Rnai oligonucleotide conjugates

Abstract

Lipid-conjugated oligonucleotide are provided herein that inhibit or reduce expression of target genes. Also provided are compositions including the same and uses thereof, particularly uses relating to treating diseases, disorders and / or conditions associated with an RNAi trigger induced decrease in target gene expression.

Description

[0001] RNAi OLIGONUCLEOTIDE CONJUGATES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 277,097 filed November 8, 2021, and U.S. Provisional Patent Application No. 63 / 340,291 filed May 10, 2022, each of which are incorporated by reference herein in their entirety.

[0004] TECHNICAL FIELD

[0005] The disclosure relates to oligonucleotides linked to lipid moieties useful in the inhibition of target genes in a variety of tissues. 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 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.

[0006] BACKGROUND OF THE DISCLOSURE

[0007] 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).

[0008] Dicer processed RNAi technologies utilize short double-stranded RNA (dsRNA) of approximately 21 base pair length with a two nucleotide (nt) 3 ’-overhang for the silencing of genes. These dsRNAs are generally called small interfering RNA (siRNA). siRNA 12 to 22 nucleotides in length are the active agent in RNAi. The siRNA duplex serves as a guide for mRNA degradation. Upon siRNA incorporation into the RNA-induced silencing complex (RISC) the complex interacts with a specific mRNA and ultimately suppresses the mRNA signal. The sense strand or passenger strand of siRNA is typically cleaved at the 9th nucleotide downstream from the 5 ’-end of the sense strand by Argonaute 2 (Ago2) endonuclease. The activated RISC complex containing the antisense strand or guide strand binds to the target mRNA through Watson-Crick base pairing causing degradation or translational blocking of the targeted RNA.

[0009] However, the in vivo use of RNAi or siRNA molecules as pharmaceuticals has remained difficult due to obstacles encountered such as low biostability and unacceptable toxicity possibly caused by off-target effects. Various types of chemical modifications to improve the pharmacokinetics and to overcome bio-instability problems have been investigated over the years to improve the stability and specificity of the RNAi duplexes. In some cases, the chemical modification in siRNAs has improved the serum stability of siRNAs. However, often RNAi activity was lost, but the careful placement of some specific modified residues enables enhanced siRNA biostability without loss of siRNA potency. Some of these modifications have reduced siRNA side effects, such as the induction of recipient immune responses and inherent off-targeting effects and have even enhanced siRNA potency. Various chemically modified siRNAs have been investigated, among them were bridged nucleic acids (BNA’s) such as 2’,4’- methylene bridged nucleic acid 2’,4’-BNAs, also known as locked nucleic acid or LNA’s. Some of these modified siRNAs showed promising effects.

[0010] 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) IOURNAL OF HEPATOLOGY 59: 1354-59). Technological hurdles remain for the development and clinical use of RNAi oligonucleotides in extrahepatic cells, tissues, and organs. 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. 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.

[0011] Over the past decade, synthetic RNAi triggers such as double stranded RNAs have become ubiquitous tools in biological research, and extensive basic and clinical development efforts have recently culminated in the FDA approval of ONPATTROtm, the first RNAi drug. Despite a burgeoning drug development pipeline and an extensive compendium of excipients targeting ligands and delivery techniques, the difficulty of delivering RNAi agents to specific populations of disease related cells and or tissues, particularly outside the liver continues to limit the potential of RNAi therapy. Repeated attempts over the past several years to develop useful, active, and persistent RNAi agents and structures for use based on known liver delivery technology have not convincingly demonstrated the intended effects outside the liver. Thus, new dsRNA’s with variant structures have been developed to overcome the limitations in the field.

[0012] BRIEF SUMMARY OF THE DISCLOSURE

[0013] The present disclosure is based in part on the discovery of lipid-conjugated RNAi oligonucleotides that are capable of inhibiting expression of a target gene in hepatic and extrahepatic tissues. As demonstrated herein, lipid-conjugated RNAi oligonucleotides having a stem-loop at the 5’ end of the oligonucleotide showed comparable efficacy in reducing target gene expression in several regions of the central nervous system as a lipid-conjugated RNAi oligonucleotide having a stem-loop at the 3’ end of the oligonucleotide. Further, the presence of a stable stem-loop (e.g., UACG) improved reduction of target gene expression compared to a relatively less stable stem-loop (e.g., GAAA). In some aspects, the presence of a Tm- increasing nucleotide (e.g., locked nucleic acid) and / or truncation of a sense strand at the 3’ terminus improved reduction of target gene expression.

[0014] It is further shown herein that lipid-conjugated RNAi oligonucleotides having a “double-overhang” (e.g., an overhang of at least one nucleotide at each of the 5’ and 3’ termini of an antisense strand) reduced target gene expression in the central nervous system at comparable levels relative to an RNAi oligonucleotide having only one overhang, i.e., at the 3 ’terminus of the antisense strand. Up to three nucleotide truncations at the 3 ’terminus of the sense strand was tolerated, whereas introduction of a Tm-increasing nucleotide allowed for truncations of up to four nucleotides.

[0015] It has been demonstrated lipid-conjugated RNAi oligonucleotides delivered to the eye can effectively reduce expression of an ocular mRNA. Specifically, double-overhang RNAi oligonucleotides having a lipid conjugated to the 5’ terminal nucleotide of the sense strand reduced expression of a target gene in the optic nerve and retina.

[0016] Further shown herein, are lipid-conjugated RNAi oligonucleotides capable of reducing expression of a target gene in a macrophage of the liver. Specifically, RNAi oligonucleotides having a blunt end comprising the 3’ terminus of the sense strand and the 5 ’terminus of the antisense strand, and an overhang of up to seven nucleotides on the 3 ’terminus of the antisense strand, resulted in reduced expression of a gene expressed in macrophages. Further, lipid- conjugated RNAi oligonucleotides having a double-overhang, with or without Tm-increasing nucleotides, similarly reduced expression of the macrophage target gene. Macrophages make up about 33% of all liver cells and are believed to play a role in liver inflammation. Accordingly, without wishing to be bound by theory, the lipid-conjugated RNAi oligonucleotides disclosed herein are useful for targeting macrophages and treating liver diseases, including, but not limited to, drug or alcohol toxicity, steatosis, infection (e.g., viral infection), inflammatory liver diseases, fibrosis, hepatocellular carcinoma, and cirrhosis.

[0017] Also shown according to the current disclosure are the bases of the lipid-conjugated RNAi oligonucleotides modified by the addition of LNA, 2'-O-methyl modification, or phosphorothioate (PS) modification; either or both termini of the sense strand are modified with PS modification, 2'-O-methyl modification, or both; or the single strand overhang of the antisense sense strand is modified by LNA modification, 2'-O-methyl modification, PS modification, or any combination thereof. In some embodiments, the sense strand and the antisense strand of the RNAi trigger are modified by different chemical modifications.

[0018] Also shown herein are lipid-conjugated RNAi oligonucleotides having either a blunt end or a stem-loop at the 3 ’terminus of the oligonucleotides, with truncated sense strands (e.g., having an overhang at the 3 ’terminus of the antisense strand) and Tm-increasing nucleotides, are capable of reducing target gene expression in several tissues, including the liver, skeletal muscle, adipose and adrenal.

[0019] Without wishing to be bound by theory, the lipid-conjugated RNAi oligonucleotides described herein are useful for reducing expression of a target gene in both hepatic and extrahepatic tissues.

[0020] In some aspects, the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of about 20-22 nucleotides in length and a sense strand of about 8-20 nucleotides in length, wherein the antisense and sense strands form a duplex region of about 8-20 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang of at least one nucleotide and a 3’ overhang of at least one nucleotide, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, and wherein the sense strand comprises at least one lipid moiety conjugated to a nucleotide on the sense strand. In some aspects, the 5’overhang is about 1-10 nucleotides. In some aspects, the 5’overhang is about 2-10 nucleotides. In some aspects, the 5’overhang is about 1-6 nucleotides. In some aspects, the 3’overhang is about 2-8 nucleotides. In some aspects, the 3’overhang is about 2-12 nucleotides. In some aspects, the 5’overhang is 2 nucleotides and the 3’overhang is about 3-7 nucleotides. In some aspects, the 3’overhang is 2 nucleotides, and the 5’overhang is about 2-8 nucleotides. In some aspects, the 3’overhang is 6-8 nucleotides, and the 5’overhang is about 2-4 nucleotides.

[0021] In any of the foregoing or related aspects,

[0022] (i) the sense strand is 18 nucleotides, the duplex region is 18 nucleotides, the 5’overhang is 2 nucleotides and the 3’overhang is 2 nucleotides;

[0023] (ii) the sense strand is 17 nucleotides, the duplex region is 17 nucleotides, the 5’overhang is 3 nucleotides and the 3’overhang is 2 nucleotides;

[0024] (iii) the sense strand is 16 nucleotides, the duplex region is 16 nucleotides, the 5’overhang is 4 nucleotides and the 3’overhang is 2 nucleotides; or

[0025] (iv) the sense strand is 13 nucleotides, the duplex region is 13 nucleotides, the 5’ overhang is 2 nucleotides and the 3’overhang is 7 nucleotides.

[0026] In any of the foregoing or related aspects,

[0027] (i) the sense strand is 12 nucleotides, the duplex region is 12 nucleotides, the 5’overhang is 2 nucleotides, and the 3’overhang is 8 nucleotides;

[0028] (ii) the sense strand is 12 nucleotides, the duplex region is 12 nucleotides, the 5’overhang is 3 nucleotides, and the 3’overhang is 7 nucleotides; or

[0029] (iii) the sense strand is 10 nucleotides, the duplex region is 10 nucleotides, the 5’overhang is 1 nucleotide, and the 3’overhang is 11 nucleotides.

[0030] In some aspects, the sense strand is 13 nucleotides, the duplex region is 13 nucleotides, the 5’ overhang is 2 nucleotides, and the 3’ overhang is 7 nucleotides. In other aspects, the sense strand is 12 nucleotides, the duplex region is 12 nucleotides, the 5’overhang is 2 nucleotides, and the 3’overhang is 8 nucleotides. In other aspects, the sense strand is 12 nucleotides, the duplex region is 12 nucleotides, the 5’overhang is 3 nucleotides, and the 3’overhang is 7 nucleotides. In some aspects, the sense strand is 10 nucleotides, the duplex region is 10 nucleotides, the 5’overhang is 1 nucleotide, and the 3’overhang is 11 nucleotides.

