Lipid conjugation for targeting oligodendrocytes of the central nervous system

Abstract

Oligonucleotide conjugates are provided herein that inhibit or reduce expression of target genes in the oligodendrocytes of the central nervous system. Also provided are compositions including the same and uses thereof, particularly uses relating to treating diseases, disorders and / or conditions associated with expression of an oligodendrocyte target gene in the CNS.

Description

[0001] LIPID CONJUGATION FOR TARGETING OLIGODENDROCYTES OF THE

[0002] CENTRAL NERVOUS SYSTEM

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 276,409 filed November 5, 2021, which is incorporated by reference herein in its entirety.

[0005] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0006] The contents of the electronic sequence listing (DICN_014_001WO_SeqList_ST26.xml; Size: 735,272 bytes; and Date of Creation: November 2, 2022) are herein incorporated by reference in its entirety.

[0007] TECHNICAL FIELD

[0008] The disclosure relates to oligonucleotides linked to lipid moieties useful in the inhibition of target genes in oligodendrocytes of the central nervous system. Specifically, the present disclosure relates to oligonucleotide-lipid conjugates, methods to prepare them, their chemical configuration, and methods to modulate (e.g., inhibit or reduce) the expression of a target gene in an oligodendrocyte of the central nervous system (abbreviated “CNS” hereinafter) e.g., tissue, or region of the CNS) using the conjugated nucleic acids and oligonucleotides according to the description provided herein. The disclosure also provides pharmaceutically acceptable compositions comprising the conjugates of the present description and methods of using said compositions in the treatment of various diseases or disorders.

[0009] BACKGROUND OF THE DISCLOSURE

[0010] Regulation of gene expression by modified nucleic acids shows great potential as both a research tool in the laboratory and a therapeutic approach in the clinic. Several classes of oligonucleotide or nucleic acid-based therapeutics have been under the clinical investigation, including antisense oligonucleotides (ASO), short interfering RNA (siRNA), double-stranded nucleic acids (dsNA), aptamers, ribozymes, exon-skipping and splice-altering oligonucleotides, immunomodulatory oligonucleotides, mRNAs, and CRISPR. Chemical modifications in the relevant molecules to allow functionality in various tissues, organs and / or cell types play a key role in overcoming challenges of oligonucleotide therapeutics, including improving nuclease stability, RNA-binding affinity, and pharmacokinetics. Various chemical modification strategies for oligonucleotides have been developed in the past three decades including modification of the sugars, nucleobases, and phosphodiester backbone to improve and optimize performance and therapeutic efficacy (Deleavey and Darma, CHEM. BIOL. 2012, 19(8):937-54; Wan and Seth, J. MED. CHEM. 2016, 59(21):9645-67; and Egli and Manoharan, Acc. CHEM. RES. 2019, 54(4): 1036-47).

[0011] Therapeutic gene silencing mediated by RNAi oligonucleotide-based therapeutics comprising siRNAs or double-stranded nucleic acids (dsNAs) offer the potential for considerable expansion of the druggable target space and the possibility for treating orphan diseases that may be therapeutically unapproachable by other drug modalities (e.g., antibodies and / or small molecules). RNAi oligonucleotide-based therapeutics that inhibit or reduce expression of specific target genes in the liver have been developed and are currently in clinical use (Sehgal et al., (2013) JOURNAL OF HEPATOLOGY 59: 1354-59). Technological hurdles remain for the development and clinical use of RNAi oligonucleotides in extrahepatic cells, tissues, and organs (e.g., the CNS). Therapeutic gene silencing mediated by RNAi oligonucleotide-based therapeutics in the CNS is of particular interest to treat neurological diseases (Boudreau & Davidson (2010) BRAIN RESEARCH 1338: 112-21). Thus, an ongoing need exists in the art for the successful development of new and effective RNAi oligonucleotides to modulate the expression of a target genes in extrahepatic cells, tissues, and / or organs (e.g., the CNS). This is complicated by the variant nature of the cell types in extrahepatic as well as concerns about circulatory patterns and cell membrane constituents such as receptor types.

[0012] BRIEF SUMMARY OF THE DISCLOSURE

[0013] The mammalian CNS is a complex system of tissues, including cells, fluids and chemicals that interact in concert to enable a wide variety of functions, including movement, navigation, cognition, speech, vision, and emotion. Unfortunately, a variety of diseases and disorders of the CNS are known (e.g., neurological disorders) and affect or disrupt some or all of these functions. Typically, treatments for diseases and disorders of the CNS have been limited to small molecule drugs, antibodies and / or to adaptive or behavioral therapies. There exists an ongoing need to develop treatment of diseases and disorders of the CNS associated with inappropriate gene expression.

[0014] The present disclosure is based, at least in part, on the discovery of lipid-conjugated RNAi oligonucleotides that effectively reduce target gene expression in oligodendrocytes of the CNS. Exemplary lipid-conjugated RNAi oligonucleotides provided herein have demonstrated reduction of target gene expression of oligodendrocyte-specific mRNA in the CNS following a single administration. Further, exemplary lipid-conjugated RNAi oligonucleotides provided herein have demonstrated pharmacological activity in multiple regions throughout the CNS, including difficult to reach areas such as the hippocampus and frontal cortex. Without being bound by theory, the hydrophobic moiety (e.g., lipid) facilitates delivery and distribution of the lipid-conjugated RNAi oligonucleotides into the CNS, thereby increasing efficacy and durability of gene knockdown in oligodendrocytes. Accordingly, the disclosure provides methods of treating a disease or disorder by modulating expression of an oligodendrocyte gene in the CNS using the lipid-conjugated RNAi oligonucleotides, and pharmaceutically acceptable compositions thereof, described herein. The disclosure further provides methods of using the lipid-conjugated RNAi oligonucleotides in the manufacture of a medicament for treating a disease or disorder by modulating expression of an oligodendrocyte gene in the CNS.

[0015] Accordingly, in some aspects, the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 15-30 nucleotides in length and a sense strand of 15-50 nucleotides in length, wherein the antisense and sense strands form a duplex region of 15-30 base pairs, wherein the antisense strand comprises a region of complementarity to an oligodendrocyte mRNA target sequence, and wherein the sense strand comprises at least one lipid moiety conjugated to a nucleotide of the sense strand.

[0016] In some aspects, the lipid moiety is selected from

[0017] In some aspects, the lipid moiety is a hydrocarbon chain. In some aspects, the hydrocarbon chain is a C8-C30 hydrocarbon chain. In some aspects, the hydrocarbon chain is a C16 hydrocarbon chain. In some aspects, the C16 hydrocarbon chain is represented by In any of the foregoing or related aspects, the lipid moiety is conjugated to the 2' carbon of the ribose ring of the nucleotide.

[0018] In any of the foregoing or related aspects, the oligonucleotide is blunt ended. In some aspects, the oligonucleotide is blunt ended at the 3' terminus of the oligonucleotide. In some aspects, the oligonucleotide comprises a blunt end. In some aspects, the blunt end comprises the 3’ terminus of the sense strand. In some aspects, the sense strand is 20-22 nucleotides. In some aspects, the at least one lipid moiety is conjugated to a nucleotide at position 1, position 4, position 8, position 12, position 13, position 18, or position 20 of the sense strand, wherein positions are numbered 5’ to 3’. In some aspects, the oligodendrocyte mRNA target is expressed in the spinal cord, wherein the at least one lipid moiety is conjugated to a nucleotide at position 2, position 3, position 6, position 13, position 14, position 15, position 19 or position 20 of the sense strand, and wherein positions are numbered 5’ to 3’. In some aspects, the oligodendrocyte mRNA target is expressed in the spinal cord, wherein the at least one lipid moiety is conjugated to a nucleotide at position 1, position 2, position 3, position 5, position 6, position 7, position 9, position 13, position 14, position 15, position 17, position 19, or position 20 of the sense strand, and wherein positions are numbered 5’ to 3’. In other aspects, the oligodendrocyte mRNA target is expressed in the medulla, wherein the at least one lipid moiety is conjugated to a nucleotide at position 2, position 14 or position 15 of the sense strand, and wherein positions are numbered 5’ to 3’. In other aspects, the oligodendrocyte mRNA target is expressed in the medulla, wherein the at least one lipid moiety is conjugated to a nucleotide at position 2, position 7, position 9, position 14, position 15, or position 19 of the sense strand, and wherein positions are numbered 5’ to 3’. In other aspects, the oligodendrocyte mRNA target is expressed in the brain stem, wherein the at least one lipid moiety is conjugated to a nucleotide at position 2, position 7, position 9, position 14, position 15, or position 19 of the sense strand, and wherein positions are numbered 5’ to 3’. In further aspects, the oligodendrocyte mRNA target is expressed in the hippocampus, wherein the at least one lipid moiety is conjugated to a nucleotide at position 3 of the sense strand, and wherein positions are numbered 5’ to 3’. In some aspects, the oligodendrocyte mRNA target is expressed in the frontal cortex, wherein the at least one lipid moiety is conjugated to a nucleotide at position 14 of the sense strand, and wherein positions are numbered 5’ to 3’. In some aspects, the oligodendrocyte mRNA target is expressed in the hypothalamus, wherein the at least one lipid moiety is conjugated to a nucleotide at position 7 of the sense strand, and wherein positions are numbered 5’ to 3’.

[0019] In any of the foregoing or related aspects, the sense strand comprises a stem-loop at the 3 ’end, wherein the stem-loop comprises a nucleotide sequence represented by the formula: 5’-Sl-L-S2-3’, wherein SI is complementary to S2, and wherein L forms a loop between SI and S2. In some aspects, the sense strand is 36-38 nucleotides. In some aspects, the at least one lipid moiety is conjugated to a nucleotide of the stem-loop or a nucleotide proximal to the stem-loop. In some aspects, the nucleotide proximal to the stem-loop is located 1-3 nucleotides from the 5’ end of the stem-loop. In some aspects, the oligodendrocyte mRNA target is expressed in the spinal cord, wherein the at least one lipid moiety is conjugated to a nucleotide at position 2, position 3, position 6, position 13, position 14, position 15, position 19, position 20, position 23, position 28, position 29 or position 30 of the sense strand, and wherein positions are numbered 5’ to 3’. In other aspects, the oligodendrocyte mRNA target is expressed in the medulla, wherein the at least one lipid moiety is conjugated to a nucleotide at position 19, position 20, position 23 or position 28 of the sense strand, and wherein positions are numbered 5’ to 3’. In further aspects, the oligodendrocyte mRNA target is expressed in the hippocampus, wherein the at least one lipid moiety is conjugated to a nucleotide at position 2 of the sense strand, and wherein positions are numbered 5’ to 3’. In some aspects, the oligodendrocyte mRNA target is expressed in the frontal cortex, wherein the at least one lipid moiety is conjugated to a nucleotide at position 14, position 15, position 19, position 20, position 23, position 28, position 29 or position 30 of the sense strand, and wherein positions are numbered 5’ to 3’.

[0020] In any of the foregoing or related aspects, the antisense strand is 22-24 nucleotides. In some aspects, the duplex region is 20-22 base pairs.

[0021] In any of the foregoing or related aspects, the antisense strand comprises a 1-4 nucleotide overhang at the 3' terminus. In some aspects, the overhang comprises purine nucleotides. In some aspects, the overhang sequence is 2 nucleotides in length. In some aspects, the overhang is selected from AA, GG, AG, and GA. In some aspects, the overhang is GG or AA. In some aspects, the overhang is GG.

[0022] In any of the foregoing or related aspects, the region of complementarity is complementary to at least 15 consecutive nucleotides of the oligodendrocyte mRNA target sequence. In some aspects, the region of complementarity is complementary to 19 consecutive nucleotides of the oligodendrocyte mRNA target sequence. In some aspects, the region of complementarity is fully complementary to the oligodendrocyte mRNA target sequence. In some aspects, the region of complementarity is partially complementary to the oligodendrocyte mRNA target sequence. In some aspects, the region of complementarity comprises no more than four mismatches to the oligodendrocyte mRNA target sequence. In some aspects, the region of complementarity comprises up to four mismatches to the oligodendrocyte mRNA target sequence.

[0023] In any of the foregoing or related aspects, the oligonucleotide comprises at least one modified nucleotide. In some aspects, the modified nucleotide comprises a 2'- modification. In some aspects, each of the nucleotides of the sense strand and the antisense strand comprise a 2'-modification except the nucleotide of the sense strand conjugated to the at least one lipid moiety. In some aspects, the 2 '-modification is a modification selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid. In some aspects, about 10-20%, 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 aspects, 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 aspects, about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35% of the nucleotides of the oligonucleotide comprise a 2'-fluoro modification. In some aspects, the sense strand comprises 20 nucleotides with positions 1-20 from 5' to 3', wherein each of positions 8-11 comprise a 2'-fluoro modification. In some aspects, the sense strand comprises 20 nucleotides with positions 1-20 from 5' to 3', wherein each of positions 9-11 comprise a 2'-fluoro modification. In some aspects, the sense strand comprises 36 nucleotides with positions 1-36 from 5' to 3', wherein each of positions 8-11 comprise a 2'-fluoro modification. In some aspects, the sense strand comprises 36 nucleotides with positions 1-36 from 5' to 3', wherein each of positions 9-11 comprise a 2'-fluoro modification. In some aspects, the antisense strand comprises 22 nucleotides with positions 1-22 from 5' to 3', and wherein each of positions 2, 3, 4, 5, 7, 10 and 14 comprise a 2'-fluoro modification. In some aspects, the remaining nucleotides comprise a 2'-O-methyl modification except the nucleotide of the sense strand conjugated to the at least one lipid moiety.

[0024] In any of the foregoing or related aspects, the oligonucleotide comprises at least one modified internucleotide linkage. In some aspects, the at least one modified internucleotide linkage is a phosphorothioate linkage. In some aspects, the antisense strand comprises a phosphorothioate linkage (i) between positions 1 and 2, and between positions 2 and 3; or (ii) between positions 1 and 2, between positions 2 and 3, and between positions 3 and 4, wherein positions are numbered 1-4 from 5’ to 3’. In some aspects, the antisense strand is 22 nucleotides in length, and wherein the antisense strand comprises a phosphorothioate linkage between positions 20 and 21 and between positions

[0025] 21 and 22, wherein positions are numbered 1-22 from 5' to 3'. In some aspects, the sense strand comprises a phosphorothioate linkage between position 1 and 2, wherein positions are numbered 1-2 from 5' to 3'. In some aspects, the sense strand is 20 nucleotides in length, and wherein the sense strand comprises a phosphorothioate linkage between positions 18 and 19, and between positions 19 and 20, wherein positions are numbered 1-

[0026] 22 from 5' to 3'.

[0027] In any of the foregoing or related aspects, the antisense strand comprises a phosphorylated nucleotide at the 5' terminus, wherein the phosphorylated nucleotide is selected from uridine and adenosine. In some aspects, the phosphorylated nucleotide is uridine. In some aspects, the 4'-carbon of the sugar of the 5 '-nucleotide of the antisense strand comprises a phosphate analog. In some aspects, the phosphate analog is oxymethyl phosphonate, vinyl phosphonate or malonyl phosphonate.

[0028] In any of the foregoing or related aspects, the region of complementary is fully complementary to the oligodendrocyte mRNA target sequence at nucleotide positions 2-8 of the antisense strand, wherein nucleotide positions are numbered 5' to 3'. In some aspects, the region of complementary is fully complementary to the oligodendrocyte mRNA target sequence at nucleotide positions 2-11 of the antisense strand, wherein nucleotide positions are numbered 5' to 3'.

[0029] In any of the foregoing or related aspects, the oligonucleotide is a Dicer substrate. In some aspects, the oligonucleotide is a Dicer substrate that, upon endogenous Dicer processing, yields double-stranded nucleic acids of 19-21 nucleotides in length capable of reducing a oligodendrocyte mRNA expression in a mammalian cell.

[0030] In any of the foregoing or related aspects, the oligodendrocyte mRNA target sequence is located in a region of the central nervous system (CNS). In some aspects, the region of the CNS is selected from the frontal cortex, spinal cord, lumbar spinal cord, cervical spinal cord, thoracic spinal cord, medulla, cerebellum, hypothalamus, hippocampus, and a combination thereof. In any of the foregoing or related aspects, the oligonucleotide reduces expression of a target mRNA in an oligodendrocyte or population of oligodendrocytes in vitro and / or in vivo. In some aspects, the disclosure provides a pharmaceutical composition comprising an oligonucleotide described herein, and a pharmaceutically acceptable carrier, delivery agent or excipient.

[0031] In other aspects, the disclosure provides a method for treating a subject having a disease, disorder or condition associated with expression of an oligodendrocyte mRNA, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide or pharmaceutical composition described herein, thereby treating the subject.

[0032] In further aspects, the disclosure provides a method of delivering an oligonucleotide to an oligodendrocyte or a population of oligodendrocytes in a subject, the method comprising administering a pharmaceutical composition described herein to the subject. In some aspects, the oligodendrocyte or a population of oligodendrocytes is located in a region of the CNS. In some aspects, the region of the CNS is selected from the frontal cortex, spinal cord, lumbar spinal cord, cervical spinal cord, thoracic spinal cord, medulla, cerebellum, hypothalamus, hippocampus, and a combination thereof.

[0033] In yet further aspects, the disclosure provides a method for reducing expression of an oligodendrocyte mRNA in a cell, a population of cells or a subject, the method comprising the step of: i. contacting the cell or the population of cells with an oligonucleotide or pharmaceutical composition described herein, optionally wherein the cell or population of cells is an oligodendrocyte or a population of oligodendrocytes; or ii. administering to the subject an oligonucleotide or pharmaceutical composition described herein. In some aspects, reducing expression of the oligodendrocyte mRNA comprises reducing an amount or level of mRNA, an amount or level of protein, or both. In some aspects, the subject has a disease, disorder or condition associated with expression of the oligodendrocyte mRNA. In some aspects, the cell or population of cells is located in a region of the CNS. In some aspects, the region of the CNS is selected from the frontal cortex, spinal cord, lumbar spinal cord, cervical spinal cord, thoracic spinal cord, medulla, cerebellum, hypothalamus, hippocampus, and a combination thereof.

[0034] In any of the foregoing or related aspects, the methods comprise administering via intrathecal administration. In some aspects, the disclosure provides a method of reducing expression of a target mRNA expressed in an oligodendrocyte in a tissue of the CNS of a subject, comprising administering to the subject a double-stranded oligonucleotide comprising an antisense strand of 15-30 nucleotides in length and a sense strand of 15-50 nucleotides in length, wherein the antisense and sense strands form a duplex region of 15-30 base pairs, wherein the antisense strand comprises a region of complementarity to a target sequence in the target mRNA, and wherein the sense strand comprises at least one lipid moiety conjugated to a nucleotide of the sense strand. In some aspects, the lipid moiety is a C16 hydrocarbon.

[0035] In any of the foregoing or related aspects of the methods described herein, the oligonucleotide is blunt ended at the 3' terminus of the oligonucleotide. In some aspects, the oligonucleotide comprises a blunt end. In some aspects, the blunt end comprises the 3 ’ terminus of the sense strand. In some aspects, the sense strand is 22-24 nucleotides. In some aspects, the tissue is the spinal cord, wherein the at least one lipid moiety is conjugated to a nucleotide at position 2, position 3, position 6, position 13, position 14, position 15, position 19 or position 20 of the sense strand, and wherein positions are numbered 5’ to 3’. In other aspects, the tissue is the spinal cord, wherein the at least one lipid moiety is conjugated to a nucleotide at position 1, position 2, position 3, position 5, position 6, position 7, position 9, position 13, position 14, position 15, position 17, position 19, or position 20 of the sense strand, and wherein positions are numbered 5’ to 3’. In other aspects, the tissue is the medulla, wherein the at least one lipid moiety is conjugated to a nucleotide at position 2, position 14 or position 15 of the sense strand, and wherein positions are numbered 5’ to 3’. In other aspects, the tissue is the medulla, wherein the at least one lipid moiety is conjugated to a nucleotide at position 2, position 7, position 14, position 15, or position 19 of the sense strand, and wherein positions are numbered 5’ to 3’. In other aspects, the tissue is the brain stem, wherein the at least one lipid moiety is conjugated to a nucleotide at position 2, position 7, position 14, position 15, or position 19 of the sense strand, and wherein positions are numbered 5’ to 3’. In further aspects, the tissue is the hippocampus, wherein the at least one lipid moiety is conjugated to a nucleotide at position 3 of the sense strand, and wherein positions are numbered 5’ to 3’. In some aspects, the tissue is the frontal cortex, wherein the at least one lipid moiety is conjugated to a nucleotide at position 14 of the sense strand, and wherein positions are numbered 5’ to 3’. In some aspects, the tissue is the hypothalamus, wherein the at least one lipid moiety is conjugated to a nucleotide at position 7 of the sense strand, and wherein positions are numbered 5’ to 3’.

[0036] In any of the foregoing or related aspects of the methods described herein, the sense strand comprises a stem-loop at the 3 ’end, wherein the stem-loop comprises a nucleotide sequence represented by the formula: 5’-Sl-L-S2-3’, wherein SI is complementary to S2, and wherein L forms a loop between SI and S2. In some aspects, the sense strand is 36-38 nucleotides. In some aspects, the at least one lipid moiety is conjugated to a nucleotide of the stem-loop or a nucleotide proximal to the stem-loop. In some aspects, the nucleotide proximal to the stem-loop is located 1-3 nucleotides from the 5’ end of the stem-loop. In some aspects, the tissue is the spinal cord, wherein the at least one lipid moiety is conjugated to a nucleotide at position 2, position 3, position 6, position 13, position 14, position 15, position 19, position 20, position 23, position 28, position 29 or position 30 of the sense strand, and wherein positions are numbered 5’ to 3’. In other aspects, the tissue is the medulla, wherein the at least one lipid moiety is conjugated to a nucleotide at position 19, position 20, position 23 or position 28 of the sense strand, and wherein positions are numbered 5’ to 3’. In further aspects, the tissue is the hippocampus, wherein the at least one lipid moiety is conjugated to a nucleotide at position 2 of the sense strand, and wherein positions are numbered 5’ to 3’. In some aspects, the tissue is the frontal cortex, wherein the at least one lipid moiety is conjugated to a nucleotide at position 14, position 15, position 19, position 20, position 23, position 28, position 29 or position 30 of the sense strand, and wherein positions are numbered 5’ to 3’.

[0037] In other aspects, the disclosure provides a kit comprising an oligonucleotide described herein, an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administration to a subject having a disease, disorder or condition associated with expression of an oligodendrocyte mRNA. In some aspects, the package insert comprises instructions for intrathecal administration.

[0038] In further aspects, the disclosure provides use of an oligonucleotide or pharmaceutical composition described herein, in the manufacture of a medicament for the treatment of a disease, disorder or condition associated with expression of an oligodendrocyte mRNA.

[0039] In some aspects, the disclosure provides an oligonucleotide or pharmaceutical composition described herein for use, or adaptable for use, in the treatment of a disease, disorder or condition associated with expression of an oligodendrocyte mRNA.

[0040] BRIEF DESCRIPTION OF FIGURES

[0041] FIG. 1 provides a graph depicting the concentration response of GalN Ac-conjugated UGT8 oligonucleotides. The percent (%) of human UGT8 mRNA remaining in the liver of mice exogenously expressing human UGT8 (hydrodynamic injection model) after treatment with GalN Ac-conjugated UGT8 oligonucleotides (Table 2) at two concentrations (0.3 mg / kg and 1 mg / kg) was measured. Three days following administration, mice were hydrodynamically injected (HDI) with a DNA plasmid encoding human UGT8. The level of human UGT8 mRNA was determined from livers collected 18 hours later. The x-axis indicates the oligonucleotide administered and the concentration (e.g., administration of UGT8-277 at 0.3 mg / kg is shown as “277-0.3”).

[0042] FIGs. 2A-2F provide graphs depicting the concentration response of a UGT8 oligonucleotide having a 2’0Me tetraloop in the central nervous system (CNS). The percent (%) of murine UGT8 mRNA remaining in lumbar spinal cord (FIG. 2A), cervical spinal cord (FIG. 2B), brainstem (FIG. 2C), cerebellum (FIG. 2D), hypothalamus (FIG. 2E), and frontal cortex (FIG. 2F) was determined. Mice were treated with 10, 32, 100, 320, or 500 pg of UGT8-277 oligonucleotide formulated in artificial cerebrospinal fluid (aCSF) via intrathecal (i.t.) injection was measured. Seven (7) days following intrathecal injection, the level of UGT8 mRNA was measured and normalized to Ribosomal Protein L23 (RPL23) mRNA and overall expression was determined between tissue types relative to control mice treated with (aCSF). ‘GalXC-UGT8’ refers to UGT8-277 oligonucleotide having SEQ ID NO: 56 (sense strand) and SEQ ID NO: 46 (antisense strand).

[0043] FIG. 3 provides a graph depicting the percent (%) murine UGT8 mRNA remaining in different tissues of the central nervous system (CNS) based on the results in FIGs. 2A-2F.

[0044] FIGs. 4A-4D provide graphs depicting the percent (%) murine UGT8 mRNA remaining in lumbar spinal cord (FIG. 4A), medulla (FIG. 4B), hippocampus (FIG. 4C), and frontal cortex (FIG. 4D) of mice after treatment with lipid-conjugated UGT8 tetraloop oligonucleotides. Mice were treated with 300 pg of the indicated UGT8 lipid-conjugated tetraloop oligonucleotides in Table 3 formulated in artificial cerebrospinal fluid (aCSF) via intrathecal (i.t.) injection into the lumbar spine. Seven (7) days following intrathecal injection, the level of UGT8 mRNA was normalized to Ribosomal Protein L23 (RPL23) mRNA and overall expression was determined between tissue types relative to control mice treated with (aCSF).

[0045] FIG. 5 provides schematics of UGT8 lipid-conjugated blunt-end oligonucleotides having the structures of the compounds in Table 4. The nucleotide position on the sense strand having a conjugated lipid is indicated as “P#”, e.g., a lipid conjugated at position 2 of the sense strand from 5’ to 3’ is indicated as “P2”.

