phosphate group. In some embodiments, the modification is a modified internucleoside linkage. In some embodiments, the modification is a modified base. In some embodiments, the modification is a reversible modification. In some embodiments, the oligonucleotides described herein can include any one of the modifications described herein or any combination thereof. For example, in some embodiments, the oligonucleotides described herein include at least one modified sugar, a 5'-terminal phosphate group, at least one modified internucleoside linkage, at least one modified base, and at least one reversible modification.

[0145] The number of modifications in an oligonucleotide (e.g., an RNAi oligonucleotide) and the location of those nucleotide modifications can affect the properties of the oligonucleotide. For example, oligonucleotides can be delivered in vivo by conjugating them with or surrounding them with lipid nanoparticles (LNPs) or similar carriers. However, if the oligonucleotide is not protected by an LNP or similar carrier, it may be advantageous for at least some of the nucleotides to be modified. Thus, in some embodiments, all or substantially all nucleotides of an oligonucleotide are modified. In some embodiments, more than half of the nucleotides are modified. In some embodiments, less than half of the nucleotides are modified. In some embodiments, the sugar moieties of all nucleotides comprising the oligonucleotide are modified at the 2' position. The modifications can be reversible or irreversible. In some embodiments, the oligonucleotides disclosed herein have a sufficient number and type of modified nucleotides to cause the desired characteristics (e.g., protection from enzymatic degradation, ability to target desired cells after in vivo administration, and / or thermodynamic stability).

[0146] sugar modification In some embodiments, the RNAi oligonucleotide conjugates described herein include modified sugars. In some embodiments, modified sugars (also referred to herein as sugar analogs) include modified deoxyribose or ribose moieties, for example, where one or more modifications occur at the 2', 3', 4', and / or 5' carbon positions of the sugar. In some embodiments, modified sugars can also include unnatural alternative carbon structures, such as those present in locked nucleic acids ("LNAs", see e.g., Koshkin et al. (1998) TETRAHEDON 54:3607-30), non-locked nucleic acids ("UNAs", see e.g., Snead et al. (2013) MOL. THER-NUCL. ACIDS 2:e103), and bridged nucleic acids ("BNAs", see e.g., Imanishi & Obika (2002) CHEM COMMUN. (CAMB) 21:1653-59).

[0147] In some embodiments, the nucleotide modification at the sugar comprises a 2'-modification. In some embodiments, the 2'-modification can be 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, 2'-fluoro (2'-F), 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), or 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid (2'-FANA). In some embodiments, the modification is 2'-F, 2'-OMe, or 2'-MOE. In some embodiments, the modification at the sugar comprises a modification of the sugar ring, which can comprise a modification of one or more carbons of the sugar ring. For example, modifications of the sugar of a nucleotide may include linking the 2'-oxygen of the sugar to the 1'-carbon or 4'-carbon of the sugar, or linking the 2'-oxygen to the 1'-carbon or 4'-carbon via an ethylene or methylene bridge. In some embodiments, modified nucleotides have acyclic sugars that lack a 2'-carbon to 3'-carbon bond. In some embodiments, modified nucleotides have a thiol group, for example, at the 4' position of the sugar.

[0148] In some embodiments, the RNAi oligonucleotide conjugates described herein comprise at least about one modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, or more). In some embodiments, the sense strand of the RNAi oligonucleotide conjugate comprises at least about one modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, or more). In some embodiments, the antisense strand of the RNAi oligonucleotide conjugate comprises at least about one modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, or more).

[0149] In some embodiments, all nucleotides of the sense strand of the RNAi oligonucleotide conjugate are modified. In some embodiments, all nucleotides of the antisense strand of the RNAi oligonucleotide conjugate are modified. In some embodiments, all nucleotides of the RNAi oligonucleotide conjugate (i.e., both sense strand and antisense strand) are modified. In some embodiments, modified nucleotides include 2'-modifications (e.g., 2'-F or 2'-OMe, 2'-MOE, and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid).

[0150] In some embodiments, the present disclosure provides the RNAi oligonucleotide conjugate with different modification patterns.In some embodiments, the modified RNAi oligonucleotide conjugate comprises the sense strand sequence with the modification pattern shown in the examples and sequence listing, and the antisense strand with the modification pattern shown in the examples and sequence listing.

[0151] In some embodiments, the RNAi oligonucleotide conjugates disclosed herein comprise an antisense strand with 2'-F modified nucleotides. In some embodiments, the RNAi oligonucleotide conjugates disclosed herein comprise an antisense strand with 2'-F and 2'-OMe modified nucleotides. In some embodiments, the RNAi oligonucleotide conjugates disclosed herein comprise a sense strand with 2'-F modified nucleotides. In some embodiments, the RNAi oligonucleotide conjugates disclosed herein comprise a sense strand with 2'-F and 2'-OMe modified nucleotides.

[0152] In some embodiments, the oligonucleotides described herein comprise a sense strand in which about 10-25%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the nucleotides of the sense strand comprise a 2'-fluoro modification. In some embodiments, about 11% of the nucleotides of the sense strand comprise a 2-fluoro modification. In some embodiments, about 20% of the nucleotides of the sense strand comprise a 2-fluoro modification. In some embodiments, the oligonucleotides described herein comprise an antisense strand in which about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% of the nucleotides of the antisense strand comprise a 2'-fluoro modification. In some embodiments, about 32% of the nucleotides of the antisense strand comprise a 2-fluoro modification. In some embodiments, the oligonucleotide has about 15-25%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of its nucleotides containing a 2'-fluoro modification. In some embodiments, about 19% of the nucleotides in the oligonucleotide contain a 2'-fluoro modification. In some embodiments, about 26% of the nucleotides in the oligonucleotide contain a 2'-fluoro modification.

[0153] In some embodiments, these oligonucleotides are modified with a 2'-F group at one or more of positions 8, 9, 10, or 11 of the sense strand. In some embodiments, these oligonucleotides are modified with a 2'-OMe group at the sugar moiety of each of the nucleotides at positions 1-7 and 12-20 of the sense strand. In some embodiments, these oligonucleotides are modified with a 2'-OMe group at the sugar moiety of each of the nucleotides at positions 1-7, 12-27, and 29-36 of the sense strand.

[0154] In some embodiments, these oligonucleotides are modified with 2'-F groups at one or more of positions 3, 5, 8, 10, 12, 13, 15, and 17 of the sense strand. In some embodiments, these oligonucleotides are modified with 2'-OMe at the sugar moiety of each of the nucleotides at positions 1, 2, 4, 6, 7, 9, 11, 14, 16, 18-27, and 29-36 of the sense strand.

[0155] In some embodiments, the antisense strand has three nucleotides in which the 2'-position of the sugar moiety is modified with 2'-F. In some embodiments, the sugar moieties of the nucleotides at positions 2, 5, and 14 of the antisense strand, and optionally up to three of the nucleotides at positions 1, 3, 7, and 10 of the antisense strand, are modified with 2'-F. In other embodiments, the sugar moieties of each of the nucleotides at positions 2, 5, and 14 of the antisense strand are modified with 2'-F. In other embodiments, the sugar moieties of each of the nucleotides at positions 1, 2, 5, and 14 of the antisense strand are modified with 2'-F. In yet other embodiments, the sugar moieties of each of the nucleotides at positions 1, 2, 3, 5, 7, and 14 of the antisense strand are modified with 2'-F. In yet other embodiments, the sugar moieties of each of the nucleotides at positions 1, 2, 3, 5, 7, and 14 of the antisense strand are modified with 2'-F. In yet other embodiments, the sugar moieties of each of the nucleotides at positions 1, 2, 3, 5, 10, and 14 of the antisense strand are modified with 2'-F. In another embodiment, the sugar moiety of each of the nucleotides at positions 2, 3, 5, 7, 10, and 14 of the antisense strand is modified with 2'-F. In some embodiments, the antisense strand has 9 nucleotides in which the 2'-position of the sugar moiety is modified with 2'-F. In certain additional embodiments, the sugar moiety of each of the nucleotides at positions 2, 3, 4, 5, 7, 10, 14, 16, and 19 of the antisense strand is modified with 2'-F.

[0156] In some embodiments, the RNAi oligonucleotide conjugates provided herein comprise an antisense strand having sugar moieties at positions 2 and 14 modified with 2'-F. In some embodiments, the RNAi oligonucleotide conjugates provided herein comprise an antisense strand having sugar moieties at positions 2, 5, and 14 modified with 2'-F. In some embodiments, the RNAi oligonucleotide conjugates provided herein comprise an antisense strand having sugar moieties at positions 1, 2, 5, and 14 modified with 2'-F. In some embodiments, the RNAi oligonucleotide conjugates provided herein comprise an antisense strand having sugar moieties at positions 1, 2, 3, 5, 7, and 14 modified with 2'-F. In some embodiments, the RNAi oligonucleotide conjugates provided herein comprise an antisense strand having sugar moieties at positions 1, 2, 3, 5, 10, and 14 modified with 2'-F. In some embodiments, the RNAi oligonucleotide conjugates provided herein comprise an antisense strand having sugar moieties at positions 2, 3, 4, 5, 7, 10, 14, 16, and 19 modified with 2'-F.

[0157] In some embodiments, an RNAi oligonucleotide conjugate provided herein comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 2, 5, and 14 of the antisense strand modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid (2'-FANA).

[0158] In some embodiments, an RNAi oligonucleotide conjugate provided herein comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 2, 3, 4, 5, 7, 10, 14, 16, and 19 of the antisense strand modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid (2'-FANA).

[0159] In some embodiments, an RNAi oligonucleotide conjugate provided herein comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 1, 2, 5, and 14 of the antisense strand modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid (2'-FANA).

[0160] In some embodiments, an RNAi oligonucleotide conjugate provided herein comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 1, 2, 3, 5, 7, and 14 of the antisense strand modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid (2'-FANA).

[0161] In some embodiments, an RNAi oligonucleotide conjugate provided herein comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 1, 2, 3, 5, 10, and 14 of the antisense strand modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid (2'-FANA).

[0162] In some embodiments, an RNAi oligonucleotide conjugate provided herein comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 2, 3, 5, 7, 10, and 14 of the antisense strand modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid (2'-FANA).

[0163] In some embodiments, an RNAi oligonucleotide conjugate provided herein comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 2, 3, 4, 5, 7, 10, 14, 16, and 19 of the antisense strand modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid (2'-FANA).

[0164] In some embodiments, the RNAi oligonucleotide conjugates provided herein comprise an antisense strand having a sugar moiety at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 modified with 2'-F.

[0165] In some embodiments, RNAi oligonucleotide conjugates provided herein comprise an antisense strand having a sugar moiety at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 that is 2'-OMe modified.

[0166] In some embodiments, an RNAi oligonucleotide conjugate provided herein comprises an antisense strand having a sugar moiety at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid (2'-FANA).

[0167] In some embodiments, the RNAi oligonucleotide conjugates provided herein comprise a sense strand having sugar moieties at positions 8-11 modified with 2'-F. In some embodiments, the RNAi oligonucleotide conjugates provided herein comprise a sense strand having sugar moieties at positions 3, 5, 8, 10, 12, 13, 15, and 17 modified with 2'-F. In some embodiments, the RNAi oligonucleotide conjugates provided herein comprise a sense strand having sugar moieties at positions 1-7 and 12-17 or 12-20 modified with 2'OMe. In some embodiments, the RNAi oligonucleotide conjugates provided herein comprise a sense strand having sugar moieties at positions 1, 2, 4, 6, 7, 9, 11, 14, 16, and 18-20 modified with 2'OMe. In some embodiments, an RNAi oligonucleotide conjugate provided herein comprises a sense strand having a sugar moiety at each of nucleotides 1-7 and 12-17 or 12-20 of the sense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid (2'-FANA). In some embodiments, an RNAi oligonucleotide conjugate provided herein comprises a sense strand having sugar moieties at positions 1, 2, 4, 6, 7, 9, 11, 14, 16, and 18-20 of the sense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid (2'-FANA).

[0168] In some embodiments, RNAi oligonucleotide conjugates provided herein comprise a sense strand having a sugar moiety at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 that is modified with 2'-F.

[0169] In some embodiments, RNAi oligonucleotide conjugates provided herein comprise a sense strand having a sugar moiety at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 that is 2'-OMe modified.

[0170] In some embodiments, the RNAi oligonucleotide conjugates provided herein include 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro- and a sense strand having sugar moieties at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 modified with a modification selected from the group consisting of β-d-arabinonucleic acid (2'-FANA).

[0171] 5'-Terminal Phosphate In some embodiments, the RNAi oligonucleotide conjugates described herein comprise a 5'-terminal phosphate. In some embodiments, the 5'-terminal phosphate group of the RNAi oligonucleotide conjugate enhances the interaction with Ago2. However, oligonucleotides that comprise a 5'-phosphate group may be susceptible to degradation via phosphatases or other enzymes, which may limit their bioavailability in vivo. In some embodiments, the RNAi oligonucleotide conjugates herein comprise an analog of the 5' phosphate that is resistant to such degradation. In some embodiments, the phosphate analog is an oxymethylphosphonate, vinylphosphonate, or malonylphosphonate, or a combination thereof. In certain embodiments, the 5' end of the RNAi oligonucleotide conjugate strand is attached to a chemical moiety ("phosphate mimic") that mimics the electrostatic and steric properties of the natural 5' phosphate group.

[0172] In some embodiments, the RNAi oligonucleotide conjugates herein have a phosphate analog at the 4'-carbon position of the sugar (referred to as a "4'-phosphate analog"). See, for example, International Patent Application Publication No. 2018 / 045317. In some embodiments, the RNAi oligonucleotide conjugates herein include a 4' phosphate analog at the 5'-terminal nucleotide. In some embodiments, the phosphate analog is an oxymethylphosphonate in which the oxygen atom of the oxymethyl group is attached to the sugar moiety (e.g., its 4' carbon) or an analog thereof. In other embodiments, the 4'-phosphate analog is a thiomethylphosphonate or an aminomethylphosphonate in which the sulfur atom of the thiomethyl group or the nitrogen atom of the aminomethyl group is attached to the 4'-carbon of the sugar moiety or an analog thereof. In certain embodiments, the 4'-phosphate analog is an oxymethylphosphonate. In some embodiments, the oxymethylphosphonate is represented by the formula -O-CH2-PO(OH)2, -O-CH2-PO(OR)2, or -O-CH2-POOH(R), where R is independently selected from H, CH3, an alkyl group, CH2CH2CN, CH2OCOC(CH3)3, CHOCH2CH2Si(CH3)3, or a protecting group. In certain embodiments, the alkyl group is CH2CH3. More typically, R is independently selected from H, CH3, or CH2CH3. In some embodiments, R is CH3. In some embodiments, the 4'-phosphate analog is 5'-methoxyphosphonate-4'-oxy. In some embodiments, the 4'-phosphate analog is 4'-oxymethylphosphonate.

[0173] In some embodiments, an RNAi oligonucleotide conjugate provided herein comprises an antisense strand that comprises a 4'-phosphate analog at the 5' terminal nucleotide, wherein the 5' terminal nucleotide comprises the following structure: [ka] 4'-O-monomethylphosphonate-2'-O-methyluridine phosphorothioate [Mephosphonate-4O-mUs]

[0174] Modified Internucleoside Linkages In some embodiments, the RNAi oligonucleotide conjugates provided herein comprise modified internucleoside linkages. In some embodiments, the phosphate modifications or substitutions result in an oligonucleotide comprising at least about one (e.g., at least one, at least two, at least three, or at least five) modified internucleoside linkages. In some embodiments, any one of the oligonucleotides disclosed herein comprises about one to about ten (e.g., 1-10, 2-8, 4-6, 3-10, 5-10, 1-5, 1-3, or 1-2) modified internucleoside linkages. In some embodiments, any one of the oligonucleotides disclosed herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified internucleoside linkages.

[0175] The modified internucleotide bond can be a phosphorodithioate bond, a phosphorothioate bond, a phosphotriester bond, a thionoalkylphosphonate bond, a thionoalkylphosphonate bond, a phosphoramidite bond, a phosphonate bond or a boranophosphate bond. In some embodiments, at least one modified internucleotide bond of any one of the oligonucleotides disclosed herein is a phosphorothioate bond.

[0176] In some embodiments, the RNAi oligonucleotide conjugates provided herein have phosphorothioate bonds between one or more of positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 3 and 4 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand. In some embodiments, the oligonucleotides described herein have phosphorothioate bonds between each of positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand.

[0177] In some embodiments, the oligonucleotide conjugates described herein comprise peptide nucleic acids (PNAs). PNAs are oligonucleotide mimics in which the sugar-phosphate backbone is replaced by a pseudopeptide backbone composed of N-(2-aminoethyl)glycine units. Nucleobases are linked to this backbone via diatomic carboxymethyl spacers. In some embodiments, the oligonucleotide conjugates described herein comprise morpholino oligomers (PMOs) that comprise an internucleotide linkage backbone of methylene morpholine rings linked via phosphorodiamidate groups.

[0178] Base Modification In some embodiments, the RNAi oligonucleotide conjugate herein comprises one or more modified nucleobases. In some embodiments, the modified nucleobase (also referred to herein as base analog) is linked to the 1' position of the nucleotide sugar moiety. In certain embodiments, the modified nucleobase is a nitrogenous base. In some embodiments, the modified nucleobase does not contain a nitrogen atom. See, for example, US Patent Application Publication No. 2008 / 0274462. In some embodiments, the modified nucleotide comprises a universal base. In some embodiments, the modified nucleotide does not contain a nucleobase (abasic).

[0179] In some embodiments, a universal base is a heterocyclic moiety located at the 1' position of a nucleotide sugar moiety of a modified nucleotide, or at an equivalent position of a nucleotide sugar moiety substitution, and when present in a duplex, can be positioned opposite multiple types of bases without substantially altering the structure of the duplex. In some embodiments, compared to a reference single-stranded nucleic acid (e.g., an oligonucleotide) that is fully complementary to a target nucleic acid, a single-stranded nucleic acid containing a universal base has a lower T than a duplex formed with a complementary nucleic acid. m In some embodiments, when compared to a reference single-stranded nucleic acid in which the universal base is replaced with a base that creates a single mismatch, the single-stranded nucleic acid containing the universal base has a higher T than the duplex formed with the nucleic acid containing the mismatched base. m The nucleic acid forms a duplex with the target nucleic acid.

[0180] Non-limiting examples of universal binding nucleotides include, but are not limited to, inosine, 1-β-D-ribofuranosyl-5-nitroindole, and / or 1-β-D-ribofuranosyl-3-nitropyrrole (see, e.g., U.S. Patent Application Publication No. 2007 / 0254362, Van Aerschot et al. (1995) NUCLEIC ACIDS RES. 23:4363-4370, Loakes et al. (1995) NUCLEIC ACIDS RES. 23:2361-66, and Loakes & Brown (1994) NUCLEIC ACIDS RES. 22:4039-43).

[0181] Targeting Ligands In some embodiments, it is desirable to target the oligonucleotides (e.g., RNAi oligonucleotide conjugates) of the present disclosure to one or more cells or one or more organs. Such a strategy can help to avoid undesirable effects in other organs or to avoid unnecessary loss of oligonucleotides to cells, tissues or organs that would not benefit from the oligonucleotide. Thus, in some embodiments, the RNAi oligonucleotide conjugates disclosed herein are modified to facilitate targeting and / or delivery to specific tissues, cells or organs (e.g., to facilitate delivery of the conjugate to the CNS). In some embodiments, the RNAi oligonucleotide conjugates comprise at least one nucleotide (e.g., 1, 2, 3, 4, 5, 6 or more nucleotides) conjugated to one or more targeting ligands.

[0182] In some embodiments, the targeting ligand comprises carbohydrate, amino sugar, cholesterol, peptide, polypeptide, protein, or protein part (e.g., antibody or antibody fragment), or lipid. In some embodiments, the targeting ligand is an aptamer. For example, the targeting ligand may be RGD peptide used to target tumor vasculature or glioma cells, CREKA peptide to target tumor vasculature or stoma, transferring, lactoferrin, or aptamer to target transferrin receptor expressed in CNS vasculature, or anti-EGFR antibody to target EGFR on glioma cells. In certain embodiments, the targeting ligand is one or more GalNAc moieties.

[0183] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides of the RNAi oligonucleotide conjugates disclosed herein are each conjugated to a separate targeting ligand. In some embodiments, 2-4 nucleotides of the RNAi oligonucleotide conjugates herein are each conjugated to a separate targeting ligand. In some embodiments, the targeting ligand is conjugated to 2-4 nucleotides at both ends of the sense or antisense strand (e.g., the targeting ligand is conjugated to an overhang or extension of 2-4 nucleotides on the 5' or 3' end of the sense or antisense strand) such that the targeting ligand resembles the bristles of a toothbrush and the RNAi oligonucleotide conjugate resembles a toothbrush. For example, the RNAi oligonucleotide conjugate may include a stem-loop at either the 5' or 3' end of the sense strand, and 1, 2, 3, or 4 nucleotides of the stem loop may be individually conjugated to a targeting ligand. In some embodiments, the RNAi oligonucleotide conjugates provided by the present disclosure comprise a stem-loop at the 3' end of the sense strand, wherein the loop of the stem-loop comprises a tri- or tetra-loop, and the three or four nucleotides comprising the tri- or tetra-loop are respectfully individually conjugated to a targeting ligand.