[0031] In any of the foregoing or related aspects, the lipid moiety is selected from:

[0032]

[0033] 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

[0034] . In some aspects, the hydrocarbon chain is a C22 hydrocarbon chain. In some aspects, the C22 hydrocarbon chain is represented by

[0035] In any of the foregoing or related aspects, the lipid moiety is conjugated to the 5 ’terminal nucleotide of the sense strand. In some aspects, the lipid moiety is conjugated to the 2’ carbon of the ribose ring of the nucleotide.

[0036] In any of the foregoing or related aspects, the antisense strand is 22 nucleotides, wherein positions are numbered 1-22 from 5’ to 3’. In some aspects, the nucleotide conjugated to the lipid moiety forms a base pair with a nucleotide at position 14 of the antisense strand, wherein positions are numbered 5’ to 3’. In some aspects, the nucleotide conjugated to the lipid moiety forms a base pair with a nucleotide at position 12, 14, or 16 of the antisense strand, wherein positions are numbered 5’ to 3’.

[0037] In any of the foregoing or related aspects, the region of complementarity is fully complementary to the mRNA target sequence. In other aspects, the region of complementarity is partially complementary to the mRNA target sequence. In some aspects, the region of complementarity comprises no more than four mismatches to the mRNA target sequence. In some aspects, the region of complementarity comprises up to four mismatches to the mRNA target sequence.

[0038] In some aspects, the region of complementarity comprises no more than four mismatches to the mRNA target sequence.

[0039] In any of the foregoing or related aspects, the mRNA target sequence is a central nervous system (CNS) target sequence. In some aspects, the CNS target sequence is a neuronal mRNA target sequence or an ocular mRNA target sequence. In some aspects, the mRNA target sequence is a liver mRNA target sequence. In some aspects, the liver mRNA target sequence is a liver macrophage mRNA target sequence. In some aspects, the liver mRNA target sequence is a liver hepatocyte mRNA target sequence. In some aspects, the liver mRNA target sequence is a liver sinusoidal endothelial cell mRNA target sequence. In other aspects, the mRNA target sequence is an ocular mRNA target sequence.

[0040] 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. 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, the sense strand comprises nucleotide positions numbered 5’ to 3’, wherein each of positions 8-11 comprise a 2'-fluoro modification. In some aspects, the sense strand comprises a 2’ -fluoro modification at each of nucleotides forming a base pair with nucleotides at positions 10-13 of the antisense strand, wherein positions are numbered 5’ to 3’. 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, provided that the nucleotide of the sense strand conjugated to the at least one lipid moiety does not comprise a 2’-O-methyl modification.

[0041] 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 21 and 22, wherein positions are numbered 1-22 from 5' to 3'. In some aspects, the antisense strand comprises a phosphorothioate linkage between positions 13 and 14, and between positions 14 and 15. In some aspects, the sense strand comprises a phosphorothioate linkage between positions 1 and 2, between the penultimate nucleotide and third nucleotide from the 3’ end, and between the penultimate nucleotide and ultimate nucleotide.

[0042] 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. In some aspects, the phosphorylated nucleotide is 4’-O-monomethylphosphonate-2’-O-methyl uridine.

[0043] In any of the foregoing or related aspects, the sense strand comprises at least one Tm- increasing nucleotide. In some aspects, the sense strand comprises up to four Tm-increasing nucleotides. In some aspects, the Tm-increasing nucleotide is a bicyclic nucleotide. In some aspects, the Tm-increasing nucleotide is a locked nucleic acid.

[0044] In other aspects, the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 18 nucleotides in length, wherein the antisense and sense strands form a duplex region of 18 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising at least two nucleotides and a 3’ overhang comprising at least two nucleotides, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to a 5’ terminal nucleotide of the sense strand, and wherein each of the antisense and sense strands comprise at least one 2’ -modified nucleotide and at least one modified intemucleotide linkage.

[0045] In some aspects, the sense strand comprises a 2’-fluoro modification at positions 8-11, numbered 5’ to 3’. In some aspects, the antisense strand comprises a 2’-fluoro modification at positions 2-5, 7, 10 and 14, numbered 5’ to 3’. In some aspects, the remaining nucleotides comprise a 2'-O-methyl modification, provided that the nucleotide of the sense strand conjugated to the at least one lipid moiety does not comprise a 2’-O-methyl modification. In some aspects, the at least one modified internucleotide linkage is a phosphorothioate linkage. In some aspects, the sense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 16 and 17, and 17 and 18, numbered 5’ to 3’. In some aspects, the antisense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22, numbered 5’ to 3’. In some aspects, the antisense strand comprises a phosphorylated uridine at position 1, numbered 5’ to 3’. In some aspects, the phosphorylated uridine is 4’-O-monomethylphosphonae-2’-O-methyl uridine. In some aspects, the lipid moiety is a C16 hydrocarbon represented by:

[0046] . In some aspects, the lipid moiety is a C22 hydrocarbon represented by: some aspects, the lipid moiety is conjugated to the 2’ carbon of the ribose ring of the nucleotide. In some aspects, the 5’overhang is 2 nucleotides and the 3’overhang is 2 nucleotides. In some aspects, the region of complementarity is fully complementary to the mRNA target sequence. In some aspects, the region of complementarity is partially complementary to the mRNA target sequence. In some aspects, the region of complementarity comprises no more than four mismatches to the mRNA target sequence. In some aspects, the mRNA target sequence is a central nervous system (CNS) target sequence, optionally a neuronal mRNA target sequence or an ocular mRNA target sequence. In some aspects, the mRNA target sequence is a liver mRNA target sequence, optionally a liver macrophage mRNA target sequence, a liver hepatocyte mRNA target sequence, or a liver sinusoidal endothelial cell mRNA target sequence. In some aspects, the mRNA target sequence is an ocular mRNA target sequence. In some aspects, the mRNA target sequence is an astrocyte mRNA target sequence.

[0047] In some aspects, the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 18 nucleotides in length, wherein the antisense and sense strands form a duplex region of 18 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising two nucleotides and a 3’ overhang comprising two nucleotides, wherein the antisense strand comprises a region of complementarity to a central nervous system mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to a 5’ terminal nucleotide of the sense strand, wherein the sense strand comprises a 2’ -fluoro modification at positions 8-11 and a 2’-O-methyl modification at positions 2-7 and 12-18, wherein the sense strand comprises a phosphorothioate linkage between positions 1 and 2, positions 16 and 17, and positions 17 and 18, wherein the antisense strand comprises a 2’ -fluoro modification at positions 2-5, 7, 10 and 14 and a 2’-O-methyl modification at positions 1, 6, 8, 9, 11-13, and 15-22, wherein the antisense strand comprises phosphorothioate linkages between positions 1 and 2, positions 2 and 3, positions 3 and 4, positions 20 and 21, and positions 21 and 22, and wherein the antisense strand comprises a 5 ’terminal phosphorylated uridine.

[0048] In some aspects, the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 18 nucleotides in length, wherein the antisense and sense strands form a duplex region of 18 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising two nucleotides and a 3’ overhang comprising two nucleotides, wherein the antisense strand comprises a region of complementarity to an ocular mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to a 5’ terminal nucleotide of the sense strand, wherein the sense strand comprises a 2’ -fluoro modification at positions 8-11 and a 2’-O-methyl modification at positions 2-7 and 12-18, wherein the sense strand comprises a phosphorothioate linkage between positions 1 and 2, positions 16 and 17, and positions 17 and 18, wherein the antisense strand comprises a 2’ -fluoro modification at positions 2-5, 7, 10 and 14 and a 2’-O-methyl modification at positions 1, 6, 8, 9, 11-13, and 15-22, wherein the antisense strand comprises phosphorothioate linkages between positions 1 and 2, positions 2 and 3, positions 3 and 4, positions 20 and 21, and positions 21 and 22, and wherein the antisense strand comprises a 5 ’terminal phosphorylated uridine.

[0049] In some aspects, the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 18 nucleotides in length, wherein the antisense and sense strands form a duplex region of 18 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising two nucleotides and a 3’ overhang comprising two nucleotides, wherein the antisense strand comprises a region of complementarity to a macrophage mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to a 5’ terminal nucleotide of the sense strand, wherein the sense strand comprises a 2’ -fluoro modification at positions 8-11 and a 2’-O- methyl modification at positions 2-7 and 12-18, wherein the sense strand comprises a phosphorothioate linkage between positions 1 and 2, positions 16 and 17, and positions 17 and 18, wherein the antisense strand comprises a 2’ -fluoro modification at positions 2-5, 7, 10 and 14 and a 2’-O-methyl modification at positions 1, 6, 8, 9, 11-13, and 15-22, wherein the antisense strand comprises phosphorothioate linkages between positions 1 and 2, positions 2 and 3, positions 3 and 4, positions 20 and 21, and positions 21 and 22, and wherein the antisense strand comprises a 5 ’terminal phosphorylated uridine.

[0050] In other aspects, the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 17 nucleotides in length, wherein the antisense and sense strands form a duplex region of 17 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising at least three nucleotides and a 3’ overhang comprising at least two nucleotides, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to a 5’ terminal nucleotide of the sense strand, and wherein each of the antisense and sense strands comprise at least one 2’ -modified nucleotide and at least one modified intemucleotide linkage. In some aspects, the sense strand comprises a 2’-fluoro modification at positions 8-11, numbered 5’ to 3’. In some aspects, the antisense strand comprises a 2’-fluoro modification at positions 2-5, 7, 10 and 14, numbered 5’ to 3’. In some aspects, the remaining nucleotides comprise a 2'-O-methyl modification, provided that the nucleotide of the sense strand conjugated to the at least one lipid moiety does not comprise a 2’-O-methyl modification. In some aspects, the at least one modified internucleotide linkage is a phosphorothioate linkage. In some aspects, the sense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 15 and 16, and 16 and 17, numbered 5’ to 3’. In some aspects, the antisense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22, numbered 5’ to 3’. In some aspects, the antisense strand comprises a phosphorylated uridine at position 1, numbered 5’ to 3’. In some aspects, the phosphorylated uridine is 4’-O-monomethylphosphonae-2’-O-methyl uridine. In some aspects, the lipid moiety is a C16 hydrocarbon represented by:

[0051] . In some aspects, the lipid moiety is conjugated to the 2’ carbon of the ribose ring of the nucleotide. In some aspects, the 5’overhang is 3 nucleotides and the 3’overhang is 2 nucleotides. In some aspects, the region of complementarity is fully complementary to the mRNA target sequence. In some aspects, the region of complementarity is partially complementary to the mRNA target sequence. In some aspects, the region of complementarity comprises no more than four mismatches to the mRNA target sequence. In some aspects, the mRNA target sequence is a central nervous system (CNS) target sequence, optionally a neuronal mRNA target sequence.