[0046] FIGs. 6A-6D provide graphs depicting the percent (%) murine UGT8 mRNA remaining in lumbar spinal cord (FIG. 6A), medulla (FIG. 6B), hippocampus (FIG. 6C), and frontal cortex (FIG. 6D) of mice after treatment with the lipid-conjugated UGT8 blunt-end oligonucleotides of FIG. 5. Mice were treated with 300 pg of the indicated UGT8 lipid- conjugated blunt-end oligonucleotides in Table 4 formulated in artificial cerebrospinal fluid (aCSF) via intrathecal injection into the lumbar spine. Seven (7) days following intrathecal injection, the level of UGT8 mRNA was normalized to Ribosomal Protein L23 (RPL23) mRNA and overall expression was determined between tissue types relative to control mice treated with (aCSF).

[0047] FIGs. 7A-7B provide schematics of UGT8 lipid-conjugated blunt-end oligonucleotides having the structures of the compounds in Table 5. The nucleotide position on the sense strand having a conjugated lipid is indicated as “P#”, e.g., a lipid conjugated at position 1 of the sense strand from 5’ to 3’ is indicated as “Pl”.

[0048] FIGs. 8A-8E provide graphs depicting the percent (%) murine UGT8 mRNA remaining in frontal cortex (FIG. 8A), hippocampus (FIG. 8B), hypothalamus (FIG. 8C), brainstem (FIG. 8D), and lumbar spinal cord (FIG. 8E) of mice after treatment with lipid- conjugated UGT8 blunt-end oligonucleotides. Mice were treated with 300 pg of the indicated UGT8 lipid-conjugated blunt-end oligonucleotides in Table 5 formulated in artificial cerebrospinal fluid (aCSF) via intrathecal injection into the lumbar spine. Twenty-eight (28) days following intrathecal injection, the level of UGT8 mRNA was normalized to Ribosomal Protein L23 (RPL23) mRNA and overall expression was determined between tissue types relative to control mice treated with (aCSF).

[0049] DETAILED DESCRIPTION

[0050] In some aspects, the disclosure provides oligonucleotide-lipid conjugates (e.g., RNAi oligonucleotide-lipid conjugates) that reduce expression of a target gene expressed in oligodendrocytes in the central nervous system (CNS). In other aspects, the disclosure provides methods of treating a disease or disorder associated with expression of an oligodendrocyte mRNA (e.g., a disease of the CNS). In other aspects, the disclosure provides methods of treating a disease or disorder (e.g., a neurological disease and / or by inappropriate gene expression) associated with expression of an oligodendrocyte mRNA using the lipid- conjugated RNAi oligonucleotides, or pharmaceutically acceptable compositions thereof, described herein. In other aspects, the disclosure provides methods of using the lipid- conjugated RNAi oligonucleotides described herein in the manufacture of a medicament for treating a disease or disorder associated with expression of an oligodendrocyte mRNA. In other aspects, the lipid-conjugated RNAi oligonucleotides provided herein are used to treat a neurological disease or disorder by modulating (e.g., inhibiting or reducing) expression of an oligodendrocyte an oligodendrocyte target gene associated with the neurological disease or disorder in the CNS. In some aspects, the disclosure provides methods of treating a neurological disease or disorder by reducing expression of an oligodendrocyte target gene associated with the neurological disease or disorder in the CNS (e.g., in cells, tissues or regions of the CNS).

[0051] Lipid-Conjugated RNAi Oligonucleotides

[0052] The disclosure provides, inter alia, lipid-conjugated RNAi oligonucleotides (e.g., RNAi oligonucleotide-lipid conjugates) that reduce expression of an oligodendrocyte target gene in the CNS. In some embodiments, a lipid-conjugated RNAi oligonucleotide provided by the disclosure is targeted to an mRNA encoding the target gene. Messenger RNA (mRNA) that encodes a target gene and is targeted by a lipid-conjugated RNAi oligonucleotide of the disclosure is referred to herein as “target mRNA”. mRNA Target Sequences

[0053] In some embodiments, the lipid-conjugated RNAi oligonucleotide is targeted to a target sequence comprising a target oligodendrocyte mRNA. In some embodiments, the lipid- conjugated RNAi oligonucleotide is targeted to a target sequence within a target oligodendrocyte mRNA. In some embodiments, the lipid-conjugated RNAi oligonucleotide, or a portion, fragment, or strand thereof (e.g., an antisense strand or a guide strand of a doublestranded oligonucleotide) binds or anneals to a target sequence comprising a target oligodendrocyte mRNA, thereby reducing target gene expression. In some embodiments, the lipid-conjugated RNAi oligonucleotide is targeted to a target sequence comprising target oligodendrocyte mRNA for the purpose of reducing expression of an oligodendrocyte target gene in vivo. In some embodiments, the amount or extent of reduction of target gene expression by an lipid-conjugated RNAi oligonucleotide targeted to a specific oligodendrocyte target sequence correlates with the potency of the lipid-conjugated RNAi oligonucleotide. In some embodiments, the amount or extent of reduction of target gene expression by an lipid- conjugated RNAi oligonucleotide targeted to a specific oligodendrocyte target sequence correlates with the amount or extent of therapeutic benefit in a subject or patient having a disease, disorder or condition associated with target gene expression treated with the lipid- conjugated RNAi oligonucleotide.

[0054] Through examination of the nucleotide sequence of mRNAs encoding target genes, including mRNAs of multiple 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 and certain systemic modifications to those oligonucleotides are more amenable than others to RNAi oligonucleotide-mediated reduction and are thus useful as part of oligonucleotides that are otherwise targeted to specific gene target sequences. In some embodiments, a sense strand of a lipid-conjugated RNAi oligonucleotide, or a portion or fragment thereof, described herein, comprises a nucleotide sequence that is similar (e.g. , having no more than 4 mismatches) or is identical to a target sequence comprising an oligodendrocyte target mRNA. In some embodiments, a portion or region of the sense strand of a doublestranded oligonucleotide described herein comprises a target sequence comprising an oligodendrocyte target mRNA.

[0055] In some embodiments, the oligodendrocyte mRNA target sequence is associated with a neurological disorder. In some embodiments, the oligodendrocyte mRNA target sequence is an mRNA expressed in oligodendrocytes of at least one region of the CNS. In some embodiments, the oligodendrocytes mRNA target sequence is an mRNA expressed in oligodendrocytes of the spinal cord. In some embodiments, the oligodendrocytes mRNA target sequence is an mRNA expressed in oligodendrocytes of the thoracic spinal cord. In some embodiments, the oligodendrocytes mRNA target sequence is an mRNA expressed in oligodendrocytes of the cervical spinal cord. In some embodiments, the oligodendrocytes mRNA target sequence is an mRNA expressed in oligodendrocytes of the lumbar spinal cord. In some embodiments, the oligodendrocytes mRNA target sequence is an mRNA expressed in oligodendrocytes of the lumbar dorsal root ganglion. In some embodiments, the oligodendrocytes mRNA target sequence is an mRNA expressed in oligodendrocytes of the medulla. In some embodiments, the oligodendrocytes mRNA target sequence is an mRNA expressed in oligodendrocytes of the hippocampus. In some embodiments, the oligodendrocytes mRNA target sequence is an mRNA expressed in oligodendrocytes of the frontal cortex. In some embodiments, the oligodendrocytes mRNA target sequence is an mRNA expressed in oligodendrocytes of the hypothalamus. In some embodiments, the oligodendrocytes mRNA target sequence is an mRNA expressed in oligodendrocytes of the brainstem. In some embodiments, the oligodendrocytes mRNA target sequence is an mRNA expressed in oligodendrocytes of the cerebellum. In some embodiments, the oligodendrocyte mRNA target sequence is an mRNA associated with a disease, disorder, or condition of the CNS.

[0056] Uridine diphosphate Glycosyltransferase 8 (UGT8) is an enzyme involved in synthesizing galactosylceramide which is a glycosphingolipid of myelin. This product is primarily produced by oligodendrocytes in the CNS, and thus sufficient knockdown of UGT8 indicates ability of oligonucleotides described herein to reduce expression of target genes expressed in oligodendrocytes. In some embodiments, an oligodendrocyte mRNA is UGT8. In some aspects, the disclosure provides oligonucleotides for reducing expression of UGT8. In some embodiments, the oligonucleotides herein have regions of complementarity to UGT8 mRNA. In some embodiments, the target sequence is within a UGT8 mRNA for purposes of targeting the mRNA in cells and inhibiting its expression. In some embodiments, the oligonucleotides herein comprise a UGT8 targeting sequence (e.g., an antisense strand or a guide strand of a double-stranded oligonucleotide) having a region of complementarity that binds or anneals to a UGT8 target sequence by complementary (Watson-Crick) base pairing. In some embodiments, a portion or region of the sense strand of a double-stranded oligonucleotide described herein comprises a UGT8 target sequence. In some embodiments, a UGT8 target sequence comprises, or consists of, a nucleotide sequence of any one of SEQ ID NOs: 1-9.

[0057] RNAi Oligonucleotide Targeting Sequences

[0058] In some embodiments, the lipid-conjugated RNAi oligonucleotides provided by the disclosure comprise a targeting sequence. As used herein, the term “targeting sequence” refers to a nucleotide sequence having a region of complementarity to a specific nucleotide sequence comprising an mRNA (e.g., an oligodendrocyte target mRNA). In some embodiments, the lipid-conjugated RNAi oligonucleotides provided by the disclosure comprise a gene targeting sequence having a region of complementarity to a nucleotide sequence comprising a target sequence of a target mRNA. In some embodiments, the targeting sequence is an oligodendrocyte mRNA target sequence.

[0059] The targeting sequence imparts the lipid-conjugated RNAi oligonucleotide 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 lipid-conjugated RNAi oligonucleotides herein (or a strand thereof, e.g., an antisense strand or a guide strand of a double-stranded oligonucleotide) comprise a targeting sequence having a region of complementarity that binds or anneals to a target sequence comprising an oligodendrocyte target mRNA by complementary (Watson-Crick) base pairing. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein (or a strand thereof, e.g., an antisense strand or a guide strand of a double-stranded oligonucleotide) comprise a targeting sequence having a region of complementarity that binds or anneals to a target sequence within an oligodendrocyte target mRNA by complementary (Watson-Crick) base pairing. The targeting sequence is generally of suitable length and base content to enable binding or annealing of the lipid-conjugated RNAi oligonucleotide (or a strand thereof) to a specific target mRNA (e.g., oligodendrocyte mRNA) for purposes of inhibiting target gene expression. In some embodiments, the 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, the targeting sequence is at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or atleast 20 nucleotides. 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.

[0060] In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a targeting sequence that is fully complementary to a target sequence comprising an oligodendrocyte target mRNA. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a targeting sequence that is fully complementary to a target sequence within an oligodendrocyte target mRNA. In some embodiments, the targeting sequence is partially complementary to a target sequence comprising a target mRNA. In some embodiments, the targeting sequence is partially complementary to a target sequence within an oligodendrocyte target mRNA. In some embodiments, the targeting sequence comprises a region of contiguous nucleotides comprising the antisense strand.

[0061] In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a targeting sequence that is complementary to a contiguous sequence of nucleotides comprising an oligodendrocyte target mRNA, wherein the contiguous sequence of nucleotides is 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 lipid-conjugated RNAi oligonucleotides comprise a targeting sequence that is complementary to a contiguous sequence of nucleotides comprising an oligodendrocyte target mRNA, wherein the contiguous sequence of nucleotides is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides comprise a targeting sequence that is complementary to a contiguous sequence of nucleotides comprising a target mRNA, wherein the contiguous sequence of nucleotides is 15 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides comprise a targeting sequence that is complementary to a contiguous sequence of nucleotides comprising a target mRNA, wherein the contiguous sequence of nucleotides is 19 nucleotides in length.

[0062] In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a targeting sequence that is complementary to a contiguous sequence of nucleotides comprising an oligodendrocyte target mRNA, wherein the contiguous sequence of nucleotides is 15 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a targeting sequence that is complementary to a contiguous sequence of nucleotides comprising an oligodendrocyte target mRNA, wherein the contiguous sequence of nucleotides is 19 nucleotides in length.

[0063] In some embodiments, a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is fully complementary (e.g., having no mismatches) to a target sequence comprising an oligodendrocyte target mRNA and comprises the entire length of an antisense strand. In some embodiments, a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is fully complementary (e.g., having no mismatches) to a target sequence comprising an oligodendrocyte target mRNA and comprises a portion of the entire length of an antisense strand. In some embodiments, a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is fully complementary (e.g., having no mismatches) to a target sequence comprising an oligodendrocyte target mRNA and comprises 10 to 20 nucleotides of the antisense strand. In some embodiments, a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is fully complementary (e.g., having no mismatches) to a target sequence comprising an oligodendrocyte target mRNA and comprises 15 to 19 nucleotides of the antisense strand. In some embodiments, a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is fully complementary (e.g., having no mismatches) to a target sequence comprising an oligodendrocyte target mRNA and comprises 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, or 22 nucleotides of the antisense strand. In some embodiments, a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is fully complementary (e.g., having no mismatches) to a target sequence comprising an oligodendrocyte target mRNA and comprises 19 nucleotides of the antisense strand. In some embodiments, a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is fully complementary (e.g., having no mismatches) to a target sequence comprising an oligodendrocyte target mRNA and comprises 20 nucleotides of the antisense strand.

[0064] In some embodiments, a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is partially complementary e.g. , having no more than 4 mismatches) to a target sequence comprising an oligodendrocyte target mRNA and comprises the entire length of an antisense strand. In some embodiments, a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is partially complementary e.g. , having no more than 4 mismatches) to a target sequence comprising an oligodendrocyte target mRNA and comprises a portion of the entire length of an antisense strand. In some embodiments, a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is partially complementary (e.g., having no more than 4 mismatches) to a target sequence comprising an oligodendrocyte target mRNA and comprises 10 to 20 nucleotides of the antisense strand. In some embodiments, a targeting sequence of a lipid- conjugated RNAi oligonucleotide herein is partially complementary (e.g., having no more than 4 mismatches) to a target sequence comprising an oligodendrocyte target mRNA and comprises 15 to 19 nucleotides of the antisense strand. In some embodiments, a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is partially complementary (e.g., having no more than 4 mismatches) to a target sequence comprising an oligodendrocyte target mRNA and comprises 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, or 22 nucleotides of the antisense strand. In some embodiments, a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is partially complementary (e.g., having no more than 4 mismatches) to a target sequence comprising an oligodendrocyte target mRNA and comprises 19 nucleotides of the antisense strand. In some embodiments, a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is partially complementary (e.g., having no more than 4 mismatches) to a target sequence comprising an oligodendrocyte target mRNA and comprises 20 nucleotides of the antisense strand.

[0065] In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a targeting sequence having one or more base pair (bp) mismatches with the corresponding target sequence comprising an oligodendrocyte target mRNA. In some embodiments, the targeting sequence has a 1 bp mismatch, a 2 bp mismatch, a 3 bp mismatch, a 4 bp mismatch, or a 5 bp mismatch with the corresponding target sequence comprising an oligodendrocyte target mRNA provided that the ability of the targeting sequence to bind or anneal to the target sequence under appropriate hybridization conditions and / or the ability of the lipid-conjugated RNAi oligonucleotide 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, no more than 2, no more than 3, no more than 4, or no more than 5 bp mismatches with the corresponding target sequence comprising an oligodendrocyte target mRNA provided that the ability of the targeting sequence to bind or anneal to the target sequence under appropriate hybridization conditions and / or the ability of the lipid-conjugated RNAi oligonucleotide to inhibit or reduce target gene expression is maintained. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a targeting sequence having 1 mismatch with the corresponding target sequence. In some embodiments, the lipid- conjugated RNAi oligonucleotide comprises a targeting sequence having 2 mismatches with the corresponding target sequence. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a targeting sequence having 3 mismatches with the corresponding target sequence. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a targeting sequence having 4 mismatches with the corresponding target sequence. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a targeting sequence having 5 mismatches with the corresponding target sequence. In some embodiments, the lipid- conjugated RNAi oligonucleotide comprises a targeting sequence having more than one mismatch (e.g., 2, 3, 4, 5 or more mismatches) with the corresponding target sequence, wherein at least 2 (e.g., all) of the mismatches are positioned consecutively (e.g., 2, 3, 4, 5 or more mismatches in a row), or wherein the mismatches are interspersed in any position throughout the targeting sequence. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a targeting sequence having more than one mismatch (e.g., 2, 3, 4, 5 or more mismatches) with the corresponding target sequence, wherein at least 2 (e.g., all) of the mismatches are positioned consecutively (e.g., 2, 3, 4, 5 or more mismatches in a row), or wherein at least one or more non-mismatched base pair is located between the mismatches, or a combination thereof.

[0066] Types of Oligonucleotides

[0067] A variety of RNAi oligonucleotide types and / or structures are useful for reducing target gene expression (e.g., reducing expression of a target gene expressed in a oligodendrocytes) in the methods herein. Any of the RNAi oligonucleotide types described herein or elsewhere are contemplated for use as a framework to incorporate a targeting sequence herein for the purposes of inhibiting or reducing corresponding target gene expression in an oligodendrocyte in the CNS.

[0068] In some embodiments, the lipid-conjugated RNAi oligonucleotides herein inhibit target gene expression by engaging with RNA interference (RNAi) pathways upstream or downstream of Dicer involvement. For example, RNAi oligonucleotides have been developed with each strand having sizes of about 19-25 nucleotides with at least one 3' overhang of 1 to 5 nucleotides (see, e.g., US Patent No. 8,372,968). Longer oligonucleotides also have been developed that are processed by Dicer to generate active RNAi products (see, e.g., US Patent No. 8,883,996). Further work produced extended double-stranded oligonucleotides where at least one end of at least one strand is extended beyond a duplex targeting region, including structures where one of the strands includes a thermodynamically-stabilizing tetraloop structure (see, e.g., US Patent Nos. 8,513,207 and 8,927,705, as well as Inti. Patent Application Publication No. WO 2010 / 033225). Such structures may include single-stranded extensions (on one or both sides of the molecule) as well as double-stranded extensions.

[0069] In some embodiments, the RNAi oligonucleotides conjugates herein engage with the RNAi pathway downstream of the involvement of Dicer (e.g., Dicer cleavage). In some embodiments, the oligonucleotides described herein are Dicer substrates. In some embodiments, upon endogenous Dicer processing, double-stranded nucleic acids of 19-23 nucleotides in length capable of reducing expression of an oligodendrocyte target mRNA are produced. In some embodiments, the lipid-conjugated RNAi oligonucleotide has an overhang (e.g., of 1, 2, or 3 nucleotides in length) in the 3' end of the sense strand. In some embodiments, the lipid-conjugated RNAi oligonucleotide (e.g., siRNA conjugate) comprises a 21 -nucleotide guide strand that is antisense to an oligodendrocyte target mRNA and a complementary passenger strand, in which both strands anneal to form a 19-bp duplex and 2 nucleotide overhangs at either or both 3' ends. Longer oligonucleotide designs also are contemplated including oligonucleotides having a guide strand of 23 nucleotides and a passenger strand of 21 nucleotides, where there is a blunt end on the right side of the molecule (3' end of passenger strand / 5' end of guide strand) and a two nucleotide 3 '-guide strand overhang on the left side of the molecule (5' end of the passenger strand / 3' end of the guide strand). In such molecules, there is a 21 bp duplex region. See, e.g., US Patent Nos. 9,012,138; 9,012,621 and 9,193,753.

[0070] In some embodiments, the RNAi oligonucleotides conjugates disclosed herein comprise sense and antisense strands that are both in the range of about 17 to 26 (e.g., 17 to 26, 20 to 25 or 21-23) nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides disclosed herein comprise a sense and antisense strand that are both in the range of about 19-22 nucleotides in length. In some embodiments, the sense and antisense strands are of equal length. In some embodiments, the lipid-conjugated RNAi oligonucleotides disclosed herein comprise sense and antisense strands, such that there is a 3 '-overhang on either the sense strand or the antisense strand, or both the sense and antisense strand. In some embodiments, for lipid-conjugated RNAi oligonucleotides that have sense and antisense strands that are both in the range of about 21-23 nucleotides in length, a 3' overhang on the sense, antisense, or both sense and antisense strands is 1 or 2 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide has a guide strand of 22 nucleotides and a passenger strand of 20 nucleotides, where there is 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 the passenger strand / 3' end of the guide strand). In such molecules, there is a 20 bp duplex region.

[0071] Other RNAi 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., having 19 bp or shorter stems; see, e.g., Moore et al. (2010) METHODS MOL. BIOL. 629: 141-58), blunt siRNAs (e.g., of 19 bps in length; see, e.g., Kraynack & Baker (2006) RNA 12: 163-76), asymmetrical siRNAs (aiRNA; see, e.g., Sun et al. (2008) NAT. BIOTECHNOL. 26: 1379-82), asymmetric shorter-duplex siRNA (see, e.g., Chang et al. (2009) MOL. THER. 17:725-32), fork siRNAs (see, e.g., Hohjoh (2004) FEBS Lett. 557: 193-98), , and small internally segmented interfering RNA (siRNA; see, e.g., Bramsen et al. (2007) NUCLEIC ACIDS RES. 35:5886-97). Further non-limiting examples of an oligonucleotide structure that may be used in some embodiments to reduce or inhibit the expression of a target gene are microRNA (miRNA), short hairpin RNA (shRNA) and short siRNA (see, e.g., Hamilton etal. (2002) EMBO J. 21 :4671-79; see also, US Patent Application Publication No. 2009 / 0099115).

[0072] Antisense Strands

[0073] In some embodiments, an antisense strand of a lipid-conjugated RNAi oligonucleotide is referred to as a “guide strand.” For example, an antisense strand that engages with RNA-induced silencing complex (RISC) and binds to an Argonaute protein such as Ago2, or engages with or binds to one or more similar factors, and directs silencing of a target gene, the antisense strand is referred to as a guide strand. In some embodiments, a sense strand complementary to a guide strand is referred to as a “passenger strand.” In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises an antisense strand of 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, a lipid-conjugated RNAi oligonucleotide herein comprises an antisense 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 22, at least 25, at least 27, at least 30, at least 35 or at least 38 nucleotides in length). In some embodiments, a herein comprises an antisense strand in a range of about 12 to about 40 (e.g., 12 to 40, 12 to 36, 12 to 32, 12 to 28, 15 to 40, 15 to 36, 15 to 32, 15 to 30, 15 to 28, 17 to 22, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to 40 or 32 to 40) nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises an antisense strand of 15 to 30 nucleotides in length. In some embodiments, an antisense strand of any one of the lipid-conjugated RNAi oligonucleotide disclosed herein is of 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, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 19-23 nucleotides in length. In some embodiments, a lipid- conjugated RNAi oligonucleotide comprises an antisense strand of 19 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 20 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 21 nucleotides in length. In some embodiments, a lipid- conjugated RNAi oligonucleotide comprises an antisense strand of 22 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 23 nucleotides in length.

[0074] In some embodiments, an oligonucleotide disclosed herein for targeting UGT8 comprises an antisense strand comprising or consisting of a sequence as set forth in any one SEQ ID NOs: 10-18. In some embodiments, an oligonucleotide disclosed herein for targeting UGT8 comprises an antisense strand comprising or consisting of a sequence as set forth in any one of SEQ ID NOs: 28-36.

[0075] Sense Strands

[0076] In some embodiments, a lipid-conjugated RNAi oligonucleotide disclosed herein comprises 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, a lipid-conjugated RNAi oligonucleotide herein comprises 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, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand in a range of about 12 to about 50 (e.g., 12 to 50, 12 to 40, 12 to 36, 12 to 32, 12 to 28, 15 to 40, 15 to 36, 15 to 32, 15 to 28, 17 to 21, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to 40 or 32 to 40) nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand 15 to 50 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand 18 to 36 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 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, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 17-21 nucleotides in length. In some embodiments, a lipid- conjugated RNAi oligonucleotide herein comprises a sense strand of 17 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 18 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 19 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 20 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 21 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 22 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 23 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 24 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 25 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 26 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 27 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 28 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 29 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 30 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 31 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 32 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 33 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 34 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 35 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 36 nucleotides in length.

[0077] In some embodiments, an oligonucleotide disclosed herein for targeting UGT8 comprises a sense strand comprising or consisting of a sequence as set forth in any one SEQ ID NOs: 1- 9. In some embodiments, an oligonucleotide disclosed herein for targeting UGT8 comprises a sense strand comprising or consisting of a sequence as set forth in any one of SEQ ID NOs: 19-27. In some embodiments, an oligonucleotide disclosed herein for targeting UGT8 comprises a sense strand comprising or consisting of a sequence as set forth in SEQ ID NO: 71.

[0078] In some embodiments, a 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, a sense strand comprises a stem-loop structure at its 5' end. In some embodiments, a stem is a duplex of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 nucleotides in length. . In some embodiments, the stem of the stem-loop comprises a duplex of 2 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 3 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 4 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 5 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 6 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 7 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 8 nucleotides in length. In some embodiments, the stem of the stemloop comprises a duplex of 9 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 10 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 11 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 12 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 13 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 14 nucleotides in length.

[0079] In some embodiments, a stem-loop provides the lipid-conjugated RNAi oligonucleotide protection against degradation (e.g., enzymatic degradation), facilitates or improves targeting and / or delivery to a target cell, tissue, or organ, or both. For example, in some embodiments, the loop of a stem-loop provides nucleotides comprising one or more modifications that facilitate, improve, or increase targeting to a target mRNA (e.g., a target mRNA expressed in the CNS), inhibition of target gene expression, and / or delivery to a target cell, tissue, or organ (e.g., the CNS), or a combination thereof. In some embodiments, the stemloop itself or modification(s) to the stem-loop do not substantially affect the inherent gene expression inhibition activity of the lipid-conjugated RNAi oligonucleotide, but facilitates, improves, or increases stability (e.g., provides protection against degradation) and / or delivery of the lipid-conjugated RNAi oligonucleotide to a target cell, tissue, or organ (e.g., the CNS). In certain embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand comprising (e.g., at its 3' end) a stem-loop set forth as: S1-L-S2, in which SI is complementary to S2, and in which L forms a single-stranded loop between SI and S2 of up to about 10 nucleotides in length (e.g., 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in length). In some embodiments, the loop (L) is 3 nucleotides in length. In some embodiments, the loop (L) is 4 nucleotides in length.