[0184] GalNAc is a high affinity ligand for ASGPR, which is mainly expressed on the sinusoidal surface of hepatocytes and plays a major role in binding, internalization, and subsequent removal of circulating glycoproteins that contain terminal galactose or GalNAc residues (asialoglycoproteins).Conjugation of GalNAc moieties (either indirectly or directly) to the oligonucleotides of the present disclosure can be used to target these oligonucleotides to ASGPR expressed on cells.In some embodiments, the oligonucleotides of the present disclosure are conjugated with at least one or more GalNAc moieties, and the GalNAc moieties target the oligonucleotides to ASGPR expressed on human liver cells (e.g., human hepatocytes).In some embodiments, the GalNAc moieties target the oligonucleotides to the liver.

[0185] In some embodiments, the oligonucleotide of the present disclosure is directly or indirectly conjugated with monovalent GalNAc moiety.In some embodiments, the oligonucleotide is directly or indirectly conjugated with multiple monovalent GalNAc moieties (i.e., conjugated with 2, 3 or 4 monovalent GalNAc moieties, typically conjugated with 3 or 4 monovalent GalNAc moieties).In some embodiments, the oligonucleotide is conjugated with one or more divalent GalNAc, trivalent GalNAc or tetravalent GalNAc moieties.

[0186] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides of the oligonucleotide are each conjugated to a GalNAc moiety. In some embodiments, 2-4 nucleotides of the tetraloop are each conjugated to a separate GalNAc. In some embodiments, 1-3 nucleotides of the triloop are each conjugated to a separate GalNAc. In some embodiments, the targeting ligand is conjugated to 2-4 nucleotides at both ends of the sense or antisense strand (e.g., the ligand is conjugated to an overhang or extension of 2-4 nucleotides on the 5' or 3' end of the sense or antisense strand) such that the GalNAc moieties resemble toothbrush bristles and the oligonucleotide resembles a toothbrush. In some embodiments, the GalNAc moiety is conjugated to a nucleotide of the sense strand. For example, four GalNAc moieties can be conjugated to nucleotides in the tetraloop of the sense strand, with each GalNAc moiety being conjugated to one nucleotide.

[0187] In some embodiments, the tetraloop is any combination of adenine and guanine nucleotides.

[0188] In some embodiments, the tetraloop (L) has a monovalent GalNAc moiety attached to any one or more guanine nucleotides of the tetraloop via any linker described herein, as shown below (X=heteroatom). [ka]

[0189] In some embodiments, the tetraloop (L) has a monovalent GalNAc attached to any one or more adenine nucleotides of the tetraloop via any linker described herein, as shown below (X=heteroatom). [ka]

[0190] In some embodiments, the RNAi oligonucleotide conjugates herein comprise a monovalent GalNAc attached to a guanine nucleotide designated [ademG-GalNAc] or 2'-aminodiethoxymethanol-guanine-GalNAc, as shown below. [ka]

[0191] In some embodiments, the RNAi oligonucleotide conjugates herein comprise a monovalent GalNAc moiety attached to an adenine nucleotide, designated [ademG-GalNAc] or 2'-aminodiethoxymethanol-adenine-GalNAc, as shown below. [ka]

[0192] An example of such a conjugate is shown below for a loop containing the nucleotide sequence GAAA (L=linker, X=heteroatom) 5' to 3'. Such a loop can be, for example, at positions 27-30 of the sense strand listed in Tables 3 or 5 and shown in Figures 12-13. In the formula: [ka] is used to describe the point of attachment to the oligonucleotide chain. [ka]

[0193] The targeting ligand can be attached to the nucleotide using a suitable method or chemistry (e.g., click chemistry). In some embodiments, the targeting ligand is conjugated to the nucleotide using a click linker. In some embodiments, the targeting ligand is conjugated to any one of the nucleotides of the oligonucleotides described herein using an acetal-based linker. Acetal-based linkers are disclosed, for example, in International Patent Application Publication No. 2016 / 100401. In some embodiments, the linker is a labile linker. However, in other embodiments, the linker is stable. An example of a loop containing the nucleotide GAAA from 5' to 3' is shown below, where the GalNAc moiety is attached to the nucleotide of the loop using an acetal linker. Such a loop can be present, for example, at positions 27-30 of any one of the sense strands listed in Tables 3 or 5 and shown in Figures 12-13. In the chemical formula: [ka] is the point of attachment to the oligonucleotide chain. [ka] [ka]

[0194] As mentioned above, the targeting ligand can be attached to the nucleotide by using various suitable methods or chemical synthesis techniques (e.g., click chemistry). In some embodiments, the targeting ligand is conjugated to the nucleotide by using a click linker. In some embodiments, the targeting ligand is conjugated to any one of the nucleotides of the oligonucleotides described herein by using an acetal-based linker. Acetal-based linkers are disclosed, for example, in International Patent Application Publication No. 2016 / 100401. In some embodiments, the linker is an unstable linker. However, in other embodiments, the linker is a stable linker.

[0195] In some embodiments, a double-stranded extension (e.g., up to 3, 4, 5, or 6 bp in length) is provided between the targeting ligand (e.g., GalNAc moiety) and the RNAi oligonucleotide conjugate. In some embodiments, the RNAi oligonucleotide conjugate herein does not have GalNAc conjugated thereto.

[0196] Exemplary RNAi Oligonucleotide Conjugates In some embodiments, an RNAi oligonucleotide conjugate comprises an oligonucleotide conjugated to one or more lipid moieties. In some embodiments, the one or more lipid moieties comprise a fatty acid. In some embodiments, the fatty acid is a saturated fatty acid. In some embodiments, the fatty acid is an unsaturated fatty acid. In some embodiments, the one or more lipid moieties comprise a hydrocarbon chain. In some embodiments, the hydrocarbon chain is saturated. In some embodiments, the hydrocarbon chain is unsaturated. In some embodiments, the one or more lipid moieties comprise a hydrocarbon chain of 50 carbons (C50) or shorter (e.g., about 50 carbons (C50), 40 carbons (C40), 30 carbons (C30), 25 carbons (C25), 22 carbons (C22), 20 carbons (C20), 18 carbons (C18), 16 carbons (C16), 14 carbons (C14), 12 carbons (C12), 10 carbons (C10), 8 carbons (C8), 6 carbons (C6) in length. In some embodiments, the one or more lipid moieties comprise a hydrocarbon chain of 16 carbons to 50 carbons (e.g., For example, C16-C50, C18-C50, C20-C50, C22-C50, C16, C18, C20, or C22). In some embodiments, one or more lipid moieties comprise a hydrocarbon chain of 15 carbons or less (e.g., C6-C15, C6-C14, C6-C12, C6-C10, C8-C14, C8-C12, C8-C10, C10-C14, C10-C12, C15, C14, C13, C12, C11, C10, C9, C8, C7, or C6).

[0197] In some embodiments, one or more lipid moieties comprise a C8 hydrocarbon chain. In some embodiments, the C8 hydrocarbon chain comprises at least one double bond. In some embodiments, one or more lipid moieties comprise a C10 hydrocarbon chain. In some embodiments, the C10 hydrocarbon chain is unsaturated (i.e., C10:0). In some embodiments, the C10 hydrocarbon chain comprises at least one double bond. In some embodiments, one or more lipid moieties comprise a C12 hydrocarbon chain. In some embodiments, the C12 hydrocarbon chain is unsaturated (i.e., C12:0). In some embodiments, the C12 hydrocarbon chain comprises at least one double bond. In some embodiments, one or more lipid moieties comprise a C14 hydrocarbon chain. In some embodiments, the C14 hydrocarbon chain is unsaturated (i.e., C14:0). In some embodiments, the C14 hydrocarbon chain comprises at least one double bond. In some embodiments, one or more lipid moieties comprise a C16 hydrocarbon chain. In some embodiments, the C16 hydrocarbon chain is unsaturated (i.e., C16:0). In some embodiments, the C16 hydrocarbon chain comprises at least one double bond. In some embodiments, one or more lipid moieties comprises a C18 hydrocarbon chain. In some embodiments, the C18 hydrocarbon chain is unsaturated (i.e., C18:0). In some embodiments, the C18 hydrocarbon chain comprises at least one double bond (e.g., C18:1, C18:2, C18:3, C18:4, C18:5, or C18:6). In some embodiments, the C18 hydrocarbon chain comprises two double bonds (e.g., C18:2). In some embodiments, one or more lipid moieties comprises a C22 hydrocarbon chain. In some embodiments, the C22 hydrocarbon chain comprises at least one double bond (e.g., C22:1, C22:2, C22:3, C22:4, C22:5, or C22:6). In some embodiments, one or more lipid moieties comprises a C24 hydrocarbon chain. In some embodiments, the C24 hydrocarbon chain contains at least one double bond (e.g., C24:1, C24:2, C24:3, C24:4, C24:5, or C24:6).

[0198] In some embodiments, the oligonucleotide of the RNAi oligonucleotide conjugate comprises a tetraloop and at least one nucleotide of the tetraloop is conjugated to a C8 lipid. In some embodiments, the second nucleotide of the tetraloop is conjugated to a C8 lipid. In some embodiments, the oligonucleotide of the RNAi oligonucleotide conjugate comprises a tetraloop and at least one nucleotide of the tetraloop is conjugated to a C10 lipid. In some embodiments, the second nucleotide of the tetraloop is conjugated to a C10 lipid. In some embodiments, the oligonucleotide of the RNAi oligonucleotide conjugate comprises a tetraloop and at least one nucleotide of the tetraloop is conjugated to a C12 lipid. In some embodiments, the second nucleotide of the tetraloop is conjugated to a C12 lipid. In some embodiments, the oligonucleotide of the RNAi oligonucleotide conjugate comprises a tetraloop and at least one nucleotide of the tetraloop is conjugated to a C14 lipid. In some embodiments, the second nucleotide of the tetraloop is conjugated to a C14 lipid. In some embodiments, the oligonucleotide of the RNAi oligonucleotide conjugate comprises a tetraloop, and at least one nucleotide of the tetraloop is conjugated with a C16 lipid. In some embodiments, the second nucleotide of the tetraloop is conjugated with a C16 lipid. In some embodiments, the oligonucleotide of the RNAi oligonucleotide conjugate comprises a tetraloop, and at least one nucleotide of the tetraloop is conjugated with a C18 lipid (e.g., C18:0, C18:1, or C18:2). In some embodiments, the second nucleotide of the tetraloop is conjugated with a C18 lipid (e.g., C18:0, C18:1, or C18:2).In some embodiments, the oligonucleotide of the RNAi oligonucleotide conjugate comprises a tetraloop, and at least one nucleotide of the tetraloop is conjugated to a C22 lipid (e.g., C22:0, C22:1, C22:2, C22:3, C22:4, C22:5, or C22:6). In some embodiments, the second nucleotide of the tetraloop is conjugated to a C22 lipid (e.g., C22:0, C22:1, C22:2, C22:3, C22:4, C22:5, or C22:6). In some embodiments, the oligonucleotide of the RNAi oligonucleotide conjugate comprises a tetraloop, and at least one nucleotide of the tetraloop is conjugated to a C24 lipid (e.g., C24:0, C24:1, C24:2, C24:3, C24:4, C24:5, or C24:6). In some embodiments, the second nucleotide of the tetraloop is conjugated to a C24 lipid (e.g., C24:0, C24:1, C24:2, C24:3, C24:4, C24:5, or C24:6).

[0199] In some embodiments, the RNAi oligonucleotide conjugate comprises a nucleotide sequence having at least one modified nucleoside. In some embodiments, the oligonucleotide-ligand conjugate comprises an antisense strand and a sense strand, each strand comprising at least one modified nucleoside.

[0200] In some embodiments, the RNAi oligonucleotide conjugate is represented by the following formula with modifications as shown in Table 1: Sense strand: [mXs][mX][fX][mX][fX][mX][mX][fX][mX][fX][mX][fX][fX][mX][fX][mX][fX][mX][m [mX] This hybridizes to: Antisense strand: [Mephosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX][fX][mX][mX][fX][mX][fX][mX][fX][mX][mXs][mXs][mXs] or Sense strand: [mXs][mX][fX][mX][fX][mX][mX][fX][mX][fX][mX][fX][fX][mX][fX][mX][fX][mX][m [mX] This hybridizes to: Antisense strand: [Mephosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX][fX][mX][mX][fX][mX][fX][mX][fX][mX][mXs][mXs][mXs] [Table 1]

[0201] In some embodiments, the oligonucleotide of the RNAi oligonucleotide conjugate is conjugated to a C8 lipid as shown below: [ka]

[0202] In some embodiments, the oligonucleotide of the RNAi oligonucleotide conjugate is conjugated to a C10 lipid as shown below: [ka]

[0203] In some embodiments, the oligonucleotide of the RNAi oligonucleotide conjugate is conjugated to a C12 lipid as shown below: [ka]

[0204] In some embodiments, the oligonucleotide of the RNAi oligonucleotide conjugate is conjugated to a C14 lipid as shown below: [ka]

[0205] In some embodiments, the oligonucleotide of the RNAi oligonucleotide conjugate is conjugated to a C16 lipid as shown below: [ka]

[0206] In some embodiments, the oligonucleotide of the RNAi oligonucleotide conjugate is conjugated to a C18:0 lipid, as shown below: [ka]

[0207] In some embodiments, the oligonucleotide of the RNAi oligonucleotide conjugate is conjugated to a C18:1 lipid, as shown below: [ka]

[0208] In some embodiments, the oligonucleotide of the RNAi oligonucleotide conjugate is conjugated to a C18:2 lipid, as shown below: [ka]

[0209] In some embodiments, the oligonucleotide of the RNAi oligonucleotide conjugate is conjugated to a C22:0 lipid, as shown below: [ka]

[0210] In some embodiments, the oligonucleotide of the RNAi oligonucleotide conjugate is conjugated to a C22:6 lipid, as shown below: [ka]

[0211] In some embodiments, the RNAi oligonucleotide conjugate reduces target mRNA in the CNS (e.g., in the somatosensory cortex (SS cortex), hippocampus (HP), striatum, frontal cortex, cerebellum, hypothalamus (HY), cervical spinal cord (CSC), thoracic spinal cord (TSC), and / or lumbar spinal cord (LSC)).

[0212] In some embodiments, the RNAi oligonucleotide conjugates reduce target mRNA in the CNS (e.g., in SS cortex, HP, HY, CSC, TSC, and / or LSC) without reducing expression of the target mRNA outside the CNS. In some embodiments, the RNAi oligonucleotide conjugates reduce target mRNA in the CNS (e.g., in SS cortex, HP, HY, CSC, TSC, and / or LSC) without reducing expression of the target mRNA outside the CNS and without reducing expression of the target mRNA in the liver.

[0213] In some embodiments, the RNAi oligonucleotide conjugate comprises a tetraloop, and at least one nucleotide of the tetraloop is conjugated to a C8 lipid, and the RNAi oligonucleotide conjugate reduces target mRNA in the CNS (e.g., in SS cortex, HP, HY, CSC, TSC, and / or LSC) without reducing expression of the target mRNA outside the CNS. In some embodiments, the RNAi oligonucleotide conjugate reduces target mRNA in the CNS (e.g., in SS cortex, HP, HY, CSC, TSC, and / or LSC) without reducing expression of the target mRNA in the liver and without reducing expression of the target mRNA outside the CNS. In some embodiments, the C8 lipid is conjugated to the second nucleotide of the tetraloop. In some embodiments, the tetraloop is composed of 5'-GAAA-3'.

[0214] In some embodiments, the RNAi oligonucleotide conjugate comprises a tetraloop, at least one nucleotide of the tetraloop is conjugated to a C10 lipid, and the RNAi oligonucleotide conjugate reduces target mRNA in the CNS (e.g., in SS cortex, HP, HY, CSC, TSC, and / or LSC) without reducing expression of the target mRNA outside the CNS. In some embodiments, the RNAi oligonucleotide conjugate reduces target mRNA in the CNS (e.g., in SS cortex, HP, HY, CSC, TSC, and / or LSC) without reducing expression of the target mRNA outside the CNS and without reducing expression of the target mRNA in the liver. In some embodiments, the C10 lipid is conjugated to the second nucleotide of the tetraloop. In some embodiments, the tetraloop is composed of 5'-GAAA-3'.

[0215] In some embodiments, the RNAi oligonucleotide conjugate comprises a tetraloop, at least one nucleotide of the tetraloop is conjugated to a C12 lipid, and the RNAi oligonucleotide conjugate reduces target mRNA in the CNS (e.g., in SS cortex, HP, HY, CSC, TSC, and / or LSC) without reducing expression of the target mRNA outside the CNS. In some embodiments, the RNAi oligonucleotide conjugate reduces target mRNA in the CNS (e.g., in SS cortex, HP, HY, CSC, TSC, and / or LSC) without reducing expression of the target mRNA outside the CNS and without reducing expression of the target mRNA in the liver. In some embodiments, the C12 lipid is conjugated to the second nucleotide of the tetraloop. In some embodiments, the tetraloop is composed of 5'-GAAA-3'.

[0216] In some embodiments, the RNAi oligonucleotide conjugate comprises a tetraloop, at least one nucleotide of the tetraloop is conjugated to a C14 lipid, and the RNAi oligonucleotide conjugate reduces target mRNA in the CNS (e.g., in SS cortex, HP, HY, CSC, TSC, and / or LSC) without reducing expression of the target mRNA outside the CNS. In some embodiments, the RNAi oligonucleotide conjugate reduces target mRNA in the CNS (e.g., in SS cortex, HP, HY, CSC, TSC, and / or LSC) without reducing expression of the target mRNA outside the CNS and without reducing expression of the target mRNA in the liver. In some embodiments, the C14 lipid is conjugated to the second nucleotide of the tetraloop. In some embodiments, the tetraloop is composed of 5'-GAAA-3'.

[0217] General Methods for Providing Nucleic Acids and Their Analogues The nucleic acid and its analogs comprising lipid conjugates described herein can be made using various synthesis methods known in the art, including standard phosphoramidite methods.Any phosphoramidite synthesis method can be used to synthesize the nucleic acid provided in the present disclosure.In certain embodiments, phosphoramidites are used in solid-phase synthesis to obtain reactive intermediate phosphite compounds, which are then oxidized using known methods to generate phosphonate-modified oligonucleotides, typically with phosphodiester or phosphorothioate internucleotide linkages.The oligonucleotide synthesis of the present disclosure can be carried out in either direction, from 5' to 3' or from 3' to 5', using methods known in the art.

[0218] In certain embodiments, a method for synthesizing a nucleic acid is provided, comprising: (a) attaching a nucleoside or analog thereof to a solid support via a covalent bond; (b) coupling a nucleoside phosphoramidite or analog thereof to a reactive hydroxyl group on the nucleoside or analog of step (a) to form an internucleotide bond therebetween, wherein any uncoupled nucleosides or analogs on the solid support are capped with a capping reagent; (c) oxidizing the internucleotide bond with an oxidizing agent; and (d) iteratively repeating steps (b)-(c) with a subsequent nucleoside phosphoramidite or analog thereof to form a nucleic acid or analog thereof, wherein at least one of the nucleoside or analog thereof of step (a), the nucleoside phosphoramidite or analog thereof of step (b), or the subsequent nucleoside phosphoramidite or analog thereof of step (d) comprises a lipid-conjugated moiety as described herein. Typically, the coupling, capping / oxidation, and optionally deprotection steps are repeated until the oligonucleotide reaches the desired length and / or sequence, and is then cleaved from the solid support. In certain embodiments, oligonucleotides are prepared that contain 1-3 nucleic acids or analogs thereof that contain lipid-conjugated units on a tetraloop.

[0219] In Scheme A below, certain protecting groups, leaving groups, or transformation conditions are depicted, however, one of skill in the art will understand that other protecting groups, leaving groups, and transformation conditions are suitable and contemplated. Certain reactive functional groups envisioned in the genus of Scheme A (e.g., -N(H)-, -OH, etc.) that require additional protecting group strategies are also contemplated and will be understood by those of skill in the art. Such groups and transformations are described in March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, M.B. Smith and J. March, 5 th Edition,John Wiley&Sons,2001,COMPREHENSIVE ORGANIC TRANSFORMATIONS,(RCLarock,2 nd Edition, John Wiley & Sons, 1999), and PROTECTING GROUPS IN ORGANIC SYNTHESIS, (TW Greene and PG M Huts, 3 rd , edition, John Wiley & Sons, 1999), each of which is incorporated herein by reference in its entirety.