[0052] In other aspects, the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 17 nucleotides in length, wherein the antisense and sense strands form a duplex region of 17 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising three nucleotides and a 3’ overhang comprising two nucleotides, wherein the antisense strand comprises a region of complementarity to a central nervous system mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to a 5’ terminal nucleotide of the sense strand, wherein the sense strand comprises a 2’-fluoro modification at positions 8-11 and a 2-O-methyl modification at positions 2-7 and 12-17, wherein the sense strand comprises phosphorothioate linkages between positions 1 and 2, positions 15 and 16, and positions 16 and 17, wherein the antisense strand comprises a 2’- fluoro modification at positions 2-5, 7, 10 and 14 and a 2-O-methyl modification at positions 1, 6, 8, 9, 11-13, and 15-22, wherein the antisense strand comprises phosphorothioate linkages between positions 1 and 2, positions 2 and 3, positions 3 and 4, positions 20 and 21, and positions 21 and 22, and wherein the antisense strand comprises a 5 ’terminal phosphorylated uridine.

[0053] In further aspects, the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 16 nucleotides in length, wherein the antisense and sense strands form a duplex region of 16 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising at least four nucleotides and a 3’ overhang comprising at least two nucleotides, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to a 5’ terminal nucleotide of the sense strand, wherein the sense strand comprises up to five Tm-increasing nucleotides, and wherein each of the antisense and sense strands comprise at least one 2’ -modified nucleotide and at least one modified internucleotide linkage.

[0054] In some aspects, the sense strand comprises a 2’-fluoro modification at positions 8-11, numbered 5’ to 3’. In some aspects, the antisense strand comprises a 2’-fluoro modification at positions 2-5, 7, 10 and 14, numbered 5’ to 3’. In some aspects, the remaining nucleotides comprise a 2'-O-methyl modification, provided that the nucleotide of the sense strand conjugated to the at least one lipid moiety does not comprise a 2’-O-methyl modification. In some aspects, the at least one modified internucleotide linkage is a phosphorothioate linkage. In some aspects, the sense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 14 and 15, and 15 and 16, numbered 5’ to 3’. In some aspects, the antisense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22, numbered 5’ to 3’. In some aspects, the antisense strand comprises a phosphorylated uridine at position 1, numbered 5’ to 3’. In some aspects, the phosphorylated uridine is 4’-O-monomethylphosphonae-2’-O-methyl uridine. In some aspects, the lipid moiety is a C16 hydrocarbon represented by:

[0055] . In some aspects, the lipid moiety is conjugated to the 2’ carbon of the ribose ring of the nucleotide. In some aspects, the 5’overhang is 4 nucleotides and the 3’overhang is 2 nucleotides. In some aspects, the sense strand comprises 1-5, 1-4, 1-3, or 1-2 Tm-increasing nucleotides. In some aspects, the sense strand comprises 1, 2, 3, 4 or 5 Tm-increasing nucleotides. In some aspects, the sense strand comprises up to three Tm-increasing nucleotides. In some aspects, the sense strand comprises a Tm-increasing nucleotide at positions 2, 15 and 16, numbered 5’ to 3’. In some aspects, the Tm-increasing nucleotide is a bicyclic nucleotide, optionally, a locked nucleic acid (LNA). In some aspects, the region of complementarity is fully complementary to the mRNA target sequence. In some aspects, the region of complementarity is partially complementary to the mRNA target sequence. In some aspects, the region of complementarity comprises no more than four mismatches to the mRNA target sequence. In some aspects, the mRNA target sequence is a central nervous system (CNS) target sequence, optionally a neuronal mRNA target sequence or an ocular mRNA target sequence.

[0056] In further aspects, the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 16 nucleotides in length, wherein the antisense and sense strands form a duplex region of 16 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising at least four nucleotides and a 3’ overhang comprising at least two nucleotides, wherein the antisense strand comprises a region of complementarity to a central nervous system mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to a 5’ terminal nucleotide of the sense strand, wherein the sense strand comprises a Tm-increasing nucleotide at positions 2, 15 and 16, wherein the sense strand comprises a 2’-fluoro modification at positions 8-11 and a 2-O-methyl modification at positions 3-7 and 12-13, wherein the sense strand comprises phosphorothioate linkages between positions 1 and 2, positions 14 and 15, and positions 15 and 16, wherein the antisense strand comprises a 2’ -fluoro modification at positions 2-5, 7, 10 and 14 and a 2-O-methyl modification at positions 1, 6, 8, 9, 11-13, and 15-22, wherein the antisense strand comprises phosphorothioate linkages between positions 1 and 2, positions 2 and 3, positions 3 and 4, positions 20 and 21, and positions 21 and 22, and wherein the antisense strand comprises a 5 ’terminal phosphorylated uridine.

[0057] In yet further aspects, the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 13 nucleotides in length, wherein the antisense and sense strands form a duplex region of 13 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising at least two nucleotides and a 3’ overhang comprising at least seven nucleotides, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to an internal nucleotide of the sense strand, wherein the sense strand comprises up to three Tm-increasing nucleotides, and wherein each of the antisense and sense strands comprise at least one 2’ -modified nucleotide and at least one modified internucleotide linkage.

[0058] In some aspects, the sense strand comprises a 2’-fluoro modification at positions 3-6, numbered 5’ to 3’. In some aspects, the antisense strand comprises a 2’-fluoro modification at positions 2-5, 7, 10 and 14, numbered 5’ to 3’. In some aspects, the remaining nucleotides comprise a 2'-O-methyl modification, provided that the nucleotide of the sense strand conjugated to the at least one lipid moiety does not comprise a 2’-O-methyl modification. In some aspects, the at least one modified internucleotide linkage is a phosphorothioate linkage. In some aspects, the sense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 11 and 12, and 12 and 13, numbered 5’ to 3’. In some aspects, the antisense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22, numbered 5’ to 3’. In some aspects, the antisense strand comprises a phosphorylated uridine at position 1, numbered 5’ to 3’. In some aspects, the phosphorylated uridine is 4’-O-monomethylphosphonae-2’-O-methyl uridine. In some aspects, the lipid moiety is conjugated at a nucleotide at position 2 of the sense strand, numbered 5’ to 3’. In some aspects, the lipid moiety is a C22 hydrocarbon represented by: some aspects, the lipid moiety is conjugated to the 2’ carbon of the ribose ring of the nucleotide. In some aspects, the 5’overhang is 2 nucleotides and the 3’overhang is 7 nucleotides. In some aspects, the sense strand comprises 1-3, or 1-2 Tm-increasing nucleotides. In some aspects, the sense strand comprises 1, 2, or 3 Tm-increasing nucleotides. In some aspects, the sense strand comprises a Tm-increasing nucleotide at (i) positions 1 and 10, or (ii) positions 1, 10 and 11, numbered 5’ to 3’. In some aspects, the Tm-increasing nucleotide is a bicyclic nucleotide, optionally a locked nucleic acid (LNA). In some aspects, the region of complementarity is fully complementary to the mRNA target sequence. In some aspects, the region of complementarity is partially complementary to the mRNA target sequence. In some aspects, the region of complementarity comprises no more than four mismatches to the mRNA target sequence. In some aspects, the mRNA target sequence is a liver target sequence. In some aspects, the mRNA target sequence is a hepatocyte target sequence. In some aspects, the mRNA target sequence is a liver sinusoidal endothelial cell mRNA target sequence. In some aspects, the mRNA target sequence is a macrophage mRNA target sequence, optionally a liver macrophage mRNA target sequence.

[0059] In yet further aspects, the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 13 nucleotides in length, wherein the antisense and sense strands form a duplex region of 13 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising two nucleotides and a 3’ overhang comprising seven nucleotides, wherein the antisense strand comprises a region of complementarity to a macrophage mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to a nucleotide at position 2 of the sense strand, wherein the sense strand comprises a Tm-increasing nucleotide at positions 1, and 11, wherein the sense strand comprises a 2’-fluoro modification at positions 3-6 and a 2-0- methyl modification at positions 7-10 and 12-13, wherein the sense strand comprises phosphorothioate linkages between positions 1 and 2, positions 11 and 12, and positions 12 and 13, wherein the antisense strand comprises a 2’-fluoro modification at positions 2-5, 7, 10 and 14 and a 2-O-methyl modification at positions 1, 6, 8, 9, 11-13, and 15-22, wherein the antisense strand comprises phosphorothioate linkages between positions 1 and 2, positions 2 and 3, positions 3 and 4, positions 20 and 21, and positions 21 and 22, and wherein the antisense strand comprises a 5 ’terminal phosphorylated uridine.

[0060] In some aspects, the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 13 nucleotides in length, wherein the antisense and sense strands form a duplex region of 13 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising two nucleotides and a 3’ overhang comprising seven nucleotides, wherein the antisense strand comprises a region of complementarity to a macrophage mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to a nucleotide at position 2 of the sense strand, wherein the sense strand comprises a Tm-increasing nucleotide at positions 1, 10, and 11, wherein the sense strand comprises a 2’-fluoro modification at positions 3-6 and a 2-O- methyl modification at positions 7-19 and 12-13, wherein the sense strand comprises phosphorothioate linkages between positions 1 and 2, positions 11 and 12, and positions 12 and 13, wherein the antisense strand comprises a 2’-fluoro modification at positions 2-5, 7, 10 and 14 and a 2-O-methyl modification at positions 1, 6, 8, 9, 11-13, and 15-22, wherein the antisense strand comprises phosphorothioate linkages between positions 1 and 2, positions 2 and 3, positions 3 and 4, positions 20 and 21, and positions 21 and 22, and wherein the antisense strand comprises a 5 ’terminal phosphorylated uridine.

[0061] In some aspects, the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 12 nucleotides in length, wherein the antisense and sense strands form a duplex region of 12 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising at least two nucleotides and a 3’ overhang comprising at least seven nucleotides, wherein the antisense strand comprises a region of complementarity to an mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to a 5 ’terminal nucleotide of the sense strand, and wherein each of the antisense and sense strands comprise at least one 2’ -modified nucleotide and at least one modified internucleotide linkage. In some aspects, the sense strand comprises a 2’-fluoro modification at positions 3-6 or 4-7, numbered 5’ to 3’. In some aspects, the at least one modified internucleotide linkage is a phosphorothioate linkage. In some aspects, the sense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 10 and 11, and 11 and 12, numbered 5’ to 3’. In some aspects, the antisense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 12 and 14, 14 and 15, 20 and 21, and 21 and 22, numbered 5’ to 3’. In some aspects, the 5’overhang is 2 nucleotides and the 3 ’overhang is 8 nucleotides. In some aspects, the 5’overhang is 3 nucleotides and the 3 ’overhang is 7 nucleotides. In some aspects, the oligonucleotide comprises (i) 1-3 or 1-2 Tm-increasing nucleotides, or (ii) 1, 2 or 3 Tm-increasing nucleotides. In some aspects, the sense strand comprises a Tm-increasing nucleotide at (i) positions 2, 10 and 11, or (ii) positions 2, 11 and 12, numbered 5’ to 3’. In some aspects, the Tm-increasing nucleotide is a bicyclic nucleotide, optionally a locked nucleic acid (LNA).