[0080] 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: 81).

[0081] In some embodiments, a loop (L) of a stem-loop having the structure S1-L-S2 as described above is a triloop. In some embodiments, the triloop comprises ribonucleotides, deoxyribonucleotides, modified nucleotides, delivery ligands, and combinations thereof.

[0082] In some embodiments, a loop (L) of a stem-loop having the structure S1-L-S2 as described above is a tetraloop (e.g., within a nicked tetraloop structure). In some embodiments, the tetraloop comprises ribonucleotides, deoxyribonucleotides, modified nucleotides, delivery ligands, and combinations thereof.

[0083] In some embodiments, a loop (L) of a stem-loop having the structure S1-L-S2 as described above is a tetraloop as described in US Patent No. 10,131,912, incorporated herein by reference (e.g., within a nicked tetraloop structure).

[0084] Duplex Length

[0085] In some embodiments, a duplex formed between a sense and 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, a duplex formed between a sense and antisense strand is in the range of 12-30 nucleotides in length (e.g., 12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30 or 21 to 30 nucleotides in length). In some embodiments, a duplex formed between a sense and 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, a duplex formed between a sense and antisense strand is 15-30 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand is 17-21 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand is 17 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand is 18 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand is 19 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand is 20 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand is 21 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand does not span the entire length of the sense strand and / or antisense strand. In some embodiments, a duplex between a sense and antisense strand spans the entire length of either the sense or antisense strands. In some embodiments, a duplex between a sense and antisense strand spans the entire length of both the sense strand and the antisense strand.

[0086] Oligonucleotide Ends

[0087] In some embodiments, a lipid-conjugated RNAi oligonucleotide disclosed herein comprises sense and antisense strands, such that there is a 3 ’-overhang on either the sense strand or the antisense strand, or both the sense and antisense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein has one 5 ’end that is thermodynamically less stable compared to the other 5’ end. In some embodiments, an asymmetric lipid-conjugated RNAi oligonucleotide conjugate is provided that includes a blunt end at the 3 ’end of a sense strand and overhang at the 3’ end of the antisense strand. In some embodiments, a 3’ overhang on an antisense strand is 1-4 nucleotides in length (e.g., 1, 2, 3, or 4 nucleotides in length).

[0088] In some embodiments, the 3’-overhang is about one (1) to twenty (20) nucleotides in length (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 nucleotides in length). In some embodiments, the 3’ overhang is about one (1) to nineteen (19), one (1) to eighteen (18), one (1) to seventeen (17), one (1) to sixteen (16), one (1) to fifteen (15), one (1) to fourteen (14), one (1) to thirteen (13), one (1) to twelve (12), one (1) to eleven (11), one (1) to ten (10), one (1) to nine (9), one (1) to eight (8), one (1) to seven (7), one (1) to six (6), one (1) to five (5), one (1) to four (4), one (1) to three (3), or about one (1) to two (2) nucleotides in length. In some embodiments, the 3 ’-overhang is (1) nucleotide in length. In some embodiments, the 3 ’-overhang is two (2) nucleotides in length. In some embodiments, the 3 ’-overhang is three (3) nucleotides in length. In some embodiments, the 3 ’-overhang is four (4) nucleotides in length. In some embodiments, the 3 ’-overhang is five (5) nucleotides in length. In some embodiments, the 3 ’-overhang is six (6) nucleotides in length. In some embodiments, the 3 ’-overhang is seven (7) nucleotides in length. In some embodiments, the 3 ’-overhang is eight (8) nucleotides in length. In some embodiments, the 3’-overhang is nine (9) nucleotides in length. In some embodiments, the 3’-overhang is ten (10) nucleotides in length. In some embodiments, the 3’-overhang is eleven (11) nucleotides in length. In some embodiments, the 3 ’-overhang is twelve (12) nucleotides in length. In some embodiments, the 3’-overhang is thirteen (13) nucleotides in length. In some embodiments, the 3’-overhang is fourteen (14) nucleotides in length. In some embodiments, the 3’-overhang is fifteen (15) nucleotides in length. In some embodiments, the 3’-overhang is sixteen (16) nucleotides in length. In some embodiments, the 3’-overhang is seventeen (17) nucleotides in length. In some embodiments, the 3 ’-overhang is eighteen (18) nucleotides in length. In some embodiments, the 3 ’-overhang is nineteen (19) nucleotides in length. In some embodiments, the 3 ’-overhang is twenty (20) nucleotides in length.

[0089] Typically, an oligonucleotide for RNAi has a two (2) nucleotide overhang on the 3’ end of the antisense (guide) strand. However, other overhangs are possible. In some embodiments, an overhang is a 3’ overhang comprising a length of between one and four nucleotides, optionally one to four, one to three, one to two, two to four, two to three, or one, two, three, or four nucleotides. In some embodiments, the overhang is a 5’ overhang comprising a length of between one and four nucleotides, optionally one to four, one to three, one to two, two to four, two to three, or one, two, three, or four nucleotides.

[0090] In some embodiments, an oligonucleotide herein comprises a sense strand and an antisense strand, wherein the 5’ terminus of either or both strands comprise a 5 ’-overhang comprising one or more nucleotides. In some embodiments, an oligonucleotide herein comprises a sense strand and an antisense strand, wherein the sense strand comprises a 5’- overhang comprising one or more nucleotides. In some embodiments, an oligonucleotide herein comprises a sense strand and an antisense strand, wherein the antisense strand comprises a 5 ’-overhang comprising one or more nucleotides. In some embodiments, an oligonucleotide herein comprises a sense strand and an antisense strand, wherein both the sense strand and the antisense strand comprises a 5 ’-overhang comprising one or more nucleotides.

[0091] In some embodiments, the 5’-overhang is about one (1) to twenty (20) nucleotides in length (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 nucleotides in length). In some embodiments, the 5’ overhang is about one (1) to nineteen (19), one (1) to eighteen (18), one (1) to seventeen (17), one (1) to sixteen (16), one (1) to fifteen (15), one (1) to fourteen (14), one (1) to thirteen (13), one (1) to twelve (12), one (1) to eleven (11), one (1) to ten (10), one (1) to nine (9), one (1) to eight (8), one (1) to seven (7), one (1) to six (6), one (1) to five (5), one (1) to four (4), one (1) to three (3), or about one (1) to two (2) nucleotides in length. In some embodiments, the 5’-overhang is (1) nucleotide in length. In some embodiments, the 5’-overhang is two (2) nucleotides in length. In some embodiments, the 5 ’-overhang is three (3) nucleotides in length. In some embodiments, the 5 ’-overhang is four (4) nucleotides in length. In some embodiments, the 5 ’-overhang is five (5) nucleotides in length. In some embodiments, the 5 ’-overhang is six (6) nucleotides in length. In some embodiments, the 5 ’-overhang is seven (7) nucleotides in length. In some embodiments, the 5 ’-overhang is eight (8) nucleotides in length. In some embodiments, the 5’-overhang is nine (9) nucleotides in length. In some embodiments, the 5’-overhang is ten (10) nucleotides in length. In some embodiments, the 5’-overhang is eleven (11) nucleotides in length. In some embodiments, the 5’-overhang is twelve (12) nucleotides in length. In some embodiments, the 5’-overhang is thirteen (13) nucleotides in length. In some embodiments, the 5’-overhang is fourteen (14) nucleotides in length. In some embodiments, the 5’-overhang is fifteen (15) nucleotides in length. In some embodiments, the 5’-overhang is sixteen (16) nucleotides in length. In some embodiments, the 5’-overhang is seventeen (17) nucleotides in length. In some embodiments, the 5’-overhang is eighteen (18) nucleotides in length. In some embodiments, the 5 ’-overhang is nineteen (19) nucleotides in length. In some embodiments, the 5 ’-overhang is twenty (20) nucleotides in length.

[0092] In some embodiments, one or more (e.g., 2, 3, or 4) terminal nucleotides of the 3’ end or 5’ end of a sense and / or antisense strand are modified. For example, in some embodiments, one or two terminal nucleotides of the 3’ end of the antisense strand are modified. In some embodiments, the last nucleotide at the 3’ end of an antisense strand is modified, e.g., comprises 2’ modification, e.g, a 2’-O-methoxyethyl. In some embodiments, the last one or two terminal nucleotides at the 3’ end of an antisense strand are complementary with the target. In some embodiments, the last one or two nucleotides at the 3’ end of the antisense strand are not complementary with the target.

[0093] In some embodiments, an 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, an RNAi oligonucleotide conjugate herein comprises a nicked tetraloop structure, wherein 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.

[0094] In some embodiments, the overhang is selected from AA, GG, AG, and GA. In some embodiments, the overhang is AA. In some embodiments, the overhang is AG. In some embodiments, the overhang is GA. 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 with the target.

[0095] In some embodiments, the 5’ end and / or the 3’end of a sense or antisense strand has an inverted cap nucleotide.

[0096] In some embodiments, one or more (e.g., 2, 3, 4, 5, 6) modified internucleotide linkages are provided between terminal nucleotides of the 3’ end or 5’ end of a sense and / or antisense strand. In some embodiments, modified internucleotide linkages are provided between overhang nucleotides at the 3’ end or 5’ end of a sense and / or antisense strand.

[0097] Oligonucleotide Modifications

[0098] In some embodiments, an RNAi oligonucleotide conjugate disclosed herein comprises one or more modifications. Oligonucleotides (e.g., RNAi oligonucleotides) may be modified in various ways to improve or control specificity, stability, delivery, bioavailability, resistance from nuclease degradation, immunogenicity, base-pairing properties, RNA distribution and cellular uptake and other features relevant to therapeutic research use.

[0099] 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, an oligonucleotide described herein can comprise any one of the modifications described herein or any combination thereof. For example, in some embodiments, an oligonucleotide described herein comprises at least one modified sugar, a 5’- terminal phosphate group, at least one modified intemucleoside linkage, and at least one modified base.

[0100] The number of modifications on an oligonucleotide (e.g., an RNAi oligonucleotide) and the position of those nucleotide modifications may influence the properties of an oligonucleotide. For example, oligonucleotides may be delivered in vivo by conjugating them to or encompassing them in a lipid nanoparticle (LNP) or similar carrier. However, when an oligonucleotide is not protected by an LNP or similar carrier, it may be advantageous for at least some of the nucleotides to be modified. Accordingly, in some embodiments, all or substantially all of the nucleotides of an oligonucleotides 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 moiety of all nucleotides comprising the oligonucleotide is modified at the 2’ position. In some embodiments, the sugar moiety of all nucleotides comprising the oligonucleotide is modified at the 2’ position, except for the nucleotide conjugated to a lipid (e.g., the 5 ’-terminal nucleotide of the sense strand). The modifications may be reversible or irreversible. In some embodiments, an oligonucleotide as disclosed herein has a number and type of modified nucleotides sufficient to cause the desired characteristics (e.g., protection from enzymatic degradation, capacity to target a desired cell after in vivo administration, and / or thermodynamic stability).

[0101] Sugar Modifications

[0102] In some embodiments, a nucleotide modification in a sugar comprises a 2'- modification. In some embodiments, a 2'-modification may be 2'-O-propargyl, 2'-O- propylamin, 2'-amino, 2'-ethyl, 2'-fluoro (2'-F), 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'- O-methoxyethyl (2'-M0E), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-0-NMA) or 2'-deoxy-2'- fluoro-P-d-arabinonucleic acid (2'-FANA). In some embodiments, the modification is 2'-F, 2'- OMe or 2'-M0E. In some embodiments, a modification in a sugar comprises a modification of the sugar ring, which may comprise modification of one or more carbons of the sugar ring. For example, a modification of a sugar of a nucleotide may comprise a 2'-oxygen of a sugar is linked to a l'-carbon or 4'-carbon of the sugar, or a 2'-oxygen is linked to the l'-carbon or d'carbon via an ethylene or methylene bridge. In some embodiments, a modified nucleotide has an acyclic sugar that lacks a 2'-carbon to 3 '-carbon bond. In some embodiments, a modified nucleotide has a thiol group, e.g., in the 4' position of the sugar.

[0103] In some embodiments, a lipid-conjugated RNAi oligonucleotide described herein comprises at least about 1 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 lipid-conjugated RNAi oligonucleotide comprises at least about 1 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 lipid-conjugated RNAi oligonucleotide comprises at least about 1 modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, or more). In some embodiments, all the nucleotides of the sense strand of the lipid-conjugated RNAi oligonucleotide are modified. In some embodiments, all the nucleotides of the antisense strand of the lipid-conjugated RNAi oligonucleotide are modified. In some embodiments, all the nucleotides of the lipid-conjugated RNAi oligonucleotide (i.e., both the sense strand and the antisense strand) are modified. In some embodiments, the modified nucleotide comprises a 2'-modification (e.g., a 2'-F or 2'-OMe, 2'-M0E, and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid).

[0104] In some embodiments, the disclosure provides lipid-conjugated RNAi oligonucleotides having different modification patterns. In some embodiments, the modified lipid-conjugated RNAi oligonucleotides comprise a sense strand sequence having a modification pattern as set forth in the Examples and Sequence Listing and an antisense strand having a modification pattern as set forth in the Examples and Sequence Listing.

[0105] In some embodiments, a lipid-conjugated RNAi oligonucleotide disclosed herein comprises an antisense strand having nucleotides that are modified with 2'-F. In some embodiments, a lipid-conjugated RNAi oligonucleotide disclosed herein comprises an antisense strand comprises nucleotides that are modified with 2'-F and 2'-0Me. In some embodiments, a lipid-conjugated RNAi oligonucleotide disclosed herein comprises a sense strand having nucleotides that are modified with 2'-F. In some embodiments, a lipid-conjugated RNAi oligonucleotide disclosed herein comprises a sense strand comprises nucleotides that are modified with 2'-F and 2'-0Me.

[0106] In some embodiments, an oligonucleotide described herein comprises a sense strand with about 10-25%, 10%, 11%, 12%, 13%, 14% 15%, 16%, 17%, 18%, 19% or 20% of the nucleotides of the sense strand comprising 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, an oligonucleotide described herein comprises an antisense strand with about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35% of the nucleotides of the antisense strand comprising 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 comprising a 2’-fluoro modification. In some embodiments, about 19% of the nucleotides in the oligonucleotide comprise a 2’ -fluoro modification. In some embodiments, about 26% of the nucleotides in the oligonucleotide comprise a 2’ -fluoro modification. In some embodiments, one or more of positions 8, 9, 10 or 11 of the sense strand is modified with a 2'-F group. In some embodiments, the sugar moiety at each of nucleotides not modified with a 2’-F group or conjugated to a lipid in the sense strand is modified with a 2'- OMe. In some embodiments, the sugar moiety at each of nucleotides at positions 1-7 and 12- 20 in the sense strand is modified with a 2'-OMe. In some embodiments, the sugar moiety at each of nucleotides at positions 2-7 and 12-20 in the sense strand is modified with a 2'-OMe. In some embodiments, the sugar moiety at each of nucleotides at positions 1, 3-7 and 12-20 in the sense strand is modified with a 2'-OMe. In some embodiments, the sugar moiety at each of nucleotides at positions 1-2, 4-7, and 12-20 in the sense strand is modified with a 2'-OMe. In some embodiments, the sugar moiety at each of nucleotides at positions 1-4, 6, 7, and 12-20 in the sense strand is modified with a 2'-OMe. In some embodiments, the sugar moiety at each of nucleotides at positions 1-5, 7, and 12-20 in the sense strand is modified with a 2'-OMe. In some embodiments, the sugar moiety at each of nucleotides at positions 1-6, and 12-20 in the sense strand is modified with a 2'-OMe. In some embodiments, the sugar moiety at each of nucleotides at positions 1-7, 12, and 14-20 in the sense strand is modified with a 2'-OMe. In some embodiments, the sugar moiety at each of nucleotides at positions 1-7, 12-13, and 15-20 in the sense strand is modified with a 2'-OMe. In some embodiments, the sugar moiety at each of nucleotides at positions 1-7, 12-14, and 16-20 in the sense strand is modified with a 2'-OMe. In some embodiments, the sugar moiety at each of nucleotides at positions 1-7, 12-16, and 18- 20 in the sense strand is modified with a 2'-OMe. In some embodiments, the sugar moiety at each of nucleotides at positions 1-7, 12-18 and 20 in the sense strand is modified with a 2'- OMe. In some embodiments, the sugar moiety at each of nucleotides at positions 1-7 and 12- 19 in the sense strand is modified with a 2'-OMe.

[0107] In some embodiments, the sugar moiety at each of nucleotides at positions 1-7 and 12- 36 in the sense strand is modified with a 2’-OMe. In some embodiments, the sugar moiety at each of nucleotides at positions 1, 3-7 and 12-36 in the sense strand is modified with a 2’-OMe. In some embodiments, the sugar moiety at each of nucleotides at positions 1-2, 4-7 and 12-36 in the sense strand is modified with a 2’-OMe. In some embodiments, the sugar moiety at each of nucleotides at positions 1-5, 7, and 12-36 in the sense strand is modified with a 2’-OMe. In some embodiments, the sugar moiety at each of nucleotides at positions 1-7, 12, and 14-36 in the sense strand is modified with a 2’-OMe. In some embodiments, the sugar moiety at each of nucleotides at positions 1-7, 12-13, and 15-36 in the sense strand is modified with a 2’-OMe. In some embodiments, oligonucleotides the sugar moiety at each of nucleotides at positions 1- 7, 12-18, and 20-36 in the sense strand is modified with a 2’-OMe. In some embodiments, the sugar moiety at each of nucleotides at positions 1-7 and 12-19 and 21-36 in the sense strand is modified with a 2’-OMe. In some embodiments, the sugar moiety at each of nucleotides at positions 1-7 and 12-22 and 24-36 in the sense strand is modified with a 2’-OMe. In some embodiments, the sugar moiety at each of nucleotides at positions 1-7 and 12-27 and 29-36 in the sense strand is modified with a 2’-OMe. In some embodiments, the sugar moiety at each of nucleotides at positions 1-7 and 12-28 and 30-36 in the sense strand is modified with a 2’-OMe. In some embodiments, the sugar moiety at each of nucleotides at positions 1-7 and 12-29 and 31-36 in the sense strand is modified with a 2’-OMe.

[0108] In some embodiments, the sense strand comprises at least one 2’-F modified nucleotide wherein the remaining nucleotides not modified with a 2’-F group or conjugated to a lipid are modified with a 2’-OMe.

[0109] In some embodiments, the antisense strand has 7 nucleotides that are modified at the 2’ position of the sugar moiety with a 2’-F. In some embodiments, the sugar moiety at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand are modified with a 2’-F. In some embodiments, the antisense strand has 14 nucleotides that are modified at the 2’ position of the sugar moiety with a 2’-OMe. In some embodiments, the sugar moiety at positions 6, 8, 9, 11, 12, 13, 15, 16,

[0110] 17, 18, 19, 20, 21, and 22 of the antisense strand are modified with a 2’-OMe.

[0111] In some embodiments, the sense strand has 4 nucleotides that are modified at the 2’ position of the sugar moiety with a 2’-F. In some embodiments, the sugar moiety at positions 2, 3, 8, 9, 10, and 11 of the sense strand are modified with a 2’-F. In some embodiments, the sense strand has 15 nucleotides that are modified at the 2’ position of the sugar moiety with a 2’-OMe. In some embodiments, the sugar moiety at positions 6, 8, 9, 11, 12, 13, 15, 16, 17,

[0112] 18, 19, 20, 21, and 22 of the antisense strand are modified with a 2’-OMe.

[0113] In some embodiments, the antisense strand has 3 nucleotides that are modified at the 2'-position of the sugar moiety with a 2'-F. In some embodiments, the sugar moiety at positions 2, 5 and 14 and optionally up to 3 of the nucleotides at positions 1, 3, 7 and 10 of the antisense strand are modified with a 2'-F. In some embodiments, the sugar moiety at each of the positions at positions 2, 5 and 14 of the antisense strand is modified with the 2'-F. In other embodiments, the sugar moiety at each of the positions at positions 1, 2, 5 and 14 of the antisense strand is modified with the 2'-F. In other embodiments, the sugar moiety at each of the positions at positions 2, 4, 5 and 14 of the antisense strand is modified with the 2'-F. In some embodiments, the sugar moiety at each of the positions at positions 1, 2, 3, 5, 7 and 14 of the antisense strand is modified with the 2'-F. In some embodiments, the sugar moiety at each of the positions at positions 2, 3, 4, 5, 7 and 14 of the antisense strand is modified with the 2'-F. In some embodiments, the sugar moiety at each of the positions at positions 1, 2, 3, 5, 10 and 14 of the antisense strand is modified with the 2'-F. In some embodiments, the sugar moiety at each of the positions at positions 2, 3, 4, 5, 10 and 14 of the antisense strand is modified with the 2'-F. In some embodiments, the sugar moiety at each of the positions at positions 2, 3, 5, 7, 10 and 14 of the antisense strand is modified with the 2'-F. In some embodiments, the sugar moiety at each of the positions at positions 2, 3, 4, 5, 7, 10 and 14 of the antisense strand is modified with the 2'-F. In some embodiments, the antisense strand has 9 nucleotides that are modified at the 2'-position of the sugar moiety with a 2'-F. In some embodiments, the sugar moiety at each of the positions at positions 2, 3, 4, 5, 7, 10, 14, 16 and 19 of the antisense strand is modified with the 2'-F.

[0114] In some embodiments, a lipid-conjugated RNAi oligonucleotide 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-propylamin, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2’-O-methyl (2'-0Me), 2’-O-methoxyethyl (2'-M0E), 2'-O-[2-(methylamino)-2-oxoethyl] (2'- 0-NMA), and 2’-deoxy-2’-fluoro-P-d-arabinonucleic acid (2'-FANA).

[0115] In some embodiments, a lipid-conjugated RNAi oligonucleotide 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-propylamin, 2'-amino, 2'-ethyl, 2'- aminoethyl (EA), 2’-O-methyl (2'-0Me), 2’-O-methoxyethyl (2'-M0E), 2'-O-[2- (methylamino)-2-oxoethyl] (2'-0-NMA), and 2’-deoxy-2’-fluoro-P-d-arabinonucleic acid (2'- FANA).

[0116] In some embodiments, a lipid-conjugated RNAi oligonucleotide 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-propylamin, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-0Me), 2'-O-methoxyethyl (2'-M0E), 2'-O-[2-(methylamino)-2-oxoethyl] (2'- 0-NMA), and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid (2'-FANA).

[0117] In some embodiments, a lipid-conjugated RNAi oligonucleotide 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-propylamin, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-M0E), 2'-O-[2-(methylamino)-2- oxoethyl] (2'-0-NMA), and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid (2'-FANA).

[0118] In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein comprises an antisense strand having the sugar moiety of each of the nucleotides at positions

[0119] 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-propylamin, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-0Me), 2'-O-methoxyethyl (2'-M0E), 2'-O-[2-(methylamino)-2- oxoethyl] (2'-0-NMA), and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid (2'-FANA).

[0120] In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein comprises an antisense strand having the sugar moiety of each of the nucleotides at positions

[0121] 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-propylamin, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-0Me), 2'-O-methoxyethyl (2'-M0E), 2'-O-[2-(methylamino)-2- oxoethyl] (2'-0-NMA), and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid (2'-FANA).

[0122] In some embodiments, a lipid-conjugated RNAi oligonucleotide 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-propylamin, 2'-amino, 2'-ethyl, 2'- aminoethyl (EA), 2'-O-methyl (2'-0Me), 2'-O-methoxyethyl (2'-M0E), 2'-O-[2- (methylamino)-2-oxoethyl] (2'-0-NMA), and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid (2'- FANA).

[0123] In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 2, 3, 4, 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-propylamin, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-0Me), 2'-O-methoxyethyl (2'-M0E), 2'-O-[2-(methylamino)-2- oxoethyl] (2'-0-NMA), and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid (2'-FANA). In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein comprises an antisense strand having the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, or position 22 modified with 2'-F.

[0124] In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein comprises an antisense strand having the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, or position 22 modified with 2'-0Me.

[0125] In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein comprises an antisense strand having the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, or position 22 modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, 2’-aminoethyl (EA), 2'-O-methyl (2'-0Me), 2'-O-methoxyethyl (2'-M0E), 2'-O-[2-(methylamino)-2-oxoethyl] (2'- 0-NMA), and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid (2'-FANA).

[0126] In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety at positions 8-11 modified with 2'-F. In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety at positions 3, 5, 8, 10, 12, 13, 15 and 17 modified with 2'-F. In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety at positions 1-7 and 12-17 or 12-20 modified with 2’0Me. In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety at positions 2-7 and 12-17 or 12-20 modified with 2’0Me. In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety at positions 1-6 and 12-17 or 12-20 modified with 2’0Me. In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety at positions 1, 2, 4, 6, 7, 9, 11, 14, 16 and 18-20 modified with 2’0Me. In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety of each of the nucleotides at positions 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-propylamin, 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-P-d-arabinonucleic acid (2'-FANA). In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety of each of the nucleotides at positions 2-7 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- propylamin, 2'-amino, 2'-ethyl, 2’-aminoethyl (EA), 2'-O-methyl (2'-0Me), 2'-O- methoxyethyl (2'-M0E), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-0-NMA), and 2'-deoxy-2'- fluoro-P-d-arabinonucleic acid (2'-FANA). In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety of each of the nucleotides at positions 1-6 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-propylamin, 2'-amino, 2'-ethyl, 2’ -aminoethyl (EA), 2'-O-methyl (2'-0Me), 2 '-O-m ethoxy ethyl (2'-M0E), 2'-O-[2- (methylamino)-2-oxoethyl] (2'-0-NMA), and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid (2'- FANA). In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety 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-propylamin, 2'-amino, 2'-ethyl, 2’ -aminoethyl (EA), 2'-O-methyl (2'-0Me), 2 '-O-m ethoxy ethyl (2'-M0E), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-0-NMA), and 2'-deoxy- 2'-fluoro-P-d-arabinonucleic acid (2 -FANA).