[0220] In certain embodiments, the nucleic acids and analogs thereof of the present disclosure are generally prepared according to Scheme A, Scheme A1, and Scheme B, as described below.

[0221] Scheme A: Synthesis of Ligand-Conjugated Oligonucleotides of the Present Disclosure [ka] Scheme A1: Synthesis of lipid-conjugated oligonucleotides of the present disclosure [ka] As shown in Scheme A and Scheme A1 above, the nucleic acid of formula I-1 or its analogue is conjugated with one or more ligands / lipophilic compounds to form a compound of formula I or Ia that includes one or more ligands / lipid conjugates.Typically, the conjugation is carried out in series or in parallel via esterification or amidation reaction between the nucleic acid of formula I-1 or I-1a or its analogue and one or more adamantyl compounds and / or lipophilic compounds (e.g., fatty acids) by techniques known in the art.The nucleic acid of formula I or Ia or its analogue can then be deprotected to form a compound of formula I-2 or I-2a, and protected with a suitable hydroxyl protecting group (e.g., DMTr) to form a compound of formula I-3 or I-3a. In one embodiment, the nucleic acid-ligand conjugate of formula I-3 or I-3a can be covalently attached (e.g., via a succinic acid linking group) to a solid support to form a solid support nucleic acid-ligand conjugate of formula I-4 or I-4a or its analogue, which contains one or more adamantyl and / or lipid conjugates. In another embodiment, the nucleic acid-ligand conjugate of formula I-3 or I-3a can be reacted with a P(III)-forming reagent (e.g., 2-cyanoethyl N,N-di-isopropylchlorophosphoramidite) to form a nucleic acid of formula I-5 or I-5a or its analogue, which contains a P(III) group. The nucleic acid-ligand conjugate of formula I-5 or I-5a or its analogue can then be subjected to oligomerization forming conditions, which are preformed using known and commonly applied processes for preparing oligonucleotides in the art. For example, the compound of formula I-5 or I-5a is attached to a solid-supported nucleic acid-ligand conjugate or its analogue, which has a 5'-hydroxyl group. Further steps may include one or more of deprotection, coupling, phosphite oxidation, and / or cleavage from the solid support to provide oligonucleotides of various nucleotide lengths comprising one or more lipid-conjugated nucleotide units represented by the compound of formula II-1 or II-Ia. B, E, L, Ligand, LC, n, PG 1 , P.G. 2 , P.G. 4, R 1 , R 2 , R 3 , X, X 1 , X 2 , X 3 Each of Z is as defined above and described herein.

[0222] Scheme B: Post-synthetic lipid conjugation of oligonucleotides of the present disclosure. [ka] As shown in Scheme B above, the nucleic acid of formula I-1 or its analogue can be deprotected to form a compound of formula I-6, protected with a suitable hydroxyl protecting group (e.g., DMTr) to form a compound of formula I-7, and reacted with a P(III)-forming reagent (e.g., 2-cyanoethyl N,N-di-isopropylchlorophosphoramidite) to form a nucleic acid of formula I-8 or its analogue containing a P(III) group. The nucleic acid of formula I-8 or its analogue can then be subjected to preformed oligomerization conditions using known and commonly applied processes for preparing oligonucleotides in the art. For example, the compound of formula I-8 is coupled to a solid-supported nucleic acid or its analogue bearing a 5'-hydroxyl group. Further steps can include one or more of deprotection, coupling, phosphite oxidation, and / or cleavage from the solid support to provide oligonucleotides of various nucleotide lengths, represented by the compound of formula II-2. The oligonucleotide of formula II-2 can then be conjugated with one or more ligands, such as adamantyl, or lipophilic compounds (e.g., fatty acids) to form a compound of formula II-1 containing one or more ligand conjugates. Typically, the conjugation is carried out in series or in parallel via esterification or amidation reaction between the nucleic acid of formula II-2 or its analog and one or more adamantyl compounds or fatty acids by techniques known in the art. B, E, L, ligand, LC, n, PG 1 , P.G. 2 , P.G. 4 , R 1 , R2 , R 3 , X, X 1 , X 2 , X 3 Each of Z is as defined above and described herein.

[0223] In certain embodiments, the nucleic acids and analogs thereof of the present disclosure are prepared according to Scheme C and Scheme D described below.

[0224] Scheme C: Synthesis of lipid-conjugated oligonucleotides of the present disclosure. [ka] As depicted in Scheme C above, the nucleic acid of formula C1 or its analogue is protected to form a compound of formula C2. The nucleic acid of formula C2 or its analogue is then alkylated (e.g., using DMSO and acetic acid via Pummerer rearrangement) to form a monothioacetal compound of formula C3. The nucleic acid of formula C3 or its analogue is then combined with C4 under appropriate conditions (e.g., mild oxidizing conditions) to form a nucleic acid of formula C5 or its analogue. The nucleic acid of formula C5 or its analogue can then be deprotected to form a compound of formula C6, which can be combined with a ligand of formula C7 (adamantyl or a lipophilic compound (e.g., fatty acid)) under appropriate amide forming conditions (e.g., HATU, DIPEA) to form a nucleic acid-ligand conjugate of formula Ib or its analogue, including lipid conjugates of the present disclosure. The nucleic acid-ligand conjugate of formula Ib or its analogue can then be deprotected to form a compound of formula C8, which can then be protected with a suitable hydroxyl protecting group (e.g., DMTr) to form a compound of formula C9. In one embodiment, the nucleic acid of formula C9 or its analogue can be covalently attached (e.g., via a succinic acid linking group) to a solid support to form a solid support nucleic acid-ligand conjugate of formula C10 or its analogue, which contains the ligand conjugate (adamantyl or lipid moiety) of the present disclosure. In another embodiment, the nucleic acid-ligand conjugate of formula C9 or its analogue can be reacted with a P(III)-forming reagent (e.g., 2-cyanoethyl N,N-di-isopropylchlorophosphoramidite) to form a nucleic acid-ligand conjugate of formula C11 or its analogue, which contains a P(III) group. The nucleic acid-ligand conjugate of formula C11 or its analogue can then be subjected to oligomerization forming conditions, which are preformed using known and commonly applied processes for preparing oligonucleotides in the art. For example, the compound of formula C11 is attached to a solid-supported nucleic acid-ligand conjugate or its analogue, which has a 5'-hydroxyl group.Further steps may include one or more of deprotection, coupling, phosphite oxidation, and / or cleavage from the solid support to provide oligonucleotides of various nucleotide lengths containing one or more adamantyl and / or lipid-conjugated nucleotide units represented by compounds of formula II-b-3, B, E, L. 2 , P.G. 1 , P.G. 2 , P.G. 3 , P.G. 4 , R 1 , R 2 , R 3 , R 4 , R 5 , X 1 , X 2 , X 3 Each of V, W, and Z is as defined above and described herein.

[0225] Scheme D: Post-synthetic lipid conjugation of oligonucleotides of the present disclosure. [ka] B, E, L 2 , P.G. 1 , P.G. 2 , P.G. 3 , P.G. 4 , R 1 , R 2 , R 3 , R 4 , R 5 , X 1 , X 2 , X 3Each of , V, W, and Z is as defined above and described herein. As shown in Scheme D above, the nucleic acid of formula C5 or its analogue can be selectively deprotected to form a compound of formula D1, protected with a suitable hydroxyl protecting group (e.g., DMTr) to form a compound of formula D2, and reacted with a P(III)-forming reagent (e.g., 2-cyanoethyl N,N-di-isopropylchlorophosphoramidite) to form a nucleic acid of formula D3 or its analogue. The nucleic acid of formula D3 or its analogue can then be subjected to preformed oligomerization conditions using known and commonly applied processes for preparing oligonucleotides in the art. For example, the compound of formula D3 is coupled to a solid-supported nucleic acid or its analogue bearing a 5'-hydroxyl group. Further steps can include one or more of deprotection, coupling, phosphite oxidation, and / or cleavage from the solid support to provide oligonucleotides of various nucleotide lengths, represented by compounds of formula D4. The oligonucleotide of formula D4 can then be deprotected to form a compound of formula D5 and coupled with a hydrophobic ligand (e.g., adamantyl or a lipophilic moiety) to form a compound of formula C7 (e.g., adamantyl or fatty acid) under appropriate amide forming conditions (e.g., HATU, DIPEA) to form an oligonucleotide of formula II-b-3 containing a ligand (e.g., fatty acid) conjugate of the present disclosure.

[0226] Those skilled in the art will appreciate that the various functional groups present in the nucleic acids or analogs thereof of the present disclosure, such as aliphatic groups, alcohols, carboxylic acids, esters, amides, aldehydes, halogens, and nitriles, can be interconverted by techniques well known in the art, including, but not limited to, reduction, oxidation, esterification, hydrolysis, partial oxidation, partial reduction, halogenation, dehydration, partial hydration, and hydration. See, e.g., "MARCH'S ADVANCED ORGANIC CHEMISTRY", (5 thEd., Ed.: Smith, MB and March, J., John Wiley & Sons, New York: 2001), each of which is incorporated herein by reference in its entirety. Such interconversions may require one or more of the techniques described above, and particular methods for synthesizing the nucleic acids provided in this disclosure are described below by way of example.

[0227] In some embodiments, the present disclosure provides a method for preparing an oligonucleotide comprising one or more lipid conjugates, wherein the lipid conjugate unit is represented by formula II-a-1 or: [ka] or a pharma- ceutically acceptable salt thereof, and the method comprises: (a) a nucleic acid of formula I-5a or an analog thereof, [ka] or a salt thereof; (b) oligomerizing said compound of formula I-5a to form a compound of formula II-1a, wherein B, E, L, LC, n, PG 4 , R 1 , R 2 , R 3 , X, X 1 , X 2 , X 3 Each of A, E, and Z is as defined above and described herein.

[0228] In the above step (b), oligomerization refers to preforming oligomerization conditions using known and commonly applied processes for preparing oligonucleotides in the art.For example, the compound of formula I-5a is bound to the solid-supported nucleic acid or its analogue with 5'-hydroxyl group.Further steps can include one or more of deprotection, coupling, phosphite oxidation, and cleavage from solid support to provide oligonucleotides of various nucleotide lengths, represented by the compound of formula II-1a with lipid conjugate of the present disclosure.

[0229] In some embodiments, the present disclosure provides a method for preparing an oligonucleotide comprising one or more lipid conjugates, the method comprising: [ka] or a salt thereof, (a) a nucleic acid of formula Ia or an analog thereof, [ka] or a salt thereof; (b) deprotecting the nucleic acid of formula Ia or its analogue to give a compound of formula I-2a, [ka] or a salt thereof; (c) protecting the nucleic acid of formula I-2 or its analogue to give a compound of formula I-3a, [ka] or a salt thereof; (d) treating said nucleic acid of formula I-3a or analog thereof with a P(III)-forming reagent to form a nucleic acid of formula I-5a or analog thereof, wherein B, E, L, LC, n, PG 4 , R 1 , R 2 , R3 , X, X 1 , X 2 , X 3 Each of A, E, and Z is as defined above and described herein.

[0230] In step (b) above, the PG of the compound of formula Ia 1 and P.G. 2 contains silyl ethers or cyclic silylene derivatives that can be removed under acidic conditions or with fluoride anions. Examples of reagents that provide fluoride anions for the removal of silicon-based protecting groups include hydrofluoric acid, hydrogen fluoride pyridine, triethylamine trihydrofluoride, tetra-N-butylammonium fluoride, and the like.

[0231] In step (c) above, the compound of formula I-2a is protected with a suitable hydroxyl protecting group. In certain embodiments, the protecting group PG is used to protect the 5'-hydroxyl group of the compound of formula I-2a. 4 include acid labile protecting groups such as trityl, 4-methyoxytrityl, 4,4'-dimethyoxytrityl, 4,4',4"-trimethyoxytrityl, 9-phenyl-xanthen-9-yl, 9-(p-tolyl)xanthen-9-yl, pixyl, 2,7-dimethylpixyl, and the like. In certain embodiments, the acid labile protecting groups are suitable for deprotection in both solution and solid phase synthesis of acid sensitive nucleic acids or analogs thereof, e.g., using dichloroacetic acid or trichloroacetic acid.

[0232] In step (d) above, the compound of formula I-3a is treated with a P(III)-forming reagent to obtain a compound of formula I-5a. In the context of the present disclosure, the P(III)-forming reagent is a phosphorus reagent that reacts to obtain a phosphorus(III) compound. In some embodiments, the P(III)-forming reagent is 2-cyanoethyl N,N-diisopropylchlorophosphoramidite or 2-cyanoethyl phosphorodichloridate. In certain embodiments, the P(III)-forming reagent is 2-cyanoethyl N,N-diisopropylchlorophosphoramidite. Those skilled in the art will be able to easily determine the X of the compound of formula I-3a. 1 It will be appreciated that the displacement of the leaving group in the P(III)-forming reagent by is accomplished in the presence or absence of a suitable base. Such suitable bases are well known in the art and include organic and inorganic bases. In certain embodiments, the base is a tertiary amine, such as triethylamine or diisopropylethylamine. In other embodiments, step (d) above is preformed using N,N-dimethylphosphoramic dichloride as the P(V)-forming reagent.

[0233] In some embodiments, the present disclosure provides a method for preparing an oligonucleotide comprising one or more lipid conjugates, the method comprising: [ka] or a salt thereof, (a) a nucleic acid of formula I-1 or an analog thereof, [ka] or a salt thereof; (b) conjugating one or more lipophilic compounds to the nucleic acid of formula I-1 or its analogue to form a nucleic acid of formula Ia or its analogue comprising one or more lipid conjugates, wherein B, E, L, LC, n, PG 1 , P.G. 2 , R1, R 2 , X, X 1Each of Z is as defined above and described herein.

[0234] In the above step (b), the nucleic acid of formula I-1a or its analogue is conjugated with one or more lipophilic compounds to form a compound of formula Ia comprising one or more lipid conjugates of the present disclosure. Typically, the conjugation is carried out in series or in parallel via esterification or amidation reaction between the nucleic acid of formula I-1a or its analogue and one or more fatty acids by techniques known in the art. In certain embodiments, the conjugation is carried out under suitable amide forming conditions to obtain a compound of formula I comprising one or more lipid conjugates. Suitable amide forming conditions can include, but are not limited to, the use of amide coupling reagents known in the art, such as HATU, PyBOP, DCC, DIC, EDC, HBTU, HCTU, PyAOP, PyBrOP, BOP, BOP-Cl, DEPBT, T3P, TATU, TBTU, TNTU, TOTU, TPTU, TSTU, or TDBTU. Alternatively, conjugation of the lipophilic compound can be achieved by any one of the cross-coupling techniques described in Table A herein.

[0235] In some embodiments, the present disclosure provides a method for preparing an oligonucleotide comprising one or more lipid conjugates, wherein the lipid conjugate unit is represented by formula II-1 or: [ka] or a pharma- ceutically acceptable salt thereof, and the method comprises: (a) an oligonucleotide of formula II-2, [ka] or a salt thereof; (b) one or more lipophilic compounds are conjugated to the oligonucleotide of formula II-2 to form the oligonucleotide of formula II-1 comprising one or more lipid conjugates. In the above step (b), the oligonucleotide of formula II-2 is conjugated to one or more lipophilic compounds to form the oligonucleotide of formula II-1 comprising one or more lipid conjugates of the present disclosure.

[0236] Typically, conjugation is carried out in series or parallel through esterification or amidation reaction between the oligonucleotide of formula II-2 and one or more fatty acids by techniques known in the art.In certain embodiments, conjugation is carried out under suitable amide forming conditions to obtain the oligonucleotide of formula II-1 comprising one or more lipid conjugates.Suitable amide forming conditions can include, but are not limited to, the use of amide coupling reagents known in the art, such as HATU, PyBOP, DCC, DIC, EDC, HBTU, HCTU, PyAOP, PyBrOP, BOP, BOP-Cl, DEPBT, T3P, TATU, TBTU, TNTU, TOTU, TPTU, TSTU or TDBTU.Alternatively, conjugation of lipophilic compounds can be achieved by any one of the cross-coupling techniques described in Table A herein.

[0237] In some embodiments, the present disclosure provides a unit represented by formula II-2: [ka] or a pharma- ceutically acceptable salt thereof, the method comprising: (a) a nucleic acid of formula I-8 or an analog thereof, [ka] or a salt thereof; (b) oligomerizing said compound of formula I-8 to form a compound of formula II-2.

[0238] In the above step (b), oligomerization refers to preforming oligomerization conditions using known and commonly applied processes for preparing oligonucleotides in the art.For example, the compound of formula I-8 is bound to the solid-supported nucleic acid or its analogue with 5'-hydroxyl group.Further steps can include one or more of deprotection, coupling, phosphite oxidation, and cleavage from solid support to provide oligonucleotides of various nucleotide lengths, represented by the compound of formula II-2.

[0239] In some embodiments, the present disclosure provides a method for preparing a nucleic acid or analog thereof comprising one or more lipid conjugates, the method comprising: [ka] or a salt thereof, (a) a nucleic acid of formula I-1 or an analog thereof, [ka] or a salt thereof; (b) deprotecting the nucleic acid of formula I-1 or its analogue to obtain a compound of formula I-6, [ka] or a salt thereof; (c) protecting the nucleic acid of formula I-6 or its analogue to give a compound of formula I-7, [ka] or a salt thereof; (d) treating said nucleic acid of formula I-7 or its analogue with a P(III)-forming agent to form a nucleic acid of formula I-8 or its analogue, wherein in the above step (b), PG of the compound of formula I-1 is 1 and P.G. 2contains silyl ethers or cyclic silylene derivatives that can be removed under acidic conditions or with fluoride anions. Examples of reagents that provide fluoride anions for the removal of silicon-based protecting groups include hydrofluoric acid, hydrogen fluoride pyridine, triethylamine trihydrofluoride, tetra-N-butylammonium fluoride, and the like.

[0240] In step (c) above, the compound of formula I-6 is protected with a suitable hydroxyl protecting group. In certain embodiments, the protecting group PG is used to protect the 5'-hydroxyl group of the compound of formula I-6. 4 include acid labile protecting groups such as trityl, 4-methyoxytrityl, 4,4'-dimethyoxytrityl, 4,4',4"-trimethyoxytrityl, 9-phenyl-xanthen-9-yl, 9-(p-tolyl)xanthen-9-yl, pixyl, 2,7-dimethylpixyl, and the like. In certain embodiments, the acid labile protecting groups are suitable for deprotection in both solution and solid phase synthesis of acid sensitive nucleic acids or analogs thereof, e.g., using dichloroacetic acid or trichloroacetic acid.

[0241] In step (d) above, the compound of formula I-7 is treated with a P(III)-forming reagent to obtain a compound of formula I-8. In the context of the present disclosure, the P(III)-forming reagent is a phosphorus reagent that reacts to obtain a phosphorus(III) compound. In some embodiments, the P(III)-forming reagent is 2-cyanoethyl N,N-diisopropylchlorophosphoramidite or 2-cyanoethyl phosphorodichloridate. In certain embodiments, the P(III)-forming reagent is 2-cyanoethyl N,N-diisopropylchlorophosphoramidite. Those skilled in the art will be able to easily determine the X of the compound of formula I-7. 1It will be appreciated that the displacement of the leaving group in the P(III)-forming reagent by is accomplished in the presence or absence of a suitable base. Such suitable bases are well known in the art and include organic and inorganic bases. In certain embodiments, the base is a tertiary amine, such as triethylamine or diisopropylethylamine. In other embodiments, step (d) above is preformed using N,N-dimethylphosphoramic dichloride as the P(V)-forming reagent.

[0242] In some embodiments, the present disclosure provides methods for preparing oligonucleotide-ligand conjugates comprising one or more adamantyl and / or lipid moieties, wherein said conjugate units are represented by formula II-b-3 or: [ka] or a pharma- ceutically acceptable salt thereof, and the method comprises: (a) a nucleic acid-ligand conjugate of formula C11 or an analog thereof; [ka] or a salt thereof; (b) oligomerizing the compound of formula C11 to form a compound of formula II-b-3, and in the above step (b), oligomerization refers to preforming oligomerization conditions using known and commonly applied processes for preparing oligonucleotides in the art.For example, the compound of formula C11 is bound to a solid-supported nucleic acid or its analogue with a 5'-hydroxyl group.Further steps include one or more of deprotection, coupling, phosphite oxidation, and cleavage from the solid support to provide oligonucleotide-ligand conjugates of various nucleotide lengths with one or more nucleic acid-ligand conjugate units, each unit being represented by the compound of formula II-b-3 containing adamantyl or lipid moiety of the present disclosure.