[0062] In other aspects, the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 10 nucleotides in length, wherein the antisense and sense strands form a duplex region of 10 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising one nucleotide and a 3’ overhang comprising eleven nucleotides, wherein the antisense strand comprises a region of complementarity to an mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to a 5 ’terminal nucleotide of the sense strand, and wherein each of the antisense and sense strands comprise at least one 2’ -modified nucleotide and at least one modified internucleotide linkage. In some aspects, the at least one modified intemucleotide linkage is a phosphorothioate linkage. In some aspects, the sense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 8 and 9, and 9 and 10, numbered 5’ to 3’. In some aspects, the antisense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 12 and 14, 14 and 15, 20 and 21, and 21 and 22, numbered 5’ to 3’. In some aspects, the oligonucleotide comprises (i) 1-3 Tm- increasing nucleotides, or (ii) 1, 2 or 3 Tm-increasing nucleotides. In some aspects, the sense strand comprises a Tm-increasing nucleotide at positions 2, 6 and 7. In some aspects, the Tm- increasing nucleotide is a bicyclic nucleotide, optionally a locked nucleic acid (LNA).

[0063] In any of the foregoing or related aspects, the antisense strand comprises a 2’ -fluoro modification at positions 2-5, 7, 10 and 14, numbered 5’ to 3’. In some aspects, the remaining nucleotides comprise a 2'-O-methyl modification, provided that the nucleotide of the sense strand conjugated to the at least one lipid moiety does not comprise a 2’-O-methyl modification

[0064] In any of the foregoing or related aspects, the oligonucleotide is a Dicer substrate. In some aspects, the oligonucleotide reduces expression of the mRNA target sequence in a cell or population of cells in vitro and / or in vivo.

[0065] In some aspects, the disclosure provides a pharmaceutical composition comprising an oligonucleotide described herein, and a pharmaceutically acceptable carrier, delivery agent or excipient.

[0066] In other aspects, the disclosure provides a method for treating a subject having a disease, disorder or condition associated with expression of a target mRNA, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide or pharmaceutical composition described herein. In some aspects, the target mRNA is expressed in the central nervous system, optionally wherein the central nervous system comprises the frontal cortex, hippocampus, medulla, cerebellum, lumbar dorsal root ganglion, and / or lumbar spinal cord. In some aspects, the target mRNA is expressed in a neuron of the central nervous system. In some aspects, the target mRNA is expressed in a macrophage. In some aspects, the macrophage is in the liver. In some aspects, the target mRNA is expressed in the liver. In some aspects, the target mRNA is expressed in a hepatocyte. In some aspects, the target mRNA is expressed in a liver sinusoidal endothelial cell. In some aspects, the target mRNA is expressed in ocular tissue. In some aspects, the target mRNA is expressed in a tissue of the central nervous system, liver tissue, ocular tissue, adipose tissue, muscle tissue, adrenal tissue, cardiac tissue, lung tissue, or any combination thereof. In some aspects, the disclosure provides a method for treating a subject having a disease, disorder or condition associated with expression of an mRNA of the central nervous system, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide or pharmaceutical composition described herein, optionally wherein the central nervous system comprises the frontal cortex, hippocampus, medulla, cerebellum, lumbar dorsal root ganglion, and / or lumbar spinal cord. In some aspects, the mRNA of the central nervous system is a neuronal mRNA.

[0067] In other aspects, the disclosure provides a method for treating a subject having a disease, disorder or condition associated with expression of an mRNA of the liver, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide or pharmaceutical composition described herein.

[0068] In further aspects, the disclosure provides a method for treating a subject having a disease, disorder or condition associated with expression of an ocular mRNA, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide or pharmaceutical composition described herein.

[0069] In yet further aspects, the disclosure provides a method for treating a subject having a disease, disorder or condition associated with expression of a macrophage mRNA, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide or pharmaceutical composition described herein. In some aspects, the macrophage mRNA is expressed in the liver.

[0070] In some aspects, the disclosure provides a method of delivering an oligonucleotide to a cell or population of cells in the central nervous system, liver tissue or ocular tissue, the method comprising administering a pharmaceutical composition described herein.

[0071] In further aspects, the disclosure provides a method of reducing expression of a target mRNA in a subject, comprising administering to the subject an oligonucleotide or pharmaceutical composition described herein. In some aspects, the target mRNA is expressed in the central nervous system, optionally wherein the central nervous system comprises the frontal cortex, hippocampus, medulla, cerebellum, lumbar dorsal root ganglion, and / or lumbar spinal cord. In some aspects, the target mRNA is expressed in a neuron of the central nervous system. In some aspects, the target mRNA is expressed in the liver. In some aspects, the target mRNA is expressed in a hepatocyte. In some aspects, the target mRNA is expressed in a liver sinusoidal endothelial cell. In some aspects, the target mRNA is expressed in a macrophage. In some aspects, the macrophage is in the liver. In some aspects, the target mRNA is expressed in ocular tissue. 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 the overexpression of a target mRNA. In further 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 the reduction in expression of a target mRNA.

[0072] In some 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 the reduction in the expression of a target mRNA. In other 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 the overexpression of a target mRNA. In further 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 a target mRNA.

[0073] In any of the foregoing or related aspects, the target mRNA is expressed in the central nervous system, a neuron of the central system, the liver, a macrophage, optionally a macrophage in the liver, ocular tissue, or any combination thereof, optionally wherein the central nervous system comprises the frontal cortex, hippocampus, medulla, cerebellum, lumbar dorsal root ganglion, and / or lumbar spinal cord.

[0074] In some aspects, the disclosure provides a method of activating target-specific RNA interference (RNAi) in an organism comprising administering to said organism an oligonucleotide described herein, said oligonucleotide being administered in an amount sufficient for degradation of the target mRNA to occur, thereby activating target-specific RNAi in the organism. In some aspects, the target mRNA specifies the amino acid sequence of a protein involved or predicted to be involved in a human disease or disorder. In some aspects, the disease or disorder is selected from the group consisting of viral infections, bacterial infections, parasitic infections, cancers, allergies, autoimmune diseases, immunodeficiencies, and immunosuppression.

[0075] In some aspects, the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of about 15-30 nucleotides in length and a sense strand of about 15-50 nucleotides in length, wherein the antisense and sense strands form a duplex region of about 15-30 base pairs, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, wherein the sense strand comprises (i) at least one lipid moiety conjugated to a nucleotide of the sense strand, and (ii) a stem-loop, 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, and wherein the sense and antisense strands each comprise an orientation of 5’ to 3’, and wherein the stem-loop is at the 5’ end of the sense strand.

[0076] In any of the foregoing or related aspects, the oligonucleotide comprises a blunt end. In some aspects, the blunt end comprises the 3’ end of the sense strand and the 5’ end of the antisense strand.

[0077] In some aspects, the oligonucleotide comprises an overhang of at least two nucleotides. In some aspects, the overhang comprises the 5 ’end of the antisense strand.

[0078] In some aspects, the sense strand is about 28-38 nucleotides. In some aspects, the antisense strand is 22 nucleotides. In some aspects, the lipid moiety is conjugated to a nucleotide comprising the loop.

[0079] In any of the foregoing or related aspects,

[0080] (i) the sense strand is 28 nucleotides, and the lipid moiety is conjugated to a nucleotide at position 4, positions numbered 5’ to 3’;

[0081] (ii) the sense strand is 30 nucleotides, and the lipid moiety is conjugated to a nucleotide at position 4, positions numbered 5’ to 3’;

[0082] (iii) the sense strand is 34 nucleotides, and the lipid moiety is conjugated to a nucleotide at position 6 or position 15, positions numbered 5’ to 3’; or

[0083] (iv) the sense strand is 38 nucleotides, and the lipid moiety is conjugated to a nucleotide at position 8, positions numbered 5’ to 3’.

[0084] In any of the foregoing or related aspects, the lipid moiety is selected from:

[0085]

[0086] 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.

[0087] In any of the foregoing or related aspects, the region of complementarity is fully complementary to the mRNA target sequence. In other aspects, the region of complementarity is partially complementary to the mRNA target sequence. In some aspects, the region of complementarity comprises no more than four mismatches to the mRNA target sequence.

[0088] In any of the foregoing or related aspects, the mRNA target sequence is a central nervous system (CNS) target sequence, optionally a neuronal mRNA target sequence or an ocular mRNA target sequence. In some aspects, the mRNA target sequence is an ocular mRNA target sequence.

[0089] In any of the foregoing or related aspects, the loop sequence is 5’-GAAA-3’. In some aspects, the loop sequence is 5’-UNCG-3’, wherein N is any nucleotide. In some aspects, the loop sequence is 5’-UACG-3’.

[0090] 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. 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, 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,

[0091] (i) the sense strand is 38 nucleotides with positions 1-38 from 5’ to 3’, and wherein each of positions 26-29 comprise a 2’ -fluoro modification;

[0092] (ii) the sense strand is 34 nucleotides with positions 1-34 from 5’ to 3’, and wherein each of positions 22-25 comprise a 2’ -fluoro modification;

[0093] (iii) the sense strand is 30 nucleotides with positions 1-30 from 5’ to 3’, and wherein each of positions 18-21 comprise a 2’ -fluoro modification; or

[0094] (iv) the sense strand is 28 nucleotides with positions 1-28 from 5’ to 3’, and wherein each of positions 18-21 comprise a 2’ -fluoro modification.

[0095] In any of the foregoing or related aspects, 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 21 and 22, wherein positions are numbered 1-22 from 5' to 3'. In some aspects, the sense strand comprises a phosphorothioate linkage between the penultimate nucleotide and third nucleotide from the 3’ end, and between the penultimate nucleotide and ultimate nucleotide. In some aspects,

[0096] (i) the sense strand is 38 nucleotides with positions 1-38 from 5’ to 3’, and wherein the sense strand comprises a phosphorothioate linkage between positions 36 and 37, and 37 and 38;

[0097] (ii) the sense strand is 34 nucleotides with positions 1-34 from 5’ to 3’, and wherein the sense strand comprises a phosphorothioate linkage between positions 32 and 33, and 33 and 34;

[0098] (iii) the sense strand is 30 nucleotides with positions 1-30 from 5’ to 3’, and wherein the sense strand comprises a phosphorothioate linkage between positions 28 and 29, and 29 and 30; or

[0099] (iv) the sense strand is 28 nucleotides with positions 1-28 from 5’ to 3’, and wherein the sense strand comprises a phosphorothioate linkage between positions 26 and 27, and 27 and 28.