[0127] In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, position 29, position 30, position 31, position 32, position 33, position 34, position 35, or position 36 modified with 2'-F.

[0128] In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, position 29, position 30, position 31, position 32, position 33, position 34, position 35, or position 36 modified with 2'-0Me. In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, position 29, position 30, position 31, position 32, position 33, position 34, position 35, or position 36 modified with a modification selected from the group consisting of 2'-O- propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, 2’ -aminoethyl (EA), 2'-O-methyl (2'-0Me), 2'- O-methoxy ethyl (2'-M0E), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-0-NMA), and 2 '-deoxy -2'- fluoro-P-d-arabinonucleic acid (2'-FANA).

[0129] 5 ’-Terminal Phosphate

[0130] In some embodiments, a lipid-conjugated RNAi oligonucleotide described herein comprises a 5’-terminal phosphate. In some embodiments, the 5'-terminal phosphate groups of the lipid-conjugated RNAi oligonucleotide enhance the interaction with Ago2. However, oligonucleotides comprising a 5 '-phosphate group may be susceptible to degradation via phosphatases or other enzymes, which can limit their bioavailability in vivo. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises analogs of 5' phosphates that are resistant to such degradation. In some embodiments, the phosphate analog is oxymethyl phosphonate, vinyl phosphonate or malonyl phosphonate, or a combination thereof. In some embodiments, the 5' end of a lipid-conjugated RNAi oligonucleotide strand is attached to chemical moiety that mimics the electrostatic and steric properties of a natural 5'- phosphate group (“phosphate mimic”).

[0131] In some embodiments, a lipid-conjugated RNAi oligonucleotide herein has a phosphate analog at a 4'-carbon position of the sugar (referred to as a “4'-phosphate analog”). See, e.g., Inti. Patent Application Publication No. WO 2018 / 045317. In some embodiments, a lipid- conjugated RNAi oligonucleotide herein comprises a 4'-phosphate analog at a 5 '-terminal nucleotide. In some embodiments, a phosphate analog is an oxymethyl phosphonate, in which the oxygen atom of the oxymethyl group is bound to the sugar moiety (e.g., at its 4'-carbon) or analog thereof. In other embodiments, a 4'-phosphate analog is a thiomethyl phosphonate or an aminomethyl phosphonate, in which the sulfur atom of the thiomethyl group or the nitrogen atom of the amino methyl group is bound to the 4'-carbon of the sugar moiety or analog thereof. In some embodiments, a 4'-phosphate analog is an oxymethyl phosphonate. In some embodiments, an oxymethyl phosphonate is represented by the formula -O-CH2-PO(OH)2,- O-CH2-PO(OR)2, or -O-CH2-POOH(R), in which R is independently selected from H, CH3, an alkyl group, CH2CH2CN, CH2OCOC(CH3)3, ClhOCIhCIhSi (CH3)3 or a protecting group. In some embodiments, the alkyl group is CH2CH3. More typically, R is independently selected from H, CH3 or CH2CH3. In some embodiment, R is CH3. In some embodiments, the 4’- phosphate analog is 5’-methoxyphosphonate-4’-oxy. In some embodiments, the 4’ -phosphate analog is 4’-oxymethyl phosphonate.

[0132] In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein comprises an antisense strand comprising a 4'-phosphate analog at the 5 '-terminal nucleotide, wherein 5’-terminal nucleotide comprises the following structure:

[0133] 4’-O-monomethylphosphonate-2’-O-methyluridine phosphorothioate [MePhosphonate-4O-mUs]

[0134] Modified Internucleotide Linkage

[0135] In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a modified internucleoside linkage. In some embodiments, phosphate modifications or substitutions result in an oligonucleotide that comprises at least about 1 (e.g., at least 1, at least 2, at least 3 or at least 5) modified internucleotide linkage. In some embodiments, any one of the oligonucleotides disclosed herein comprises about 1 to about 10 (e.g., 1 to 10, 2 to 8, 4 to 6, 3 to 10, 5 to 10, 1 to 5, 1 to 3 or 1 to 2) modified intemucleotide linkages. In some embodiments, any one of the oligonucleotides disclosed herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 modified internucleotide linkages.

[0136] A modified internucleotide linkage may be a phosphorodithioate linkage, a phosphorothioate linkage, a phosphotriester linkage, a thionoalkylphosphonate linkage, a thionalkylphosphotriester linkage, a phosphoramidite linkage, a phosphonate linkage or a boranophosphate linkage. In some embodiments, at least one modified internucleotide linkage of any one of the oligonucleotides as disclosed herein is a phosphorothioate linkage.

[0137] In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein has a phosphorothioate linkage 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 oligonucleotide described herein has a phosphorothioate linkage 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. In some embodiments, the oligonucleotide described herein has a phosphorothioate linkage 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 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 oligonucleotide described herein has a phosphorothioate linkage between each of positions 1 and 2 of the sense strand, positions 18 and 19 of the sense strand, positions 19 and 20 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.

[0138] In some embodiments, an oligonucleotide conjugate described herein comprises a peptide nucleic acid (PNA). PNAs are oligonucleotide mimics in which the sugar-phosphate backbone has been replaced by a pseudopeptide skeleton, composed of N-(2- aminoethyljglycine units. Nucleobases are linked to this skeleton through a two-atom carboxymethyl spacer. In some embodiments, an oligonucleotide conjugate described herein comprises a morpholino oligomer (PMO) comprising an internucleotide linkage backbone of methylene morpholine rings linked through phosphorodiamidate groups.

[0139] Base Modifications

[0140] In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises one or more modified nucleobases. In some embodiments, modified nucleobases (also referred to herein as base analogs) are linked at the 1' position of a nucleotide sugar moiety. In some embodiments, a modified nucleobase is a nitrogenous base. In some embodiments, a modified nucleobase does not contain nitrogen atom. See, e.g., US Patent Application Publication No. 2008 / 0274462. In some embodiments, a modified nucleotide comprises a universal base. In some embodiments, a modified nucleotide does not contain a nucleobase (abasic).

[0141] In some embodiments, a universal base is a heterocyclic moiety located at the 1' position of a nucleotide sugar moiety in a modified nucleotide, or the equivalent position in a nucleotide sugar moiety substitution, that, when present in a duplex, can be positioned opposite more than one type of base without substantially altering structure of the duplex. In some embodiments, compared to a reference single-stranded nucleic acid (e.g., oligonucleotide) that is fully complementary to a target nucleic acid, a single-stranded nucleic acid containing a universal base forms a duplex with the target nucleic acid that has a lower Tm than a duplex formed with the complementary nucleic acid. In some embodiments, when compared to a reference single-stranded nucleic acid in which the universal base has been replaced with a base to generate a single mismatch, the single-stranded nucleic acid containing the universal base forms a duplex with the target nucleic acid that has a higher Tm than a duplex formed with the nucleic acid comprising the mismatched base.

[0142] Non-limiting examples of universal-binding nucleotides include, but are not limited to, inosine, l-P-D-ribofuranosyl-5-nitroindole and / or l-P-D-ribofuranosyl-3 -nitropyrrole (see, US Patent Application Publication No. 2007 / 0254362; Van Aerschot et al. (1995) NUCLEIC ACIDS RES. 23:4363-70; Loakes etal. (1995) NUCLEIC ACIDS RES. 23:2361-66; and Loakes & Brown (1994) NUCLEIC ACIDS RES. 22:4039-43).

[0143] Targeting Ligands

[0144] In some embodiments, it is desirable to target the oligonucleotides of the disclosure (e.g., lipid-conjugated RNAi oligonucleotides) to one or more cells or tissues of the central nervous system (CNS). Such a strategy can help to avoid undesirable effects in other organs or avoid undue loss of the oligonucleotide to cells, tissue or organs that would not benefit from the oligonucleotide. Accordingly, in some embodiments, a lipid-conjugated RNAi oligonucleotide disclosed herein is modified to facilitate targeting and / or delivery to a particular tissue, cell, or organ (e.g. , to facilitate delivery of the conjugate to the CNS). In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises at least one nucleotide (e.g., 1, 2, 3, 4, 5, 6 or more nucleotides) conjugated to one or more targeting ligand(s).

[0145] In some embodiments, 1 or more (e.g., 1, 2, 3, 4, 5 or 6) nucleotides of a lipid- conjugated RNAi oligonucleotide disclosed herein are each conjugated to a separate targeting ligand. In some embodiments, 1 nucleotide of a lipid-conjugated RNAi oligonucleotide herein is conjugated to a separate targeting ligand. In some embodiments, 2 to 4 nucleotides of a lipid- conjugated RNAi oligonucleotide herein are each conjugated to a separate targeting ligand. In some embodiments, targeting ligands are conjugated to 2 to 4 nucleotides at either ends of the sense or antisense strand (e.g., targeting ligands are conjugated to a 2 to 4 nucleotide overhang or extension on the 5' or 3' end of the sense or antisense strand) such that the targeting ligands resemble bristles of a toothbrush and the lipid-conjugated RNAi oligonucleotide resembles a toothbrush. For example, a lipid-conjugated RNAi oligonucleotide may comprise a stem-loop at either the 5' or 3' end of the sense strand and 1, 2, 3 or 4 nucleotides of the loop of the stem may be individually conjugated to a targeting ligand. In some embodiments, a lipid-conjugated RNAi oligonucleotide provided by the disclosure comprises a stem-loop at the 3' end of the sense strand, wherein the loop of the stem-loop comprises a triloop or a tetraloop, and wherein the 3 or 4 nucleotides comprising the triloop or tetraloop, respectfully, are individually conjugated to a targeting ligand.

[0146] GalNAc is a high affinity ligand for the ASGPR, which is primarily expressed on the sinusoidal surface of hepatocyte cells and has a major role in binding, internalizing and subsequent clearing circulating glycoproteins that contain terminal galactose or GalNAc residues (asialoglycoproteins). Conjugation (either indirect or direct) of GalNAc moieties to oligonucleotide of the instant disclosure can be used to target these oligonucleotides to the ASGPR expressed on cells. In some embodiments, an oligonucleotide of the instant disclosure is conjugated to at least one or more GalNAc moieties, wherein the GalNAc moieties target the oligonucleotide to an ASGPR expressed on human liver cells (e.g., human hepatocytes). In some embodiments, the GalNAc moiety target the oligonucleotide to the liver.

[0147] In some embodiments, an oligonucleotide of the instant disclosure is conjugated directly or indirectly to a monovalent GalNAc. In some embodiments, the oligonucleotide is conjugated directly or indirectly to more than one monovalent GalNAc (i.e., is conjugated to 2, 3 or 4 monovalent GalNAc moieties, and is typically conjugated to 3 or 4 monovalent GalNAc moieties). In some embodiments, an oligonucleotide is conjugated to one or more bivalent GalNAc, trivalent GalNAc or tetravalent GalNAc moieties.

[0148] In some embodiments, 1 or more (e.g., 1, 2, 3, 4, 5 or 6) nucleotides of an oligonucleotide are each conjugated to a GalNAc moiety. In some embodiments, 2 to 4 nucleotides of a tetraloop are each conjugated to a separate GalNAc. In some embodiments, 1 to 3 nucleotides of a triloop are each conjugated to a separate GalNAc. In some embodiments, targeting ligands are conjugated to 2 to 4 nucleotides at either ends of the sense or antisense strand (e.g., ligands are conjugated to a 2 to 4 nucleotide overhang or extension on the 5' or 3' end of the sense or antisense strand) such that the GalNAc moieties resemble bristles of a toothbrush and the oligonucleotide resembles a toothbrush. In some embodiments, GalNAc moieties are conjugated to a nucleotide of the sense strand. For example, four (4) GalNAc moieties can be conjugated to nucleotides in the tetraloop of the sense strand where each GalNAc moiety is conjugated to 1 nucleotide. In some embodiments, the tetraloop is any combination of adenine and guanine nucleotides.

[0149] 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 depicted below (X=heteroatom):

[0150] 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 depicted below (X=heteroatom): In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a monovalent GalNAc attached to a guanine nucleotide referred to as [ademG-GalNAc] or 2'- aminodiethoxymethanol-Guanine-GalNAc, as depicted below:

[0151] In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a monovalent GalNAc attached to an adenine nucleotide, referred to as [ademA-GalNAc] or 2'- aminodiethoxymethanol-Adenine-GalNAc, as depicted below:

[0152] An example of such conjugation is shown below for a loop comprising from 5' to 3' the nucleotide sequence GAAA (L = linker, X = heteroatom). Such a loop may be present, for example, at positions 27-30 of the sense strand. In the chemical formula, is used to describe an attachment point to the oligonucleotide strand.

[0153]

[0154] Appropriate methods or chemistry (e.g., click chemistry) can be used to link a targeting ligand to a nucleotide. In some embodiments, a targeting ligand is conjugated to a nucleotide using a click linker. In some embodiments, an acetal-based linker is used to conjugate a targeting ligand to a nucleotide of any one of the oligonucleotides described herein. Acetal- based linkers are disclosed, for example, in Inti. Patent Application Publication No. WO 2016 / 100401. In some embodiments, the linker is a labile linker. However, in other embodiments, the linker is stable. Examples are shown below for a loop comprising from 5' to 3' the nucleotides GAAA, in which GalNAc moi eties are attached to nucleotides of the loop using an acetal linker. Such a loop may be present, for example, at positions 27-30 of the sense strand. In the chemical formula, is an attachment point to the oligonucleotide strand.

[0155]

[0156] As mentioned, various appropriate methods or chemistry synthetic techniques (e.g., click chemistry) can be used to link a targeting ligand to a nucleotide. In some embodiments, a targeting ligand is conjugated to a nucleotide using a click linker. In some embodiments, an acetal-based linker is used to conjugate a targeting ligand to a nucleotide of any one of the oligonucleotides described herein. Acetal-based linkers are disclosed, for example, in Inti. Patent Application Publication No. WO 2016 / 100401. In some embodiments, the linker is a labile linker. However, in other embodiments, the linker is a stable linker.

[0157] In some embodiments, a duplex extension (e.g., of up to 3, 4, 5 or 6 bp in length) is provided between a targeting ligand (e.g., a GalNAc moiety) and a lipid-conjugated RNAi oligonucleotide. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein does not have a GalNAc conjugated thereto.

[0158] Lipid Conjugates

[0159] In some embodiments, any of the lipid moieties described herein are conjugated to a nucleotide of the sense strand of the oligonucleotide. In some embodiments, a lipid moiety is conjugated to a terminal position of the oligonucleotide. In some embodiments, the lipid moiety is conjugated to the 5’ terminal nucleotide of the sense strand. In some embodiments, the lipid moiety is conjugated to the 3’ terminal nucleotide of the sense strand.

[0160] In some embodiments, the lipid moiety is conjugated to an internal nucleotide on the sense strand. An internal position is any nucleotide position other than the two terminal positions from each end of the sense strand. In some embodiments, the lipid moiety is conjugated to one or more internal positions of the sense strand. In some embodiments, the lipid moiety is conjugated to position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position

[0161] 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, position 29, position 30, position 31, position 32, position 33, position 34, position 35, or position 36 of a sense strand. In some embodiments, the lipid moiety is conjugated to position 1 of the sense strand. In some embodiments, the lipid moiety is conjugated to position 2 of the sense strand. In some embodiments, the lipid moiety is conjugated to position 3 of the sense strand. In some embodiments, the lipid moiety is conjugated to position 5 of the sense strand. In some embodiments, the lipid moiety is conjugated to position 6 of the sense strand. In some embodiments, the lipid moiety is conjugated to position 7 of the sense strand. In some embodiments, the lipid moiety is conjugated to position 9 of the sense strand. In some embodiments, the lipid moiety is conjugated to position 13 of the sense strand. In some embodiments, the lipid moiety is conjugated to position 14 of the sense strand. In some embodiments, the lipid moiety is conjugated to position 15 of the sense strand. In some embodiments, the lipid moiety is conjugated to position 17 of the sense strand. In some embodiments, the lipid moiety is conjugated to position 19 of the sense strand. In some embodiments, the lipid moiety is conjugated to position 20 of the sense strand. In some embodiments, the lipid moiety is conjugated to position 23 of the sense strand. In some embodiments, the lipid moiety is conjugated to position 28 of the sense strand. In some embodiments, the lipid moiety is conjugated to position 29 of the sense strand. In some embodiments, the lipid moiety is conjugated to position 30 of the sense strand.

[0162] In some embodiments, a lipid-conjugated RNAi oligonucleotide described herein comprises at least one nucleotide conjugated with one or more lipid moieties. In some embodiments, the one or more lipid moieties are conjugated to the same nucleotide. In some embodiments, the one or more lipid moieties are conjugated to different nucleotides. In some embodiments, one, two, three, four, five, or six lipid moieties are conjugated to the oligonucleotide. In some embodiments, one or more lipid moieties are conjugated to an adenine nucleotide. In some embodiments, one or more lipid moieties are conjugated to a guanine nucleotide. In some embodiments, one or more lipid moieties are conjugated to a cytosine nucleotide. In some embodiments, one or more lipid moieties are conjugated to a thymine nucleotide. In some embodiments, one or more lipid moieties are conjugated to a uracil nucleotide.

[0163] In some embodiments, the lipid moiety is a hydrocarbon chain. In some embodiments, the hydrocarbon chain is saturated. In some embodiments, the hydrocarbon chain is unsaturated. In some embodiments, the hydrocarbon chain is branched. In some embodiments, the hydrocarbon chain is straight. In some embodiments, the lipid moiety is a C8-C30 hydrocarbon chain. In some embodiments, the lipid moiety is a C8:0, C10:0, Cl 1 :0, C12:0, C14:0, C16:0, C17:0, C18:0, C18:l, C18:2, C22:5, C22:0, C24:0, C26:0, C22:6, C24:l, diacyl C16:0 or diacyl C18: l.

[0164] In some embodiments, the lipid moiety is a C 16 hydrocarbon chain.

[0165] In some embodiments, the lipid moiety is conjugated to the oligonucleotide via a linker. In some embodiments, a nucleotide of the lipid-conjugated oligonucleotide is represented by formula Il-b or II-c:

[0166] Il-b

[0167] II-C or a pharmaceutically acceptable salt thereof, wherein:

[0168] L1is a covalent bond, a monovalent or a bivalent saturated or unsaturated, straight, or branched Ci-50 hydrocarbon chain, wherein 0-10 methylene units of the hydrocarbon chain are independently replaced by -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -

[0169] R4is hydrogen, RA, or a suitable amine protection group; and

[0170] R5is adamantyl, or a saturated or unsaturated, straight, or branched C 1-50 hydrocarbon chain, wherein 0-10 methylene units of the hydrocarbon chain are independently replaced by

[0171] -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR-, or -P(S)OR.

[0172] In some embodiments of the lipid-conjugated RNAi oligonucleotide, R5is selected from

[0173]

[0174] 5 In certain embodiments of the lipid-conjugated RNAi oligonucleotide,

[0175] R5is selected from In some embodiments, R5is

[0176] In some embodiments, a nucleotide of the lipid-conjugated RNAi oligonucleotide is represented by formula Il-Ib or II-Ic:

[0177] II-Ic or a pharmaceutically acceptable salt thereof; wherein

[0178] B is a nucleobase or hydrogen; m is 1-50;

[0179] X1is -O-, or -S-;

[0180] Y is hydrogen,

[0181] R3is hydrogen, or a suitable protecting group;

[0182] X2is O, or S;

[0183] X3is -O-, -S-, or a covalent bond;

[0184] Y1is a linking group attaching to the 2'- or 3 '-terminal of a nucleoside, a nucleotide, or an oligonucleotide; Y2is hydrogen, a phosphoramidite analogue, an intemucleotide linking group ataching to the 5 '-terminal of a nucleoside, a nucleotide, or an oligonucleotide, or a linking group attaching to a solid support;

[0185] R5is adamantyl, or a saturated or unsaturated, straight, or branched C1-50 hydrocarbon chain, wherein 0-10 methylene units of the hydrocarbon chain are independently replaced by -

[0186] O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR-, or -P(S)OR-; and R is hydrogen, a suitable protecting group, or an optionally substituted group selected from Ci- 6 aliphatic, phenyl, a 4-7 membered saturated or partially unsaturated heterocyclic 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.

[0187] In some embodiments, the lipid is

[0188] In some embodiments, the oligonucleotide of the oligonucleotide-ligand conjugate is a double-stranded molecule. In some embodiments, the oligonucleotide is an RNAi molecule. In some embodiments, the double stranded oligonucleotide comprises a stem loop. In some embodiments, the stem loop is set forth as S1-L-S2, wherein SI is complementary to S2, and wherein L forms a loop between SI and S2. In some embodiments, the ligand is conjugated to any of the nucleotides in the loop of the stem loop. In some embodiments, the ligand is conjugated to any of the nucleotides in the stem of the stem loop. In some embodiments, the ligand is conjugated to the first nucleotide from 5’ to 3’ in the loop. In some embodiments, the ligand is conjugated to the second nucleotide from 5’ to 3’ in the loop. In some embodiments, the ligand is conjugated to the third nucleotide from 5’ to 3’ in the loop. In some embodiments, the ligand is conjugated to the fourth nucleotide from 5’ to 3’ in the loop. In some embodiments, the ligand is conjugated to one, two, three, or four of the nucleotides in the loop. In some embodiments, the ligand is conjugated to three of the nucleotides in the stem loop.

[0189] In some embodiments, the stem loop is 16 nucleotides in length. In some embodiments, the ligand is conjugated to the third nucleotide from 5’ to 3’ in the stem loop. In some embodiments, the ligand is conjugated to the eighth nucleotide from 5’ to 3’ in the stem loop. In some embodiments, the ligand is conjugated to the ninth nucleotide from 5’ to 3’ in the stem loop. In some embodiments, the ligand is conjugated to the tenth nucleotide from 5’ to 3’ in the stem loop.

[0190] In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a sense strand of 20 nucleotides with positions numbered 1-20 from 5’ to 3’. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a lipid conjugated to position 2 of a 20- nucleotide sense strand. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a lipid conjugated to position 3 of a 20-nucleotide sense strand. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a lipid conjugated to position 5 of a 20-nucleotide sense strand. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a lipid conjugated to position 6 of a 20-nucleotide sense strand. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a lipid conjugated to position 7 of a 20-nucleotide sense strand. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a lipid conjugated to position 9 of a 20-nucleotide sense strand. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a lipid conjugated to position 13 of a 20-nucleotide sense strand. In some embodiments, the lipid- conjugated RNAi oligonucleotide comprises a lipid conjugated to position 14 of a 20- nucleotide sense strand. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a lipid conjugated to position 15 of a 20-nucleotide sense strand. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a lipid conjugated to position 17 of a 20-nucleotide sense strand. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a lipid conjugated to position 19 of a 20-nucleotide sense strand. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a lipid conjugated to position 20 of a 20-nucleotide sense strand.

[0191] In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a sense strand of 36-nucleotides with positions numbered 1-36 from 5’ to 3’. In some embodiments, the lipid conjugated RNAi oligonucleotide comprises a lipid conjugated to position 1 of a 36 nucleotide sense strand. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a lipid conjugated to position 2 of a 36-nucleotide sense strand. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a lipid conjugated to position 3 of a 36-nucleotide sense strand. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a lipid conjugated to position 6 of a 36-nucleotide sense strand. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a lipid conjugated to position 13 of a 36-nucleotide sense strand. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a lipid conjugated to position 14 of a 36-nucleotide sense strand. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a lipid conjugated to position 15 of a 36-nucleotide sense strand. In some embodiments, the lipid- conjugated RNAi oligonucleotide comprises a lipid conjugated to position 19 of a 36- nucleotide sense strand. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a lipid conjugated to position 20 of a 36-nucleotide sense strand. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a lipid conjugated to position 23 of a 36-nucleotide sense strand. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a lipid conjugated to position 28 of a 36-nucleotide sense strand. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a lipid conjugated to position 29 of a 36-nucleotide sense strand. In some embodiments, the lipid- conjugated RNAi oligonucleotide comprises a lipid conjugated to position 30 of a 36- nucleotide sense strand.

[0192] Exemplary Oligonucleotides

[0193] In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a nucleotide conjugated with 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, lipid-conjugated RNAi oligonucleotide comprises a nucleotide conjugated with a lipid. In some embodiments, the lipid is a carbon chain. In some embodiments, the carbon chain is saturated. In some embodiments, the carbon chain is unsaturated. In some embodiments, the lipid- conjugated RNAi oligonucleotide comprises a nucleotide conjugated with a 16-carbon (Cl 6) lipid. In some embodiments, the C16 lipid comprises at least one double bond.

[0194] In some embodiments, the oligonucleotide of the lipid-conjugated RNAi oligonucleotide is conjugated to a C16 lipid as shown in:

[0195] In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a sense strand of 20 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises an anti-sense strand of 22 nucleotides in length. In some embodiments, the sense strand is 20 nucleotides in length and the antisense strand in 22 nucleotides in length. In some embodiments, lipid-conjugated RNAi oligonucleotide comprises a sense strand of 20 nucleotides in length and an antisense strand in 22 nucleotides in length, wherein the sense and antisense strands form a duplex region of 20 base pairs.

[0196] In some embodiments, the 3’ end of the sense strand is a blunt end. In some embodiments, the 5’ end of the antisense strand is a blunt end. In some embodiments, the 3’ end of the antisense strand comprises an overhang. In some embodiments, the overhang is 2 nucleotides in length. In some embodiments the overhang is GG.

[0197] In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises one or more 2’ modifications. In some embodiments, the 2’ modifications are selected from 2’ -fluoro and 2 ’-methyl.

[0198] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand and a sense strand described herein, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the 5’ terminal nucleotide of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand and a sense strand described herein, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the second nucleotide of the sense strand. In some embodiments, a lipid- conjugated RNAi oligonucleotide comprises an antisense strand and a sense strand described herein, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the third nucleotide of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand and a sense strand described herein, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the sixth nucleotide of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand and a sense strand described herein, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the thirteenth nucleotide of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand and a sense strand described herein, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the fourteenth nucleotide of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand and a sense strand described herein, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the fifteenth nucleotide of the sense strand. In some embodiments, a lipid- conjugated RNAi oligonucleotide comprises an antisense strand and a sense strand described herein, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the nineteenth nucleotide of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand and a sense strand described herein, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the twentieth nucleotide of the sense strand.