[0243] In some embodiments, the method for preparing an oligonucleotide of formula II-b-3 comprising one or more lipid conjugates comprises the step of: [ka] or a salt thereof, (a) a nucleic acid-ligand conjugate of formula Ib or an analog thereof; [ka] or a salt thereof; (b) deprotecting the nucleic acid-ligand conjugate of formula Ib above or an analog thereof to obtain a compound of formula C8, [ka] or a salt thereof; (c) deprotecting the nucleic acid-ligand conjugate of formula C8 or analog thereof to give a compound of formula C9, [ka] or a salt thereof; (d) treating the nucleic acid-ligand conjugate of formula C9 or its analog with a P(III)-forming reagent to form a nucleic acid of formula C11 or its analog. In the above step (b), the PG of the compound of formula Ib is 1 and P.G. 2 contains silyl ethers or cyclic silylene derivatives that can be removed under acidic conditions or with fluoride anions. Examples of reagents that provide fluoride anions for the removal of silicon-based protecting groups include hydrofluoric acid, hydrogen fluoride pyridine, triethylamine trihydrofluoride, tetra-N-butylammonium fluoride, and the like.

[0244] In step (c) above, the compound of formula C8 is protected with a suitable hydroxyl protecting group. In certain embodiments, the protecting group PG is used to protect the 5'-hydroxyl group of the compound of formula C8. 4 include acid labile protecting groups such as trityl, 4-methyoxytrityl, 4,4'-dimethyoxytrityl, 4,4',4"-trimethyoxytrityl, 9-phenyl-xanthen-9-yl, 9-(p-tolyl)xanthen-9-yl, pixyl, 2,7-dimethylpixyl, and the like. In certain embodiments, the acid labile protecting groups are suitable for deprotection in both solution and solid phase synthesis of acid sensitive nucleic acids or analogs thereof, e.g., using dichloroacetic acid or trichloroacetic acid.

[0245] In step (d) above, the compound of formula C9 is treated with a P(III)-forming reagent to obtain a compound of formula C11. In the context of the present disclosure, the P(III)-forming reagent is a phosphorus reagent that reacts to obtain a phosphorus(III) compound. In some embodiments, the P(III)-forming reagent is 2-cyanoethyl N,N-diisopropylchlorophosphoramidite or 2-cyanoethyl phosphorodichloridate. In certain embodiments, the P(III)-forming reagent is 2-cyanoethyl N,N-diisopropylchlorophosphoramidite. Those skilled in the art will be able to easily determine the X of the compound of formula C9. 1 It will be appreciated that the displacement of the leaving group in the P(III)-forming reagent by is accomplished in the presence or absence of a suitable base. Such suitable bases are well known in the art and include organic and inorganic bases. In certain embodiments, the base is a tertiary amine, such as triethylamine or diisopropylethylamine. In other embodiments, step (d) above is preformed using N,N-dimethylphosphoramic dichloride as the P(V)-forming reagent.

[0246] In some embodiments, the disclosure provides a method for preparing an oligonucleotide-ligand conjugate of formula II-b-3, comprising one or more nucleic acid-ligand conjugate units each comprising one or more adamantyl or lipid moieties, the method comprising preparing a nucleic acid-ligand conjugate of formula Ib or an analog thereof: [ka] or a salt thereof, (a) a nucleic acid-ligand conjugate of formula C6 or an analog thereof; [ka] or a salt thereof; (b) conjugating a lipophilic compound to the nucleic acid-ligand conjugate of formula C6 or its analogue to form a nucleic acid-ligand conjugate of formula Ib or its analogue, comprising one or more adamantyl and / or lipid conjugates. In the above step (b), conjugation is carried out under suitable amide forming conditions to obtain a compound of formula Ib comprising adamantyl and / or lipid conjugates. Suitable amide forming conditions can include, but are not limited to, the use of amide coupling reagents known in the art, such as HATU, PyBOP, DCC, DIC, EDC, HBTU, HCTU, PyAOP, PyBrOP, BOP, BOP-Cl, DEPBT, T3P, TATU, TBTU, TNTU, TOTU, TPTU, TSTU, or TDBTU. In certain embodiments, the amide forming conditions include HATU and DIPEA or TEA.

[0247] In certain embodiments, the nucleic acid-ligand conjugate of formula C6 or an analog thereof is provided in a salt form (e.g., a fumarate salt) and is first converted to the free base (e.g., using sodium bicarbonate) prior to preforming the conjugation step.

[0248] In some embodiments, the present disclosure provides a method for preparing an oligonucleotide-ligand conjugate of formula II-b-3 comprising one or more nucleic acid-ligand conjugate units, the method comprising preparing a nucleic acid-ligand conjugate of formula C6 or an analog thereof: [ka] or a salt thereof, (a) a nucleic acid of formula C1 or an analogue thereof, [ka] or a salt thereof; (b) protecting the nucleic acid of formula C1 or an analog thereof to give a compound of formula C2, [ka] or a salt thereof; (c) alkylating the nucleic acid of formula C2 or an analog thereof to give a compound of formula C3, [ka] or a salt thereof; (d) converting a nucleic acid or analogue thereof of formula C3 into a compound of formula C4, [ka] or a salt thereof, [ka] or a salt thereof; (e) deprotecting the nucleic acid of formula C5 or its analogue to form a nucleic acid-ligand conjugate of formula C6 or its analogue. 1 and P.G. 2The groups, together with their intervening atoms, form a cyclic diol protecting group, such as a cyclic acetal or ketal. Such groups include methylene, ethylidene, benzylidene, isopropylidene, cyclohexylidene, and cyclopentylidene, silylene derivatives, such as di-t-butylsilylene and 1,1,3,3-tetraisopropyldisiloxanilidene, cyclic carbonates, cyclic boronates, and cyclic monophosphate derivatives based on cyclic adenosine monophosphate (i.e., cAMP). In certain embodiments, the cyclic diol protecting group is 1,1,3,3-tetraisopropyldisiloxanilidene prepared from the reaction of a diol of formula C1 with 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane under basic conditions.

[0249] In step (c) above, the nucleic acid of formula C2 or its analogue is alkylated under acidic conditions using a mixture of DMSO and acetic anhydride. In certain embodiments, when -VH is a hydroxyl group, the mixture of DMSO and acetic anhydride forms methyl (methylthio)acetate in situ via Pummerer rearrangement in the presence of acetic acid, which then reacts with the hydroxyl group of the nucleic acid of formula C2 or its analogue to provide a monothioacetal-functionalized fragment nucleic acid of formula C3 or its analogue.

[0250] In step (d) above, the replacement of the thiomethyl group of the nucleic acid of formula C3 or its analog with a nucleic acid of formula C4 or its analog results in a nucleic acid of formula C4 or its analog. In certain embodiments, the replacement occurs under mildly oxidizing and / or acidic conditions. In some embodiments, V is oxygen. In some embodiments, the mild oxidizing reagent includes a mixture of elemental iodine and hydrogen peroxide, urea hydrogen peroxide complex, silver nitrate / silver sulfate, sodium bromate, ammonium peroxodisulfate, tetrabutylammonium peroxodisulfate, Oxone®, Chloramine T, Selectfluor®, Selectfluor® II, sodium hypochlorite, or potassium iodide / sodium periodate. In certain embodiments, the mild oxidizing agent includes N-iodosuccinimide, N-bromosuccinimide, N-chlorosuccinimide, 1,3-diiodo-5,5-dimethylhydantoin, pyridinium tribromide, iodine monochloride or complexes thereof, and the like. Commonly used acids under mild oxidation conditions include sulfuric acid, p-toluenesulfonic acid, trifluoromethanesulfonic acid, methanesulfonic acid, and trifluoroacetic acid. In certain embodiments, the mild oxidation reagent comprises a mixture of N-iodosuccinimide and trifluoromethanesulfonic acid.

[0251] In step (e) above, the PG of the nucleic acid-ligand conjugate of formula C5 or its analogue is 3 and optionally R 4 Removal of (R 4 is a suitable amine protecting group) to provide a nucleic acid-ligand conjugate of formula C6 or an analog thereof or a salt thereof. In some embodiments, PG 3 and / or R 4 includes a carbamate derivative that can be removed under acidic or basic conditions. In certain embodiments, the protecting group of the nucleic acid-ligand conjugate of formula C5 or its analog (e.g., PG 3 and R 4 Both, or PG 3 or R 4(independently) is removed by acid hydrolysis. It will be understood that acid hydrolysis of the protecting group of the nucleic acid-ligand conjugate of formula C5 or its analog will form its salt of formula C6. For example, when the acid labile protecting group of the nucleic acid-ligand conjugate of formula C5 or its analog is removed by treatment with an acid such as hydrochloric acid, the resulting amine compound is formed as its hydrochloride salt. Those skilled in the art will recognize that a wide variety of acids are useful for removing acid labile amino protecting groups, and therefore a wide variety of salt forms of the nucleic acid of formula C6 or its analog are contemplated.

[0252] In other embodiments, the protecting group of the nucleic acid of formula C5 or its analogue (e.g., PG 3 and R 4 Both, or PG 3 or R 4 (either independently) is removed by base hydrolysis. For example, Fmoc and trifluoroacetyl protecting groups can be removed by treatment with base. Those skilled in the art will recognize that a wide variety of bases are useful for removing amino protecting groups that are base labile. In some embodiments, the base is piperidine. In some embodiments, the base is 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). In certain embodiments, the nucleic acid-ligand conjugate of formula C5 or its analog is deprotected under basic conditions and subsequently treated with acid to form a salt of formula C6. In certain embodiments, the acid is fumaric acid and the salt of formula C6 is a fumarate salt.

[0253] In some embodiments, the present disclosure provides a method for preparing an oligonucleotide-ligand conjugate comprising one or more nucleic acid-ligand conjugates, wherein the nucleic acid-ligand conjugate units are represented by formula II-b-3 or: [ka] or a pharma- ceutically acceptable salt thereof, and the method comprises: (a) an oligonucleotide of formula D5, [ka] or a salt thereof; (b) one or more adamantyl compounds or lipophilic compounds are conjugated to the oligonucleotide of formula D5 to form an oligonucleotide-ligand conjugate of formula II-b-3, which comprises one or more nucleic acid-ligand conjugate units. In the above step (b), conjugation is carried out under suitable amide forming conditions to obtain a compound of formula D5, which comprises an adamantyl or lipid conjugate. Suitable amide forming conditions can include, but are not limited to, the use of amide coupling reagents known in the art, such as HATU, PyBOP, DCC, DIC, EDC, HBTU, HCTU, PyAOP, PyBrOP, BOP, BOP-Cl, DEPBT, T3P, TATU, TBTU, TNTU, TOTU, TPTU, TSTU, or TDBTU. In certain embodiments, the amide forming conditions include HATU and DIPEA or TEA.

[0254] In some embodiments, the present disclosure provides an oligonucleotide-ligand conjugate comprising a unit represented by formula D5: [ka] or a salt thereof, the method comprising: (a) a nucleic acid-ligand conjugate of formula D4 or an analog thereof; [ka] or a salt thereof; (b) deprotecting the compound of formula D4 to form a compound of formula D5. In the above step (b), the PG of the oligonucleotide of formula D4 is 3 and optionally R 4 Removal of (R 4is a suitable amine protecting group) to provide an oligonucleotide-ligand conjugate of formula D5, or a salt thereof. In some embodiments, PG 3 and / or R 4 comprises a carbamate derivative that can be removed under acidic or basic conditions. In certain embodiments, the protecting group of the oligonucleotide-ligand conjugate of formula D4 (e.g., PG 3 and R 4 Both, or PG 3 or R 4 (either independently) is removed by acid hydrolysis. It will be understood that the acid hydrolysis of the protecting group of the oligonucleotide-ligand conjugate of formula D4 will form its salt of formula D5. For example, when the acid labile protecting group of the oligonucleotide of formula D4 is removed by treating with an acid such as hydrochloric acid, the resulting amine compound will be formed as its hydrochloride salt. Those skilled in the art will recognize that a wide variety of acids are useful for removing the acid labile amino protecting group, and therefore a wide variety of salt forms of the nucleic acid-ligand conjugate unit of formula D5 or its analogs are contemplated.

[0255] In other embodiments, the protecting group of the oligonucleotide-ligand conjugate of formula D4 (e.g., PG 3 and R 4 Both, or PG 3 or R 4 (either independently) are removed by base hydrolysis. For example, Fmoc and trifluoroacetyl protecting groups can be removed by treatment with base. One of skill in the art will recognize that a wide variety of bases are useful for removing amino protecting groups that are base labile. In some embodiments, the base is piperidine. In some embodiments, the base is 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).

[0256] In some embodiments, the present disclosure provides a method for preparing an oligonucleotide-ligand conjugate comprising one or more nucleic acid-ligand conjugate units having one or more adamantyl and / or lipid moieties, said conjugate units being represented by formula D4 or: [ka] or a pharma- ceutically acceptable salt thereof, and the method comprises: (a) a nucleic acid of formula D3 or an analog thereof, [ka] or a salt thereof; (b) oligomerizing said compound of formula D3 to form a compound of formula D4,

[0257] In the above step (b), oligomerization refers to preforming oligomerization conditions using known and commonly applied processes for preparing oligonucleotides in the art.For example, the nucleic acid or its analog of formula D3 is bound to the solid-supported nucleic acid or its analog with 5'-hydroxyl group.Further steps can include one or more of deprotection, coupling, phosphite oxidation, and cleavage from solid support to provide oligonucleotides of various nucleotide lengths, represented by the compound of formula D4 that includes adamantyl or lipid conjugate of the present disclosure.

[0258] In some embodiments, the present disclosure provides a method for preparing a nucleic acid or analog thereof comprising one or more lipid conjugates, the method comprising: [ka] or a salt thereof, (a) a nucleic acid of formula C5 or an analog thereof, [ka] or a salt thereof; (b) deprotecting the nucleic acid of formula C5 or an analog thereof to give a compound of formula D1, [ka] or a salt thereof; (c) protecting the nucleic acid of formula D1 or its analogue to obtain a nucleic acid of formula D2 or its analogue, [ka] or a salt thereof; (d) treating the nucleic acid or analogue of formula D2 with a P(III)-forming reagent to form a nucleic acid or analogue of formula D3. In the above step (b), the PG of the nucleic acid or analogue of formula C5 is 1 and P.G. 2 contains silyl ethers or cyclic silylene derivatives that can be removed under acidic conditions or with fluoride anions. Examples of reagents that provide fluoride anions for the removal of silicon-based protecting groups include hydrofluoric acid, hydrogen fluoride pyridine, triethylamine trihydrofluoride, tetra-N-butylammonium fluoride, and the like.

[0259] In step (c) above, the nucleic acid of formula D1 or its analogue is protected with a suitable hydroxyl protecting group. In certain embodiments, the protecting group PG is used to protect the 5'-hydroxyl group of the compound of formula D1. 4 include acid labile protecting groups such as trityl, 4-methyoxytrityl, 4,4'-dimethyoxytrityl, 4,4',4"-trimethyoxytrityl, 9-phenyl-xanthen-9-yl, 9-(p-tolyl)xanthen-9-yl, pixyl, 2,7-dimethylpixyl, and the like. In certain embodiments, the acid labile protecting groups are suitable for deprotection in both solution and solid phase synthesis of acid sensitive nucleic acids or analogs thereof, e.g., using dichloroacetic acid or trichloroacetic acid.

[0260] In step (d) above, the nucleic acid of formula D2 or its analogue is treated with a P(III)-forming reagent to obtain a compound of formula D3. In the context of the present disclosure, the P(III)-forming reagent is a phosphorus reagent that reacts to obtain a phosphorus(III) compound. In some embodiments, the P(III)-forming reagent is 2-cyanoethyl N,N-diisopropylchlorophosphoramidite or 2-cyanoethyl phosphorodichloridate. In certain embodiments, the P(III)-forming reagent is 2-cyanoethyl N,N-diisopropylchlorophosphoramidite. Those skilled in the art will be able to easily determine the X of the compound of formula D2. 1 It will be appreciated that the displacement of the leaving group in the P(III)-forming reagent by is accomplished in the presence or absence of a suitable base. Such suitable bases are well known in the art and include organic and inorganic bases. In certain embodiments, the base is a tertiary amine, such as triethylamine or diisopropylethylamine. In other embodiments, step (d) above is preformed using N,N-dimethylphosphoramic dichloride as the P(V)-forming reagent.

[0261] formulation Various formulations have been developed to facilitate the use of oligonucleotides. For example, oligonucleotides (e.g., RNAi oligonucleotide conjugates) can be delivered to a subject or cellular environment using a formulation that minimizes degradation, facilitates delivery and / or uptake, or provides another beneficial property to the oligonucleotide in the formulation. In some embodiments, compositions are provided herein that include oligonucleotides (e.g., RNAi oligonucleotide conjugates) that reduce the expression of target mRNAs (e.g., target mRNAs expressed in the CNS). Such compositions can be suitably formulated so that when administered to a subject, either within the environment surrounding the target cells or systemically, a sufficient portion of the oligonucleotides enters the cells and reduces target gene expression. Any of a variety of suitable oligonucleotide formulations can be used to deliver oligonucleotides for reducing target gene expression as disclosed herein. In some embodiments, oligonucleotides are formulated in buffers such as phosphate buffered saline, liposomes, micellar structures, and capsids.

[0262] Formulations of oligonucleotides containing cationic lipids can be used to facilitate the introduction of oligonucleotides into cells. For example, cationic lipids such as lipofectin, cationic glycerol derivatives, and polycationic molecules (e.g., polylysine) can be used. Suitable lipids include oligofectamine, lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals, Inc., Boulder, Colo.), or FuGene6 (Roche), all of which can be used according to the manufacturer's instructions.

[0263] Thus, in some embodiments, the formulation comprises a lipid nanoparticle. In some embodiments, the excipient comprises a liposome, lipid, lipid complex, microsphere, microparticle, nanosphere, or nanoparticle, or may be otherwise formulated for administration to a cell, tissue, organ, or body of a subject in need thereof (see, e.g., Remington: THE SCIENCE AND PRACTICE OF PHARMACY, 22). nd (See, e.g., U.S. Pat. No. 6,2013, edition, Pharmaceutical Press, 2013).

[0264] In some embodiments, the formulations herein include an excipient. In some embodiments, the excipient provides the composition with improved stability, improved absorption, improved solubility, and / or therapeutic enhancement of the active ingredient. In some embodiments, the excipient is a buffer (e.g., sodium citrate, sodium phosphate, Tris base, or sodium hydroxide) or a vehicle (e.g., buffer solution, petrolatum, dimethyl sulfoxide, or mineral oil). In some embodiments, the oligonucleotide is lyophilized to extend its shelf life and then put into solution before use (e.g., administration to a subject). Thus, the excipient in a composition comprising any one of the oligonucleotides described herein can be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone) or a collapse temperature modifier (e.g., dextran, Ficoll™, or gelatin). Similarly, the oligonucleotides herein may be provided in their free acid form.

[0265] In some embodiments, the pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intramuscular, intraperitoneal, intradermal, subcutaneous), oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. In some embodiments, the pharmaceutical composition is formulated for delivery to the central nervous system (e.g., intrathecal, epidural). In some embodiments, the pharmaceutical composition is formulated for delivery to the eye (e.g., ocular, intraocular, subconjunctival, intravitreal, retrobulbar, intracameral).

[0266] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water soluble) or dispersions, and sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). The carrier can be, for example, a solvent or dispersion medium, including water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. In many cases, it will be preferable to include an isotonic agent, for example, sugars, polyalcohols, such as mannitol, sorbitol, sodium chloride, in the composition. Sterile injectable solutions can be prepared by incorporating the required amount of oligonucleotide in a selected solvent, including one or a combination of the above-listed ingredients as needed, followed by filtered sterilization.

[0267] In some embodiments, the composition may contain at least about 0.1% of a therapeutic agent (e.g., an RNAi oligonucleotide conjugate herein) or more, although the percentage of active ingredient may be from about 1% to about 80% or more by weight or volume of the total composition. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations will be contemplated by those skilled in the art of preparing such pharmaceutical formulations, and therefore various dosages and treatment regimens may be desirable.