[0100] 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. In some aspects, the phosphorylated nucleotide is 4’-O-monomethylphosphonate-2’-O-methyl uridine.

[0101] In any of the foregoing or related aspects, the sense strand comprises at least one Tm- increasing nucleotide. In some aspects, the sense strand comprises 1-6, 1-5, 1-4, 1-3 or 12 Tm-increasing nucleotides. In some aspects, the sense strand comprises 1, 2, 3, 4, 5 or 6 Tm- increasing nucleotides. In some aspects, the sense strand comprises up to six Tm-increasing nucleotides. In some aspects, the Tm-increasing nucleotide is a bicyclic nucleotide. In some aspects, the Tm-increasing nucleotide is a locked nucleic acid.

[0102] In any of the foregoing or related aspects, SI and S2 each comprise 1-6 nucleotides. In some aspects, SI and S2 each comprise 4 nucleotides. In some aspects, SI and S2 each comprise 2 nucleotides. In some aspects, SI and S2 each comprise at least one Tm-increasing nucleotide. In some aspects, SI and S2 are each 4 nucleotides, wherein 1-3 nucleotides of each SI and S2 are Tm-increasing nucleotides.

[0103] In any of the foregoing or related aspects, the remaining nucleotides comprise a 2'-O- methyl modification, provided that the nucleotide of the sense strand conjugated to the at least one lipid moiety does not comprise a 2’-O-methyl modification.

[0104] In some aspects, the disclosure provides, a double-stranded oligonucleotide comprising an antisense strand of about 20-22 nucleotides in length and a sense strand of about 32-34 nucleotides in length, wherein the antisense and sense strands form a duplex region of about 20-22 base pairs and the oligonucleotide is blunt ended, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, wherein the sense strand comprises (i) a stem-loop, 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, and (ii) at least one lipid moiety conjugated to a nucleotide of the loop, wherein the sense and antisense strands each comprise an orientation of 5’ to 3’, wherein the stem-loop is at the 5’ end of the sense strand, and wherein each of the antisense and sense strands comprise at least one 2’ -modified nucleotide and at least one modified internucleotide linkage.

[0105] In some aspects, the sense strand is 34 nucleotides and comprises a 2’ -fluoro modification at positions 22-25, numbered 5’ to 3’. In some aspects, the antisense strand is 22 nucleotides and comprises a 2’-fluoro modification at positions 2-5, 7, 10 and 14, numbered 5’ to 3’. In some aspects, the remaining nucleotides comprise a 2'-O-methyl modification, provided that the nucleotide of the sense strand conjugated to the at least one lipid moiety does not comprise a 2’-O-methyl modification. In some aspects, the at least one modified internucleotide linkage is a phosphorothioate linkage. In some aspects, the sense strand is 34 nucleotides and comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 2 and 3, 32 and 33, and 33 and 34, numbered 5’ to 3’. In some aspects, the antisense strand is 22 nucleotides and comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22, numbered 5’ to 3’. In some aspects, the antisense strand comprises a phosphorylated uridine at position 1, numbered 5’ to 3’. In some aspects, the phosphorylated uridine is 4’-O-monomethylphosphonae-2’-O-methyl uridine. In some aspects, the lipid moiety is a C16 hydrocarbon represented by:

[0106] . In some aspects, the lipid moiety is conjugated to the 2’ carbon of the ribose ring of the nucleotide. In some aspects, the lipid moiety is conjugated to a nucleotide at position 6 of the sense strand, numbered 5’ to 3’. In some aspects, SI and S2 each comprise 1-6 nucleotides. In some aspects, SI and S2 are each 4 nucleotides. In some aspects, L is 4 nucleotides. In some aspects, L comprises the sequence 5’-GAAA-3’. In some aspects, the region of complementarity is fully complementary to the mRNA target sequence. In other aspects, wherein the region of complementarity is partially complementary to the mRNA target sequence. In some aspects, the region of complementarity comprises no more than four mismatches to the mRNA target sequence. In some aspects, the mRNA target sequence is a central nervous system (CNS) target sequence, optionally a neuronal mRNA target sequence or an ocular mRNA target sequence.

[0107] In some aspects, the disclosure provides, a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 34 nucleotides in length, wherein the antisense and sense strands form a duplex region of 22 base pairs and the oligonucleotide is blunt ended, wherein the antisense strand comprises a region of complementarity to a central nervous system mRNA target sequence, wherein the sense strand comprises (i) a stem-loop, wherein the stem-loop comprises a nucleotide sequence represented by the formula: 5’-Sl-L-S2-3’, wherein SI is complementary to S2, wherein SI and S2 each comprise 4 nucleotides, wherein L forms a loop between SI and S2, and wherein L comprises four nucleotides, and (ii) at least one lipid moiety conjugated to a nucleotide of the loop, wherein the sense and antisense strands each comprise an orientation of 5’ to 3’, wherein the stem-loop is at the 5’ end of the sense strand, wherein the sense strand comprises a 2’ -fluoro modification at positions 22-25 and a 2’-O-methyl modification at positions 1-5, 7-21 and 26-34, wherein the antisense strand comprises a 2’ -fluoro modification at positions 2-5, 7, 10 and 14 and a 2-O-methyl modification at positions 1, 6, 8, 9, 11-13, and 15-22, wherein the sense strand comprises phosphorothioate linkages between positions 1 and 2, 2 and 3, 32 and 33, and 33 and 34, and wherein the antisense strand comprises phosphorothioate linkages between positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22.

[0108] In some aspects, the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of about 20-22 nucleotides in length and a sense strand of about 26-28 nucleotides in length, wherein the antisense and sense strands form an asymmetric duplex region of about 20-22 base pairs comprising a 3’ terminal overhang of at least 2 nucleotides of the antisense strand, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, wherein the sense strand comprises: (i) a stemloop, wherein the stem-loop comprises a nucleotide sequence represented by the formula: 5’- S1-L-S2-3’, wherein SI is complementary to S2, wherein L forms a loop between SI and S2 and comprises the sequence UNCG, and wherein SI and S2 each comprise at least one Tm- increasing nucleotide, and (ii) at least one lipid moiety conjugated to a nucleotide of the loop, wherein the sense strand comprises an orientation of 5’ to 3’, wherein the stem-loop is at the 5’ end of the sense strand, and wherein each of the antisense and sense strands comprise at least one 2’-modified nucleotide and at least one modified internucleotide linkage.

[0109] In some aspects, the sense strand is 28 nucleotides and comprises a 2’ -fluoro modification at positions 18-21, numbered 5’ to 3’. In some aspects, the antisense strand is 22 nucleotides and comprises a 2’-fluoro modification at positions 2-5, 7, 10 and 14, numbered 5’ to 3 ’.In some aspects, the at least one modified internucleotide linkage is a phosphorothioate linkage. In some aspects, the sense strand is 28 nucleotides and comprises phosphorothioate linkages between nucleotides at positions 26 and 27, and 27 and 28, numbered 5’ to 3’. In some aspects, the antisense strand is 22 nucleotides and comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22, numbered 5’ to 3’. In some aspects, the 3 ’terminal overhang is 2 nucleotides. In some aspects, the antisense strand comprises a phosphorylated uridine at position 1, numbered 5’ to 3’. In some aspects, the phosphorylated uridine is 4’-O- monomethylphosphonae-2’-O-methyl uridine. In some aspects, the lipid moiety is a C16 hydrocarbon represented by:

[0110] . In some aspects, the lipid moiety is conjugated to the 2’ carbon of the ribose ring of the nucleotide. In some aspects, the lipid moiety is conjugated to a nucleotide at position 4 of the sense strand, numbered 5’ to 3’. In some aspects, SI and S2 each comprise 1-6 nucleotides. In some aspects, wherein SI and S2 are each 2 nucleotides. In some aspects, SI and S2 each comprise one Tm-increasing nucleotide. In some aspects, the Tm-increasing nucleotide is a bicyclic nucleotide. In some aspects, Tm-increasing nucleotide is a locked nucleic acid (LNA). In some aspects, the remaining nucleotides comprise a 2'-O-methyl modification, provided that the nucleotide of the sense strand conjugated to the at least one lipid moiety does not comprise a 2’-O-methyl modification. In some aspects, L is 4 nucleotides. In some aspects, L comprises the sequence 5’-UNCG-3’, wherein N is any nucleotide. In some aspects, L comprises the sequence 5’- UACG-3. In some aspects, the region of complementarity is fully complementary to the mRNA target sequence. In some aspects, the region of complementarity is partially complementary to the mRNA target sequence. In some aspects, the region of complementarity comprises no more than four mismatches to the mRNA target sequence. In some aspects, the mRNA target sequence is a central nervous system (CNS) target sequence, optionally a neuronal mRNA target sequence or an ocular mRNA target sequence.

[0111] In some aspects, the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 28 nucleotides in length, wherein the antisense and sense strands form an asymmetric duplex region of 22 base pairs comprising a 3’ terminal overhang of 2 nucleotides of the antisense strand, wherein the antisense strand comprises a region of complementarity to a central nervous system mRNA target sequence, wherein the sense strand comprises: (i) a stem-loop, wherein the stem-loop comprises a nucleotide sequence represented by the formula: 5’-Sl-L- S2-3’, wherein SI is complementary to S2, wherein SI and S2 each comprise 2 nucleotides, wherein L forms a loop between SI and S2 and comprises the sequence UNCG, and wherein SI and S2 each comprise at least one Tm-increasing nucleotide, and (ii) at least one lipid moiety conjugated to a nucleotide of the loop, wherein the sense strand comprises an orientation of 5’ to 3’, wherein the stem-loop is at the 5’ end of the sense strand, wherein the sense strand comprises a 2’ -fluoro modification at positions 18-21 and a 2’-O-methyl modification at positions 2-3, 5-7, 9-17 and 22-28, wherein the antisense strand comprises a 2’-fluoro modification at positions 2-5, 7, 10 and 14 and a 2-O-methyl modification at positions 1, 6, 8, 9, 11-13, and 15-22, wherein the sense strand comprises phosphorothioate linkages between positions 26 and 27, and 27 and 28, and wherein the antisense strand comprises phosphorothioate linkages between positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22.