[0199] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the first nucleotide (Position 1 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20- 22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the first nucleotide (Position from 5’ > 3’) of the sense strand.

[0200] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the second nucleotide (Position 2 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20- 22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the second nucleotide (Position 2 from 5’ > 3’) of the sense strand.

[0201] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the third nucleotide (Position 3 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20- 22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the third nucleotide (Position 3 from 5’ > 3’) of the sense strand.

[0202] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the fifth nucleotide (Position 5 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20- 22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the fifth nucleotide (Position 5 from 5’ > 3’) of the sense strand.

[0203] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the sixth nucleotide (Position 6 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20- 22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the sixth nucleotide (Position 6 from 5’ > 3’) of the sense strand.

[0204] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the seventh nucleotide (Position 7 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20- 22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the seventh nucleotide (Position 7 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the ninth nucleotide (Position 9 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20- 22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the ninth nucleotide (Position 9 from 5’ > 3’) of the sense strand.

[0205] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the eleventh nucleotide (Position 11 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20- 22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the eleventh nucleotide (Position 11 from 5’ > 3’) of the sense strand.

[0206] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the thirteenth nucleotide (Position 13 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20- 22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the thirteenth nucleotide (Position 13 from 5’ > 3’) of the sense strand.

[0207] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the fourteenth nucleotide (Position 14 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20- 22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the fourteenth nucleotide (Position 14 from 5’ > 3’) of the sense strand.

[0208] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the fifteenth nucleotide (Position 15 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20- 22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the fifteenth nucleotide (Position 15 from 5’ > 3’) of the sense strand.

[0209] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the seventeenth nucleotide (Position 17 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20- 22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the seventeenth nucleotide (Position 17 from 5’ > 3’) of the sense strand.

[0210] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the nineteenth nucleotide (Position 19 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20- 22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the nineteenth nucleotide (Position 19 from 5’ > 3’) of the sense strand.

[0211] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the twentieth nucleotide (Position 20 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20- 22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the twentieth nucleotide (Position 20 from 5’ > 3’) of the sense strand.

[0212] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the first nucleotide (Position 1 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the first nucleotide (Position 1 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20- 22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the second nucleotide (Position 2 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the second nucleotide (Position 2 from 5’ > 3’) of the sense strand.

[0213] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the third nucleotide (Position 3 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the third nucleotide (Position 3 from 5’ > 3’) of the sense strand.

[0214] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the fifth nucleotide (Position 5 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the fifth nucleotide (Position 5 from 5’ > 3’) of the sense strand.

[0215] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the sixth nucleotide (Position 6 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the sixth nucleotide (Position 6 from 5’ > 3’) of the sense strand.

[0216] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the seventh nucleotide (Position 7 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the seventh nucleotide (Position 7 from 5’ > 3’) of the sense strand.

[0217] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the ninth nucleotide (Position 9 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the ninth nucleotide (Position 9 from 5’ > 3’) of the sense strand.

[0218] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the eleventh nucleotide (Position 11 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the eleventh nucleotide (Position 11 from 5’ > 3’) of the sense strand.

[0219] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the thirteenth nucleotide (Position 13 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22- 24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the thirteenth nucleotide (Position 13 from 5’ > 3’) of the sense strand.

[0220] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the fourteenth nucleotide (Position 14 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22- 24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the fourteenth nucleotide (Position 14 from 5’ > 3’) of the sense strand.

[0221] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the fifteenth nucleotide (Position 15 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the fifteenth nucleotide (Position 15 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the seventeenth nucleotide (Position 17 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22- 24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the seventeenth nucleotide (Position 17 from 5’ > 3’) of the sense strand.

[0222] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the nineteenth nucleotide (Position 19 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22- 24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the nineteenth nucleotide (Position 19 from 5’ > 3’) of the sense strand.

[0223] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the twentieth nucleotide (Position 20 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22- 24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the twentieth nucleotide (Position 20 from 5’ > 3’) of the sense strand.

[0224] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand and a sense strand described herein, wherein the sense strand comprises at least one hydrocarbon chain conjugated to an internal nucleotide of the sense strand (e.g, nucleotide at position 6). In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand and a sense strand described herein, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to an internal nucleotide of the sense strand (e.g., nucleotide at position 6). In some embodiments, not all internal nucleotides are suitable for lipid conjugation for delivery of an RNAi oligonucleotide to an oligodendrocyte of the CNS. For example, in some embodiments, conjugation at positions 9 or 10 of a sense strand numbered from 5’ to 3’ is not suitable for delivery of an RNAi oligonucleotide to an oligodendrocyte of the CNS. In some embodiments, lipid conjugation at an internal position of a sense strand numbered from 5’ to 3’ excludes positions 9 and 10. In some embodiments, lipid conjugation at an internal position of a sense strand numbered from 5’ to 3’ excludes position 10.

[0225] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one hydrocarbon chain conjugated to an internal nucleotide of the sense strand (e.g., nucleotide at position 6). In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20- 22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to an internal nucleotide of the sense strand (e.g, nucleotide at position 6).

[0226] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one hydrocarbon chain conjugated to an internal nucleotide of the sense strand (e.g., nucleotide at position 6). In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22- 24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to an internal nucleotide of the sense strand (e.g., nucleotide at position 6).

[0227] In some embodiments, the 5’ terminal nucleotide of the sense strand comprises a mismatch with the antisense strand.

[0228] In some embodiments, position numbers described throughout are based on numbering from the 5’ end to the 3’ end, for example, the terminal nucleotide at the 5’ end is Position 1.

[0229] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte target gene comprises the modification pattern of

[0230] Sense Strand : 5 ’ - [ademXs-L] [mX] [mX] [mX] [mX] [mX] [mX] [fX] [fX] [fX] [fX] [mX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX]-3 ’ Hybridized to:

[0231] Antisense Strand: 5’- [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX] [mX] [fX] [mX] [mX] [mX] [fX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’ wherein [mXs]= 2’-( -methyl modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [fXs] =2’- fluoro modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [mX]= 2’-( -methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides, [fX] =2’- fluoro modified nucleotide with phosphodiester linkages to neighboring nucleotides, [MePhosphonate-4O-mX] = 4’-O- monomethylphosphonate-2’-O-m ethyl modified nucleotide, [AdemX-Ls] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide, and [AdemX-L] = Lipid attached to a nucleotide, optionally wherein L is a C16 lipid.

[0232] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte target gene comprises the modification pattern of

[0233] Sense Strand : 5 ’ - [mXs] [ademX-L] [mX] [mX] [mX] [mX] [mX] [fX] [fX] [fX] [fX] [mX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX]-3 ’ Hybridized to:

[0234] Antisense Strand: 5’- [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX] [mX] [fX] [mX] [mX] [mX] [fX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’ wherein [mXs]= 2’-( -methyl modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [fXs] =2’- fluoro modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [mX]= 2’-( -methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides, [fX] =2’- fluoro modified nucleotide with phosphodiester linkages to neighboring nucleotides, [MePhosphonate-4O-mX] = 4’-O- monomethylphosphonate-2’-O-m ethyl modified nucleotide, [AdemX-Ls] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide, and [AdemX-L] = Lipid attached to a nucleotide, optionally wherein L is a C16 lipid.

[0235] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte target gene comprises the modification pattern of

[0236] Sense Strand: 5’-[mXs][mX][ademX-L][mX][mX][mX][mX][fX][fX][fX] [fX] [mX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’ Hybridized to:

[0237] Antisense Strand: 5’- [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX] [mX] [fX] [mX] [mX] [mX] [fX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’ wherein [mXs]= 2’-( -methyl modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [fXs] =2’- fluoro modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [mX]= 2’-( -methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides, [fX] =2’- fluoro modified nucleotide with phosphodiester linkages to neighboring nucleotides, [MePhosphonate-4O-mX] = 4’-O- monomethylphosphonate-2’-O-m ethyl modified nucleotide, [AdemX-Ls] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide, and [AdemX-L] = Lipid attached to a nucleotide, optionally wherein L is a C16 lipid.

[0238] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte target gene comprises the modification pattern of

[0239] Sense Strand: 5’-[mXs][mX][mX][mX][ ademX-L][mX][mX][fX][fX][fX][fX][mX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX]-3 ’ Hybridized to:

[0240] Antisense Strand: 5’- [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX] [mX] [fX] [mX] [mX] [mX] [fX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’ wherein [mXs]= 2’-( -methyl modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [fXs] =2’- fluoro modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [mX]= 2’-( -methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides, [fX] =2’- fluoro modified nucleotide with phosphodiester linkages to neighboring nucleotides, [MePhosphonate-4O-mX] = 4’-O- monomethylphosphonate-2’-O-m ethyl modified nucleotide, [AdemX-Ls] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide, and [AdemX-L] = Lipid attached to a nucleotide, optionally wherein L is a C16 lipid. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte target gene comprises the modification pattern of

[0241] Sense Strand : 5 ’ - [mXs] [mX] [mX] [mX] [mX] [ademX-L] [mX] [fX] [fX] [fX] [fX] [mX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX]-3 ’ Hybridized to:

[0242] Antisense Strand: 5’- [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX] [mX] [fX] [mX] [mX] [mX] [fX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’ wherein [mXs]= 2’-O-methyl modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [fXs] =2’- fluoro modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [mX]= 2’-O-methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides, [fX] =2’- fluoro modified nucleotide with phosphodiester linkages to neighboring nucleotides, [MePhosphonate-4O-mX] = 4’-O- monomethylphosphonate-2’-O-m ethyl modified nucleotide, [AdemX-Ls] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide, and [AdemX-L] = Lipid attached to a nucleotide, optionally wherein L is a C16 lipid.

[0243] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte target gene comprises the modification pattern of

[0244] Sense Strand : 5 ’ - [mXs] [mX] [mX] [mX] [mX] [mX] [ademX-L] [fX] [fX] [fX] [fX] [mX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX]-3 ’ Hybridized to:

[0245] Antisense Strand: 5’- [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX] [mX] [fX] [mX] [mX] [mX] [fX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’ wherein [mXs]= 2’-O-methyl modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [fXs] =2’- fluoro modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [mX]= 2’-O-methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides, [fX] =2’- fluoro modified nucleotide with phosphodiester linkages to neighboring nucleotides, [MePhosphonate-4O-mX] = 4’-O- monomethylphosphonate-2’-O-m ethyl modified nucleotide, [AdemX-Ls] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide, and [AdemX-L] = Lipid attached to a nucleotide, optionally wherein L is a C16 lipid.

[0246] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte target gene comprises the modification pattern of

[0247] Sense Strand: 5’-[mXs][mX][mX][mX][mX][mX][mX][fX][ademX- L][fX][fX][mX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX]-3 ’

[0248] Hybridized to:

[0249] Antisense Strand: 5’- [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX] [mX] [fX] [mX] [mX] [mX] [fX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’ wherein [mXs]= 2’-O-methyl modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [fXs] =2’- fluoro modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [mX]= 2’-O-methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides, [f ] =2’- fluoro modified nucleotide with phosphodiester linkages to neighboring nucleotides, [MePhosphonate-4O-mX] = 4’-O- monomethylphosphonate-2’-O-m ethyl modified nucleotide, [AdemX-Ls] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide, and [AdemX-L] = Lipid attached to a nucleotide, optionally wherein L is a C16 lipid.

[0250] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte target gene comprises the modification pattern of

[0251] Sense Strand: 5’-[mXs][mX][mX][mX][mX][mX][mX][fX][fX][fX][ademX- L][mX]

[0252] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX]-3 ’

[0253] Hybridized to:

[0254] Antisense Strand: 5’- [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX] [mX] [fX] [mX] [mX] [mX] [fX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’ wherein [mXs]= 2’-O-methyl modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [fXs] =2’- fluoro modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [mX]= 2’-O-methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides, [f ] =2’- fluoro modified nucleotide with phosphodiester linkages to neighboring nucleotides, [MePhosphonate-4O-mX] = 4’-O- monomethylphosphonate-2’-O-m ethyl modified nucleotide, [AdemX-Ls] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide, and [AdemX-L] = Lipid attached to a nucleotide, optionally wherein L is a C16 lipid.

[0255] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte target gene comprises the modification pattern of

[0256] Sense Strand: 5’-

[0257] [mXs] [mX] [mX] [mX] [mX] [mX] [mX] [fX] [fX] [fX] [fX] [mX] [ademX- L] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’

[0258] Hybridized to: Antisense Strand: 5’- [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX] [mX] [fX] [mX] [mX] [mX] [fX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’ wherein [mXs]= 2’-0-methyl modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [fXs] =2’- fluoro modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [mX]= 2’-O-methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides, [f ] =2’- fluoro modified nucleotide with phosphodiester linkages to neighboring nucleotides, [MePhosphonate-4O-mX] = 4’-O- monomethylphosphonate-2’-O-m ethyl modified nucleotide, [AdemX-Ls] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide, and [AdemX-L] = Lipid attached to a nucleotide, optionally wherein L is a C16 lipid.

[0259] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte target gene comprises the modification pattern of

[0260] Sense Strand: 5’-[mXs][mX][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX] [mX] [ademX-L] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’ Hybridized to:

[0261] Antisense Strand: 5’- [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX] [mX] [fX] [mX] [mX] [mX] [fX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’ wherein [mXs]= 2’-O-methyl modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [fXs] =2’- fluoro modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [mX]= 2’-O-methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides, [fX] =2’- fluoro modified nucleotide with phosphodiester linkages to neighboring nucleotides, [MePhosphonate-4O-mX] = 4’-O- monomethylphosphonate-2’-O-m ethyl modified nucleotide, [AdemX-Ls] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide, and [AdemX-L] = Lipid attached to a nucleotide, optionally wherein L is a C16 lipid.

[0262] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte target gene comprises the modification pattern of

[0263] Sense Strand: 5’-[mXs][mX][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX] [mX] [mX] [ademX-L] [mX] [mX] [mXs] [mXs] [mX] -3 ’ Hybridized to:

[0264] Antisense Strand: 5’- [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX] [mX] [fX] [mX] [mX] [mX] [fX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’ wherein [mXs]= 2’-O-methyl modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [fXs] =2’- fluoro modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [mX]= 2’-O-methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides, [fX] =2’- fluoro modified nucleotide with phosphodiester linkages to neighboring nucleotides, [MePhosphonate-4O-mX] = 4’-O- monomethylphosphonate-2’-O-m ethyl modified nucleotide, [AdemX-Ls] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide, and [AdemX-L] = Lipid attached to a nucleotide, optionally wherein L is a C16 lipid.

[0265] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte target gene comprises the modification pattern of

[0266] Sense Strand: 5’-[mXs][mX][mX][mX][mX][mX][mX][fX][fX][fX][fX]

[0267] [mX] [mX] [mX] [mX] [mX] [ademX-L] [mXs] [mXs] [mX]-3 ’ Hybridized to:

[0268] Antisense Strand: 5’- [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX] [mX] [fX] [mX] [mX] [mX] [fX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’ wherein [mXs]= 2’-O-methyl modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [fXs] =2’- fluoro modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [mX]= 2’-O-methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides, [fX] =2’- fluoro modified nucleotide with phosphodiester linkages to neighboring nucleotides, [MePhosphonate-4O-mX] = 4’-O- monomethylphosphonate-2’-O-m ethyl modified nucleotide, [AdemX-Ls] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide, and [AdemX-L] = Lipid attached to a nucleotide, optionally wherein L is a C16 lipid.

[0269] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte target gene comprises the modification pattern of

[0270] Sense Strand: 5’-[mXs][mX][mX][mX][mX][mX][mX][fX][fX][fX][fX]

[0271] [mX] [mX] [mX] [mX] [mX] [mX] [mXs] [ademX-Ls] [mX]-3 ’ Hybridized to:

[0272] Antisense Strand: 5’- [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX] [mX] [fX] [mX] [mX] [mX] [fX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’ wherein [mXs]= 2’-O-methyl modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [fXs] =2’- fluoro modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [mX]= 2’-O-methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides, [fX] =2’- fluoro modified nucleotide with phosphodiester linkages to neighboring nucleotides, [MePhosphonate-4O-mX] = 4’-O- monomethylphosphonate-2’-O-m ethyl modified nucleotide, [AdemX-Ls] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide, and [AdemX-L] = Lipid attached to a nucleotide, optionally wherein L is a C16 lipid.

[0273] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte target gene comprises the modification pattern of

[0274] Sense Strand: 5’-[mXs] [mX][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX][mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [ademX-L] - 3 ’ Hybridized to:

[0275] Antisense Strand: 5’- [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX] [mX] [fX] [mX] [mX] [mX] [fX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’ wherein [mXs]= 2’-O-methyl modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [fXs] =2’- fluoro modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [mX]= 2’-O-methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides, [fX] =2’- fluoro modified nucleotide with phosphodiester linkages to neighboring nucleotides, [MePhosphonate-4O-mX] = 4’-O- monomethylphosphonate-2’-O-m ethyl modified nucleotide, [AdemX-Ls] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide, and [AdemX-L] = Lipid attached to a nucleotide, optionally wherein L is a C16 lipid.

[0276] In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a sense strand of 36 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises an anti-sense strand of 22 nucleotides in length. In some embodiments, the sense strand is 36 nucleotides in length and the antisense strand in 22 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises a sense strand of 36 nucleotides in length and an antisense strand in 22 nucleotides in length, wherein the sense and antisense strands form a duplex region of 20 base pairs.

[0277] In some embodiments, the 3’ end of the sense strand comprises a stem-loop. In some embodiments, the 3’ end of the sense strand comprises a tetraloop. In some embodiments, the 3’ end of the sense strand comprises a stem-loop comprising the sequence of SEQ ID NO: 21. In some embodiments, the 3’ end of the antisense strand comprises an overhang. In some embodiments, the overhang is 2 nucleotides in length. In some embodiments the overhang is GG.

[0278] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises a sense strand comprising a stem-loop at its 3’ end and at least one hydrocarbon chain conjugated to the 5’ terminal nucleotide of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises a sense strand comprising a stem-loop at its 3’ end and at least one hydrocarbon chain conjugated to a nucleotide of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises a sense strand comprising a stem-loop at its 3’ end and at least one C16 hydrocarbon chain conjugated to the 5’ terminal nucleotide of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises a sense strand comprising a stem-loop at its 3’ end and at least one C16 hydrocarbon chain conjugated to a nucleotide of the sense strand.

[0279] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the first nucleotide (Position 1 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20- 36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the second nucleotide (Position 2 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22- 24 nucleotides and a sense strand of 20-36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the third nucleotide (Position 3 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the sixth nucleotide (Position 6 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20- 36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the thirteenth nucleotide (Position 13 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22- 24 nucleotides and a sense strand of 20-36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the fourteenth nucleotide (Position 14 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the fifteenth nucleotide (Position 15 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the nineteenth nucleotide (Position 19 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the twentieth nucleotide (Position 20 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20- 36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the twenty-third nucleotide (Position 23 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the twenty-eighth nucleotide (Position 28 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20- 36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the twenty-ninth nucleotide (Position 29 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the thirtieth nucleotide (Position 30 from 5’ > 3’) of the sense strand.

[0280] In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C 16 hydrocarbon chain conjugated to the first nucleotide (Position 1 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20- 36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the second nucleotide (Position 2 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22- 24 nucleotides and a sense strand of 20-36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the third nucleotide (Position 3 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the sixth nucleotide (Position 6 from 5’ > 3’) of the sense strand. In some embodiments, a lipid- conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the thirteenth nucleotide (Position 13 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the fourteenth nucleotide (Position 14 from 5’ > 3’) of the sense strand. In some embodiments, a lipid- conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the fifteenth nucleotide (Position 15 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the nineteenth nucleotide (Position 19 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20- 36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the twentieth nucleotide (Position 20 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22- 24 nucleotides and a sense strand of 20-36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the twenty -third nucleotide (Position 23 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the twenty-eighth nucleotide (Position 28 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the twenty-ninth nucleotide (Position 29 from 5’ > 3’) of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the thirtieth nucleotide (Position 30 from 5’ > 3’) of the sense strand.

[0281] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte target gene comprises the modification pattern of

[0282] Sense Strand: 5’- [mXs][AdemX-L][mX][mX][mX][mX][mX][fX][fX][fX] [fX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX][mX][mX][mX][mX][mX] [mX][mX][mX][mX] -3’ Hybridized to:

[0283] Antisense Strand: 5’- [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX] [mX] [fX] [mX] [mX] [mX] [fX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’ wherein [mXs]= 2’-O-methyl modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [fXs] =2’- fluoro modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [mX]= 2’-O-methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides, [fX] =2’- fluoro modified nucleotide with phosphodiester linkages to neighboring nucleotides, [MePhosphonate-4O-mX] = 4’-O- monomethylphosphonate-2’-O-m ethyl modified nucleotide, [AdemX-Ls] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide, and [AdemX-L] = Lipid attached to a nucleotide optionally wherein L is a C16 lipid.

[0284] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte target gene comprises the modification pattern of

[0285] Sense Strand: 5’- [mXs][mX] [AdemX-L] [mX][mX][mX][mX][fX][fX][fX][fX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX]-3 ’ Hybridized to:

[0286] Antisense Strand: 5’- [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX] [mX] [fX] [mX] [mX] [mX] [fX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’ wherein [mXs]= 2’-( -methyl modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [fXs] =2’- fluoro modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [mX]= 2’-( -methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides, [fX] =2’- fluoro modified nucleotide with phosphodiester linkages to neighboring nucleotides, [MePhosphonate-4O-mX] = 4’-O- monomethylphosphonate-2’-O-m ethyl modified nucleotide, [AdemX-Ls] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide, and [AdemX-L] = Lipid attached to a nucleotide, optionally wherein L is a C16 lipid.

[0287] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte target gene comprises the modification pattern of

[0288] Sense Strand: 5 ’-[mXs][mX][mX][mX][mX] [AdemX-L] [mX][fX][fX][fX][fX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] -3 ’ Hybridized to:

[0289] Antisense Strand: 5’- [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX] [mX] [fX] [mX] [mX] [mX] [fX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’ wherein [mXs]= 2’-( -methyl modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [fXs] =2’- fluoro modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [mX]= 2’-( -methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides, [fX] =2’- fluoro modified nucleotide with phosphodiester linkages to neighboring nucleotides, [MePhosphonate-4O-mX] = 4’-O- monomethylphosphonate-2’-O-m ethyl modified nucleotide, [AdemX-Ls] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide, and [AdemX-L] = Lipid attached to a nucleotide, optionally wherein L is a C16 lipid. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte target gene comprises the modification pattern of

[0290] Sense Strand: 5’- [mXs][mX][mX][mX][mX][mX][mX][fX][fX][fX][fX]

[0291] [mX] [ AdemX-L] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX]- 3 ’

[0292] Hybridized to:

[0293] Antisense Strand: 5’- [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX] [mX] [fX] [mX] [mX] [mX] [fX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’ wherein [mXs]= 2’-( -methyl modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [fXs] =2’- fluoro modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [mX]= 2’-( -methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides, [fX] =2’- fluoro modified nucleotide with phosphodiester linkages to neighboring nucleotides, [MePhosphonate-4O-mX] = 4’-O- monomethylphosphonate-2’-O-m ethyl modified nucleotide, [AdemX-Ls] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide, and [AdemX-L] = Lipid attached to a nucleotide, optionally wherein L is a C16 lipid.

[0294] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte target gene comprises the modification pattern of

[0295] Sense Strand: 5’- [mXs][mX][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX][mX] [AdemX-L] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] -3 ’ Hybridized to:

[0296] Antisense Strand: 5’- [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX] [mX] [fX] [mX] [mX] [mX] [fX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’ wherein [mXs]= 2’-( -methyl modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [fXs] =2’- fluoro modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [mX]= 2’-( -methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides, [fX] =2’- fluoro modified nucleotide with phosphodiester linkages to neighboring nucleotides, [MePhosphonate-4O-mX] = 4’-O- monomethylphosphonate-2’-O-m ethyl modified nucleotide, [AdemX-Ls] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide, and [AdemX-L] = Lipid attached to a nucleotide, optionally wherein L is a C16 lipid.

[0297] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte target gene comprises the modification pattern of Sense Strand: 5’ -[mXs][mX][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX] [mX] [mX] [ AdemX-L] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX]- 3 ’ Hybridized to:

[0298] Antisense Strand: 5’- [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX] [mX] [fX] [mX] [mX] [mX] [fX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’ wherein [mXs]= 2’-( -methyl modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [f s] =2’- fluoro modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [mX]= 2’-( -methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides, [f ] =2’- fluoro modified nucleotide with phosphodiester linkages to neighboring nucleotides, [MePhosphonate-4O-mX] = 4’-0- monomethylphosphonate-2’-O-m ethyl modified nucleotide, [AdemX-Ls] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide, and [AdemX-L] = Lipid attached to a nucleotide, optionally wherein L is a C16 lipid.

[0299] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte target gene comprises the modification pattern of

[0300] Sense Strand: 5’ -[mXs][mX][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX] [mX] [mX] [mX] [mX] [mX] [mX] [AdemX-L] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] -3 ’ Hybridized to:

[0301] Antisense Strand: 5’- [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX] [mX] [fX] [mX] [mX] [mX] [fX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’ wherein [mXs]= 2’-( -methyl modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [fXs] =2’- fluoro modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [mX]= 2’-( -methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides, [fX] =2’- fluoro modified nucleotide with phosphodiester linkages to neighboring nucleotides, [MePhosphonate-4O-mX] = 4’-O- monomethylphosphonate-2’-O-m ethyl modified nucleotide, [AdemX-Ls] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide, and [AdemX-L] = Lipid attached to a nucleotide, optionally wherein L is a C16 lipid.