[0268] How to use Reduction of target gene expression In some embodiments, the present disclosure provides a method for contacting or delivering an effective amount of any of the RNAi oligonucleotide conjugates herein to a cell or cell population to reduce expression of a target gene (e.g., reduce expression of a target gene in the CNS). In some embodiments, the reduction in target gene expression is determined by measuring the reduction in the amount or level of target mRNA, protein encoded by target mRNA, or target gene (mRNA or protein) activity in the cell. These methods include those described herein and known to those skilled in the art. In some embodiments, the present disclosure provides a method for contacting or delivering an effective amount of any of the RNAi oligonucleotide conjugates herein to a cell or cell population in one or more regions of the CNS to reduce expression of a target gene in the CNS. In some embodiments, regions of the CNS include, but are not limited to, the cerebrum, prefrontal cortex, frontal cortex, motor cortex, temporal cortex, parietal cortex, occipital cortex, somatosensory cortex, hippocampus, caudate nucleus, striatum, globus pallidus, thalamus, midbrain, tegmentum, substantia nigra, pons, brainstem, cerebellar white matter, cerebellum, dentate nucleus, medulla, cervical spinal cord, thoracic spinal cord, lumbar spinal cord, cervical dorsal root ganglion, thoracic dorsal root ganglion, lumbar dorsal root ganglion, sacral dorsal root ganglion, nodose ganglion, femoral nerve, sciatic nerve, sural nerve, amygdala, hypothalamus, putamen, corpus callosum, and cranial nerves.

[0269] In some embodiments, the disclosure provides a method for reducing expression of a target mRNA in the CNS in a subject, comprising administering one or more RNAi oligonucleotide conjugates described herein. In some embodiments, the method comprises reducing expression of a target mRNA in the somatosensory cortex (SS cortex). In some embodiments, the method comprises reducing expression of a target mRNA in the hippocampus (HP). In some embodiments, the method comprises reducing expression of a target mRNA in the striatum. In some embodiments, the method comprises reducing expression of a target mRNA in the frontal cortex. In some embodiments, the method comprises reducing expression of a target mRNA in the cerebellum. In some embodiments, the method comprises reducing expression of a target mRNA in the hypothalamus (HY). In some embodiments, the method comprises reducing expression of a target mRNA in the cervical spinal cord (CSC). In some embodiments, the method comprises reducing expression of a target mRNA in the thoracic spinal cord (TSC). In some embodiments, the method comprises reducing expression of a target mRNA in the lumbar spinal cord (LSC).

[0270] The methods provided herein are useful for any suitable cell type. In some embodiments, the cell is any cell that expresses the target mRNA. In some embodiments, the cell is a primary cell obtained from a subject. In some embodiments, the primary cell has undergone a limited number of passages so that the cell substantially maintains its natural phenotypic characteristics. In some embodiments, the cell to which the oligonucleotide is delivered is ex vivo or in vitro (i.e., it can be delivered to a cell in culture or to an organism in which the cell resides).

[0271] In some embodiments, the RNAi oligonucleotide conjugate disclosed herein is delivered to cells or cell populations using nucleic acid delivery methods known in the art, including but not limited to injection of a solution or pharmaceutical composition containing the RNAi oligonucleotide conjugate, bombardment with particles covered with the RNAi oligonucleotide conjugate, exposure of cells or cell populations to a solution containing the RNAi oligonucleotide conjugate, or electroporation of cell membrane in the presence of the RNAi oligonucleotide conjugate.Other methods known in the art for delivering oligonucleotides to cells can be used, such as lipid-mediated carrier transport, chemical-mediated transport, and cationic liposome introduction such as calcium phosphate, and others.

[0272] In some embodiments, the reduction in target gene expression is determined by an assay or technique that evaluates one or more molecules, characteristics, or features of a cell or cell population that are associated with target gene expression, or by an assay or technique that evaluates a molecule (e.g., a target mRNA or protein) that directly indicates target gene expression in a cell or cell population. In some embodiments, the extent to which the RNAi oligonucleotide conjugates provided herein reduce target gene expression is evaluated by comparing target gene expression in a cell or cell population that has been contacted with the RNAi oligonucleotide conjugate to a control cell or cell population (e.g., a cell or cell population that has not been contacted with the RNAi oligonucleotide conjugate or that has been contacted with a control RNAi oligonucleotide conjugate). In some embodiments, the control amount or level of target gene expression in a control cell or cell population is predetermined so that it is not necessary to measure the control amount or level in every instance that the assay or technique is performed. The predetermined level or value can take a variety of forms. In some embodiments, the predetermined level or value can be a single cutoff value, such as a median or mean value.

[0273] In some embodiments, contacting or delivering the RNAi oligonucleotide conjugates described herein to a cell or cell population results in a reduction in target gene expression. In some embodiments, the reduction in target gene expression is relative to a control amount or level of target gene expression in a cell or cell population that has not been contacted with the RNAi oligonucleotide conjugate or that has been contacted with a control RNAi oligonucleotide conjugate. In some embodiments, the reduction in target gene expression is about 1% or less, about 5% or less, about 10% or less, about 15% or less, about 20% or less, about 25% or less, about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or less, about 60% or less, about 70% or less, about 80% or less, or about 90% or less compared to the control amount or level of target gene expression. In some embodiments, the control amount or level of target gene expression is the amount or level of target mRNA and / or protein in a cell or cell population that has not been contacted with the RNAi oligonucleotide conjugates described herein. In some embodiments, the effect of the delivery of the RNAi oligonucleotide conjugates of the present invention to a cell or cell population by the method of the present invention is evaluated after any finite period or amount of time (e.g., minutes, hours, days, weeks, months).For example, in some embodiments, target gene expression is determined in a cell or cell population at least about 4 hours, about 8 hours, about 12 hours, about 18 hours, about 24 hours, or at least about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, about 56 days, about 63 days, about 70 days, about 77 days, or about 84 days or more after the RNAi oligonucleotide conjugates are contacted or delivered to the cell or cell population. In some embodiments, target gene expression is determined in a cell or cell population at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months or more after contacting or delivering an RNAi oligonucleotide conjugate to the cell or cell population.

[0274] Tissue-specific regulation of gene expression In some embodiments, the present disclosure provides a method of contacting or delivering the RNAi oligonucleotide conjugate described herein to a cell or cell population, wherein the cell or cell population is present in one or more target tissues of a subject.In some embodiments, the method comprises administering the RNAi oligonucleotide conjugate described herein to a subject, the conjugate being distributed to one or more target tissues of the subject, and the conjugate being contacted or delivered to a cell or cell population in the one or more target tissues.

[0275] As used herein, "target tissue" refers to a tissue of a subject in which the reduction of expression of a target gene by a cell or cell population in the tissue results in one or more desirable physiological outcomes. In some embodiments, the target gene has aberrant expression in a cell or cell population in one or more target tissues, and the aberrant expression is responsible for the pathology of a disease or disorder in a subject. In some embodiments, the reduction of expression of the target gene by a cell or cell population in the target tissue serves to treat, alleviate, prevent, or alleviate a disease or disorder in a subject.

[0276] The distribution and / or function of the RNAi oligonucleotide conjugate in target tissue is desirable for reducing target gene expression in cells or cell populations present in the target tissue, while the distribution and / or function of the conjugate in non-target tissues may cause harmful effects.For example, the distribution of the conjugate in non-target tissues may limit its availability for distribution to target tissues, thereby limiting the efficacy and / or activity of the conjugate for reducing target gene expression in cells or cell populations present in the target tissue.As another example, the target tissue may have abnormal expression of the target gene, and it is beneficial to reduce target gene expression to restore normal physiological function, while the non-target tissue may require the expression of the target gene for normal physiological function.Under these circumstances, the distribution and / or function of the conjugate in non-target tissues impairs the expression of the target gene in a manner that will result in undesirable or harmful pathology.Therefore, for many in vivo therapeutic situations, it is beneficial to distribute the RNAi oligonucleotide to the target tissue in the subject, while limiting its distribution and / or function in one or more non-target tissues (e.g., liver) of the subject.

[0277] In some embodiments, the present disclosure provides a method for reducing or inhibiting the expression of a target gene in a cell population associated with one or more target tissues in a subject, comprising administering an RNAi oligonucleotide conjugate or a pharmaceutical composition thereof described herein.In some embodiments, the method comprises distributing the RNAi oligonucleotide conjugate to one or more target tissues of a subject with minimal distribution to one or more non-target tissues of the subject.In some embodiments, the RNAi oligonucleotide conjugate is contacted or delivered to a cell or cell population present in one or more target tissues of a subject with minimal contact or delivery to a cell or cell population present in one or more non-target tissues of the subject.In some embodiments, the expression of the target gene is reduced in one or more target tissues without being reduced to the same or similar level in one or more non-target tissues.

[0278] In some embodiments, the method results in (i) a reduction in expression of the target gene by a cell or cell population in one or more target tissues compared to a control expression of the target gene, and (ii) a substantially equivalent expression of the target gene by a cell or cell population in one or more non-target tissues relative to the control expression of the target gene. In some embodiments, the control expression of the target gene corresponds to the amount or level of expression of the target gene in a cell or cell population from a comparable tissue that has not been contacted with the RNAi oligonucleotide conjugate or that has been contacted with a control RNAi oligonucleotide conjugate. In some embodiments, the reduction in target gene expression is measured as a reduction in the amount or level of target mRNA transcribed from the target gene, or the amount or level of protein encoded by the target gene. In some embodiments, the method results in (i) a reduction in expression of the target mRNA in one or more target tissues compared to a control, and (ii) a substantially equivalent expression of the target mRNA in one or more non-target tissues compared to a control. In some embodiments, the method results in (i) a reduction in the level of the target protein in one or more target tissues compared to a control, and (ii) a substantially equivalent level of the target protein in one or more non-target tissues compared to a control.

[0279] In some embodiments, the present disclosure provides a method for reducing or inhibiting the expression of a target gene in a cell population associated with the CNS in a subject, comprising administering an RNAi oligonucleotide conjugate or a pharmaceutical composition thereof described herein. In some embodiments, the method comprises distributing the RNAi oligonucleotide conjugate to the CNS in a subject with minimal distribution to one or more non-target tissues (e.g., liver) of the subject. In some embodiments, the RNAi oligonucleotide conjugate is contacted or delivered to a cell or cell population present in the CNS of the subject, with minimal contact or delivery to a cell or cell population present in one or more non-target tissues (e.g., liver) of the subject. In some embodiments, the expression of the target gene is reduced in the CNS of a subject without being reduced to a similar level in one or more non-target tissues (e.g., liver).

[0280] In some embodiments, the expression of the target gene is reduced in the CNS without being reduced to a similar level in one or more non-target tissues. In some embodiments, the one or more non-target tissues include liver tissue. In some embodiments, the method results in (i) a reduction in the expression of the target gene in a cell or cell population of the CNS compared to a control expression of the target gene, and (ii) a substantially equivalent expression of the target gene in a cell or cell population of one or more non-target tissues compared to a control expression of the target gene. In some embodiments, the control expression of the target gene corresponds to the amount or level of expression of the target gene in a cell or cell population from a comparable tissue that has not been contacted with the RNAi oligonucleotide conjugate or that has been contacted with a control RNAi oligonucleotide conjugate. In some embodiments, the method results in (i) reduced expression of the target gene in a cell or cell population of the CNS compared to a control expression of the target gene (e.g., expression of the target gene in a cell or cell population of the CNS not contacted with an RNAi oligonucleotide conjugate or contacted with a control RNAi oligonucleotide conjugate), and (ii) substantially equivalent expression of the target gene in a cell or cell population of the liver compared to a control expression of the target gene (e.g., expression of the target gene in a cell or cell population of the liver not contacted with an RNAi oligonucleotide conjugate or contacted with a control RNAi oligonucleotide conjugate). In some embodiments, the method results in expression of the target gene in a cell or cell population of the CNS that is about 1% or less, about 5% or less, about 10% or less, about 15% or less, about 20% or less, about 25% or less, about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or less, about 60% or less, about 70% or less, about 80% or less, or about 90% or less compared to the control expression of the target gene. In some embodiments, expression of the target gene in the liver is comparable to the control expression of the target gene (e.g., has a difference of about ±30%, about ±25%, about ±20%, about ±15%, about ±10%, about ±5%, about ±4%, about ±3%, about ±2%, or about ±1% or less).In some embodiments, a reduction in target gene expression in the CNS is measured as a reduction in the amount or level of target mRNA transcribed from the target gene, or the amount or level of a protein encoded by the target gene.

[0281] Treatment method The present disclosure provides oligonucleotides for use as medicines, particularly for use in methods for treating diseases, disorders, and conditions associated with the CNS.The present disclosure also provides RNAi oligonucleotide conjugates for use or adapted for use to treat subjects (e.g., humans with diseases, disorders, or conditions associated with target gene expression) that may benefit from reducing target gene expression (e.g., in the CNS).In some aspects, the present disclosure provides RNAi oligonucleotide conjugates for use or adapted for use to treat subjects with diseases, disorders, or conditions associated with target gene expression.The present disclosure also provides RNAi oligonucleotide conjugates for use or adapted for use in the manufacture of medicines or pharmaceutical compositions for treating diseases, disorders, or conditions associated with target gene expression.In some embodiments, the RNAi oligonucleotide conjugates for use or adapted for use target mRNA and reduce target gene expression (e.g., via the RNAi pathway). In some embodiments, the RNAi oligonucleotide conjugates for use or adapted for use target mRNA and reduce the amount or level of the target mRNA, protein, and / or activity.

[0282] In addition, in some embodiments of the methods herein, subjects who have or are predisposed to a disease, disorder, or condition associated with target gene expression are selected for treatment with the RNAi oligonucleotide conjugates provided herein.In some embodiments, the methods include selecting individuals who have or are susceptible to a marker (e.g., biomarker) of a disease, disorder, or condition associated with target gene expression, such as but not limited to a target mRNA, protein, or combination thereof.Similarly, as detailed below, some embodiments of the methods provided by the present disclosure include, for example, measuring or obtaining a baseline value of a marker of target gene expression, and then comparing the value thus obtained with one or more other baseline values ​​or values ​​obtained after administering the RNAi oligonucleotide conjugate to the subject to evaluate the effectiveness of treatment.

[0283] The present disclosure also provides a method of treating a subject having, suspected of having, or at risk of developing a disease, disorder, or condition associated with target gene expression with an RNAi oligonucleotide conjugate provided herein. In some aspects, the present disclosure provides a method of treating or alleviating the onset or progression of a disease, disorder, or condition associated with target gene expression using an RNAi oligonucleotide conjugate provided herein. In other aspects, the present disclosure provides a method of using an RNAi oligonucleotide conjugate provided herein to achieve one or more therapeutic benefits in a subject having a disease, disorder, or condition associated with target gene expression. In some embodiments of the methods herein, the subject is treated by administering a therapeutically effective amount of any one or more of the RNAi oligonucleotide conjugates provided herein. In some embodiments, the treatment includes reducing target gene expression (e.g., in the CNS). In some embodiments, the subject is treated therapeutically. In some embodiments, the subject is treated prophylactically.

[0284] In some embodiments of the method herein, the RNAi oligonucleotide conjugate provided herein or the pharmaceutical composition comprising the RNAi oligonucleotide conjugate is administered to a subject having a disease, disorder or condition associated with target gene expression, so that target gene expression is reduced in the subject, thereby treating the subject.In some embodiments, the amount or level of target mRNA is reduced in the subject.In some embodiments, the amount or level of protein encoded by target mRNA is reduced in the subject.

[0285] In some embodiments of the methods herein, an RNAi oligonucleotide conjugate, or a pharmaceutical composition comprising an RNAi oligonucleotide conjugate provided herein, is administered to a subject having a disease, disorder, or condition associated with target gene expression such that target gene expression is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99%, when compared to target gene expression prior to administration of the RNAi oligonucleotide conjugate or pharmaceutical composition. In some embodiments, target gene expression is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99% in a subject when compared to target gene expression in a subject (e.g., a reference or control subject) that has not received the RNAi oligonucleotide conjugate or pharmaceutical composition or that has received a control RNAi oligonucleotide conjugate, pharmaceutical composition or treatment.

[0286] In some embodiments of the methods herein, an RNAi oligonucleotide conjugate herein, or a pharmaceutical composition comprising an RNAi oligonucleotide conjugate, is administered to a subject having a disease, disorder, or condition associated with target gene expression such that the amount or level of the target mRNA is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99%, when compared to the amount or level of the target mRNA before administration of the RNAi oligonucleotide conjugate or pharmaceutical composition. In some embodiments, the amount or level of target mRNA is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% in a subject when compared to the amount or level of target mRNA in a subject (e.g., a reference or control subject) that has not received the RNAi oligonucleotide conjugate or pharmaceutical composition or that has received a control RNAi oligonucleotide conjugate, pharmaceutical composition or treatment.

[0287] In some embodiments of the methods herein, an RNAi oligonucleotide conjugate herein, or a pharmaceutical composition comprising an RNAi oligonucleotide conjugate, is administered to a subject having a disease, disorder, or condition associated with target gene expression such that the amount or level of protein encoded by the target gene is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99%, compared to the amount or level of protein encoded by the target gene prior to administration of the RNAi oligonucleotide conjugate or pharmaceutical composition. In some embodiments, the amount or level of protein encoded by the target gene is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% in a subject when compared to the amount or level of protein encoded by the target gene in a subject (e.g., a reference or control subject) that has not received the RNAi oligonucleotide conjugate or pharmaceutical composition or that has received a control RNAi oligonucleotide conjugate, pharmaceutical composition or treatment.

[0288] In some embodiments of the methods herein, an RNAi oligonucleotide conjugate herein, or a pharmaceutical composition comprising an RNAi oligonucleotide conjugate, is administered to a subject having a disease, disorder, or condition associated with target gene expression such that the amount or level of target gene activity is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99%, when compared to the amount or level of target gene activity before administration of the RNAi oligonucleotide conjugate or pharmaceutical composition. In some embodiments, the amount or level of target gene activity is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99% in a subject when compared to the amount or level of target gene activity in a subject (e.g., a reference or control subject) that has not received the RNAi oligonucleotide conjugate or pharmaceutical composition or that has received a control RNAi oligonucleotide conjugate, pharmaceutical composition or treatment.

[0289] Suitable methods for determining target gene expression, the amount or level of target mRNA, the amount or level of a protein encoded by a target gene, and / or the amount or level of target gene activity in a subject or in a sample from a subject are known in the art. Additionally, the Examples provided herein set forth exemplary methods for determining target gene expression.

[0290] In some embodiments, target gene expression, amount or level of target gene mRNA, amount or level of protein encoded by target gene, amount or level of target gene activity, or any combination thereof, is reduced in a cell, a cell population or cell group (e.g., organoid), an organ (e.g., CNS), blood or its fraction (e.g., plasma), a tissue (e.g., brain tissue), a sample (e.g., CSF sample or brain biopsy sample), or any other biological material obtained or isolated from a subject.In some embodiments, target gene expression, amount or level of target gene mRNA, amount or level of protein encoded by target gene, amount or level of target gene activity, or any combination thereof, is reduced in multiple types of cells, multiple cell groups, multiple organs (e.g., brain and one or more other organs), multiple blood fractions (e.g., plasma and one or more other blood fractions), multiple types of tissues (e.g., brain tissue and one or more other types of tissues), multiple types of samples obtained or isolated from a subject (e.g., brain biopsy and one or more other types of biopsy sample).

[0291] In some embodiments, the method provides for the reduction of target gene expression in cells or cell populations in one or more target tissues (e.g., CNS) while maintaining target gene expression in cells or cell populations in one or more non-target tissues (e.g., liver). In some embodiments, the target gene expression in one or more non-target tissues contributes to the normal physiological function of the subject. In some embodiments, the method provides differential target gene expression to reduce irregular target gene expression in target tissues to treat, alleviate, or mitigate disease or disorder associated with the target gene without inducing dysregulation of the target gene in non-target tissues, which causes disruption of physiological function.

[0292] In some embodiments, the disclosure provides a method of treating a disease, disorder, or condition associated with a target gene, comprising administering to a subject in need of treatment an RNAi oligonucleotide conjugate or pharmaceutical composition thereof described herein, wherein (i) the amount or level of target gene expression in a target tissue (e.g., CNS) of the subject is reduced by at least about 30%, about 35%, about 40%, about 40%, or about 50%, as compared to a control amount or level of target gene expression. or greater than 5%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or reduced by greater than 99%, and (ii) the amount or level of target gene expression in one or more non-target tissues (e.g., liver) is comparable to a control expression level of the target gene (e.g., having a difference of about ±30%, about ±25%, about ±20%, about ±15%, about ±10%, about ±5%, about ±4%, about ±3%, about ±2%, or about ±1% or less).

[0293] In some embodiments, the amount or level of target mRNA is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% in a subject when compared to the amount or level of target mRNA in a subject (e.g., a reference or control subject) that has not received the RNAi oligonucleotide conjugate or pharmaceutical composition or that has received a control RNAi oligonucleotide conjugate, pharmaceutical composition or treatment.