[0112] In some aspects, the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of about 20-22 nucleotides in length and a sense strand of about 32-34 nucleotides in length, wherein the antisense and sense strands form a duplex region of about 20-22 base pairs, and the oligonucleotide is blunt ended, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, wherein the sense strand comprises: (i) a stem-loop, wherein the stem-loop comprises a nucleotide sequence represented by the formula: 5’-Sl-L-S2-3’, wherein SI is complementary to S2, wherein L forms a loop between SI and S2, and wherein SI and S2 each comprise at least one Tm-increasing nucleotide, and (ii) at least one lipid moiety conjugated to a nucleotide of the loop, wherein the sense strand comprises an orientation of 5’ to 3’, wherein the stem-loop is at the 5’ end of the sense strand, and wherein each of the antisense and sense strands comprise at least one 2’ -modified nucleotide and at least one modified internucleotide linkage.

[0113] In some aspects, the sense strand is 34 nucleotides and comprises a 2’ -fluoro modification at positions 22-25, numbered 5’ to 3’. In some aspects, antisense strand is 22 nucleotides and comprises a 2’-fluoro modification at positions 2-5, 7, 10 and 14, numbered 5’ to 3’. In some aspects, the at least one modified intemucleotide linkage is a phosphorothioate linkage. In some aspects, the sense strand is 34 nucleotides and comprises phosphorothioate linkages between nucleotides at positions 32 and 33, and 33 and 34, numbered 5’ to 3’. In some aspects, the antisense strand is 22 nucleotides and comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22, numbered 5’ to 3’. In some aspects, the oligonucleotide comprises a blunt end comprising the 5’ end of the antisense strand and the 3’ end of the sense strand. In some aspects, the antisense strand comprises a phosphorylated uridine at position 1, numbered 5’ to 3’. In some aspects, the phosphorylated uridine is 4’-O-monomethylphosphonae-2’-O-methyl uridine. In some aspects, the lipid moiety is a C16 hydrocarbon represented by:

[0114] . In some aspects, the lipid moiety is conjugated to the 2’ carbon of the ribose ring of the nucleotide. In some aspects, the lipid moiety is conjugated to a nucleotide at position 6 of the sense strand, numbered 5’ to 3’. In some aspects, SI and S2 each comprise 1-6 nucleotides. In some aspects, SI and S2 are each 4 nucleotides. In some aspects, SI and S2 each comprise one Tm-increasing nucleotide, some aspects, the Tm-increasing nucleotide is a bicyclic nucleotide. In some aspects, the Tm- increasing nucleotide is a locked nucleic acid (LNA). In some aspects, the remaining nucleotides comprise a 2'-O-methyl modification, provided that the nucleotide of the sense strand conjugated to the at least one lipid moiety does not comprise a 2’-O-methyl modification. In some aspects, L is 4 nucleotides. In some aspects, L comprises the sequence 5’-GAAA-3’. In some aspects, the region of complementarity is fully complementary to the mRNA target sequence. In some aspects, the region of complementarity is partially complementary to the mRNA target sequence. In some aspects, the region of complementarity comprises no more than four mismatches to the mRNA target sequence. In some aspects, the mRNA target sequence is (i) a central nervous system (CNS) mRNA target sequence, optionally a neuronal mRNA target sequence or an ocular mRNA target sequence; or (ii) an ocular tissue mRNA target sequence.

[0115] In some aspects, the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 34 nucleotides in length, wherein the antisense and sense strands form a duplex region of 22 base pairs, and the oligonucleotide is blunt ended, wherein the antisense strand comprises a region of complementarity to a central nervous system mRNA target sequence, wherein the sense strand comprises: (i) a stem-loop, wherein the stem-loop comprises a nucleotide sequence represented by the formula: 5’-Sl-L-S2-3’, wherein SI is complementary to S2, wherein SI and S2 each comprise 4 nucleotides, wherein L forms a loop between SI and S2, wherein the loop comprises 4 nucleotides, and wherein SI and S2 each comprise at least one Tm-increasing nucleotide, and (ii) at least one lipid moiety conjugated to a nucleotide of the loop, wherein the sense strand comprises an orientation of 5’ to 3’, wherein the stem-loop is at the 5’ end of the sense strand, wherein the sense strand comprises a 2’ -fluoro modification at positions 22-25 and a 2’-O-methyl modification at positions 2-5, 7-11, 12-21 and 26-34, wherein the antisense strand comprises a 2’-fluoro modification at positions 2-5, 7, 10 and 14 and a 2-O-methyl modification at positions 1, 6, 8, 9, 11-13, and 15-22, wherein the sense strand comprises phosphorothioate linkages between positions 32 and 33, and 33 and 34, and wherein the antisense strand comprises phosphorothioate linkages between positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22.

[0116] In any of the foregoing or related aspects, the oligonucleotide is a Dicer substrate. In further aspects, the oligonucleotide reduces expression of the mRNA target sequence in a cell or population of cells in vitro and / or in vivo.

[0117] In some aspects, the disclosure provides a pharmaceutical composition comprising an oligonucleotide of any of the foregoing or related aspects, and a pharmaceutically acceptable carrier, delivery agent or excipient.

[0118] In some aspects, the disclosure provides a method for treating a subject having a disease, disorder or condition associated with expression of a target mRNA, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide or pharmaceutical composition described herein. In some aspects of the methods described herein, the target mRNA is expressed in the central nervous system, optionally a neuron of the central nervous system. In some aspects of the methods described herein, the target mRNA is expressed in ocular tissue, optionally the optic nerve and / or retina.

[0119] In some aspects, the disclosure provides a method of delivering an oligonucleotide to a cell or population of cells in the central nervous system or ocular tissue, the method comprising administering a pharmaceutical composition described herein.

[0120] In some aspects, the disclosure provides a method of reducing expression of a target mRNA in a subject, comprising administering to the subject an oligonucleotide or pharmaceutical composition described herein.

[0121] In some aspects of the methods described herein, target mRNA is expressed in the central nervous system, optionally a neuron of the central nervous system. In some aspects of the methods described herein, the target mRNA is expressed in ocular tissue, optionally the optic nerve and / or retina.

[0122] In some 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 a target mRNA.

[0123] In some 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 a target mRNA.

[0124] In some aspects, the disclosure provides any 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 a target mRNA.

[0125] In some aspects of the methods described herein, the target mRNA is expressed in the central nervous system and / or a neuron of the central nervous system. In some aspects, the target mRNA is expressed in ocular tissue, optionally the retina and / or optic nerve.

[0126] In any of the foregoing or related aspects of the methods described herein the central nervous system comprises the frontal cortex, the hippocampus, the cerebellum, the brainstem, lumbar dorsal root ganglion, the lumbar spinal cord, or combinations thereof.

[0127] In any of the foregoing or related aspects, the central nervous system comprises the frontal cortex, the hippocampus, the cerebellum, the brainstem, lumbar dorsal root ganglion, the lumbar spinal cord, or combinations thereof. In some aspects, the disclosure provides a method of activating target-specific RNA interference (RNAi) in an organism comprising administering to said organism a dsRNA oligonucleotide described herein, said oligonucleotide being administered in an amount sufficient for degradation of the target mRNA to occur, thereby activating target-specific RNAi in the organism. In some aspects of, the target mRNA specifies the amino acid sequence of a protein involved or predicted to be involved in a human disease or disorder. In some aspects, the disease or disorder is selected from the group consisting of viral infections, bacterial infections, parasitic infections, cancers, allergies, autoimmune diseases, immunodeficiencies, and immunosuppression.

[0128] In some aspects, the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of about 13-30 nucleotides in length and a sense strand of about 10-50 nucleotides in length, wherein the antisense and sense strands are separate strands which form an asymmetric duplex region having an overhang of about 2-10 nucleotides at the 3’ terminus of the antisense strand, wherein the duplex region is about 10- 30 nucleotides, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, and wherein the sense strand comprises at least one lipid moiety conjugated to a nucleotide on the sense strand.

[0129] In some aspects, the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of about 13-30 nucleotides in length and a sense strand of about 10-50 nucleotides in length, wherein the antisense and sense strands are separate strands which form an asymmetric duplex region having an overhang of about 2-12 nucleotides at the 3’ terminus of the antisense strand, wherein the duplex region is about 10- 30 nucleotides, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, and wherein the sense strand comprises at least one lipid moiety conjugated to a nucleotide on the sense strand.

[0130] In some aspects, the oligonucleotide comprises a blunt end. In some aspects, the blunt end comprises the 3’ end of the sense strand and the 5’ end of the antisense strand.

[0131] In further aspects, the oligonucleotide comprises a stem loop, 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, and wherein the sense strand comprises an orientation of 5’ to 3’, and wherein the stem-loop is at the 3’ end of the sense strand.

[0132] In any of the foregoing or related aspects, (i) the sense strand is 19 nucleotides, the duplex region is 19 nucleotides, and the overhang is 3 nucleotides;

[0133] (ii) the sense strand is 18 nucleotides, the duplex region is 18 nucleotides, and the overhang is 4 nucleotides;

[0134] (iii) the sense strand is 17 nucleotides, the duplex region is 17 nucleotides, and the overhang is 5 nucleotides;

[0135] (iv) the sense strand is 16 nucleotides, the duplex region is 16 nucleotides, and the overhang is 6 nucleotides; or

[0136] (v) the sense strand is 15 nucleotides, the duplex region is 15 nucleotides, and the overhang is 7 nucleotides.

[0137] In some aspects,

[0138] (i) the sense strand is 19 nucleotides, the duplex region is 19 nucleotides, and the overhang is 3 nucleotides;

[0139] (ii) the sense strand is 18 nucleotides, the duplex region is 18 nucleotides, and the overhang is 4 nucleotides;

[0140] (iii) the sense strand is 17 nucleotides, the duplex region is 17 nucleotides, and the overhang is 5 nucleotides;

[0141] (iv) the sense strand is 16 nucleotides, the duplex region is 16 nucleotides, and the overhang is 6 nucleotides;

[0142] (v) the sense strand is 15 nucleotides, the duplex region is 15 nucleotides, and the overhang is 7 nucleotides;

[0143] (vi) the sense strand is 14 nucleotides, the duplex region is 14 nucleotides, and the overhang is 8 nucleotides;

[0144] (vii) the sense strand is 13 nucleotides, the duplex region is 13 nucleotides, and the overhang is 8 nucleotides; or

[0145] (viii) the sense strand is 12 nucleotides, the duplex region is 12 nucleotides, and the overhang is 10 nucleotides.

[0146] In some aspects, the sense strand is 10 nucleotides, the duplex region is 10 nucleotides, and the overhang is 12 nucleotides.