[0302] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte target gene comprises the modification pattern of Sense Strand: 5’ - [mXs][mX][mX][mX][mX][mX][mX][fX][fX][fX][fX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [ AdemX-L] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] -3 ’ Hybridized to:

[0303] Antisense Strand: 5’- [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX] [mX] [fX] [mX] [mX] [mX] [fX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’ wherein [mXs]= 2’-( -methyl modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [fXs] =2’- fluoro modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [mX]= 2’-( -methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides, [f ] =2’- fluoro modified nucleotide with phosphodiester linkages to neighboring nucleotides, [MePhosphonate-4O-mX] = 4’-0- monomethylphosphonate-2’-O-m ethyl modified nucleotide, [AdemX-Ls] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide, and [AdemX-L] = Lipid attached to a nucleotide, optionally wherein L is a C16 lipid.

[0304] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte target gene comprises the modification pattern of

[0305] Sense Strand: 5’ - [mXs][mX][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [AdemX-L] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] -3 ’ Hybridized to:

[0306] Antisense Strand: 5’- [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX] [mX] [fX] [mX] [mX] [mX] [fX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’ wherein [mXs]= 2’-( -methyl modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [fXs] =2’- fluoro modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [mX]= 2’-( -methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides, [fX] =2’- fluoro modified nucleotide with phosphodiester linkages to neighboring nucleotides, [MePhosphonate-4O-mX] = 4’-O- monomethylphosphonate-2’-O-m ethyl modified nucleotide, [AdemX-Ls] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide, and [AdemX-L] = Lipid attached to a nucleotide, optionally wherein L is a C16 lipid.

[0307] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte target gene comprises the modification pattern of Sense Strand: 5’ -[mXs][mX][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [ AdemX- L] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] - 3 ’ Hybridized to:

[0308] Antisense Strand: 5’- [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX] [mX] [fX] [mX] [mX] [mX] [fX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’ wherein [mXs]= 2’-( -methyl modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [f s] =2’- fluoro modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [mX]= 2’-( -methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides, [f ] =2’- fluoro modified nucleotide with phosphodiester linkages to neighboring nucleotides, [MePhosphonate-4O-mX] = 4’-0- monomethylphosphonate-2’-O-m ethyl modified nucleotide, [AdemX-Ls] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide, and [AdemX-L] = Lipid attached to a nucleotide, optionally wherein L is a C16 lipid.

[0309] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte target gene comprises the modification pattern of

[0310] Sense Strand: 5’ -[mXs][mX][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX]

[0311] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [Adem X-L] [mX] [mX] [mX] [mX] [mX] [mX] [mX] -3 ’

[0312] Hybridized to:

[0313] Antisense Strand: 5’- [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX] [mX] [fX] [mX] [mX] [mX] [fX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’ wherein [mXs]= 2’-( -methyl modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [fXs] =2’- fluoro modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [mX]= 2’-( -methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides, [fX] =2’- fluoro modified nucleotide with phosphodiester linkages to neighboring nucleotides, [MePhosphonate-4O-mX] = 4’-O- monomethylphosphonate-2’-O-m ethyl modified nucleotide, [AdemX-Ls] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide, and [AdemX-L] = Lipid attached to a nucleotide, optionally wherein L is a C16 lipid.

[0314] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte target gene comprises the modification pattern of Sense Strand: 5’ -[mXs][mX][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX][mX ] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [ AdemX-L] [mX] [mX] [mX] [mX] [mX] [mX] -3 ’ Hybridized to:

[0315] Antisense Strand: 5’- [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX] [mX] [fX] [mX] [mX] [mX] [fX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’ wherein [mXs]= 2’-( -methyl modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [f s] =2’- fluoro modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [mX]= 2’-( -methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides, [f ] =2’- fluoro modified nucleotide with phosphodiester linkages to neighboring nucleotides, [MePhosphonate-4O-mX] = 4’-0- monomethylphosphonate-2’-O-m ethyl modified nucleotide, [AdemX-Ls] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide, and [AdemX-L] = Lipid attached to a nucleotide, optionally wherein L is a C16 lipid.

[0316] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte target gene comprises the modification pattern of

[0317] Sense Strand: 5’ -[AdemX-

[0318] Ls] [mX] [mX] [mX] [mX] [mX] [mX] [fX] [fX] [fX] [fX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX]-3 ’ Hybridized to:

[0319] Antisense Strand: 5’- [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX] [mX] [fX] [mX] [mX] [mX] [fX] [mX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’ wherein [mXs]= 2’-( -methyl modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [fXs] =2’- fluoro modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [mX]= 2’-( -methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides, [fX] =2’- fluoro modified nucleotide with phosphodiester linkages to neighboring nucleotides, [MePhosphonate-4O-mX] = 4’-O- monomethylphosphonate-2’-O-m ethyl modified nucleotide, [AdemX-Ls] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide, and [AdemX-L] = Lipid attached to a nucleotide, optionally wherein L is a C16 lipid.

[0320] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and at least one hydrocarbon chain conjugated to a nucleotide at Position 2, Position 3, Position 6, Position 13, Position 14, Position 15, Position 19, Position 20, Position 23, Position 28, Position 29, or Position 30 of the sense strand.

[0321] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the medulla comprises a sense strand comprising a stem-loop at its 3’ end and at least one hydrocarbon chain conjugated to a nucleotide at Position 19, Position 20, Position 23, or Position 28 of the sense strand.

[0322] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the hippocampus comprises a sense strand comprising a stem-loop at its 3’ end and at least one hydrocarbon chain conjugated to a nucleotide at Position 2 of the sense strand.

[0323] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and at least one hydrocarbon chain conjugated to a nucleotide at Position 14, Position 15, Position 19, Position 20, Position 23, Position 28, Position 29, or Position 30 of the sense strand.

[0324] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a blunt-end at its 3’ end and at least one hydrocarbon chain conjugated to a nucleotide at Position 2, Position 3, Position 6, Position 13, Position 14, Position 15, Position 19, or Position 20 of the sense strand.

[0325] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a blunt-end at its 3’ end and at least one hydrocarbon chain conjugated to a nucleotide at Position 1, Position 2, Position 3, Position 5, Position 6, Position 7, Position 9, Position 13, Position 14, Position 15, Position 17, Position 19, or Position 20 of the sense strand.

[0326] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the medulla comprises a sense strand comprising a blunt-end at its 3’ end and at least one hydrocarbon chain conjugated to a nucleotide at Position 2, Position 14, or Position 15 of the sense strand.

[0327] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the medulla comprises a sense strand comprising a blunt-end at its 3’ end and at least one hydrocarbon chain conjugated to a nucleotide at Position 2, Position 7, Position 9, Position 14, Position 15, or Position 19 of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the hippocampus comprises a sense strand comprising a blunt-end at its 3’ end and at least one hydrocarbon chain conjugated to a nucleotide at Position 3 of the sense strand.

[0328] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the frontal cortex comprises a sense strand comprising a blunt-end at its 3’ end and at least one hydrocarbon chain conjugated to a nucleotide at Position 14 of the sense strand.

[0329] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the hypothalamus comprises a sense strand comprising a blunt-end at its 3’ end and at least one hydrocarbon chain conjugated to a nucleotide at Position 7 of the sense strand.

[0330] In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 2 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 3 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 6 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 13 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 14 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 15 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 19 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 20 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 23 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 28 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 29 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 30 of the sense strand.

[0331] In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the medulla a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 19 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the medulla comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 20 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the medulla comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 23 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the medulla comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 28 of the sense strand.

[0332] In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the hippocampus comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 2 of the sense strand.

[0333] In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the frontal cortex comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 14 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the frontal cortex comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 15 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the frontal cortex comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 19 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the frontal cortex comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 20 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the frontal cortex comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 23 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the frontal cortex comprises a sense strand comprising a stemloop at its 3’ end and a lipid conjugated to Position 28 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the frontal cortex comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 29 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the frontal cortex comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 30 of the sense strand.

[0334] In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 2 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 3 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 6 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 13 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 14 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 15 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 19 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 20 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 23 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 28 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 29 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 30 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.

[0335] In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the medulla comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 19 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the medulla comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 20 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the medulla comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 23 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the medulla comprises a sense strand comprising a stem-loop at its 3 ’ end and a lipid conjugated to Position 28 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.

[0336] In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the hippocampus comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 2 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.

[0337] In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the frontal cortex comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 14 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the frontal cortex comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 15 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the frontal cortex comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 19 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the frontal cortex comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 20 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the frontal cortex comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 23 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the frontal cortex comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 28 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the frontal cortex comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 29 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the frontal cortex comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 30 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.

[0338] In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 1 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 2 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 3 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 5 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 6 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 7 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 9 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 13 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 14 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 15 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 17 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 19 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 20 of the sense strand.

[0339] In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the medulla comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 2 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the medulla comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 7 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the medulla comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 9 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the medulla comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 14 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the medulla comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 15 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the medulla comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 19 of the sense strand.

[0340] In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the hippocampus a sense strand comprising a blunt- end at its 3’ end and a lipid conjugated to Position 3 of the sense strand.

[0341] In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the frontal cortex comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 14 of the sense strand. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the hypothalamus comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 7 of the sense strand.

[0342] In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a blunt end at its 3’ end and a lipid conjugated to Position 1 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a blunt end at its 3’ end and a lipid conjugated to Position 2 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a blunt end at its 3’ end and a lipid conjugated to Position 3 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a blunt end at its 3’ end and a lipid conjugated to Position 5 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a blunt end at its 3’ end and a lipid conjugated to Position 6 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a blunt end at its 3’ end and a lipid conjugated to Position 7 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a blunt end at its 3’ end and a lipid conjugated to Position 9 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a blunt end at its 3’ end and a lipid conjugated to Position 13 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord a sense strand comprising a blunt end at its 3’ end and a lipid conjugated to Position 14 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a blunt end at its 3’ end and a lipid conjugated to Position 15 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a blunt end at its 3’ end and a lipid conjugated to Position 17 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a blunt end at its 3’ end and a lipid conjugated to Position 19 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the spinal cord comprises a sense strand comprising a blunt end at its 3’ end and a lipid conjugated to Position 20 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.

[0343] In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the medulla comprises a sense strand comprising a blunt end at its 3’ end and a lipid conjugated to Position 2 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the medulla comprises a sense strand comprising a blunt end at its 3’ end and a lipid conjugated to Position 7 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the medulla comprises a sense strand comprising a blunt end at its 3’ end and a lipid conjugated to Position 9 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the medulla comprises a sense strand comprising a blunt end at its 3’ end and a lipid conjugated to Position 14 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the medulla comprises a sense strand comprising a blunt end at its 3’ end and a lipid conjugated to Position 15 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide. In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the medulla comprises a sense strand comprising a blunt end at its 3’ end and a lipid conjugated to Position 19 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.

[0344] In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the hippocampus comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 3 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.

[0345] In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the frontal cortex comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 14 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.

[0346] In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an oligodendrocyte mRNA in the hypothalamus comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 7 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.

[0347] In some embodiments, the disclosure provides an oligonucleotide (e.g., an RNAi oligonucleotide) for reducing UGT8 expression, wherein the oligonucleotide comprises a sense strand comprising the nucleotide sequence of SEQ ID NO: 71 and an antisense strand comprising the nucleotide sequence of SEQ ID NO: 28.

[0348] In some embodiments, the current disclosure provides an oligonucleotide (e.g., an RNAi oligonucleotide) for reducing UGT8 expression, wherein the oligonucleotide comprises a sense strand comprising the nucleotide sequence of SEQ ID NO: 19 and an antisense strand comprising the nucleotide sequence of SEQ ID NO: 28.

[0349] In some embodiments, the current disclosure provides an oligonucleotide (e.g., an RNAi oligonucleotide) for reducing UGT8 expression, wherein the oligonucleotide comprises a sense strand and an antisense strand comprising nucleotide sequences selected from the group consisting of: a) SEQ ID NOs: 19 and 28, respectively; b) SEQ ID NOs: 20 and 29, respectively; c) SEQ ID NOs: 21 and 30, respectively; d) SEQ ID NOs: 22 and 31, respectively; e) SEQ ID NOs: 23 and 32, respectively; f) SEQ ID NOs: 24 and 33, respectively; g) SEQ ID NOs: 25 and 34, respectively; h) SEQ ID NOs: 26 and 35, respectively; and, i) SEQ ID NOs: 27 and 36, respectively.

[0350] In some embodiments, the current invention provides an oligonucleotide (e.g., an RNAi oligonucleotide) for reducing UGT8 expression, wherein the oligonucleotide comprises a sense strand and an antisense strand comprising nucleotide sequences selected from the group consisting of: a) SEQ ID NOs: 72 and 55, respectively; b) SEQ ID NOs: 73 and 55, respectively; c) SEQ ID NOs: 74 and 55, respectively; d) SEQ ID NOs: 75 and 55, respectively; e) SEQ ID NOs: 76 and 55, respectively; f) SEQ ID NOs: 77 and 55, respectively; g) SEQ ID NOs: 78 and 55, respectively; h) SEQ ID NOs: 79 and 55, respectively; and, i) SEQ ID NOs: 80 and 55, respectively.

[0351] In some embodiments, the current invention provides an oligonucleotide (e.g., an RNAi oligonucleotide) for reducing UGT8 expression, wherein the oligonucleotide comprises a sense strand and an antisense strand comprising nucleotide sequences selected from the group consisting of: a) SEQ ID NOs: 72 and 55, respectively; b) SEQ ID NOs: 73 and 55, respectively; c) SEQ ID NOs: 74 and 55, respectively; d) SEQ ID NOs: 75 and 55, respectively; e) SEQ ID NOs: 76 and 55, respectively; f) SEQ ID NOs: 77 and 55, respectively; g) SEQ ID NOs: 78 and 55, respectively; h) SEQ ID NOs: 79 and 55, respectively; i) SEQ ID NOs: 80 and 55, respectively; j) SEQ ID NOs: 82 and 55, respectively; k) SEQ ID NOs: 83 and 55, respectively; l) SEQ ID NOs: 84 and 55, respectively; m) SEQ ID NOs: 85 and 55, respectively; n) SEQ ID NOs: 86 and 55, respectively; and, o) SEQ ID NOs: 87 and 55, respectively.

[0352] In some embodiments, the current invention provides an oligonucleotide (e.g., an RNAi oligonucleotide) for reducing UGT8 expression, wherein the oligonucleotide comprises a sense strand and an antisense strand comprising nucleotide sequences selected from the group consisting of: a) SEQ ID NOs: 57 and 55, respectively; b) SEQ ID NOs: 58 and 55, respectively; c) SEQ ID NOs: 59 and 55, respectively; d) SEQ ID NOs: 60 and 55, respectively; e) SEQ ID NOs: 61 and 55, respectively; f) SEQ ID NOs: 62 and 55, respectively; g) SEQ ID NOs: 63 and 55, respectively; h) SEQ ID NOs: 64 and 55, respectively; i) SEQ ID NOs: 65 and 55, respectively; j) SEQ ID NOs: 66 and 55, respectively; k) SEQ ID NOs: 67 and 55, respectively; l) SEQ ID NOs: 68 and 55, respectively; and, m) SEQ ID NOs: 69 and 55, respectively.

[0353] General Methods of Providing the Nucleic Acids and Analogues Thereof

[0354] The nucleic acids and analogues thereof comprising lipid conjugate described herein can be made using a variety of synthetic methods known in the art, including standard phosphoramidite methods. Any phosphoramidite synthesis method can be used to synthesize the provided nucleic acids of this disclosure. In certain embodiments, phosphoramidites are used in a solid phase synthesis method to yield reactive intermediate phosphite compounds, which are subsequently oxidized using known methods to produce phosphonate-modified oligonucleotides, typically with a phosphodiester or phosphorothioate internucleotide linkages. The oligonucleotide synthesis of the present disclosure can be performed in either direction: from 5' to 3' or from 3' to 5' using art known methods.

[0355] In certain embodiments, the method for synthesizing a provided nucleic acid comprises (a) attaching a nucleoside or analogue thereof to a solid support via a covalent linkage; (b) coupling a nucleoside phosphoramidite or analogue thereof to a reactive hydroxyl group on the nucleoside or analogue thereof of step (a) to form an internucleotide bond there between, wherein any uncoupled nucleoside or analogue thereof on the solid support is capped with a capping reagent; (c) oxidizing said intemucleotide bond with an oxidizing agent; and (d) repeating steps (b) to (c) iteratively with subsequent nucleoside phosphoramidites or analogue thereof to form a nucleic acid or analogue thereof, wherein at least the nucleoside or analogue thereof of step (a), the nucleoside phosphoramidite or analogue thereof of step (b) or at least one of the subsequent nucleoside phosphoramidites or analogues thereof of step (d) comprises a lipid conjugate moiety as described herein. Typically, the coupling, capping / oxidizing steps and optionally, the deprotecting steps, are repeated until the oligonucleotide reaches the desired length and / or sequence, after which it is cleaved from the solid support. In certain embodiments, an oligonucleotide is prepared comprising 1-3 nucleic acid or analogues thereof comprising lipid conjugates units on a tetraloop.

[0356] In Scheme A below, where a particular protecting group, leaving group, or transformation condition is depicted, one of ordinary skill in the art will appreciate that other protecting groups, leaving groups, and transformation conditions are also suitable and are contemplated. Certain reactive functional groups (e.g., -N(H)-, -OH, etc.) envisioned in the genera in Scheme A requiring additional protection group strategies are also contemplated and is appreciated by those having ordinary skill in the art. Such groups and transformations are described in detail in March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, M. B. Smith and J. March, 5thEdition, John Wiley & Sons, 2001, COMPREHENSIVE ORGANIC TRANSFORMATIONS, (R. C. Larock, 2ndEdition, John Wiley & Sons, 1999), and PROTECTING GROUPS IN ORGANIC SYNTHESIS, (T. W. Greene and P. G. M. Wuts, 3rdedition, John Wiley & Sons, 1999), the entirety of each of which is hereby incorporated herein by reference.

[0357] In certain embodiments, nucleic acids, and analogues thereof of the present disclosure are generally prepared according to Scheme A, Scheme Al and Scheme B set forth below:

[0358] Scheme A: Synthesis of Ligand Conjugated Oligonucleotides of the Disclosure tyl or Lipid

[0359] Scheme Al: Synthesis of Lipid Conjugated Oligonucleotides of the Disclosure

[0360]

[0361] As depicted in Scheme A and Scheme Al above, a nucleic acid or analogue thereof of formula 1-1 is conjugated with one or more ligand / lipophilic compound to form a compound of formula I or la comprising one more ligand / lipid conjugates. Typically, conjugation is performed through an esterification or amidation reaction between a nucleic acid or analogue thereof of formula 1-1 or I-la and one or more adamantyl and / or lipophilic compound (e.g., fatty acid) in series or in parallel by known techniques in the art. Nucleic acid or analogue thereof of formula I or la can then be deprotected to form a compound of formula 1-2 or I-2a and protected with a suitable hydroxyl protecting group (e.g., DMTr) to form a compound of formula 1-3 or I-3a. In one aspect, nucleic acid-ligand conjugates of formula 1-3 or I-3a can be covalently attached to a solid support (e.g., through a succinic acid linking group) to form a solid support nucleic acid-ligand conjugate or analogue thereof of formula 1-4 or I-4a comprising one or more adamantyl and / or lipid conjugate. In another aspect, a nucleic acidligand conjugates of formula 1-3 or I-3a can react with a P(III) forming reagent (e.g., 2- cyanoethyl M -di-i sopropylchlorophosphoramidite) to form a nucleic acid or analogue thereof of formula 1-5 or I-5a comprising a P(III) group. A nucleic acid-ligand conjugate or analogue thereof of formula 1-5 or I-5a can then be subjected to oligomerization forming conditions preformed using known and commonly applied processes to prepare oligonucleotides in the art. For example, the compound of formula 1-5 or I-5a is coupled to a solid supported nucleic acid-ligand conjugate or analogue thereof bearing a 5 ’-hydroxyl group. Further steps can comprise one or more deprotections, couplings, phosphite oxidation, and / or cleavage from the solid support to provide an oligonucleotide of various nucleotide lengths, including one or more lipid conjugate nucleotide units represented by a compound of formula II-l or Il-Ia. Each of B, E, L, ligand, LC, n, PG1, PG2, PG4, R1, R2, R3, X, X1, X2, X3, and Z is as defined above and described herein.

[0362] Scheme B: Post-Synthetic Lipid Conjugation of Oligonucleotides of the Disclosure

[0363] Lipid moiety (LC)

[0364] As depicted in Scheme B above, a nucleic acid or analogue thereof of formula 1-1 can be deprotected to form a compound of formula 1-6, protected with a suitable hydroxyl protecting group (e.g., DMTr) to form a compound of formula 1-7, and reacted with a P(III) forming reagent (e.g., 2-cyanoethyl A,A-di-isopropylchlorophosphoramidite) to form a nucleic acid or analogue thereof of formula 1-8 comprising a P(III) group. Next, a nucleic acid or analogue thereof of formula 1-8 is subjected to oligomerization forming conditions preformed using known and commonly applied processes to prepare oligonucleotides in the art. For example, the compound of formula 1-8 is coupled to a solid supported nucleic acid or analogue thereof bearing a 5 ’-hydroxyl group. Further steps can comprise one or more deprotections, couplings, phosphite oxidation, and / or cleavage from the solid support to provide an oligonucleotide of various nucleotide lengths represented by a compound of formula II-2. An oligonucleotide of formula II-2 can then be conjugated with one or more ligands e.g., adamantyl, or lipophilic compound (e.g, fatty acid) to form a compound of formula II-l comprising one or more ligand conjugates. Typically, conjugation is performed through an esterification or amidation reaction between a nucleic acid or analogue thereof of formula II-2 and one or more adamantyl or fatty acid in series or in parallel by known techniques in the art. Each of B, E, L, ligand, LC, n, PG1, PG2, PG4, R1, R2, R3, X, X1, X2, X3, and Z is as defined above and described herein.

[0365] In certain embodiments, nucleic acids, and analogues thereof of the present disclosure are prepared according to Scheme C and Scheme D set forth below:

[0366] Scheme C: Synthesis of Lipid Conjugated Oligonucleotides of the Disclosure As depicted in Scheme C above, a nucleic acid or analogue thereof of formula Cl is protected to form a compound of formula C2. Nucleic acid or analogue thereof of formula C2 is then alkylated (e.g., using DMSO and acetic acid via the Pummerer rearrangement) to form a monothioacetal compound of formula C3. Next, nucleic acid or analogue thereof of formula C3 is coupled with C4 under appropriate conditions (e.g., mild oxidizing conditions) to form a nucleic acid or analogue thereof of formula C5. Nucleic acid or analogue thereof of formula C5 can then be deprotected to form a compound of formula C6 and coupled with a ligand (adamantyl or lipophilic compound (e.g., a fatty acid)) of formula C7 under appropriate amide forming conditions (e.g., HATU, DIPEA), to form a nucleic acid-ligand conjugate or analogue thereof of formula I-b comprising a lipid conjugate of the disclosure. Nucleic acid-ligand conjugate or analogue thereof of formula I-b can then be deprotected to form a compound of formula C8 and protected with a suitable hydroxyl protecting group (e.g., DMTr) to form a compound of formula C9. In one aspect, nucleic acid, or analogue thereof of formula C9 can be covalently attached to a solid support (e.g., through a succinic acid linking group) to form a solid support nucleic acid-ligand conjugate or analogue thereof of formula CIO comprising a ligand conjugate (adamantyl or lipid moiety) of the disclosure. In another aspect, a nucleic acid-ligand conjugate or analogue thereof of formula C9 can reacted with a P(III) forming reagent (e.g., 2-cyanoethyl A,A-di-isopropylchlorophosphoramidite) to form a nucleic acidligand conjugate or analogue thereof of formula Cll comprising a P(III) group. A nucleic acidligand conjugate or analogue thereof of formula Cll can then be subjected to oligomerization forming conditions preformed using known and commonly applied processes to prepare oligonucleotides in the art. For example, the compound of formula Cll is coupled to a solid supported nucleic acid-ligand conjugate or analogue thereof bearing a 5 ’-hydroxyl group. Further steps can comprise one or more deprotections, couplings, phosphite oxidation, and / or cleavage from the solid support to provide an oligonucleotide of various nucleotide lengths, including one or more adamantyl and / or lipid conjugate nucleotide units represented by a compound of formula II-b-3. Each of B, E, L2, PG1, PG2, PG3, PG4, R1, R2, R3, R4, R5, X1, X2, X3, V, W, and Z is as defined above and described herein.

[0367] Scheme D: Post-Synthetic Lipid Conjugation of Oligonucleotides of the Disclosure

[0368] Each of B, E, L2, PG1, PG2, PG3, PG4, R1, R2, R3, R4, R5, X1, X2, X3, V, W, and Z is as defined above and described herein. As depicted in Scheme D above, a nucleic acid or analogue thereof of formula C5 can be selectively deprotected to form a compound of formula DI, 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 A,A-di- isopropylchlorophosphoramidite) to form a nucleic acid or analogue thereof of formula D3. Next, a nucleic acid or analogue thereof of formula D3 is subjected to oligomerization forming conditions preformed using known and commonly applied processes to prepare oligonucleotides in the art. For example, the compound of formula D3 is coupled to a solid supported nucleic acid or analogue thereof bearing a 5 ’-hydroxyl group. Further steps can comprise one or more deprotections, couplings, phosphite oxidation, and / or cleavage from the solid support to provide an oligonucleotide of various nucleotide lengths, represented by a compound of formula D4. An 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 a fatty acid) under appropriate amide forming conditions (e.g, HATU, DIPEA), to form an oligonucleotide of formula II-b-3 comprising a ligand (e.g., adamantyl or a fatty acid) conjugate of the disclosure.

[0369] One of skill in the art will appreciate that various functional groups present in the nucleic acid or analogues thereof of the 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 for example, “MARCH’S ADVANCED ORGANIC CHEMISTRY”, (5thEd., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001), the entirety of each of which is herein incorporated by reference. Such interconversions may require one or more of the aforementioned techniques, and certain methods for synthesizing the provided nucleic acids of the disclosure are described below in the Exemplification.