[0294] Examples of diseases, disorders, or conditions associated with target gene expression include progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), argyrophilic granulopathy (AGD), glioglobular tauopathy (GGT), age-related tau astrogliopathy (ARTAG), familial frontotemporal dementia 17 (FTD-17), tauopathy with respiratory failure, dementia with seizures, Pick's disease, myotonic dystrophy 1 or 2 (MD). 1 or MD2), Down syndrome, spastic paraplegia (SP), Niemann-Pick disease type C, Dementia with Lewy bodies (DLB), Lewy body dysphagia, Lewy body disease, Olivopontocerebellar atrophy, Striatonigral degeneration, Shy-Drager syndrome, Spinal muscular atrophy V (SMAV), Huntington's disease (HD), Alzheimer's disease, SCA1, SCA2, SCA3, SCA7, SCA10 (Spinocerebellar ataxia type 1, type 2, type 3) Type 7 or 10), Multiple System Atrophy (MSA), Spinal-bulbar Muscular Atrophy (SBMA, Kennedy's Disease), Friedreich's Ataxia, Fragile X-associated Tremor / Ataxia Syndrome (FXTAS), Fragile X Syndrome (FRAXA), X-linked Mental Retardation (XLMR), Parkinson's Disease, Dystonia, Spinal-bulbar Muscular Atrophy (SBMA), Neuropathic Pain Disorders, Spinal Cord Injury, Dentatorubral-Pallidoluysian Atrophy (DRPLA), Inferior These include, but are not limited to, chronic CNS disorders, ALS (amyotrophic lateral sclerosis), M2DS (MECP2 duplication syndrome), FTD (frontotemporal dementia), prion diseases, adult-onset leukodystrophy, Alexander disease, Krabbe disease, chronic traumatic encephalopathy, Pelizaeus-Merzbach disease (PMD), Lafora disease, stroke, cerebral amyloid angiopathy (CAA), and metachromatic leukodystrophy (MLD).

[0295] In some embodiments, the target gene can be a target gene from any mammal, such as a human. Any gene can be silenced according to the methods described herein.

[0296] The methods described herein typically comprise administering to subject a therapeutically effective amount of the RNAi oligonucleotide conjugates herein, i.e., the amount that can produce desired therapeutic results.The therapeutically acceptable amount can be the amount that can treat disease or disorder therapeutically.The appropriate dose for any one subject will depend on certain factors, including subject's size, body surface area, age, composition administered, active ingredient in the composition, time and route of administration, general health condition, and other drugs administered at the same time.

[0297] In some embodiments, a subject is administered any one of the compositions herein either enterally (e.g., orally, by a gastric feeding tube, by a duodenal feeding tube, via a gastrostomy, or rectally), parenterally (e.g., subcutaneous injection, intravenous injection or infusion, intraarterial injection or infusion, intraosseous injection, intramuscular injection, intracerebral injection, intraventricular injection, intrathecal injection), locally (e.g., transdermally, inhalation, via eye drops, or via a mucosa), or by direct injection into a target organ (e.g., the subject's brain).

[0298] In some embodiments, the RNAi oligonucleotide conjugates herein, or compositions thereof, are administered intrathecally into the cerebrospinal fluid (CSF) (e.g., injection or infusion into the fluid in the subarachnoid space). In some embodiments, the intrathecal administration of the RNAi oligonucleotide conjugates herein, or compositions thereof, is performed as a bolus injection into the subarachnoid space. In some embodiments, the intrathecal administration of the RNAi oligonucleotide conjugates herein, or compositions thereof, is performed as an infusion into the subarachnoid space. In some embodiments, the intrathecal administration of the RNAi oligonucleotide conjugates herein, or compositions thereof, is performed into the subarachnoid space via a catheter. In some embodiments, the intrathecal administration of the RNAi oligonucleotide conjugates herein, or compositions thereof, is performed via a pump. In some embodiments, the intrathecal administration of the RNAi oligonucleotide conjugates herein, or compositions thereof, is performed via an implantable pump. In some embodiments, the administration is performed via an implantable device that operates or functions as a reservoir.

[0299] In some embodiments, the RNAi oligonucleotide conjugates herein, or compositions thereof, are administered intrathecally into the cerebellomedullary cistern (also called the cisterna magna). Intrathecal administration into the cisterna magna is referred to as "intracisternal administration" or "intracisternal magna (icm) administration. In some embodiments, the RNAi oligonucleotide conjugates herein, or compositions thereof, are administered intrathecally into the subarachnoid space of the lumbar spinal cord. Intrathecal administration into the subarachnoid space of the lumbar spinal cord is referred to as "lumbar intrathecal (it) administration." In some embodiments, the RNAi oligonucleotide conjugates herein, or compositions thereof, are administered intrathecally into the subarachnoid space of the cervical spinal cord. Intrathecal administration into the subarachnoid space of the cervical spinal cord is referred to as "cervical intrathecal (it) administration." In some embodiments, the RNAi oligonucleotide conjugates herein, or compositions thereof, are administered intrathecally into the subarachnoid space of the thoracic spinal cord. Intrathecal administration into the subarachnoid space of the thoracic spinal cord is referred to as "thoracic intrathecal (it) administration". In some embodiments, the RNAi oligonucleotide conjugates herein, or compositions thereof, are administered by intraventricular injection or infusion into the ventricle. Intraventricular administration into the ventricle space is referred to as "intraventricular (icv) administration". In some embodiments, an Ommaya reservoir is used to administer the RNAi oligonucleotide conjugates herein, or compositions thereof, by intraventricular injection or infusion.

[0300] In some embodiments, the RNAi oligonucleotide conjugates herein, or compositions thereof, are administered annually, every six months, every four months, quarterly (every three months), bimonthly (every two months), monthly, or weekly. In some embodiments, the RNAi oligonucleotide conjugates herein, or compositions thereof, are administered weekly, or at two-week or three-week intervals. In some embodiments, the RNAi oligonucleotide conjugates herein, or compositions thereof, are administered daily. In some embodiments, the subject is administered one or more loading doses of the RNAi oligonucleotide conjugates herein, or compositions thereof, followed by one or more maintenance doses of the RNAi oligonucleotide conjugates herein, or compositions thereof.

[0301] In some embodiments, the subject to be treated is a human or non-human primate or other mammalian subject. Other exemplary subjects include domesticated animals such as dogs and cats, farm animals such as horses, cows, pigs, sheep, goats, and chickens, and animals such as mice, rats, guinea pigs, and hamsters.

[0302] kit In some embodiments, the present disclosure provides a kit comprising the RNAi oligonucleotide conjugates herein, or compositions thereof, as described herein, and instructions for use. In some embodiments, the kit comprises the RNAi oligonucleotide conjugates herein, or compositions thereof, as described herein, and a package insert comprising instructions for use of the kit and / or any of its components. In some embodiments, the kit comprises the RNAi oligonucleotide conjugates herein, or compositions thereof, as described herein, one or more controls, and various buffers, reagents, enzymes, and other standard components well known in the art, in a suitable container. In some embodiments, the container comprises at least one vial, well, test tube, flask, bottle, syringe, or other container means into which the RNAi oligonucleotide conjugates herein, or compositions thereof, are placed, and in some cases, suitably aliquoted. In some embodiments where additional components are provided, the kit comprises an additional container into which the components are placed. The kit can also comprise a means for tightly closing the RNAi oligonucleotide conjugates herein, or compositions thereof, and any other reagents for commercial sale. Such containers can include injection-molded or blow-molded plastic containers into which the desired vials are held. The containers and / or kits can include a label with instructions and / or warnings.

[0303] In some embodiments, the kit comprises an RNAi oligonucleotide conjugate as described herein, or a composition thereof, and a pharma- ceutically acceptable carrier, or a pharmaceutical composition comprising an RNAi oligonucleotide conjugate, as described herein, and instructions for treating or delaying the progression of a disease, disorder, or condition associated with a target gene in a subject in need thereof.

[0304] In some embodiments, the disclosure provides a kit comprising an RNAi oligonucleotide conjugate as described herein, or a composition thereof, and a pharma- ceutically acceptable carrier, or a pharmaceutical composition comprising an RNAi oligonucleotide conjugate, and instructions, for reducing or inhibiting expression in a subject of a target mRNA expressed by a cell population associated with the CNS.

[0305] In some embodiments, the disclosure provides a kit comprising an RNAi oligonucleotide conjugate as described herein, or a composition thereof, and a pharma- ceutically acceptable carrier, or a pharmaceutical composition comprising an RNAi oligonucleotide conjugate, and instructions for reducing or inhibiting expression of a target mRNA in a subject that is expressed by a cell population associated with the CNS without reducing expression of the target mRNA to a similar level outside the CNS.

[0306] In some embodiments, the disclosure provides a kit comprising an RNAi oligonucleotide conjugate as described herein, or a composition thereof, and a pharma- ceutically acceptable carrier, or a pharmaceutical composition comprising an RNAi oligonucleotide conjugate, and instructions, for reducing or inhibiting expression of a target mRNA in a subject that is expressed by a cell population associated with the CNS without reducing expression of the target mRNA to a similar level in cells of the liver.

[0307] In some embodiments, the disclosure provides a kit comprising an RNAi oligonucleotide conjugate as described herein, or a composition thereof, and a pharma- ceutically acceptable carrier, or a pharmaceutical composition comprising an RNAi oligonucleotide conjugate, as described herein, for reducing or inhibiting expression of a target mRNA in a subject in the CNS, where the target mRNA is associated with a disease or disorder, optionally a neurological disease or disorder.

[0308] In some embodiments, the disclosure provides a kit comprising an RNAi oligonucleotide conjugate as described herein, or a composition thereof, and a pharma- ceutically acceptable carrier, or a pharmaceutical composition comprising an RNAi oligonucleotide conjugate, as described herein, and instructions for reducing or inhibiting expression of a target mRNA in a subject in the CNS without reducing expression of the target mRNA to a similar level outside the CNS, wherein the target mRNA is associated with a disease or disorder, optionally a neurological disease or disorder.

[0309] In some embodiments, the disclosure provides a kit comprising an RNAi oligonucleotide conjugate as described herein, or a composition thereof, and a pharma- ceutically acceptable carrier, or a pharmaceutical composition comprising an RNAi oligonucleotide conjugate, as described herein, and instructions for reducing or inhibiting expression of a target mRNA in a subject in the CNS without reducing expression of the target mRNA to a similar level in the liver, wherein the target mRNA is associated with a disease or disorder, optionally a neurological disease or disorder.

[0310] Other embodiments The present disclosure relates to the following embodiments. Throughout this section, the term embodiment is abbreviated as "E" followed by an ordinal number. For example, E1 corresponds to embodiment 1.

[0311] E1. A nucleic acid-ligand conjugate represented by formula Ia: [ka] or a pharma- ceutically acceptable salt thereof, wherein B is a nucleobase or hydrogen; R 1 and R 2 are independently hydrogen, halogen, or R A, -CN, -S(O)R, -S(O)2R, -Si(OR)2R, -Si(OR)R2, or -SiR3, or R on the same carbon 1 and R 2 together with their intervening atoms form a 3-membered saturated or partially unsaturated ring having 0 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; Each R A is independent, C 1-6 an optionally substituted group selected from aliphatic; phenyl; a 4-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; Each R is independently hydrogen, a suitable protecting group, or C 1-6 an optionally substituted group selected from aliphatic; phenyl; a 4-7 membered saturated or partially unsaturated heterocycle having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or two R groups on the same atom together with their intervening atoms form a 4-7 membered saturated, partially unsaturated, or heteroaryl ring having 0-3 heteroatoms independently selected from nitrogen, oxygen, silicon, and sulfur; L A is independent, P.G. 1 or -L-ligand, PG 1 is hydrogen or a suitable hydroxyl protecting group, Each ligand is independently -(LC) n and / or an adamantyl group, Each LC can be independently saturated or unsaturated, linear or branched C 1-50a lipid conjugate moiety comprising a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently replaced by -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR-, or -P(S)OR-; Each -Cy- is independently selected from phenylenyl; 8-10 membered bicyclic arylenyl; 4-7 membered saturated or partially unsaturated carbocyclylenyl; 4-11 membered saturated or partially unsaturated spirocarbocyclylenyl; 8-10 membered bicyclic saturated or partially unsaturated carbocyclylenyl; adamantanenyl; 4-7 membered saturated or partially unsaturated heterocyclylenyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; 4-7 membered saturated or partially unsaturated heterocyclylenyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; an optionally substituted bivalent ring selected from: an 8- to 11-membered saturated or partially unsaturated spiroheterocyclylenyl; an 8- to 10-membered bicyclic saturated or partially unsaturated heterocyclylenyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; a 5- to 6-membered heteroarylenyl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or an 8- to 10-membered bicyclic heteroarylenyl having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; n is 1 to 10; L is a covalent bond or a divalent saturated or unsaturated, linear or branched C 1-50 a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently selected from the group consisting of -Cy-, -O-, -NR-, -N(R)-C(O)-, -S-, -C(O)-, -S(O)-, -S(O)2-, -P(O)OR-, -P(S)OR-, -V 1 CR 2 W 1 -or [ka] is replaced by m is 1 to 50; X 1 , V 1 , and W1 are independently -C(R)2-, -OR, -O-, -S-, -Se-, or -NR-; Z is -O-, -S-, -NR-, or -CR2-; PG 2 is hydrogen, a phosphoramidite analog, or a suitable protecting group. E2. The conjugate is represented by formula Ib or Ic: [ka] or a pharma- ceutically acceptable salt thereof, wherein: L 1 is a covalent bond or a divalent saturated or unsaturated, linear or branched C 1-50 a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR-, -P(S)OR-, or [ka] is replaced by R 4 is hydrogen, R A or a suitable amine protecting group, R 5 is adamantyl or saturated or unsaturated, linear or branched C 1-50 a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently replaced by -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR-, or -P(S)OR-. E3. A nucleic acid-ligand conjugate represented by formula I-Ib or I-Ic; [ka] or a pharma- ceutically acceptable salt thereof, wherein B is a nucleobase or hydrogen; m is 1 to 50; PG 1 and P.G. 2 are independently hydrogen, a phosphoramidite analog, or a suitable protecting group; R 5 is adamantyl or saturated or unsaturated, linear or branched C 1-50 a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently replaced by -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR-, or -P(S)OR-. E4. A nucleic acid-ligand conjugate of E3, R 5 is selected from the following: [ka] E5. A nucleic acid-ligand conjugate of E4, R 5 is selected from the following: [ka] E6. An oligonucleotide-ligand conjugate comprising one or more nucleic acid-ligand conjugates of any one of E1 to E5. E7. The oligonucleotide-ligand conjugate of E6, wherein the conjugate comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleic acid-ligand conjugates. E8. Oligonucleotide-ligand conjugates comprising one or more nucleic acid-ligand conjugates of formula II-a; [ka] or a pharma- ceutically acceptable salt thereof, wherein B is a nucleobase or hydrogen; R 1 and R 2 are independently hydrogen, halogen, or RA , -CN, -S(O)R, -S(O)2R, -Si(OR)2R, -Si(OR)R2, or -SiR3, or R on the same carbon 1 and R 2 together with their intervening atoms form a 3- to 7-membered saturated or partially unsaturated ring having 0-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; Each R A is independent, C 1-6 an optionally substituted group selected from aliphatic; phenyl; a 4-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; Each R is independently hydrogen, a suitable protecting group, or C 1-6 an optionally substituted group selected from aliphatic; phenyl; a 4-7 membered saturated or partially unsaturated heterocycle having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or two R groups on the same atom together with their intervening atoms form a 4-7 membered saturated, partially unsaturated, or heteroaryl ring having 0-3 heteroatoms independently selected from nitrogen, oxygen, silicon, and sulfur; Each LC can be independently saturated or unsaturated, linear or branched C 1-50 a lipid conjugate moiety comprising a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently replaced by -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)-OR-, -P(S)OR-; Each -Cy- is independently selected from phenylenyl; 8-10 membered bicyclic arylenyl; 4-7 membered saturated or partially unsaturated carbocyclylenyl; 4-11 membered saturated or partially unsaturated spirocarbocyclylenyl; 8-10 membered bicyclic saturated or partially unsaturated carbocyclylenyl; 4-7 membered saturated or partially unsaturated heterocyclylenyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; 4-11 membered saturated or partially unsaturated heterocyclylenyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. an optionally substituted bivalent ring selected from: a saturated or partially unsaturated spiroheterocyclylenyl; an 8-10 membered bicyclic saturated or partially unsaturated heterocyclylenyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; a 5-6 membered heteroarylenyl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or an 8-10 membered bicyclic heteroarylenyl having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; n is 1 to 10; L is a covalent bond or a divalent saturated or unsaturated, linear or branched C 1-50 a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently selected from the group consisting of -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, S(O)-, -S(O)2-, -P(O)OR-, -P(S)OR-, -V 1 CR 2 W 1 -or [ka] is replaced by m is 1 to 50; X 1 , V 1 , and W 1 are independently -C(R)2-, -OR, -O-, -S-, -Se-, or -NR-; Y is hydrogen, a suitable hydroxyl protecting group, [ka] and R 3 is hydrogen, a suitable protecting group, a suitable prodrug, or C 1-6 an optionally substituted group selected from aliphatic, phenyl, 4-7 membered saturated or partially unsaturated heterocycle having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; X 2 is O, S, or NR; X 3 is -O-, -S-, -BH2-, or a covalent bond, Y 1 is a linking group attached to the 2' or 3' end of a nucleoside, nucleotide, or oligonucleotide, Y 2 is hydrogen, a suitable protecting group, a phosphoramidite analog; an internucleotide linkage attached to the 5' end of a nucleoside, nucleotide, or oligonucleotide, or a linkage attached to a solid support; Z is -O-, -S-, -NR-, or -CR2-. E9. The conjugate is represented by formula II-b or II-c; [ka] or a pharma-ceutically acceptable salt thereof, wherein: L 1 is a covalent bond, monovalent or divalent saturated or unsaturated, linear or branched C 1-50 a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR-, -P(S)OR-, or [ka] is replaced by R4 is hydrogen, R A or a suitable amine protecting group, R 5 is adamantyl or saturated or unsaturated, linear or branched C 1-50 a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently replaced by -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR-, or -P(S)OR. E10.E9 oligonucleotide-ligand conjugate, R 5 is selected from the following: [ka] E11.E9 oligonucleotide-ligand conjugate, R 5 is selected from the following: [ka] E12. An oligonucleotide-ligand conjugate represented by formula II-Ib or II-Ic; [ka] or a pharma- ceutically acceptable salt thereof, wherein B is a nucleobase or hydrogen; m is 1 to 50; X 1 is -O- or -S-; Y is hydrogen, [ka] and R 3 is hydrogen or a suitable protecting group, X 2 is O or S, X 3 is -O-, -S-, or a covalent bond; Y 1 is a linking group attached to the 2' or 3' end of a nucleoside, nucleotide, or oligonucleotide, Y 2 is hydrogen, a phosphoramidite analog; an internucleotide linkage attached to the 5' end of a nucleoside, nucleotide, or oligonucleotide, or a linkage attached to a solid support; R 5 is adamantyl or saturated or unsaturated, linear or branched C 1-50 a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently replaced by -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR-, or -P(S)OR-; R is hydrogen, a suitable protecting group, or C 1-6 an optionally substituted group selected from aliphatic; phenyl; a 4-7 membered saturated or partially unsaturated heterocycle having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. E13. An oligonucleotide-ligand conjugate of E12, R 5 is selected from the following: [ka] E14. The oligonucleotide-ligand conjugate of any one of E8-E13, wherein the conjugate comprises from 1 to 10 nucleic acid-ligand conjugate units. E15. The oligonucleotide-ligand conjugate of any one of E8-E13, wherein the conjugate comprises 1, 2 or 3 nucleic acid-ligand conjugate units. E16. The oligonucleotide-ligand conjugate of any one of E6 to E15, wherein the oligonucleotide comprises a sense strand 10-53 nucleotides in length and an antisense strand 15-53 nucleotides in length, the antisense oligonucleotide strand having a sequence complementary to at least 15 consecutive nucleotides of a target gene sequence, and wherein the oligonucleotide conjugate reduces gene expression when introduced into a mammalian cell. E17. The oligonucleotide-ligand conjugate of E16, wherein the nucleic acid-ligand conjugate unit is in the sense strand. E18. The oligonucleotide-ligand conjugate of E16, wherein the antisense strand is 19-27 nucleotides in length. E19. The oligonucleotide-ligand conjugate of E16, wherein the sense strand is 12-40 nucleotides in length. E20. The oligonucleotide-ligand conjugate of any one of E16-E19, wherein the sense strand forms a double-stranded region with the antisense strand. E21. The oligonucleotide-ligand conjugate of E16, wherein the complementary region is perfectly complementary to the target sequence. E22. The oligonucleotide-ligand conjugate of any one of E16-E21, wherein the sense strand comprises a stem-loop at its 3' end shown as S1-L-S2, where S1 is complementary to S2, and L forms a loop 3-5 nucleotides long between S1 and S2. E23.L is an oligonucleotide-ligand conjugate of E22, wherein L is a tetraloop. E24.L is an oligonucleotide-ligand conjugate of E22 comprising the sequence shown as GAAA. E25. An oligonucleotide-ligand conjugate of any one of E16 to E24, further comprising a 3' overhang sequence on the antisense strand having a length of 2 nucleotides. E26. The oligonucleotide-ligand conjugate of any one of E16-E24, wherein the oligonucleotide comprises a 3' overhang sequence of one or more nucleotides in length, and the 3' overhang sequence is present on the antisense strand, the sense strand, or the antisense strand and the sense strand. E27. The oligonucleotide-ligand conjugate of any one of E16-E26, wherein the oligonucleotide comprises at least one modified nucleotide. E28. The oligonucleotide-ligand conjugate of E27, wherein the modified nucleotide comprises a 2'-modification. E29. The oligonucleotide-ligand conjugate of E28, wherein the 2'-modification is a modification selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, 2'-deoxy-2'-fluoro, and 2'-deoxy-2'-fluoro-β-d-arabino. E30. The oligonucleotide-ligand conjugate of any one of E16 to E26, wherein all nucleotides of the oligonucleotide are modified. E31. The oligonucleotide-ligand conjugate of any one of E16-E30, wherein the oligonucleotide comprises at least one modified internucleotide linkage. E32. The oligonucleotide-ligand conjugate of E31, wherein at least one modified internucleotide linkage is a phosphorothioate linkage. E33. The oligonucleotide-ligand conjugate of any one of E16-E30, wherein the 4'-carbon of the sugar of the 5'-nucleotide of the antisense strand comprises a phosphate analog. E34. The oligonucleotide-ligand conjugate of E33, wherein the phosphate analog is an oxymethylphosphonate, vinylphosphonate, or malonylphosphonate. E35. A composition comprising any one of the oligonucleotide-ligand conjugates of E16 to E34 and an excipient. E36. A method of delivering an oligonucleotide-ligand conjugate to a subject, the method comprising administering to the subject a composition of E35. E37. An oligonucleotide-ligand conjugate of any one of E16-E34 to reduce expression of a target gene. E38. An oligonucleotide-ligand conjugate of E37, wherein the target gene is expressed in the CNS. E39. The oligonucleotide-ligand conjugate of E38, wherein the target gene is associated with a disease or disorder, optionally a neurological disease or disorder. E40. Disease or disorder is progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), argyrophilic granulomatosis (AGD), glioglobular tauopathy (GGT), age-related tau astrogliopathy (ARTAG), familial frontotemporal dementia 17 (FTD-17), tauopathy with respiratory failure, dementia with seizures, Pick's disease, myotonic dystrophy 1 or 2 (MD1 or MD2), Down's syndrome, Spastic paraplegia (SP), Niemann-Pick disease type C, Dementia with Lewy bodies (DLB), Lewy body dysphagia, Lewy body disease, Olivopontocerebellar atrophy, Striatonigral degeneration, Shy-Drager syndrome, Spinal muscular atrophy V (SMAV), Huntington's disease (HD), Alzheimer's disease, SCA1, SCA2, SCA3, SCA7, SCA10 (Spinocerebellar ataxia types 1, 2, 3, 7 or 103), Multiple system atrophy Symptoms of muscular atrophy include spinal-bulbar muscular atrophy (MSA), spinal-bulbar muscular atrophy (SBMA, Kennedy disease), Friedreich's ataxia, fragile X-associated tremor / ataxia syndrome (FXTAS), fragile X syndrome (FRAXA), X-linked mental retardation (XLMR), Parkinson's disease, dystonia, SBMA (spinal-bulbar muscular atrophy), neuropathic pain disorders, spinal cord injury, dentatorubral-pallidoluysian atrophy (DRPLA), recessive CNS disorders, and ALS (amyotrophic lateral sclerosis). lateral sclerosis), M2DS (MECP2 duplication syndrome), FTD (frontotemporal dementia), prion disease, adult-onset leukodystrophy, Alexander disease, Krabbe disease, chronic traumatic encephalopathy, Pelizaeus-Merzbach disease (PMD), Lafora disease, stroke, cerebral amyloid angiopathy (CAA), and metachromatic leukodystrophy (MLD).