[0147] In some aspects,

[0148] (i) the sense strand is 32 nucleotides, the duplex region is 16 nucleotides, and the overhang is 6 nucleotides;

[0149] (ii) the sense strand is 30 nucleotides, the duplex region is 14 nucleotides, and the overhang is 8 nucleotides; or (iii) the sense strand is 28 nucleotides, the duplex region is 12 nucleotides, and the overhang is 10 nucleotides.

[0150] In some aspects, the antisense strand is 22 nucleotides, with positions 1-22 numbered 5’ to 3’.

[0151] In some aspects, the lipid moiety is conjugated to a nucleotide of the sense strand that forms a base pair with a nucleotide at position 14 of the antisense strand. In some aspects, the lipid moiety is conjugated to a nucleotide of the sense strand that forms a base pair with a nucleotide at position 12 of the antisense strand. In some aspects, the lipid moiety is conjugated to a nucleotide of the sense strand that forms a base pair with a nucleotide at position 20, position 19, position 18, position 17, position 16, position 15, position 14, position 13, or position 12 of the antisense strand.

[0152] In further aspects, the lipid moiety is conjugated to a nucleotide in the loop. In some aspects, the lipid moiety is conjugated to the 3 ’terminal nucleotide on the sense strand. In some aspects, the lipid moiety is conjugated to the 5’ terminal nucleotide on the sense strand.

[0153] In any of the foregoing or related aspects, the lipid moiety is selected from:

[0154]

[0155] 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

[0156] . In some aspects, the hydrocarbon chain is a C22 hydrocarbon chain. In some aspects, the C22 hydrocarbon chain is represented by

[0157] In any of the foregoing or related aspects, the lipid moiety is conjugated to the 2’ carbon of the ribose ring of the nucleotide.

[0158] In any of the foregoing or related aspects, the region of complementarity is fully complementary to the mRNA target sequence. In some aspects, the region of complementarity is partially complementary to the mRNA target sequence. In some aspects, the region of complementarity comprises no more than four mismatches to the mRNA target sequence.

[0159] In any of the foregoing or related aspects, the mRNA target sequence is a liver mRNA target sequence, optionally a liver macrophage mRNA target sequence, a liver hepatocyte mRNA target sequence, or a liver sinusoidal endothelial cell mRNA target sequence. In some aspects, the mRNA target sequence is an ocular mRNA target sequence. In some aspects, the mRNA target sequence is expressed in liver tissue, skeletal muscle tissue, adipose tissue and / or adrenal tissue. In other aspects, the mRNA target sequence is expressed in at least one tissue of the central nervous system. In some aspects, the at least one tissue of the central nervous system is selected from frontal cortex, medulla, hippocampus, hypothalamus, cerebellum, lumbar spinal cord, lumbar dorsal root ganglion, and any combination thereof.

[0160] In any of the foregoing or related aspects, 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. 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, the antisense strand is 22 nucleotides with positions 1- 22 numbered 5’ to 3’, and wherein the sense strand comprises a 2’-fluoro modification at each of nucleotides forming a base pair with nucleotides at positions 10-13 of the antisense strand. In some aspects, the antisense strand is 22 nucleotides with positions 1-22 numbered 5’ to 3’, and wherein the sense strand comprises a 2’-fluoro modification at each of nucleotides forming a base pair with nucleotides at positions 10, 11, 12, 13, or any combination thereof, of the antisense strand. 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, provided that the nucleotide of the sense strand conjugated to the at least one lipid moiety does not comprise a 2’-O-methyl modification.

[0161] 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 21 and 22, wherein positions are numbered 1-22 from 5' to 3'. In some aspects, the antisense strand comprises a phosphorothioate linkage between positions 13 and 14 and between positions 14 and 15. In some aspects, the antisense strand comprises a phosphorothioate linkage between positions 16 and 17, between positions 17 and 18, between positions 18 and 19, and between positions 19 and 20. In some aspects, the antisense strand comprises a phosphorothioate linkage between positions 15 and 16, between positions 16 and 17, between positions 17 and 18, between positions 18 and 19, and between positions 19 and 20. In some aspects, the antisense strand comprises a phosphorothioate linkage between positions 12 and 13, between positions 15 and 16, between positions 16 and 17, between positions 17 and 18, between positions 18 and 19, and between positions 19 and 20. In some aspects, the sense strand comprises a phosphorothioate linkage between positions 1 and 2. In some aspects, the sense strand comprises a phosphorothioate linkage between positions 1 and 2, between the penultimate nucleotide and third nucleotide from the 3’ end, and between the penultimate nucleotide and ultimate nucleotide.

[0162] 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. In some aspects, the phosphorylated nucleotide is 4’-O-monomethylphosphonate-2’-O-methyl uridine.

[0163] In any of the foregoing or related aspects, the sense strand comprises at least one Tm- increasing nucleotide. In some aspects, the sense strand comprises up to nine Tm-increasing nucleotides. In some aspects, the sense strand comprises 1-3 Tm-increasing nucleotides. In some aspects, SI and S2 each comprise at least one Tm-increasing nucleotide. In some aspects, SI and S2 are each 3 nucleotides, and each nucleotide is a Tm-increasing nucleotide. In some aspects, the sense strand comprises up to three Tm-increasing nucleotides at nucleotide positions, provided the nucleotide positions are not in the stem-loop. In some aspects, wherein the sense strand comprises up to five Tm-increasing nucleotides.

[0164] In any of the foregoing or related aspects, the Tm-increasing nucleotide is a bicyclic nucleotide. In some aspects, the Tm-increasing nucleotide is a locked nucleic acid. In some aspects, the sense strand is 20 nucleotides in length, wherein the nucleotides are numbered 1- 20 5’ to 3’, and wherein the sense strand comprises a locked nucleic acid at a nucleotide located at:

[0165] (i) position 2;

[0166] (ii) position 2 and position 15;

[0167] (iii) position 2, position 15, and position 16;

[0168] (iv) position 2, position 15, position 16, and position 18; or,

[0169] (v) position 2, position 15, position 16, position 18, and position 19. In some aspects, the sense strand is 16 nucleotides in length, wherein the nucleotides are numbered 1-16 5’ to 3’, and wherein the sense strand comprises a locked nucleic acid at a nucleotide located at:

[0170] (i) position 2;

[0171] (ii) position 2 and position 11;

[0172] (iii) position 2, position 11, and position 12;

[0173] (iv) position 2, position 11, position 12, and position 14; or,

[0174] (v) position 2, position 11, position 12, position 14, and position 15.

[0175] In some aspects, the sense strand is 14 nucleotides in length, wherein the nucleotides are numbered 1-14 5’ to 3, and wherein the sense strand comprises a locked nucleic acid at a nucleotide located at:

[0176] (i) position 2;

[0177] (ii) position 2 and position 9;

[0178] (iii) position 2, position 9, and position 10; or,

[0179] (iv) position 2, position 9, position 10, position 12, and position 13.

[0180] In some aspects, the sense strand is 12 nucleotides in length, wherein the nucleotides are numbered 1-12 5’ to 3’, and wherein the sense strand comprises a locked nucleic acid at a nucleotide located at:

[0181] (i) position 2;

[0182] (ii) position 2 and position 7;

[0183] (iii) position 2, position 7, and position 8;

[0184] (iv) position 2, position 7, position 8, and position 10;

[0185] (v) position 2, position 7, position 8, position 10, and position 11.

[0186] In some aspects, the sense strand comprises a locked nucleic acid at a nucleotide that forms a base pair with a nucleotide at a position of the antisense strand selected from:

[0187] (i) position 2;

[0188] (ii) position 3;

[0189] (iii) position 5;

[0190] (iv) position 6; and

[0191] (v) any combination of (i) - (iv).

[0192] In any of the foregoing or related aspects, the oligonucleotide is a Dicer substrate. In some aspects, the oligonucleotide reduces expression of the mRNA target sequence in a cell or population of cells in vitro and / or in vivo. In some aspects, the disclosure provides a pharmaceutical composition comprising any oligonucleotide described herein, and a pharmaceutically acceptable carrier, delivery agent or excipient.

[0193] In some aspects, the disclosure provides a method of delivering an oligonucleotide to a cell or population of cells in the central nervous system, liver tissue, muscle tissue, adipose tissue, adrenal tissue and / or ocular tissue, the method comprising administering a pharmaceutical composition described herein.

[0194] In some aspects, the disclosure provides a method for treating a subject having a disease, disorder or condition associated with expression of a target mRNA, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide or pharmaceutical composition described herein.

[0195] In some aspects, the disclosure provides a method of reducing expression of a target mRNA in a subject, comprising administering to the subject an oligonucleotide or pharmaceutical composition described herein.

[0196] In some aspects of the methods described herein, the target mRNA is expressed in the liver. In some aspects, the target mRNA is expressed in a hepatocyte. In some aspects, the target mRNA is expressed in a liver sinusoidal endothelial cell. In some aspects of the methods described herein, the target mRNA is expressed in a macrophage of the liver. In some aspects, the target mRNA is expressed in at least one cell type of the central nervous system. In some aspects, the at least one cell type of the central nervous system is an astrocyte, a neuron, or an oligodendrocyte. In some aspects, the target mRNA is expressed in an astrocyte, a neuron, an oligodendrocyte, or any combination thereof. In some aspects, the target mRNA is expressed in the frontal cortex, medulla, hippocampus, hypothalamus, cerebellum, lumbar spinal cord, lumbar dorsal root ganglion, or any combination thereof. In some aspects of the methods described herein, the target mRNA is expressed in ocular tissue, optionally the retina or optic nerve. In some aspects of the methods described herein, the target mRNA is expressed in liver tissue, skeletal muscle tissue, adipose tissue and / or adrenal tissue.

[0197] In some 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 a target mRNA. In some 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 a target mRNA.

[0198] 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 a target mRNA.

[0199] In any of the foregoing or related aspects, the target mRNA is expressed in the liver, optionally wherein the target mRNA is expressed in a liver macrophage, a liver hepatocyte, or a liver sinusoidal endothelial cell. In any of the foregoing or related aspects, the target mRNA is expressed in ocular tissue, optionally the retina or optic nerve. In any of the foregoing or related aspects, the target mRNA is expressed in liver tissue, skeletal muscle tissue, adipose tissue and / or adrenal tissue. In some aspects, the target mRNA is expressed in at least one tissue of the central nervous system. In some aspects, the at least one tissue of the central nervous system is selected from the frontal cortex, medulla, hippocampus, hypothalamus, cerebellum, lumbar spinal cord, lumbar dorsal root ganglion, and any combinations thereof.