[0370] In some embodiments, the present disclosure provides a method for preparing an oligonucleotide comprising one or more lipid conjugate, said lipid conjugate unit represent by formula II-a-1: or a pharmaceutically acceptable salt thereof, comprising the steps of:

[0371] (a) providing a nucleic acid or analogue thereof of formula I-5a:

[0372] I-5a or salt thereof, and

[0373] (b) oligomerizing said compound of formula I-5a to form a compound of formula Il-la, wherein each of B, E, L, LC, n, PG4, R1, R2, R3, X, X1, X2, X3, E, and Z is as defined above and described herein.

[0374] In step (b) above, oligomerizing refers to preforming oligomerization forming conditions using known and commonly applied processes to prepare oligonucleotides in the art. For example, the compound of formula I-5a is coupled to a solid supported nucleic acid or analogue thereof bearing a 5 ’-hydroxyl group. Further steps can comprise one or more deprotections, couplings, phosphite oxidation, and cleavage from the solid support to provide an oligonucleotide of various nucleotide lengths, represented by a compound of formula Il-la comprising a lipid conjugate of the disclosure.

[0375] In some embodiments, the present disclosure provides a method for preparing an oligonucleotide comprising one or more lipid conjugate, further comprising preparing a nucleic acid or analogue thereof of formula I-5a: or a salt thereof, comprising the steps of:

[0376] (a) providing a nucleic acid or analogue thereof of formula la: or salt thereof,

[0377] (b) deprotecting said nucleic acid or analogue thereof of formula la to form a compound of formula I-2a: or salt thereof,

[0378] (c) protecting said nucleic acid or analogue thereof of formula 1-2 to form a compound of formula I-3a: or salt thereof, and

[0379] (d) treating said nucleic acid or analogue thereof of formula I-3a with a P(III) forming reagent to form a nucleic acid or analogue thereof of formula I-5a, wherein each of B, E, L, LC, n, PG4, R1, R2, R3, X, X1, X2, X3, E, and Z is as defined above and described herein.

[0380] In step (b) above, PG1and PG2of a compound of formula la comprise silyl ethers or cyclic silylene derivatives that can be removed under acidic conditions or with fluoride anion. Examples of reagents providing fluoride anion for the removal of silicon-based protecting groups include hydrofluoric acid, hydrogen fluoride pyridine, triethylamine trihydrofluoride, tetra-A-butylammonium fluoride, and the like.

[0381] In step (c) above, a compound of formula I-2a is protected with a suitable hydroxyl protecting group. In certain embodiments, the protecting group PG4used for protection of the 5 ’-hydroxyl group of a compound of formula I-2a includes an acid labile protecting group 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 group is suitable for deprotection during both solutionphase and solid-phase synthesis of acid-sensitive nucleic acids or analogues thereof using for example, dichloroacetic acid or trichloroacetic acid.

[0382] In step (d) above, a compound of formula I-3a is treated with a P(III) forming reagent to afford a compound of formula I-5a. In the context of the present disclosure, a P(III) forming reagent is a phosphorus reagent that is reacted to for a phosphorus (III) compound. In some embodiments, the P(IH) forming reagent is 2-cyanoethyl N,N- diisopropylchlorophosphoramidite or 2-cyanoethyl phosphorodichloridate. In certain embodiments, the P(IH) forming reagent is 2-cyanoethyl N,N- diisopropylchlorophosphoramidite. One of ordinary skill would recognize that the displacement of a leaving group in a P(III) forming reagent by X1of a compound of formula I-3a is achieved either with or without the presence 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 7V,7V-dimethylphosphoramic dichloride as a P(V) forming reagent.

[0383] In some embodiments, the present disclosure provides a method for preparing an oligonucleotide comprising one or more lipid conjugates, further comprising preparing a nucleic acid-lipid conjugate or analogue thereof of formula la: or a salt thereof, comprising the steps of:

[0384] (a) providing a nucleic acid or analogue thereof of formula 1-1:

[0385] 1-1 or salt thereof, and,

[0386] (b) conjugating one or more lipophilic compounds to a nucleic acid or analogue thereof of formula 1-1 to form a nucleic acid or analogue thereof of formula la comprising one or more lipid conjugates, wherein : each of B, E, L, LC, n, PG1, PG2, Rl, R2, X, X1, and Z is as defined above and described herein.

[0387] In step (b) above, a nucleic acid or analogue thereof of formula I-la is conjugated with one or more lipophilic compounds to form a compound of formula la comprising one more lipid conjugates of the disclosure. Typically, conjugation is performed through an esterification or amidation reaction between a nucleic acid or analogue thereof of formula I-la and one or more fatty acids in series or in parallel by known techniques in the art. In certain embodiments, conjugation is performed under suitable amide forming conditions to afford a compound of formula I comprising one more lipid conjugates. Suitable amide forming conditions can include the use of an amide coupling reagent known in the art such as, but not limited to HATU, PyBOP, DCC, DIC, EDC, HBTU, HCTU, PyAOP, PyBrOP, BOP, BOP-CI, DEPBT, T3P, TATU, TBTU, TNTU, TOTU, TPTU, TSTU, or TDBTU. Alternatively, conjugation of a lipophilic compound can be accomplished by any one of the cross-coupling technologies described in Table A herein.

[0388] In some embodiments, the present disclosure provides a method for preparing an oligonucleotide comprising one or more lipid conjugate, said lipid conjugate unit represent by formula II-l: or a pharmaceutically acceptable salt thereof, comprising the steps of:

[0389] (a) providing an oligonucleotide of formula II-2: or salt thereof, and,

[0390] (b) conjugating one or more lipophilic compounds to an oligonucleotide of formula II-2 to form an oligonucleotide of formula II-l comprising one or more lipid conjugates. In step (b) above, an oligonucleotide of formula II-2 is conjugated with one or more lipophilic compounds to form an oligonucleotide of formula II-l comprising one more lipid conjugates of the disclosure. Typically, conjugation is performed through an esterification or amidation reaction between an oligonucleotide of formula II-2 and one or more fatty acids in series or in parallel by known techniques in the art. In certain embodiments, conjugation is performed under suitable amide forming conditions to afford an oligonucleotide of formula II-l comprising one more lipid conjugates. Suitable amide forming conditions can include the use of an amide coupling reagent known in the art such as, but not limited to HATU, PyBOP, DCC, DIC, EDC, HBTU, HCTU, PyAOP, PyBrOP, BOP, BOP-CI, DEPBT, T3P, TATU, TBTU, TNTU, TOTU, TPTU, TSTU, or TDBTU. Alternatively, conjugation of a lipophilic compound can be accomplished by any one of the cross-coupling technologies described in Table A herein.

[0391] In some embodiments, the present disclosure provides a method for preparing an oligonucleotide comprising a unit represent by formula II-2: or a pharmaceutically acceptable salt thereof, comprising the steps of:

[0392] (a) providing a nucleic acid or analogue thereof of formula 1-8:

[0393] 1-8 or salt thereof, and

[0394] (b) oligomerizing said compound of formula 1-8 to form a compound of formula II-2.

[0395] In step (b) above, oligomerizing refers to preforming oligomerization forming conditions using known and commonly applied processes to prepare oligonucleotides in the art. For example, the compound of formula 1-8 is coupled to a solid supported nucleic acid or analogue thereof bearing a 5 ’-hydroxyl group. Further steps can comprise one or more deprotections, couplings, phosphite oxidation, and cleavage from the solid support to provide an oligonucleotide of various nucleotide lengths, represented by a compound of formula II-2.

[0396] In some embodiments, the present disclosure provides a method for preparing a nucleic acid or analogue thereof comprising one or more lipid conjugate, further comprising preparing a nucleic acid or analogue thereof of formula 1-8:

[0397] 1-8 or a salt thereof, comprising the steps of:

[0398] (a) providing a nucleic acid or analogue thereof of formula 1-1: or salt thereof,

[0399] (b) deprotecting said nucleic acid or analogue thereof of formula 1-1 to form a compound of formula 1-6: or salt thereof,

[0400] (c) protecting said nucleic acid or analogue thereof of formula 1-6 to form a compound of formula 1-7: or salt thereof, and

[0401] (d) treating said nucleic acid or analogue thereof of formula 1-7 with a P(III) forming reagent to form a nucleic acid or analogue thereof of formula 1-8, In step (b) above, PG1and PG2of a compound of formula 1-1 comprise silyl ethers or cyclic silylene derivatives that can be removed under acidic conditions or with fluoride anion. Examples of reagents providing fluoride anion for the removal of silicon-based protecting groups include hydrofluoric acid, hydrogen fluoride pyridine, triethylamine trihydrofluoride, tetra-7V-butylammonium fluoride, and the like.

[0402] In step (c) above, a compound of formula 1-6 is protected with a suitable hydroxyl protecting group. In certain embodiments, the protecting group PG4used for protection of the 5 ’-hydroxyl group of a compound of formula 1-6 includes an acid labile protecting group 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 group is suitable for deprotection during both solutionphase and solid-phase synthesis of acid-sensitive nucleic acids or analogues thereof using for example, dichloroacetic acid or trichloroacetic acid.

[0403] In step (d) above, a compound of formula 1-7 is treated with a P(III) forming reagent to afford a compound of formula 1-8. In the context of the present disclosure, a P(III) forming reagent is a phosphorus reagent that is reacted to for a phosphorus (III) compound. In some embodiments, the P(IH) forming reagent is 2-cyanoethyl N,N- diisopropylchlorophosphoramidite or 2-cyanoethyl phosphorodichloridate. In certain embodiments, the P(IH) forming reagent is 2-cyanoethyl N,N- diisopropylchlorophosphoramidite. One of ordinary skill would recognize that the displacement of a leaving group in a P(III) forming reagent by X1of a compound of formula 1-7 is achieved either with or without the presence 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 7V,7V-dimethylphosphoramic dichloride as a P(V) forming reagent.

[0404] In some embodiments, the present disclosure provides a method for preparing an oligonucleotide-ligand conjugate comprising one or more adamantyl and / or lipid moieties, said conjugate unit represented by formula II-b-3:

[0405] II-b-3 or a pharmaceutically acceptable salt thereof, comprising the steps of:

[0406] (a) providing a nucleic acid-ligand conjugate or analogue thereof of formula Cll: or salt thereof, and (b) oligomerizing said compound of formula Cll to form a compound of formula II-b-3, In step (b) above, oligomerizing refers to preforming oligomerization forming conditions using known and commonly applied processes to prepare oligonucleotides in the art. For example, the compound of formula Cll is coupled to a solid supported nucleic acid or analogue thereof bearing a 5 ’-hydroxyl group. Further steps can comprise one or more deprotections, couplings, phosphite oxidation, and cleavage from the solid support to provide an oligonucleotide-ligand conjugate of various nucleotide lengths, with one or more nucleic acid-ligand conjugate units, wherein each unit is represented by a compound of formula II-b-3 comprising an adamantyl or lipid moiety of the disclosure.

[0407] In some embodiments, the method for preparing an oligonucleotide of formula II-b-3 comprising one or more lipid conjugate, further comprises preparing a nucleic acid-ligand conjugate or analogue thereof of formula Cll: or a salt thereof, comprising the steps of:

[0408] (a) providing a nucleic acid-ligand conjugate or analogue thereof of formula I-b:

[0409] I-b or salt thereof,

[0410] (b) deprotecting said nucleic acid-ligand conjugate or analogue thereof of formula I-b to form a compound of formula C8: or salt thereof, (c) protecting said nucleic acid-ligand conjugate or analogue thereof of formula C8 to form a compound of formula C9: or salt thereof, and

[0411] (d) treating said nucleic acid-ligand conjugate or analogue thereof of formula C9 with a P(III) forming reagent to form a nucleic acid or analogue thereof of formula Cll. In step (b) above, PG1and PG2of a compound of formula I-b comprise silyl ethers or cyclic silylene derivatives that can be removed under acidic conditions or with fluoride anion. Examples of reagents providing fluoride anion for the removal of silicon-based protecting groups include hydrofluoric acid, hydrogen fluoride pyridine, triethylamine trihydrofluoride, tetra-7V- butylammonium fluoride, and the like.

[0412] In step (c) above, a compound of formula C8 is protected with a suitable hydroxyl protecting group. In certain embodiments, the protecting group PG4used for protection of the 5 ’-hydroxyl group of a compound of formula C8 includes an acid labile protecting group 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 group is suitable for deprotection during both solutionphase and solid-phase synthesis of acid-sensitive nucleic acids or analogues thereof using for example, dichloroacetic acid or trichloroacetic acid.

[0413] In step (d) above, a compound of formula C9 is treated with a P(III) forming reagent to afford a compound of formula Cll. In the context of the present disclosure, a P(III) forming reagent is a phosphorus reagent that is reacted to for a phosphorus (III) compound. In some embodiments, the P(IH) forming reagent is 2-cyanoethyl N,N- diisopropylchlorophosphoramidite or 2-cyanoethyl phosphorodichloridate. In certain embodiments, the P(IH) forming reagent is 2-cyanoethyl N,N- diisopropylchlorophosphoramidite. One of ordinary skill would recognize that the displacement of a leaving group in a P(III) forming reagent by X1of a compound of formula C9 is achieved either with or without the presence 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 A,A-dimethylphosphoramic dichloride as a P(V) forming reagent.

[0414] 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 each comprising one or more adamantyl or lipid moieties, further comprising preparing a nucleic acid-ligand conjugate or analogue thereof of formula I-b:

[0415] I-b or a salt thereof, comprising the steps of:

[0416] (a) providing a nucleic acid-ligand conjugate or analogue thereof of formula C6: or salt thereof, and,

[0417] (b) conjugating a lipophilic compound to a nucleic acid or analogue thereof of formula C6 to form a nucleic acid-ligand conjugate or analogue thereof of formula I-b comprising one or more adamantyl and / or lipid conjugates. In step (b) above, conjugation is performed under suitable amide forming conditions to afford a compound of formula I-b comprising an adamantyl and / or lipid conjugate. Suitable amide forming conditions can include the use of an amide coupling reagent known in the art such as, but not limited to HATU, PyBOP, DCC, DIC, EDC, HBTU, HCTU, PyAOP, PyBrOP, BOP, BOP-CI, DEPBT, T3P, TATU, TBTU, TNTU, TOTU, TPTU, TSTU, or TDBTU. In certain embodiments, the amide forming conditions comprise HATU and DIPEA or TEA.

[0418] In certain embodiments, a nucleic acid-ligand conjugate or analogue thereof of formula C6 is provided in salt form (e.g., a fumarate salt) and is first converted to the free base (e.g., using sodium bicarbonate) before preforming the conjugation step.

[0419] 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, further comprises preparing a nucleic acid-ligand conjugate or analogue thereof of formula C6:

[0420] C6 or a salt thereof, comprising the steps of:

[0421] (a) providing a nucleic acid or analogue thereof of formula Cl: or salt thereof, and,

[0422] (b) protecting said nucleic acid or analogue thereof of formula Cl to form a compound of formula C2: or salt thereof,

[0423] (c) alkylating said nucleic acid or analogue thereof of formula C2 to form a compound of formula C3: or salt thereof,

[0424] (d) substituting said nucleic acid or analogue thereof of formula C3 with a compound of formula C4:

[0425] C4 or salt thereof, to form a compound of formula C5:

[0426] C5 or salt thereof,

[0427] (e) deprotecting said nucleic acid or analogue thereof of formula C5 to form a nucleic acidligand conjugate or analogue thereof of formula C6. In step (b) above, PG1and PG2groups of formula C2 are taken together with their intervening atoms to 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-tetraisopropylidisiloxanylidene, a cyclic carbonate, a cyclic boronate, and cyclic monophosphate derivatives based on cyclic adenosine monophosphate (i.e., cAMP). In certain embodiments, the cyclic diol protection group is 1, 1,3,3- tetraisopropylidisiloxanylidene prepared from the reaction of a diol of formula Cl and 1,3- dichloro-l,l,3,3-tetraisopropyldisiloxane under basic conditions.

[0428] In step (c) above, a nucleic acid or analogue thereof of formula C2 is alkylated with a mixture of DMSO and acetic anhydride under acidic conditions. In certain embodiments, when -V-H is a hydroxyl group, the mixture of DMSO and acetic anhydride in the presence of acetic acid forms (methylthio)methyl acetate in situ via the Pummerer rearrangement which then reacts with the hydroxyl group of the nucleic acid or analogue thereof of formula C2 to provide a monothioacetal functionalized fragment nucleic acid or analogue thereof of formula C3.

[0429] In step (d) above, substitution of the thiomethyl group of a nucleic acid or analogue thereof of formula C3 using a nucleic acid or analogue thereof of formula C4 affords a nucleic acid or analogue thereof of formula C4. In certain embodiments, substitution occurs under mild oxidizing and / or acidic conditions. In some embodiments, V is oxygen. In some embodiments, the mild oxidation reagent includes a mixture of elemental iodine and hydrogen peroxide, urea hydrogen peroxide complex, silver nitrate / silver sulfate, sodium bromate, ammonium peroxodi sulfate, tetrabutylammonium peroxy di sulfate, Oxone®, Chloramine T, Selectfluor®, Selectfluor® II, sodium hypochlorite, or potassium iodate / sodium periodiate. In certain embodiments, the mild oxidizing agent includes N-iodosuccinimide, N- bromosuccinimide, N-chlorosuccinimide, l,3-diiodo-5,5-dimethylhydantion, pyridinium tribromide, iodine monochloride or complexes thereof, etc. Acids that are typically used under mild oxidizing condition include sulfuric acid, p-toluenesulfonic acid, trifluoromethanesulfonic acid, methanesulfonic acid, and trifluoroacetic acid. In certain embodiments, the mild oxidation reagent includes a mixture of N-iodosuccinimide and trifluoromethanesulfonic acid.

[0430] In step (e) above, removal of PG3and optionally R4(when R4is a suitable amine protecting group) of a nucleic acid-ligand conjugate or analogue thereof of formula C5 affords a nucleic acid-ligand conjugate or analogue thereof of formula C6 or a salt thereof. In some embodiments, PG3and / or R4comprise carbamate derivatives that can be removed under acidic or basic conditions. In certain embodiments, the protecting groups (e.g., both PG3and R4or either of PG3or R4independently) of a nucleic acid-ligand conjugate or analogue thereof of formula C5 are removed by acid hydrolysis. It will be appreciated that upon acid hydrolysis of the protecting groups of a nucleic acid-ligand conjugate or analogue thereof of formula C5, a salt of formula C6 thereof is formed. For example, when an acid-labile protecting group of a nucleic acid-ligand conjugate or analogue thereof of formula C5 is removed by treatment with an acid such as hydrochloric acid, then the resulting amine compound would be formed as its hydrochloride salt. One of ordinary skill in the art would recognize that a wide variety of acids are useful for removing amino protecting groups that are acid-labile and therefore a wide variety of salt forms of a nucleic acid or analogue thereof of formula C6 are contemplated.

[0431] In other embodiments, the protecting groups (e.g., both PG3and R4or either of PG3or R4independently) of a nucleic acid or analogue thereof of formula C5 are removed by base hydrolysis. For example, Fmoc and trifluoroacetyl protecting groups can be removed by treatment with base. One of ordinary skill in the art would recognize that a wide variety of bases are useful for removing amino protecting groups that are base-labile. In some embodiments, a base is piperidine. In some embodiments, a base is 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU). In certain embodiments, a nucleic acid-ligand conjugate or analogue thereof of formula C5 is deprotected under basic conditions followed by treating with an acid to form a salt of formula C6. In certain embodiments, the acid is fumaric acid the salt of formula C6 is the fumarate.

[0432] In some embodiments, the present disclosure provides a method for preparing an oligonucleotide-ligand conjugate comprising one or more nucleic acid-ligand conjugate, said nucleic acid-ligand conjugate unit represented by formula II-b-3:

[0433] II-b-3 or a pharmaceutically acceptable salt thereof, comprising the steps of:

[0434] (a) providing an oligonucleotide of formula D5: or salt thereof, and,

[0435] (b) conjugating one or more adamantyl or lipophilic compounds to an oligonucleotide of formula D5 to form an oligonucleotide-ligand conjugate of formula II-b-3 comprising one or more nucleic acid-ligand conjugate units. In step (b) above, conjugation is performed under suitable amide forming conditions to afford a compound of formula D5 comprising an adamantyl or lipid conjugate. Suitable amide forming conditions can include the use of an amide coupling reagent known in the art such as, but not limited to HATU, PyBOP, DCC, DIC, EDC, HBTU, HCTU, PyAOP, PyBrOP, BOP, BOP-CI, DEPBT, T3P, TATU, TBTU, TNTU, TOTU, TPTU, TSTU, or TDBTU. In certain embodiments, the amide forming conditions comprise HATU and DIPEA or TEA.

[0436] In some embodiments, the present disclosure provides a method for preparing an oligonucleotide-ligand conjugate comprising a unit represent by formula D5:

[0437] D5 or a salt thereof, comprising the steps of: (a) providing a nucleic acid-ligand conjugate or analogue thereof of formula D4: or salt thereof, and

[0438] (b) deprotecting said compound of formula D4 to form a compound of formula D5. In step (b) above, removal of PG3and optionally R4(when R4is a suitable amine protecting group) of an oligonucleotide of formula D4 affords an oligonucleotide-ligand conjugate of formula D5 or a salt thereof. In some embodiments, PG3and / or R4comprise carbamate derivatives that can be removed under acidic or basic conditions. In certain embodiments, the protecting groups (e.g., both PG3and R4or either of PG3or R4independently) of an oligonucleotide-ligand conjugate of formula D4 are removed by acid hydrolysis. It will be appreciated that upon acid hydrolysis of the protecting groups of an oligonucleotide-ligand conjugate of formula D4, a salt of formula D5 thereof is formed. For example, when an acid-labile protecting group of an oligonucleotide of formula D4 is removed by treatment with an acid such as hydrochloric acid, then the resulting amine compound would be formed as its hydrochloride salt. One of ordinary skill in the art would recognize that a wide variety of acids are useful for removing amino protecting groups that are acid-labile and therefore a wide variety of salt forms of a nucleic acid-ligand conjugate unit or analogue thereof of formula D5 are contemplated.

[0439] In other embodiments, the protecting groups (e.g., both PG3and R4or either of PG3or R4independently) of an oligonucleotide-ligand conjugate of formula D4 are removed by base hydrolysis. For example, Fmoc and trifluoroacetyl protecting groups can be removed by treatment with base. One of ordinary skill in the art would recognize that a wide variety of bases are useful for removing amino protecting groups that are base-labile. In some embodiments, a base is piperidine. In some embodiments, a base is 1,8- diazabicy clo[5.4.0]undec-7-ene (DBU) .

[0440] In some embodiments, the present disclosure provides a method for preparing an oligonucleotide-ligand conjugate comprising one or more nucleic acid-ligand conjugate unit with one or more adamantyl and / or lipid moiety, said conjugate unit represented by formula D4: or a pharmaceutically acceptable salt thereof, comprising the steps of:

[0441] (a) providing a nucleic acid or analogue thereof of formula D3:

[0442] D3 or salt thereof, and

[0443] (b) oligomerizing said compound of formula D3 to form a compound of formula D4,

[0444] In step (b) above, oligomerizing refers to preforming oligomerization forming conditions using known and commonly applied processes to prepare oligonucleotides in the art. For example, the nucleic acid or analogue thereof of formula D3 is coupled to a solid supported nucleic acid or analogue thereof bearing a 5 ’-hydroxyl group. Further steps can comprise one or more deprotections, couplings, phosphite oxidation, and cleavage from the solid support to provide an oligonucleotide of various nucleotide lengths, represented by a compound of formula D4 comprising an adamantyl or lipid conjugate of the disclosure.

[0445] In some embodiments, the present disclosure provides a method for preparing a nucleic acid or analogue thereof comprising one or more lipid conjugate, further comprising preparing a nucleic acid or analogue thereof of formula D3:

[0446] D3 or a salt thereof, comprising the steps of:

[0447] (a) providing a nucleic acid or analogue thereof of formula C5:

[0448] C5 or salt thereof,

[0449] (b) deprotecting said nucleic acid or analogue thereof of formula C5 to form a compound of formula DI:

[0450] DI or salt thereof,

[0451] (c) protecting said nucleic acid or analogue thereof of formula DI to form a nucleic acid or analogue thereof of formula D2:

[0452] D2 or salt thereof, and

[0453] (d) treating said nucleic acid or analogue thereof of formula D2 with a P(III) forming reagent to form a nucleic acid or analogue thereof of formula D3. In step (b) above, PG1and PG2of a nucleic acid or analogue thereof of formula C5 comprise silyl ethers or cyclic silylene derivatives that can be removed under acidic conditions or with fluoride anion. Examples of reagents providing fluoride anion for the removal of silicon-based protecting groups include hydrofluoric acid, hydrogen fluoride pyridine, triethylamine trihydrofluoride, tetra-7V- butylammonium fluoride, and the like.

[0454] In step (c) above, a nucleic acid or analogue thereof of formula DI is protected with a suitable hydroxyl protecting group. In certain embodiments, the protecting group PG4used for protection of the 5 ’-hydroxyl group of a compound of formula DI includes an acid labile protecting group 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 group is suitable for deprotection during both solution-phase and solid-phase synthesis of acid-sensitive nucleic acids or analogues thereof using for example, di chloroacetic acid or trichloroacetic acid.

[0455] In step (d) above, a nucleic acid or analogue thereof of formula D2 is treated with a P(III) forming reagent to afford a compound of formula D3. In the context of the present disclosure, a P(III) forming reagent is a phosphorus reagent that is reacted to for 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(IH) forming reagent is 2-cyanoethyl N,N- diisopropylchlorophosphoramidite. One of ordinary skill would recognize that the displacement of a leaving group in a P(III) forming reagent by X1of a compound of formula D2 is achieved either with or without the presence 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 7V,7V-dimethylphosphoramic dichloride as a P(V) forming reagent.