[0312] definition As used herein, "approximately" or "about" as applied to one or more subject values ​​refers to a value similar to the stated reference value.In certain embodiments, "about" refers to a range of values ​​that is within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater or less) of the stated reference value, unless otherwise specified or clear from the context (except when such number exceeds 100% of possible values).

[0313] As used herein, "administer," "administering," "administration," and the like refer to providing a substance (e.g., an RNAi oligonucleotide conjugate) to a subject in a pharmacologically useful manner (e.g., to treat a condition in the subject).

[0314] As used herein, "asialoglycoprotein receptor" or "ASGPR" refers to a bipartite C-type lectin formed by a major 48 kDa subunit (ASGPR-1) and a minor 40 kDa subunit (ASGPR-2). ASGPR is expressed primarily on the sinusoidal surface of hepatocytes and plays a major role in binding, internalization, and subsequent clearance of circulating glycoproteins that contain terminal galactose or GalNAc residues (asialoglycoproteins).

[0315] As used herein, "alleviate", "alleviating", "alleviation" and the like refer to reducing or effectively stopping. As a non-limiting example, one or more treatments herein can reduce or effectively stop the onset or progression of a disease (e.g., a neurological disease or disorder) associated with target gene expression in a subject. This attenuation can be exemplified, for example, by a reduction in one or more aspects of the disease associated with target gene expression (e.g., symptoms, tissue characteristics, and cellular, inflammatory, or immune activity, etc.), no detectable progression (worsening) of one or more aspects of the disease, or no detectable aspects of the disease in a subject when they would otherwise be expected.

[0316] As used herein, "complementary" refers to a structural relationship between two nucleotides (e.g., on two opposing nucleic acids or on opposing regions of a single nucleic acid strand) that allows the two nucleotides to base pair with each other. For example, purine nucleotides of one nucleic acid that are complementary to pyrimidine nucleotides of an opposing nucleic acid may base pair together by forming hydrogen bonds with each other. In some embodiments, complementary nucleotides may base pair in a Watson-Crick manner or in any other manner that allows the formation of a stable duplex. In some embodiments, two nucleic acids may have a region of multiple nucleotides that are complementary to each other, forming a region of complementarity, as described herein.

[0317] As used herein, "CNS mRNA" and "CNS gene" refer to any gene, mRNA and / or protein encoded / expressed by a gene in a cell or tissue of the central nervous system.

[0318] As used herein, "deoxyribonucleotide" refers to a nucleotide that, when compared to a ribonucleotide, has a hydrogen instead of a hydroxyl at the 2' position of its pentose sugar. Modified deoxyribonucleotides are deoxyribonucleotides that have one or more modifications or substitutions of atoms other than the 2' position, including modifications or substitutions at or in the sugar, phosphate group, or base.

[0319] As used herein, "double-stranded oligonucleotide" or "ds oligonucleotide" refers to an oligonucleotide that is in a substantially double-stranded form. In some embodiments, the complementary base pairing of the double-stranded region of the double-stranded oligonucleotide is formed between the antiparallel sequence of nucleotides of the covalently separated nucleic acid strands. In some embodiments, the complementary base pairing of the double-stranded region of the double-stranded oligonucleotide is formed between the antiparallel sequence of nucleotides of the covalently linked nucleic acid strands. In some embodiments, the complementary base pairing of the double-stranded region of the double-stranded oligonucleotide is formed from a single nucleic acid strand that folds back (e.g., via a hairpin) to provide a complementary antiparallel sequence of nucleotides that base pair together. In some embodiments, the double-stranded oligonucleotide comprises two covalently separated nucleic acid strands that are fully double-stranded with each other. However, in some embodiments, the double-stranded oligonucleotide comprises two covalently separated nucleic acid strands that are partially double-stranded (e.g., have an overhang at one or both ends). In some embodiments, the double-stranded oligonucleotide comprises an antiparallel sequence of nucleotides that are partially complementary, and thus may have one or more mismatches, which may include internal or terminal mismatches.

[0320] As used herein, "duplex" with respect to a nucleic acid (eg, an oligonucleotide) refers to the structure formed through complementary base pairing of two antiparallel sequences of nucleotides.

[0321] As used herein, "excipient" refers to a non-therapeutic agent that may be included in a composition, for example, to provide or contribute a desired consistency or stabilizing effect.

[0322] As used herein, a "labile linker" refers to a linker that can be cleaved (e.g., by acidic pH). A "fairly stable linker" refers to a linker that cannot be cleaved.

[0323] As used herein, a "loop" refers to an unpaired region of a nucleic acid (e.g., an oligonucleotide) that is flanked by two antiparallel regions of the nucleic acid that are sufficiently complementary to each other such that under appropriate hybridization conditions (e.g., intracellularly, in phosphate buffer), the two antiparallel regions flanking the unpaired regions hybridize to form a duplex (called a "stem").

[0324] As used herein, "modified internucleotide bond" refers to an internucleotide bond that has one or more chemical modifications when compared to a reference internucleotide bond that includes a phosphodiester bond. In some embodiments, the modified nucleotide is a bond that does not occur naturally. Typically, the modified internucleotide bond confers one or more desirable properties to the nucleic acid in which the modified internucleotide bond is present. For example, the modified nucleotide may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, biological activity, reduced immunogenicity, etc.

[0325] As used herein, "modified nucleotide" refers to a nucleotide that has one or more chemical modifications when compared to a corresponding reference nucleotide selected from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymidine deoxyribonucleotide. In some embodiments, the modified nucleotide is a non-naturally occurring nucleotide. In some embodiments, the modified nucleotide has one or more chemical modifications in its sugar, nucleobase, and / or phosphate group. In some embodiments, the modified nucleotide has one or more chemical moieties conjugated to the corresponding reference nucleotide. Typically, the modified nucleotide confers one or more desirable properties to the nucleic acid in which the modified nucleotide is present. For example, the modified nucleotide may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, biological activity, reduced immunogenicity, etc.

[0326] As used herein, "nicked tetraloop structure" refers to a structure of an RNAi oligonucleotide (e.g., an RNAi oligonucleotide conjugate) characterized by separate sense (passenger) and antisense (guide) strands, where the sense strand has a region of complementarity with the antisense strand, and at least one of the strands, typically the sense strand, has a tetraloop configured to stabilize an adjacent stem region formed in at least one strand.

[0327] As used herein, "oligonucleotide" refers to a short nucleic acid (e.g., less than about 100 nucleotides in length). Oligonucleotides can be single-stranded (ss) or ds. Oligonucleotides can have or not have double-stranded regions. As a set of non-limiting examples, oligonucleotides can be, but are not limited to, small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), dicer substrate interfering RNA (dsiRNA), antisense oligonucleotide, short siRNA, or single-stranded siRNA. In some embodiments, double-stranded oligonucleotides are RNAi oligonucleotides.

[0328] As used herein, "overhang" refers to the terminal non-base-paired nucleotides that originate from one strand or region that extends beyond the end of the complementary strand that forms a duplex.In some embodiments, the overhang comprises one or more unpaired nucleotides that extend from the double-stranded region at the 5'-end or 3'-end of the double-stranded oligonucleotide.In certain embodiments, the overhang is the 3'- or 5'-overhang on the antisense strand or the sense strand of the double-stranded oligonucleotide.

[0329] As used herein, "phosphate analog" refers to a chemical moiety that mimics the electrostatic and / or steric properties of the phosphate group. In some embodiments, the phosphate analog is positioned at the 5'-terminal nucleotide of an oligonucleotide in place of the 5'-phosphate, which is often susceptible to enzymatic removal. In some embodiments, the 5' phosphate analog comprises a phosphatase-resistant linkage. Examples of phosphate analogs include, but are not limited to, 5' phosphonates, such as 5' methylene phosphonate (5'-MP) and 5'-(E)-vinyl phosphonate (5'-VP). In some embodiments, an oligonucleotide has a phosphate analog at the 4'-carbon position of the sugar at the 5'-terminal nucleotide (referred to as a "4'-phosphate analog"). An example of a 4'-phosphate analog is an oxymethyl phosphonate, in which the oxygen atom of the oxymethyl group is attached to the sugar moiety (e.g., at its 4' carbon) or an analog thereof. See, e.g., U.S. Provisional Patent Application Nos. 62 / 383,207, filed September 2, 2016, and 62 / 393,401, filed September 12, 2016. Other modifications to the 5' end of oligonucleotides have been developed (see, e.g., International Patent Application No. 2011 / 133871, U.S. Patent No. 8,927,513, and Prakash et al. (2015) NUCLEIC ACIDS RES. 43:2993-3011).

[0330] As used herein, "reducing expression" of a target gene refers to a reduction in the amount or level of an RNA transcript (e.g., target mRNA) or protein encoded by a target gene, and / or a reduction in the amount or level of activity of the gene in a cell, cell population, sample, or subject, when compared to an appropriate reference (e.g., a reference cell, a reference cell population, a reference sample, or a reference subject). For example, the act of contacting a cell with an oligonucleotide or conjugate herein (e.g., an RNAi oligonucleotide conjugate comprising an antisense strand having a nucleotide sequence complementary to a nucleotide sequence comprising a target mRNA) can result in a reduction in the amount or level of target mRNA, the amount or level of protein encoded by a target gene, and / or the amount or level of target gene activity (e.g., by inactivation and / or degradation of target mRNA by the RNAi pathway), when compared to a cell that is not treated with a double-stranded oligonucleotide. Similarly, as used herein, "reducing expression" refers to an action that results in a reduction in the expression of a target gene.

[0331] As used herein, "reduced target gene expression" refers to a decrease in the amount or level of target mRNA, the protein encoded by the target gene, and / or target gene activity in a cell, a cell population, a sample, or a subject when compared to an appropriate reference (e.g., a reference cell, a reference cell population, a reference sample, or a reference subject).

[0332] As used herein, "complementary region" refers to a nucleotide sequence of a nucleic acid (e.g., a double-stranded oligonucleotide or an RNAi oligonucleotide conjugate described herein) that is sufficiently complementary to an antiparallel sequence of nucleotides to allow hybridization between the two sequences of nucleotides under suitable hybridization conditions (e.g., in a phosphate buffer, in a cell, etc.). In some embodiments, the oligonucleotide herein comprises a targeting sequence that has a region that is complementary to an mRNA target sequence.

[0333] As used herein, "ribonucleotide" refers to a nucleotide having ribose as its pentose sugar, containing a hydroxyl group at its 2' position. A modified ribonucleotide is a ribonucleotide that has one or more modifications or substitutions of atoms other than the 2' position, including modifications or substitutions of the ribose, phosphate group, or base.

[0334] As used herein, an "RNAi oligonucleotide" refers to either (a) a double-stranded oligonucleotide having a sense strand (passenger) and an antisense strand (guide), where the antisense strand, or a portion of the antisense strand, uses Argonaute 2 (Ago2) endonuclease in cleaving a target mRNA (e.g., a target mRNA expressed in the CNS), or (b) a single-stranded oligonucleotide having a single antisense strand, where the antisense strand (or a portion of the antisense strand) uses Ago2 endonuclease in cleaving a target mRNA (e.g., a target mRNA expressed in the CNS).

[0335] As used herein, a "strand" refers to a single, contiguous sequence of nucleotides linked together through internucleotide bonds (e.g., phosphodiester or phosphorothioate bonds). In some embodiments, a strand has two free ends (e.g., a 5' end and a 3' end).

[0336] As used herein, "subject" refers to any mammal, including mice, rabbits, and humans. In one embodiment, the subject is a human or NHP. Furthermore, "individual" or "patient" may be used interchangeably with "subject."

[0337] As used herein, "synthetic" refers to a nucleic acid or other molecule that is artificially synthesized (e.g., using a machine (e.g., a solid-state nucleic acid synthesizer)) or is not otherwise derived from a natural source (e.g., a cell or organism) that normally produces the molecule.

[0338] As used herein, a "targeting ligand" refers to a molecule (e.g., a carbohydrate, an amino sugar, cholesterol, a polypeptide, or a lipid) that selectively binds to a cognate molecule (e.g., a receptor) of a tissue or cell of interest and can be conjugated to another substance for the purpose of targeting the tissue or cell of interest and other substances. For example, in some embodiments, a targeting ligand can be conjugated to an oligonucleotide for the purpose of targeting the oligonucleotide to a specific tissue or cell of interest. In some embodiments, a targeting ligand selectively binds to a cell surface receptor. Thus, in some embodiments, a targeting ligand, when conjugated to an oligonucleotide, promotes delivery of an oligonucleotide to a specific cell through selective binding to a receptor expressed on the surface of the cell and endosomal internalization by the cell of a complex comprising the oligonucleotide, the targeting ligand, and the receptor. In some embodiments, a targeting ligand is conjugated to an oligonucleotide via a linker that is cleaved after or during cellular internalization so that the oligonucleotide is released from the targeting ligand within the cell.

[0339] As used herein, "tetraloop" refers to a loop that increases the stability of adjacent duplexes formed by hybridization of adjacent sequences of nucleotides. The increase in stability is determined by the average increase in the T of the predicted adjacent stem duplexes from a set of loops of equivalent length composed of randomly selected nucleotide sequences. m The melting temperature (T m For example, a tetraloop can be detected in a hairpin comprising a duplex of at least 2 base pairs (bp) in length at a T of at least about 50° C., at least about 55° C., at least about 56° C., at least about 58° C., at least about 60° C., at least about 65° C., or at least about 75° C. in 10 mM NaHPO4. mIn some embodiments, the tetraloop can stabilize the bp of the adjacent stem duplex by stacking interactions. Additionally, interactions between nucleotides in the tetraloop include, but are not limited to, non-Watson-Crick base pairing, stacking interactions, hydrogen bonds, and contact interactions (Cheong et al. (1990) Nature 346:680-682, Heus & Pardi (1991) Science 253:191-94). In some embodiments, the tetraloop comprises or consists of 3-6 nucleotides, typically 4-5 nucleotides. In certain embodiments, the tetraloop comprises or consists of 3, 4, 5, or 6 nucleotides, which may or may not be modified (e.g., conjugated to a targeting moiety). In one embodiment, the tetraloop consists of 4 nucleotides. Any nucleotide may be used in the tetraloop, and the standard IUPAC-IUB symbols for such nucleotides may be used as described in Cornish-Bowden (1985) NUCLEIC ACIDS RES. 13:3021-30. For example, the letter "N" may be used to mean that any base may be at that position, the letter "R" may be used to indicate that A (adenine) or G (guanine) may be at that position, and "B" may be used to indicate that C (cytosine), G (guanine), T (thymine), or U (uracil) may be at that position. Examples of tetraloops include the UNCG family of tetraloops (e.g., UUCG), the GNRA family of tetraloops (e.g., GAAA), and the CUUG tetraloop (Woese et al. (1990) PROC. NATL. ACAD. SCI. USA 87:8467-71; Antao et al. (1991) NUCLEIC ACIDS RES. 19:5901-05).Examples of DNA tetraloops include the d(GNNA) family of tetraloops (e.g., d(GTTA), d(GNRA)), the d(GNAB) family of tetraloops, the d(CNNG) family of tetraloops, and the d(TNCG) family of tetraloops (e.g., d(TTCG)). See, e.g., Nakano et al. (2002) BIOCHEM. 41:4281-92; Shinji et al. (2000) Nippon Kagaku Kai Koen Yokusyu Kogakushu 78:731. In some embodiments, the tetraloop is contained within a nicked tetraloop structure.

[0340] As used herein, "treat" or "treating" refers to the act of providing care to a subject in need of care, for example, by administering a therapeutic agent (e.g., an RNAi oligonucleotide conjugate herein) to the subject for the purpose of improving the health and / or well-being of the subject with respect to an existing condition (e.g., disease, disorder) or preventing or reducing the likelihood of the occurrence of a condition. In some embodiments, treatment includes reducing the frequency or severity of at least one sign, symptom, or contributing factor of a condition (e.g., disease, disorder) experienced by the subject. EXAMPLES

[0341] In order that the invention described herein may be more fully understood, the following examples are set forth. The examples described in this application are provided to illustrate the methods, compositions, and systems provided herein, and should not be construed as limiting the scope thereof in any way.

[0342] Abbreviation Ac: Acetyl AcOH: acetic acid ACN: Acetonitrile Ad: Adamantly AIBN: 2,2'-azobisisobutyronitrile Anhydride: Anhydrous Aq: Water-based B2Pin2: Bis(pinacolato)diboron-4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) BINAP: 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl BH3: Volan Bn: Benzyl Boc: tert-butoxycarbonyl Boc2O: Di-tert-butyl dicarbonate BPO: Benzoyl peroxide n BuOH: n-butanol CDI: Carbonyldiimidazole COD: Cyclooctadiene d:day DABCO: 1,4-diazobicyclo[2.2.2]octane DAST: Diethylaminosulfur trifluoride dba: dibenzylideneacetone DBU: 1,8-diazobicyclo[5.4.0]undec-7-ene DCE: 1,2-dichloroethane DCM: dichloromethane DEA: Diethylamine DHP: Dihydropyran DIBAL-H: Diisobutylaluminum hydride DIPA: Diisopropylamine DIPEA or DIEA: N,N-Diisopropylethylamine DMA: N,N-Dimethylacetamide DME: 1,2-dimethoxyethane DMAP: 4-dimethylaminopyridine DMF: N,N-dimethylformamide DMP: Dess-Martin periodinane DMSO - Dimethyl sulfoxide DMTr: 4,4'-Dimethyloxytrityl DPPA: Diphenylphosphoryl azide dppf: 1,1'-bis(diphenylphosphino)ferrocene EDC or EDCI: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ee: enantiomeric excess ESI: electrospray ionization EA: Ethyl acetate EtOAc: ethyl acetate EtOH: Ethanol FA: Formic acid h or hrs: hours HATU: N,N,N',N'-Tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate HCl: Hydrochloric acid HPLC: High-performance liquid chromatography HOAc: acetic acid IBX: 2-iodoxybenzoic acid IPA: Isopropyl alcohol KHMDS: Potassium hexamethyldisilazide K2CO3: Potassium carbonate LAH: Lithium aluminum hydride LDA: Lithium diisopropylamide L-DBTA: Dibenzoyl-L-tartaric acid m-CPBA: meta-chloroperbenzoic acid M: mole MeCN: Acetonitrile MeOH: Methanol Me2S: Dimethyl sulfide MeONa: Sodium methylate MeI: Iodomethane min:minutes mL: milliliter mM: millimolar mmol: millimolar MPa: Megapascals MOMCl: Methyl chloromethyl ether MsCl: methanesulfonyl chloride MTBE: Methyl tert-butyl ether nBuLi: n-butyl lithium NaNO2: Sodium nitrite NaOH: Sodium hydroxide Na2SO4: Sodium sulfate NBS: N-bromosuccinimide NCS: N-chlorosuccinimide NFSI: N-fluorobenzenesulfonimide NMO: N-methylmorpholine N-oxide NMP: N-methylpyrrolidine NMR: nuclear magnetic resonance ℃: Celsius degree Pd / C: Palladium on carbon Pd(OAc)2: Palladium acetate PBS: Phosphate-buffered saline PE: Petroleum ether POCl3: Phosphorus oxychloride PPh3: Triphenylphosphine PyBOP: (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate Rel: Relative RT or rt: room temperature s or sec: seconds sat: saturation SEMCl: Chloromethyl-2-trimethylsilyl ethyl ether SFC: Supercritical Fluid Chromatography SOCl2: Sulfur dichloride tBuOK: Potassium tert-butoxide TBAB: Tetrabutylammonium bromide TBAF: Tetrabutylammonium fluoride TBAI: Tetrabutylammonium iodide TEA: Triethylamine Tf: Trifluoromethanesulfonate TfAA, TFMSA or Tf2O: Trifluoromethanesulfonic anhydride TFA: Trifluoroacetic acid TIBSCl: 2,4,6-triisopropylbenzenesulfonyl chloride TIPS: Triisopropylsilyl THF: tetrahydrofuran THP: Tetrahydropyran TLC: Thin Layer Chromatography TMEDA: Tetramethylethylenediamine pTSA: paratoluenesulfonic acid UPLC: Ultra-high performance liquid chromatography wt: weight Xantphos: 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene

[0343] General synthesis method The following examples are intended to illustrate the present disclosure and should not be construed as limiting the present disclosure. Temperatures are given in degrees Celsius (C). Unless otherwise stated, all evaporations are carried out under reduced pressure, preferably at about 15 mmHg to 100 mmHg (=20 to 133 mbar). The structures of final products, intermediates and starting materials were confirmed by standard analytical methods, e.g., microanalysis and spectroscopic characteristics, e.g., MS, IR, NMR. The abbreviations used are those commonly used in the art.

[0344] All starting materials, building blocks, reagents, acids, bases, dehydrating agents, solvents, and catalysts utilized in the synthesis of the nucleic acids or analogs thereof of the present disclosure are commercially available or can be produced by organic synthesis methods known to those skilled in the art (METHODS OF ORGANIC SYNTHESIS, Thieme, Volume 21 (Houben-Weyl 4th Ed. 1952)). In addition, the nucleic acids or analogs thereof of the present disclosure can be produced by organic synthesis methods known to those skilled in the art, as shown in the following examples.

[0345] Unless otherwise specified, all reactions are carried out under nitrogen or argon.

[0346] Proton NMR ( 11 H NMR) was performed in a deuterated solvent. Certain nucleic acids or analogs thereof disclosed herein include one or more 1 The H shifts overlap with residual proteosolvent signals, which are not reported in the experiments presented below.

[0347] As shown in the Examples below, in certain exemplary embodiments, nucleic acids or analogs thereof were prepared according to the following general procedures: While the general methods are shown to synthesize specific nucleic acids or analogs thereof of the present disclosure, it will be understood that the following general methods, as well as other methods known to those of skill in the art, can be applied to all nucleic acids or analogs thereof, as well as each subclass and species of these nucleic acids or analogs thereof, as described herein.

[0348] A general method for preparing double-stranded RNAi oligonucleotides Oligonucleotide synthesis and purification The double-stranded RNAi (dsRNA) oligonucleotides described in the Examples below are chemically synthesized using methods described herein. Generally, dsRNAi oligonucleotides are synthesized using solid-phase oligonucleotide synthesis methods described for 19-23 mer siRNAs (see, e.g., Scaringe et al. (1990) NUCLEIC ACIDS 2001; Hughes and Ellington (2017) COLD SPRING HARB PERSPECT BIOL. 9(1):a023812; Beaucage SL, Caruthers MHSTUDIES ON NUCLEOTIDE CHEMISTRY V: Deoxynucleoside Phosphoramidites-A New Class of Key Intermediates for Deoxypolynucleotide Synthesis. TETRAHEDRON LETT. (1981); 22:1859-1862. doi:10.1016 / S0040-4039(01)90461-7), in addition to using known phosphoramidite synthesis. RES. 18:5433-5441, and Usman et al. (1987) J. AM. CHEM. Soc. 109:7845-7845, see also U.S. Patent Nos. 5,804,683, 5,831,071, 5,998,203, 6,008,400, 6,111,086, 6,117,657, 6,353,098, 6,362,323, 6,437,117, and 6,469,158).

[0349] Individual RNA strands were synthesized and HPLC-purified according to standard methods (Integrated DNA Technologies; Coralville, IA). For example, RNA oligonucleotides were synthesized using solid-phase phosphoramidite chemistry using standard techniques, deprotected and desalted on a NAP-5 column (Amersham Pharmacia Biotech, Piscataway, NJ) (Damha & Olgivie (1993) METHODS MOL. BIOL. 20:81-114; Wincott et al. (1995) NUCLEIC ACIDS RES. 23:2677-2684). Oligomers were purified using ion-exchange high-performance liquid chromatography (IE-HPLC) on an Amersham Source15Q column (1.0 cm x 25 cm, Amersham Pharmacia Biotech) using a 15-minute linear step gradient. The gradient varied from 90:10 buffer A:B to 52:48 buffer A:B, where buffer A was 100 mM Tris pH 8.5 and buffer B was 100 mM Tris pH 8.5, 1 M NaCl. Samples were monitored at 260 nm and peaks corresponding to full-length oligonucleotide species were collected, pooled, desalted on a NAP-5 column and lyophilized to dryness.

[0350] The purity of each oligomer was determined by capillary electrophoresis (CE) on a Beckman PACE5000 (Beckman Coulter, Inc., Fullerton, CA). The CE capillary had an inner diameter of 100 μm and contained ssDNA100R Gel (Beckman-Coulter). Typically, approximately 0.6 nmole of oligonucleotide was injected into the capillary, run at an electric field of 444 V / cm, and detected by UV absorbance at 260 nm. Denaturing Tris-borate-7M-urea running buffer was purchased from Beckman-Coulter. Oligoribonucleotides that were at least 90% pure as assessed by CE for use in the experiments described below were obtained. Compound identity was confirmed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry on a Voyager DE™ Biospectometry Work Station (Applied Biosystems; Foster City, CA) following the manufacturer's recommended protocol. The relative molecular masses of all oligomers were obtained, in most cases within 0.2% of the predicted molecular mass.

[0351] Preparation of double strands The single-stranded RNA oligomers were resuspended (e.g., at a concentration of 100 μM) in a duplex buffer consisting of 100 mM potassium acetate, 30 mM HEPES, pH 7.5. The complementary sense and antisense strands were mixed in equimolar amounts to obtain a final solution of, e.g., 50 μM duplex. The samples were heated at 100° C. for 5 minutes in RNA buffer (IDT) and allowed to cool to room temperature before use. The dsRNA oligonucleotides were stored at −20° C. The single-stranded RNA oligomers were stored lyophilized or in nuclease-free water at −80° C.

[0352] Example 1: Synthesis of 2-(2-((((6aR,8R,9R,9aR)-8-(6-benzamido-9H-purin-9-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-fluoro[3,2-f][1,3,5,2,4]trioxadisilosin-9-yl)oxy)methoxy)ethoxy)ethane-1-ammonium formate (1-6) [ka] A solution of compound 1-1 (25.00 g, 67.38 mmol) in 20 mL of DMF was treated with pyridine (11 mL, 134.67 mmol) and tetraisopropyldisiloxane dichloride (22.63 mL, 70.75 mmol) at 10° C. The resulting mixture was stirred at 25° C. for 3 h and quenched with 20% citric acid (50 mL). The aqueous layer was extracted with EtOAc (3×50 mL) and the combined organic layers were concentrated in vacuo. The crude residue was recrystallized from a mixture of MTBE and n-heptane (1:15, 320 mL) to give compound 1-2 (37.20 g, 90%) as a white oily solid.

[0353] A solution of compound 1-2 (37.00 g, 60.33 mmol) in 20 mL of DMSO was treated with AcOH (20 mL, 317.20 mmol) and Ac2O (15 mL, 156.68 mmol). The mixture was stirred at 25° C. for 15 h. The reaction was diluted with EtOAc (100 mL) and quenched with saturated K2CO3 (50 mL). The aqueous layer was extracted with EtOAc (3×50 mL). The combined organic layers were concentrated and recrystallized with ACN (30 mL) to give compound 1-3 (15.65 g, 38.4%) as a white solid.

[0354] A solution of compound 1-3 (20.00 g, 29.72 mmol) in 120 mL of DCM was treated with Fmoc-amino-ethoxyethanol (11.67 g, 35.66 mmol) at 25° C. The mixture was stirred to obtain a clear solution and then treated with 4 Å molecular sieves (20.0 g), N-iodosuccinimide (8.02 g, 35.66 mmol), and TfOH (5.25 mL, 59.44 mmol). The mixture was stirred at 30° C. until HPLC analysis showed >95% consumption of compound 1-3. The reaction was quenched with TEA (6 mL) and filtered. The filtrate was diluted with EtOAc, washed with saturated NaHCO3 (2X100 mL), saturated Na2SO3 (2X100 mL), and water (2X100 mL), and concentrated in vacuo to give crude compound 1-4 (26.34 g, 93.9%) as a yellow solid, which was used directly in the next step without further purification.

[0355] A solution of compound 1-4 (26.34 g, 27.62 mmol) in a mixture of DCM / water (10:7, 170 mL) was treated with DBU (7.00 mL, 45.08 mmol) at 5 °C. The mixture was stirred at 5-25 °C for 1 h. The organic layer was then separated, washed with water (100 mL) and diluted with DCM (130 mL). The solution was treated with fumaric acid (7.05 g, 60.76 mmol) and 4 Å molecular sieves (26.34 g) in four portions. The mixture was stirred for 1 h, concentrated and recrystallized from a mixture of MTBE and DCM (5:1) to give compound 1-6 (14.74 g, 62.9%) as a white solid. 1 H NMR(400MHz,d6-DMSO)8.73(s,1H), 8.58(s,1H), 8.15-8.02(m,2H), 7.65-7.60(m,1H), 7.59-7.51(m,2H), 6.52(s,2H), 6.15 (s,1H), 5.08-4.90(m,3H), 4.83-4.78(m,1H), 4.15-3.90(m,3H), 3.79-3.65(m,2H), 2.98-2.85(m,6H), 1.20-0.95(m,28H).

[0356] Example 2: Synthesis of (2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((2-(2-[lipid]-amidoethoxy)ethoxy)methoxy)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite (2-4a to 2-4e) [ka] A solution of compound 1-6 (50.00 g, 59.01 mmol) in 150 mL of 2-methyltetrahydrofuran was washed with ice-cold aqueous K2HPO4 (6%, 100 mL) and brine (20%, 2X100 mL). The organic layer was separated and treated with hexanoic acid (10.33 mL, 82.61 mmol), HATU (33.66 g, 88.52 mmol), and DMAP (10.81 g, 147.52 mmol) at 0°C. The resulting mixture was warmed to 25°C and stirred for 1 h. The solution was washed with water (2x100 mL), brine (100 mL), and concentrated in vacuo to give the crude residue. Flash chromatography on silica gel (1:1 hexane / acetone) afforded compound 2-1a (34.95 g, 71.5%) as a white solid.

[0357] A mixture of compound 2-1a (34.95 g, 42.19 mmol) and TEA (9.28 mL, 126.58 mmol) in 80 mL...

Claims

1. 1. An oligonucleotide-ligand conjugate for reducing expression of a target mRNA in the central nervous system (CNS), comprising: (i) a double-stranded oligonucleotide comprising an antisense strand 15-30 nucleotides in length and a sense strand 15-40 nucleotides in length, the antisense strand and the sense strand each comprising a 5' end and a 3' end, the antisense strand and the sense strand forming a double-stranded region, the antisense strand having a region of complementarity to a target sequence of the target mRNA in the CNS, the region of complementarity being at least 15 contiguous nucleotides in length, and the oligonucleotide comprising a stem loop; (ii) one or more lipid moieties conjugated to said stem loop.

2. 2. The oligonucleotide-ligand conjugate of claim 1, wherein said sense strand comprises said stem loop at its 3' end, said stem loop comprising a nucleotide sequence represented by the formula: 5'-S1-L-S2-3', where S1 is complementary to S2, L forms a loop between S1 and S2, S1 and S2 are 1-8 nucleotides in length, and L is 3-5 nucleotides in length.

3. The oligonucleotide-ligand conjugate of claim 2, wherein the one or more lipid moieties are conjugated to the S1 or S2 nucleotide.

4. 3. The oligonucleotide-ligand conjugate of claim 2, wherein L is a tetraloop, optionally L is 4 nucleotides in length, L comprises a nucleotide sequence shown as 5'-GAAA-3', and said one or more lipid moieties are conjugated to the nucleotides of L. (i) L is three nucleotides in length and has, from 5' to 3', a first, second, and third nucleotide, and the oligonucleotide-ligand conjugate comprises a lipid moiety, the lipid moiety being conjugated to the first, second, or third nucleotide of L; or (ii) the oligonucleotide-ligand conjugate of claim 2, wherein L is 4 nucleotides in length and has, from 5' to 3', a first, a second, a third, and a fourth nucleotide, said oligonucleotide-ligand conjugate comprises one lipid moiety, said lipid moiety being conjugated to said first, second, third, or fourth nucleotide of L; and L consists of 5'-GAAA-3' and said lipid moiety being conjugated to said second nucleotide of L. (i) the antisense strand is 19-27 nucleotides in length, optionally the antisense strand is 22 nucleotides in length; and (ii) the sense strand is 19-40 nucleotides in length, optionally wherein the sense strand is 36 nucleotides in length, and the double-stranded region is at least 19 nucleotides in length, optionally wherein the double-stranded region is 20 nucleotides in length.

7. The oligonucleotide-ligand conjugate of claim 1, wherein the oligonucleotide comprises a 3' overhang sequence, the 3' overhang sequence being on the antisense strand, and the 3' overhang sequence being 2 nucleotides in length.

8. The method of claim 7, wherein the oligonucleotide comprises at least one modified nucleotide, the modified nucleotide comprising a 2'-modification selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, 2'-deoxy-2'-fluoro, and 2'-deoxy-2'-fluoro-β-d-arabino; (ii) all nucleotides of the oligonucleotide are modified; (iii) the oligonucleotide comprises at least one modified internucleotide linkage, and the at least one modified internucleotide linkage is a phosphorothioate linkage; and (iv) the oligonucleotide-ligand conjugate of claim 1, wherein the 4'-carbon of the sugar of the 5'-nucleotide of the antisense strand comprises a phosphate analog, and the phosphate analog is an oxymethylphosphonate, vinylphosphonate, or malonylphosphonate.

9. the one or more lipid moieties comprise a saturated C8, C9, C10, C11, C12, C13, or C14 hydrocarbon chain; 2. The oligonucleotide-ligand conjugate of claim 1, wherein the oligonucleotide-ligand conjugate reduces expression of the target mRNA in the CNS without reducing expression of the target mRNA outside the CNS, the target mRNA being expressed in liver cells, and the oligonucleotide-ligand conjugate does not reduce expression of the target mRNA in liver cells to the same or similar levels as in cells of the CNS.

10. 2. The oligonucleotide-ligand conjugate of claim 1, wherein said lipid moiety comprises a saturated or unsaturated C16, C17, C18, C19, C20, C21, or C22 hydrocarbon chain, and optionally, said lipid moiety comprises C22:

6.

11. The target gene is selected from the group consisting of progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), argyrophilic granulomatosis (AGD), glioglobular tauopathy (GGT), age-related tauastrogliopathy (ARTAG), familial frontotemporal dementia 17 (FTD-17), tauopathy with respiratory failure, dementia with seizures, Pick's disease, myotonic dystrophy 1 or 2 (MD1 or MD2), Down's syndrome, spastic paraplegia (SP), and Niemann's syndrome. Pick's disease type C, dementia with Lewy bodies (DLB), Lewy body dysphagia, Lewy body disease, olivopontocerebellar atrophy, striatonigral degeneration, Shy-Drager syndrome, spinal muscular atrophy V (SMAV), Huntington's disease (HD), Alzheimer's disease, SCA1, SCA2, SCA3, SCA7, SCA10 (spinocerebellar ataxia type 1, 2, 3, 7, or 103), multiple system atrophy (MSA), spinal-bulbar muscular atrophy (SBMA, Kennedy disease), Friedreich's ataxia, Fragile X-associated tremor / ataxia syndrome (FXTAS), Fragile X syndrome (FRAXA), X-linked mental retardation (XLMR), Parkinson's disease, dystonia, SBMA (Spinal-bulbar muscular atrophy), neuropathic pain disorders, spinal cord injury, Dentatorubral-Pallidoluysian atrophy (DRPLA), recessive CNS disorders, and ALS (Amyotrophic Lateral Sclerosis), M2DS (MECP2 Duplication Syndrome), FTD (Frontotemporal Degeneration), and 2. The oligonucleotide-ligand conjugate of claim 1, wherein the oligonucleotide-ligand conjugate is associated with a disease or disorder, optionally a neurological disease or disorder, selected from: chronic temporal dementia, prion disease, adult-onset leukodystrophy, Alexander disease, Krabbe disease, chronic traumatic encephalopathy, Pelizaeus-Merzbach disease (PMD), Lafora disease, stroke, cerebral amyloid angiopathy (CAA), and metachromatic leukodystrophy (MLD).

12. 1. An oligonucleotide-ligand conjugate for reducing expression of a target mRNA in the CNS, comprising: (i) a double-stranded oligonucleotide comprising an antisense strand 15-30 nucleotides in length and a sense strand 15-40 nucleotides in length, the antisense strand and the sense strand each comprising a 5' end and a 3' end, the antisense strand and the sense strand forming a double-stranded region, the antisense strand having a region of complementarity to a target sequence of the target mRNA in the CNS, the region of complementarity being at least 15 contiguous nucleotides in length, and the oligonucleotide comprising a stem loop; (ii) one or more lipid moieties conjugated to said stem loop, said one or more lipid moieties being selected from a saturated or unsaturated C8-14 hydrocarbon chain; An oligonucleotide-ligand conjugate, wherein expression of the target mRNA in the liver is not reduced to the same or similar levels as in the CNS.

13. A pharmaceutical composition comprising the oligonucleotide-ligand conjugate of any one of claims 1 to 12 and a pharma- ceutically acceptable carrier.

14. 14. Use of an oligonucleotide-ligand conjugate according to any one of claims 1 to 12 or a pharmaceutical composition according to claim 13 for reducing or inhibiting expression of a target mRNA in a subject expressed by a cell population associated with the CNS of the subject, wherein the expression level of the target mRNA is reduced in the cell population associated with the CNS compared to a control cell population.

15. The use of claim 14, wherein the expression level of the target mRNA is not reduced in a cell population located outside the CNS compared to a control cell population, and the cell population located outside the CNS is in the liver.

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