[0456] Formulations

[0457] Various formulations have been developed to facilitate oligonucleotide use. For example, oligonucleotides (e.g., lipid-conjugated RNAi oligonucleotides) can be delivered to a subject or a cellular environment using a formulation that minimizes degradation, facilitates delivery and / or uptake, or provides another beneficial property to the oligonucleotides in the formulation. In some embodiments, provided herein are compositions comprising oligonucleotides (e.g., lipid-conjugated RNAi oligonucleotides) reduce the expression of a target mRNA (e.g., a target mRNA expressed in an oligodendrocyte of the CNS). Such compositions can be suitably formulated such that when administered to a subject, either into the immediate environment of a target cell or systemically, a sufficient portion of the oligonucleotides enter the cell to reduce target gene expression. Any variety of suitable oligonucleotide formulations can be used to deliver oligonucleotides for the reduction of oligodendrocyte target gene expression as disclosed herein. In some embodiments, an oligonucleotide is formulated in buffer solutions such as phosphate buffered saline solutions, liposomes, micellar structures, and capsids.

[0458] In some embodiments, the formulations herein comprise an excipient. In some embodiments, an excipient confers to a composition improved stability, improved absorption, improved solubility and / or therapeutic enhancement of the active ingredient. In some embodiments, an excipient is a buffering agent (e.g., sodium citrate, sodium phosphate, a tris base, or sodium hydroxide) or a vehicle (e.g., a buffered solution, petrolatum, dimethyl sulfoxide, or mineral oil). In some embodiments, an oligonucleotide is lyophilized for extending its shelf-life and then made into a solution before use (e.g., administration to a subject). Accordingly, an excipient in a composition comprising any one of the oligonucleotides described herein may be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol or polyvinylpyrrolidone) or a collapse temperature modifier (e.g., dextran, Ficoll™ or gelatin). Likewise, the oligonucleotides herein may be provided in the form of their free acids.

[0459] In some embodiments, a 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, intrathecal), oral (e.g., inhalation), transdermal (e.g., topical), transmucosal and rectal administration. In some embodiments, a pharmaceutical composition is formulated for delivery to the central nervous system (e.g., intrathecal, epidural). In some embodiments, a pharmaceutical composition is formulated for delivery to the eye (e.g., ophthalmic, intraocular, subconjunctival, intravitreal, retrobulbar, intracam eral).

[0460] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, 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 isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Sterile injectable solutions can be prepared by incorporating the oligonucleotides in a required amount in a selected solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.

[0461] In some embodiments, a composition may contain at least about 0.1% of the therapeutic agent (e.g., an lipid-conjugated RNAi oligonucleotide herein) or more, although the percentage of the active ingredient(s) may be between about 1% to about 80% or more of the weight or volume of the total composition. Factors such as solubility, bioavailability, biological halflife, route of administration, product shelflife, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

[0462] Methods of Use

[0463] Reducing Target Gene Expression

[0464] In some embodiments, the disclosure provides methods for contacting or delivering to a cell or population of cells an effective amount of any of the lipid-conjugated RNAi oligonucleotides herein to reduce expression of a target gene in an oligodendrocyte in the CNS. In some embodiments, expression of an oligodendrocyte target gene is reduced in a region of the CNS. In some embodiments, regions of the CNS include, but are not limited to, cerebrum, prefrontal cortex, frontal cortex, motor cortex, temporal cortex, parietal cortex, occipital cortex, somatosensory cortex, hippocampus, caudate, 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 ganglia, femoral nerve, sciatic nerve, sural nerve, amygdala, hypothalamus, putamen, corpus callosum, and cranial nerve. In some embodiments, the region of the CNS is selected from the spinal cord, lumbar spinal cord, lumbar dorsal root ganglion, thoracic spinal cord, cervical spinal cord, medulla, hippocampus, frontal cortex, hypothalamus, brain stem, cerebellum, and a combination thereof. In some embodiments, a lipid-conjugated RNAi oligonucleotide described herein reduces expression of a target gene in an oligodendrocyte in the spinal cord. In some embodiments, a lipid-conjugated RNAi oligonucleotide described herein reduces expression of a target gene in an oligodendrocyte in the lumbar spinal cord. In some embodiments, a lipid-conjugated RNAi oligonucleotide described herein reduces expression of a target gene in an oligodendrocyte in the thoracic spinal cord. In some embodiments, a lipid-conjugated RNAi oligonucleotide described herein reduces expression of a target gene in an oligodendrocyte of the cervical spinal cord. In some embodiments, a lipid- conjugated RNAi oligonucleotide described herein reduces expression of a target gene in an oligodendrocyte in lumbar dorsal root ganglion. In some embodiments, a lipid-conjugated RNAi oligonucleotide described herein reduces expression of a target gene in an oligodendrocyte in the medulla. In some embodiments, a lipid-conjugated RNAi oligonucleotide described herein reduces expression of a target gene in an oligodendrocyte in the hippocampus. In some embodiments, a lipid-conjugated RNAi oligonucleotide described herein reduces expression of a target gene in an oligodendrocyte in the hypothalamus. In some embodiments, a lipid-conjugated RNAi oligonucleotide described herein reduces expression of a target gene in an oligodendrocyte in the frontal cortex. In some embodiments, a lipid- conjugated RNAi oligonucleotide described herein reduces expression of a target gene in an oligodendrocyte in the brainstem. In some embodiments, a lipid-conjugated RNAi oligonucleotide described herein reduces expression of a target gene in an oligodendrocyte in the cerebellum.

[0465] In some embodiments, the disclosure provides methods for contacting or delivering to a cell or population of cells an effective amount of any of the oligonucleotides (e.g., RNAi oligonucleotides) herein to reduce UGT8 expression. In some embodiments, a reduction of UGT8 expression is determined by measuring a reduction in the amount or level of UGT8 mRNA, UGT8 protein, or UGT8 activity in a cell. The methods include those described herein and known to one of ordinary skill in the art.

[0466] In some embodiments, the disclosure provides methods for reducing UGT8 expression in the central nervous system. In some embodiments, the central nervous system comprises the brain and spinal cord. In some embodiments, UGT8 expression is reduced in at least one region of the brain. In some embodiments, regions of the brain include spinal cord, lumbar spinal cord, lumbar dorsal root ganglion, thoracic spinal cord, cervical spinal cord, medulla, hippocampus, frontal cortex, hypothalamus, brain stem, and cerebellum.

[0467] In some embodiments, a method for reducing expression of a target gene in an oligodendrocyte of the spinal cord in a subject comprises administering to the subject a lipid- conjugated RNAi oligonucleotide comprising a stem-loop and at least one lipid moiety conjugated at Position 2, Position 3, Position 6, Position 13, Position 14, Position 15, Position 19, Position 20, Position 23, Position 28, Position 39, or Position 30 of the sense strand.

[0468] In some embodiments, a method for reducing expression of a target gene in an oligodendrocyte of the medulla in a subject comprises administering to the subject a lipid- conjugated RNAi oligonucleotide comprising a stem-loop and at least one lipid moiety conjugated at Position 19, Position 20, Position 23, or Position 28 of the sense strand.

[0469] In some embodiments, a method for reducing expression of a target gene in an oligodendrocyte of the hippocampus in a subject comprises administering to the subject a lipid- conjugated RNAi oligonucleotide comprising a stem-loop and at least one lipid moiety conjugated at Position 2 of the sense strand.

[0470] In some embodiments, a method for reducing expression of a target gene in an oligodendrocyte of the frontal cortex in a subject comprises administering to the subject a lipid- conjugated RNAi oligonucleotide comprising a stem-loop and at least one lipid moiety conjugated at Position 14, Position 15, Position 19, Position 20, Position 23, Position 28, Position 29, or Position 30 of the sense strand.

[0471] In some embodiments, a method for reducing expression of a target gene in an oligodendrocyte of the spinal cord in a subject comprises administering to the subject a lipid- conjugated RNAi oligonucleotide comprising a blunt-end and at least one lipid moiety conjugated at Position 2, Position 3, Position 6, Position 13, Position 14, Position 15, Position 19, or Position 20 of the sense strand.

[0472] In...

Claims

CLAIMS1. A double-stranded oligonucleotide comprising an antisense strand of 15-30 nucleotides in length and a sense strand of 15-50 nucleotides in length, wherein the antisense and sense strands form a duplex region of 15-30 base pairs, wherein the antisense strand comprises a region of complementarity to an oligodendrocyte mRNA target sequence, and wherein the sense strand comprises at least one lipid moiety conjugated to a nucleotide of the sense strand.

3. The oligonucleotide of claim 1, wherein the lipid moiety is a hydrocarbon chain.

4. The oligonucleotide of claim 3, wherein the hydrocarbon chain is a C8-C30 hydrocarbon chain.

5. The oligonucleotide of claim 3 or 4, wherein the hydrocarbon chain is a C16 hydrocarbon chain.

6. The oligonucleotide of claim 5 wherein the C 16 hydrocarbon chain is represented by7. The oligonucleotide of any one of claims 1-6, wherein the lipid moiety is conjugated to the 2' carbon of the ribose ring of the nucleotide.

8. The oligonucleotide of any one of claims 1-7, wherein the oligonucleotide is blunt ended.

9. The oligonucleotide of claim 8, wherein the oligonucleotide is blunt ended at the 3' terminus of the oligonucleotide.

10. The oligonucleotide of any one of claims 1-7, wherein the oligonucleotide comprises a blunt end.

11. The oligonucleotide of claim 11, wherein the blunt end comprises the 3’ terminus of the sense strand12. The oligonucleotide of any one of claims 8-11, wherein the sense strand is 20-22 nucleotides.

13. The oligonucleotide of claim 12, wherein the at least one lipid moiety is conjugated to a nucleotide at position 1, position 4, position 8, position 12, position 13, position 18, or position 20 of the sense strand, wherein positions are numbered 5’ to 3’.

14. The oligonucleotide of claim 12, wherein the oligodendrocyte mRNA target is expressed in the spinal cord, wherein the at least one lipid moiety is conjugated to a nucleotide at position 1, position 2, position 3, position 5, position 6, position 7, position 9, position 13, position 14, position 15, position 17, position 19, or position 20 of the sense strand, and wherein positions are numbered 5’ to 3’.

15. The oligonucleotide of claim 12, wherein the oligodendrocyte mRNA target is expressed in the medulla, wherein the at least one lipid moiety is conjugated to a nucleotide at position 2, position 7, position 9, position 14, position 15, or position 19 of the sense strand, and wherein positions are numbered 5’ to 3’.

16. The oligonucleotide of claim 12, wherein the oligodendrocyte mRNA target is expressed in the hippocampus, wherein the at least one lipid moiety is conjugated to a nucleotide at position 3 of the sense strand, and wherein positions are numbered 5’ to 3’.

17. The oligonucleotide of claim 12, wherein the oligodendrocyte mRNA target is expressed in the frontal cortex, wherein the at least one lipid moiety is conjugated to a nucleotide at position 14 of the sense strand, and wherein positions are numbered 5’ to 3’.

18. The oligonucleotide of claim 12, wherein the oligodendrocyte mRNA target is expressed in the hypothalamus, wherein the at least one lipid moiety is conjugated to a nucleotide at position 7 of the sense strand, and wherein positions are numbered 5’ to 3’.

19. The oligonucleotide of any one of claims 1-7, wherein the sense strand comprises a stem-loop at the 3 ’end, wherein the stem-loop comprises a nucleotide sequence represented by the formula: 5’-Sl-L-S2-3’, wherein SI is complementary to S2, and wherein L forms a loop between SI and S2.18620. The oligonucleotide of any one of claims 1-7 and 19, wherein the sense strand is 36-38 nucleotides.

21. The oligonucleotide of claim 19 or 20, wherein the at least one lipid moiety is conjugated to a nucleotide of the stem-loop or a nucleotide proximal to the stem-loop.

22. The oligonucleotide of claim 21, wherein the nucleotide proximal to the stem-loop is located 1-3 nucleotides from the 5’ end of the stem-loop.

23. The oligonucleotide of claim 20, wherein the oligodendrocyte mRNA target is expressed in the spinal cord, wherein the at least one lipid moiety is conjugated to a nucleotide at position 2, position 3, position 6, position 13, position 14, position 15, position 19, position 20, position 23, position 28, position 29 or position 30 of the sense strand, and wherein positions are numbered 5’ to 3’.

24. The oligonucleotide of claim 20, wherein the oligodendrocyte mRNA target is expressed in the medulla, wherein the at least one lipid moiety is conjugated to a nucleotide at position 19, position 20, position 23 or position 28 of the sense strand, and wherein positions are numbered 5’ to 3’.

25. The oligonucleotide of claim 20, wherein the oligodendrocyte mRNA target is expressed in the hippocampus, wherein the at least one lipid moiety is conjugated to a nucleotide at position 2 of the sense strand, and wherein positions are numbered 5’ to 3’.

26. The oligonucleotide of claim 20, wherein the oligodendrocyte mRNA target is expressed in the frontal cortex, wherein the at least one lipid moiety is conjugated to a nucleotide at position 14, position 15, position 19, position 20, position 23, position 28, position 29 or position 30 of the sense strand, and wherein positions are numbered 5’ to 3’.

27. The oligonucleotide of any one of claims 1-26, wherein the antisense strand is 22- 24 nucleotides.18728. The oligonucleotide of any one of claims 1-27, wherein the duplex region is 20-22 base pairs.

29. The oligonucleotide of any one of claims 1-28, wherein the antisense strand comprises a 1-4 nucleotide overhang at the 3' terminus.

30. The oligonucleotide of claim 29, wherein the overhang comprises purine nucleotides.

31. The oligonucleotide of claim 29 or 30, wherein the overhang sequence is 2 nucleotides in length.

32. The oligonucleotide of claim 30 or 31, wherein the overhang is selected from AA, GG, AG, and GA.

33. The oligonucleotide of claim 32, wherein the overhang is GG.

34. The oligonucleotide of any one of claims 1-33, wherein the region of complementarity is complementary to at least 15 consecutive nucleotides of the oligodendrocyte mRNA target sequence.

35. The oligonucleotide of any one of claims 1-34, wherein the region of complementarity is complementary to 19 consecutive nucleotides of the oligodendrocyte mRNA target sequence.

36. The oligonucleotide of any one of claims 1-35, wherein the region of complementarity is fully complementary to the oligodendrocyte mRNA target sequence.

37. The oligonucleotide of any one of claims 1-35, wherein the region of complementarity is partially complementary to the oligodendrocyte mRNA target sequence.

38. The oligonucleotide of claim 37, wherein the region of complementarity comprises no more than four mismatches to the oligodendrocyte mRNA target sequence.18839. The oligonucleotide of any one of claims 1-38, wherein the oligonucleotide comprises at least one modified nucleotide.

40. The oligonucleotide of claim 39, wherein the modified nucleotide comprises a 2'- modification.

41. The oligonucleotide of claim 40, wherein each of the nucleotides of the sense strand and the antisense strand comprise a 2'-modification except the nucleotide of the sense strand conjugated to the at least one lipid moiety.

42. The oligonucleotide of claim 40 or 41, wherein the 2'-modification is a modification selected from 2 '-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy-2'- fluoro-P-d-arabinonucleic acid.

43. The oligonucleotide of any one of claims 40-42, wherein about 10-20%, 10%,11%, 12%, 13%, 14% 15%, 16%, 17%, 18%, 19% or 20% of the nucleotides of the sense strand comprise a 2'-fluoro modification.

44. The oligonucleotide of any one of claims 40-43, wherein 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.

45. The oligonucleotide of any one of claims 40-44, wherein about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35% of the nucleotides of the oligonucleotide comprise a 2'-fluoro modification.

46. The oligonucleotide of any one of claims 40-45, wherein the sense strand comprises 20 nucleotides with positions 1-20 from 5' to 3', wherein each of positions 8-11 comprise a 2'-fluoro modification.

47. The oligonucleotide of any one of claims 40-45, wherein the sense strand comprises 20 nucleotides with positions 1-20 from 5' to 3', wherein each of positions 9-11 comprise a 2'-fluoro modification.18948. The oligonucleotide of any one of claims 40-45, wherein the sense strand comprises 36 nucleotides with positions 1-36 from 5' to 3', wherein each of positions 8-11 comprise a 2'-fluoro modification.

49. The oligonucleotide of any one of claims 40-45, wherein the sense strand comprises 36 nucleotides with positions 1-36 from 5' to 3', wherein each of positions 9-11 comprise a 2'-fluoro modification.

50. The oligonucleotide of any one of claims 40-49, wherein the antisense strand comprises 22 nucleotides with positions 1-22 from 5' to 3', and wherein each of positions 2, 3, 4, 5, 7, 10 and 14 comprise a 2'-fluoro modification.

51. The oligonucleotide of any one of claims 41-50, wherein the remaining nucleotides comprise a 2'-O-methyl modification except the nucleotide of the sense strand conjugated to the at least one lipid moiety.

52. The oligonucleotide of any one of the preceding claims, wherein the oligonucleotide comprises at least one modified intemucleotide linkage.

53. The oligonucleotide of claim 52, wherein the at least one modified intemucleotide linkage is a phosphorothioate linkage.

54. The oligonucleotide of claim 53, wherein the antisense strand comprises a phosphorothioate linkage (i) between positions 1 and 2, and between positions 2 and 3; or (ii) between positions 1 and 2, between positions 2 and 3, and between positions 3 and 4, wherein positions are numbered 1-4 from 5’ to 3’.

55. The oligonucleotide of claim 53 or 54 , wherein the antisense strand is 22 nucleotides in length, and wherein the antisense strand comprises a phosphorothioate linkage between positions 20 and 21 and between positions 21 and 22, wherein positions are numbered 1-22 from 5' to 3'.19056. The oligonucleotide of any one of claims 53-55, wherein the sense strand comprises a phosphorothioate linkage between position 1 and 2, wherein positions are numbered 1-2 from 5' to 3'.

57. The oligonucleotide of any one of claims 53-56, wherein the sense strand is 20 nucleotides in length, and wherein the sense strand comprises a phosphorothioate linkage between positions 18 and 19, and between positions 19 and 20, wherein positions are numbered 1-22 from 5' to 3'.

58. The oligonucleotide of any one of claims 1-57, wherein the antisense strand comprises a phosphorylated nucleotide at the 5' terminus, wherein the phosphorylated nucleotide is selected from uridine and adenosine.

59. The oligonucleotide of claim 58, wherein the phosphorylated nucleotide is uridine.

60. The oligonucleotide of any one of the preceding claims, wherein the 4'-carbon of the sugar of the 5 '-nucleotide of the antisense strand comprises a phosphate analog.

61. The oligonucleotide of claim 60, wherein the phosphate analog is oxymethyl phosphonate, vinyl phosphonate or malonyl phosphonate.

62. The oligonucleotide of any one of claims 1-61, wherein the region of complementary is fully complementary to the oligodendrocyte mRNA target sequence at nucleotide positions 2-8 of the antisense strand, wherein nucleotide positions are numbered 5' to 3'.

63. The oligonucleotide of any one of claims 1-61, wherein the region of complementary is fully complementary to the oligodendrocyte mRNA target sequence at nucleotide positions 2-11 of the antisense strand, wherein nucleotide positions are numbered 5' to 3'.

64. The oligonucleotide of any one of claims 1-63, wherein the oligonucleotide is a Dicer substrate.19165. The oligonucleotide of any one of claims 1-63, wherein the oligonucleotide is a Dicer substrate that, upon endogenous Dicer processing, yields double-stranded nucleic acids of 19- 21 nucleotides in length capable of reducing a oligodendrocyte mRNA expression in a mammalian cell.

66. The oligonucleotide of any one of claims 1-65, wherein the oligodendrocyte mRNA target sequence is located in a region of the central nervous system (CNS).

67. The oligonucleotide of claim 66, wherein the region of the CNS is selected from the frontal cortex, spinal cord, lumbar spinal cord, cervical spinal cord, thoracic spinal cord, medulla, cerebellum, hypothalamus, hippocampus, and a combination thereof.

68. The oligonucleotide of any one of claims 1-67, wherein the oligonucleotide reduces expression of a target mRNA in an oligodendrocyte or population of oligodendrocytes in vitro and / or in vivo.

69. A pharmaceutical composition comprising the oligonucleotide of any one of claims 1- 68, and a pharmaceutically acceptable carrier, delivery agent or excipient.

70. A method for treating a subject having a disease, disorder or condition associated with expression of an oligodendrocyte mRNA, the method comprising administering to the subject a therapeutically effective amount of the oligonucleotide of any one of claims 1-68 or the pharmaceutical composition of claim 69, thereby treating the subject.

71. A method of delivering an oligonucleotide to an oligodendrocyte or a population of oligodendrocytes in a subject, the method comprising administering the pharmaceutical composition of claim 69 to the subject.

72. The method of claim 71, wherein the oligodendrocyte or a population of oligodendrocytes is located in a region of the CNS.

73. The method of claim 72 wherein the region of the CNS is selected from the frontal cortex, spinal cord, lumbar spinal cord, cervical spinal cord, thoracic spinal cord, medulla, cerebellum, hypothalamus, hippocampus, and a combination thereof.

74. A method for reducing expression of an oligodendrocyte mRNA in a cell, a population of cells or a subject, the method comprising the step of: i. contacting the cell or the population of cells with the oligonucleotide of any one of claims 1 to 68, or the pharmaceutical composition of claim 69, optionally wherein the cell or population of cells is an oligodendrocyte or a population of oligodendrocytes; or ii. administering to the subject the oligonucleotide of any one of claims 1 to 68, or the pharmaceutical composition of claim 69.

75. The method of claim 74, wherein reducing expression of the oligodendrocyte mRNA comprises reducing an amount or level of mRNA, an amount or level of protein, or both.

76. The method of claim 74 or 75, wherein the subject has a disease, disorder or condition associated with expression of the oligodendrocyte mRNA.

77. The method of any one of claims 74-76, wherein the cell or population of cells is located in a region of the CNS.

78. The method of claim 77, wherein the region of the CNS is selected from the frontal cortex, spinal cord, lumbar spinal cord, cervical spinal cord, thoracic spinal cord, medulla, cerebellum, hypothalamus, hippocampus, and a combination thereof.

79. The method of any one of claims 70-78, wherein administering is intrathecal.

80. A method of reducing expression of a target mRNA expressed in an oligodendrocyte in a tissue of the CNS of a subject, comprising administering to the subject a double-stranded oligonucleotide comprising an antisense strand of 15-30 nucleotides in length and a sense strand of 15-50 nucleotides in length, wherein the antisense and sense strands form a duplex region of 15-30 base pairs, wherein the antisense strand comprises a region of complementarity to a target sequence in the target mRNA, and wherein the sense strand comprises at least one lipid moiety conjugated to a nucleotide of the sense strand.

81. The method of claim 80, wherein the lipid moiety is a C16 hydrocarbon.

82. The method of any one of claims 80-81, wherein the oligonucleotide is blunt-ended at the 3' terminus of the oligonucleotide.

83. The method of claim 82, wherein the sense strand is 22-24 nucleotides.

84. The method of claim 83, wherein the tissue is the spinal cord, wherein the at least one lipid moiety is conjugated to a nucleotide at position 1, position 2, position 3, position 5, position 6, position 7, position 9, position 13, position 14, position 15, position 17, position 19, or position 20 of the sense strand, and wherein positions are numbered 5’ to 3’85. The method of claim 83, wherein the tissue is the medulla, wherein the at least one lipid moiety is conjugated to a nucleotide at position 2, position 7, position 14, position 15, or position 19 of the sense strand, and wherein positions are numbered 5’ to 3’.

86. The method of claim 83, wherein the tissue is the hippocampus, wherein the at least one lipid moiety is conjugated to a nucleotide at position 3 of the sense strand, and wherein positions are numbered 5’ to 3’.

87. The method of claim 83, wherein the tissue is the frontal cortex, wherein the at least one lipid moiety is conjugated to a nucleotide at position 14 of the sense strand, and wherein positions are numbered 5’ to 3’.

88. The method of claim 83, wherein the tissue is the hypothalamus, wherein the at least one lipid moiety is conjugated to a nucleotide at position 7 of the sense strand, and wherein positions are numbered 5’ to 3’.

89. The method of any one of claims 80-81, wherein the sense strand comprises a stemloop at the 3 ’end, wherein the stem-loop comprises a nucleotide sequence represented by the formula: 5’-Sl-L-S2-3’, wherein SI is complementary to S2, and wherein L forms a loop between SI and S2.

90. The method of any one of claims 80-81 and 89, wherein the sense strand is 36-38 nucleotides.19491. The method of claim 89 or 90, wherein the at least one lipid moiety is conjugated to a nucleotide of the stem-loop or a nucleotide proximal to the stem-loop.

92. The method of claim 91, wherein the nucleotide proximal to the stem-loop is located 1-3 nucleotides from the 5’ end of the stem-loop.

93. The method of claim 90, wherein the tissue is the spinal cord, wherein the at least one lipid moiety is conjugated to a nucleotide at position 2, position 3, position 6, position 13, position 14, position 15, position 19, position 20, position 23, position 28, position 29 or position 30 of the sense strand, and wherein positions are numbered 5’ to 3’.

94. The method of claim 90, wherein the tissue is the medulla, wherein the at least one lipid moiety is conjugated to a nucleotide at position 19, position 20, position 23 or position 28 of the sense strand, and wherein positions are numbered 5’ to 3’.

95. The method of claim 90, wherein the tissue is the hippocampus, wherein the at least one lipid moiety is conjugated to a nucleotide at position 2 of the sense strand, and wherein positions are numbered 5’ to 3’.

96. The method of claim 90, wherein the tissue is the frontal cortex, wherein the at least one lipid moiety is conjugated to a nucleotide at position 14, position 15, position 19, position 20, position 23, position 28, position 29 or position 30 of the sense strand, and wherein positions are numbered 5’ to 3’.

97. A kit comprising the oligonucleotide of any one of claims 1-68, an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administration to a subject having a disease, disorder or condition associated with expression of an oligodendrocyte mRNA.

98. The kit of claim 97, wherein the package insert comprises instructions for intrathecal administration.19599. Use of the oligonucleotide of any one of claims 1-68 or the pharmaceutical composition of claim 69, in the manufacture of a medicament for the treatment of a disease, disorder or condition associated with expression of an oligodendrocyte mRNA.

100. The oligonucleotide of any one of claims 1-68 or the pharmaceutical composition of claim 69, for use, or adaptable for use, in the treatment of a disease, disorder or condition associated with expression of an oligodendrocyte mRNA.196

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