[0200] In any of the foregoing or related aspects of the methods described herein, the disease or disorder is selected from the group consisting of viral infections, bacterial infections, parasitic infections, cancers, allergies, autoimmune diseases, immunodeficiencies, and immunosuppression.

[0201] In some aspects, the disclosure provides a double-stranded oligonucleotide comprising:

[0202] (i) a sense strand 14-20 nucleotides in length, wherein the sense strand comprises at least one lipid moiety conjugated to the 5’ terminus, and wherein the sense strand comprises at least one locked nucleic acid, and

[0203] (ii) an antisense strand 22 nucleotides in length, wherein the antisense strand comprises a region of complementarity to an astrocyte target mRNA, and wherein the sense and antisense strand are separate strands which form an asymmetric duplex region having an overhang of about 2-8 nucleotides at the 3’ terminus of the antisense strand, wherein the duplex region is about 14-20 nucleotides.

[0204] In other aspects, the disclosure provides a double-stranded oligonucleotide comprising: (i) a sense strand 14-20 nucleotides in length, wherein the sense strand comprises at least one lipid moiety conjugated to the 5’ terminus, and wherein the sense strand comprises at least one locked nucleic acid, and

[0205] (ii) an antisense strand 22 nucleotides in length, wherein the antisense strand comprises a region of complementarity to a neuron target mRNA, and wherein the sense and antisense strand are separate strands which form an asymmetric duplex region having an overhang of about 2-8 nucleotides at the 3’ terminus of the antisense strand, wherein the duplex region is about 14-20 nucleotides.

[0206] In further aspects, the disclosure provides a double-stranded oligonucleotide comprising:

[0207] (i) a sense strand 14-20 nucleotides in length, wherein the sense strand comprises at least one lipid moiety conjugated to the 5’ terminus, and wherein the sense strand comprises at least one locked nucleic acid, and

[0208] (ii) an antisense strand 22 nucleotides in length, wherein the antisense strand comprises a region of complementarity to an oligodendrocyte target mRNA, and wherein the sense and antisense strand are separate strands which form an asymmetric duplex region having an overhang of about 2-8 nucleotides at the 3’ terminus of the antisense strand, wherein the duplex region is about 14-20 nucleotides.

[0209] In any of the foregoing or related aspects, the sense strand is 14 nucleotides in length. In some aspects, the sense strand comprises a locked nucleic acid at one or more of position 2, position 9, position 10, position 12, or position 13 with positions numbered 5’ to 3’. In some aspects, the antisense strand comprises a phosphorothioate linkage between positions 1 and 2, between positions 2 and 3, between positions 3 and 4, between positions 13 and 14, between positions 14 and 15, between positions 20 and 21, and between positions 21 and 22.

[0210] In any of the foregoing or related aspects, the sense strand is 20 nucleotides in length. In some aspects, the sense strand comprises a locked nucleic acid at one or more of position 2, position 15, or position 16 with positions numbered 5’ to 3’. In some aspects, the antisense strand comprises a phosphorothioate linkage between positions 1 and 2, between positions 2 and 3, between positions 3 and 4, between positions 20 and 21, and between positions 21 and 22.

[0211] In other aspects, the disclosure provides a method of reducing expression of a target mRNA in an astrocyte, comprising administering a double-stranded oligonucleotide wherein the oligonucleotide comprises: (i) a sense strand, wherein the sense strand is 14-20 nucleotides in length, wherein the sense strand comprises at least one lipid moiety conjugated to the 5’ terminus, and wherein the sense strand comprises at least one locked nucleic acid, and

[0212] (ii) an antisense strand, wherein the antisense strand is 22 nucleotides in length, and wherein the antisense strand comprises a region of complementarity to a target mRNA in the astrocyte, and wherein the sense and antisense strand are separate strands which form an asymmetric duplex region having an overhang of about 2-8 nucleotides at the 3’ terminus of the antisense strand, wherein the duplex region is about 14-20 nucleotides, thereby reducing expression of the target mRNA in the astrocyte. In some aspects, the oligonucleotide comprises a blunt end comprising the 3’ end of the sense strand and the 5’ end of the antisense strand. In some aspects, the sense strand comprises no more than 3 locked nucleic acids. In some aspects, the sense strand is 20 nucleotides in length, and wherein reduction of the target mRNA in an astrocyte is increased compared to reduction in a neuron. In some aspects, reduction of the target mRNA is increased by at least 5%. In some aspects, reduction of the target mRNA is increased by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45% or by at least 50%. In some aspects, the sense strand is 14 nucleotides in length, and wherein reduction of the target mRNA in an astrocyte is increased compared to reduction in an oligodendrocyte. In some aspects, reduction of the target mRNA is increased by at least 5%. In some aspects, reduction of the target mRNA is increased by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45% or by at least 50%. In some aspects, the sense strand is 20 nucleotides in length, and wherein the target mRNA is reduced in an astrocyte and an oligodendrocyte to the same or similar level. In some aspects, the sense strand is 14 nucleotides in length, wherein the target mRNA is reduced in an astrocyte and in a neuron to the same or similar level, and wherein reduction of the target mRNA in an astrocyte and in a neuron is increased compared to reduction in an oligodendrocyte. In some aspects, reduction of the target mRNA is increased by at least 5%. In some aspects, reduction of the target mRNA is increased by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45% or by at least 50%.

[0213] In other aspects, the disclosure provides a method of reducing expression of a target mRNA in an oligodendrocyte, comprising administering a double-stranded oligonucleotide wherein the oligonucleotide comprises: (i) a sense strand, wherein the sense strand is 14-40 nucleotides in length, wherein the sense strand comprises at least one lipid moiety conjugated to the 5’ terminus, and wherein the sense strand comprises at least one locked nucleic acid, and

[0214] (ii) an antisense strand, wherein the antisense strand is 22 nucleotides in length, and wherein the antisense strand comprises a region of complementarity to a target mRNA in the oligodendrocyte, and wherein the sense and antisense strand are separate strands which form an asymmetric duplex region having an overhang of about 2-8 nucleotides at the 3’ terminus of the antisense strand, wherein the duplex region is about 14-20 nucleotides, thereby reducing expression of the target mRNA in the oligodendrocyte. In some aspects, the oligonucleotide comprises a blunt end comprising the 3’ end of the sense strand and the 5’ end of the antisense strand. In some aspects, the sense strand is 20 nucleotides in length. In some aspects, the sense strand in 36 nucleotides in length, and wherein the oligonucleotide comprises a stem-loop. In some aspects, the target mRNA is reduced in an astrocyte and the oligodendrocyte to the same or similar level, and wherein reduction of the target mRNA is increased in the astrocyte and the oligodendrocyte compared to reduction in a neuron. In some aspects, reduction of the target mRNA is increased by at least 5%. In some aspects, reduction of the target mRNA is increased by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45% or by at least 50%.

[0215] In other aspects, the disclosure provides a method of reducing expression of a target mRNA in a neuron, comprising administering a double-stranded oligonucleotide wherein the oligonucleotide comprises:

[0216] (i) a sense strand, wherein the sense strand is 14-20 nucleotides in length, wherein the sense strand comprises at least one lipid moiety conjugated to the 5’ terminus, and wherein the sense strand comprises at least one locked nucleic acid, and

[0217] (ii) an antisense strand, wherein the antisense strand is 22 nucleotides in length, and wherein the antisense strand comprises a region of complementarity to a target mRNA in the neuron, and wherein the sense and antisense strand are separate strands which form an asymmetric duplex region having an overhang of about 2-8 nucleotides at the 3’ terminus of the antisense strand, wherein the duplex region is about 14-20 nucleotides, thereby reducing expression of the target mRNA in the neuron. In some aspects, the oligonucleotide comprises a blunt end comprising the 3’ end of the sense strand and the 5’ end of the antisense strand. In some aspects, the sense strand comprises no more than 5 locked nucleic acids. In some aspects, the sense strand is 14 nucleotides in length.

[0218] BRIEF DESCRIPTION OF FIGURES

[0219] FIGs 1A-1C provide schematics of RNAi oligonucleotide-lipid conjugates targeting TUBB3 mRNA having the structures of Compounds 1-14.

[0220] FIGs 2A-2F provide graphs measuring percent (%) of murine TUBB3 mRNA remaining in frontal cortex (FIG. 2A), hippocampus (FIG. 2B), cerebellum (FIG. 2C), brain stem (FIG. 2D), lumbar dorsal root ganglion (DRG) (FIG. 2E), and lumbar spinal cord (FIG. 2F) of control mice (group A) or mice administered Compounds 1-14 (respectively groups B-O) via lumbar intrathecal injection.

[0221] FIG. 3 provides a schematic of RNAi oligonucleotide-lipid conjugates targeting TUBB3 mRNA having the structures of Compounds 15-18.

[0222] FIGs. 4A-4F provide graphs measuring percent (%) of murine TUBB3 mRNA remaining in frontal cortex (FIG. 4A), hippocampus (FIG. 4B), medulla (FIG. 4C) cerebellum (FIG. 4D), lumbar dorsal root ganglion (DRG) (FIG. 4E), and lumbar spinal cord (FIG. 4F) of control mice (group A) or mice administered Compounds 7-9 (respectively groups B-D) or Compounds 15-18 (respectively groups E-H) via lumbar intrathecal injection.

[0223] FIG. 5 provides a schematic of RNAi oligonucleotide-lipid conjugates targeting TUBB3 mRNA having the structures of Compounds 18-28.

[0224] FIGs. 6A-6B provide graphs measuring percent (%) of murine TUBB3 mRNA remaining in cerebellum (FIG. 6A) and lumbar dorsal root ganglion (DRG) (FIG. 6B) of control mice (group A) or mice administered Compounds 18-28 (respectively groups B-L).

[0225] FIG. 7 provides a schematic of RNAi oligonucleotide-lipid conjugates targeting TUBB3 mRNA having the structures of Compounds 18 and 29-38.

[0226] FIGs. 8A-8B provide graphs measuring percent (%) of murine TUBB3 mRNA remaining in cerebellum (FIG. 8A) and lumbar dorsal root ganglion (DRG) (FIG. 8B) of control mice (group A) or mice administered Compounds 18 or 29-38 (respectively groups B- L).

[0227] FIG. 9 provides a schematic of RNAi oligonucleotide-lipid conjugates targeting reticulon 4 (RTN4) mRNA having the structures of Compounds 39-49. FIGs. 10A-10B provide graphs measuring percent (%) of rat RTN4 mRNA remaining in retina (FIG. 10A) and optic nerve (FIG. 8B) tissues of control rats (group A) or rats administered Compounds 39-49 (respectively groups B-L) via intravitreal injection.

[0228] FIG. 10C provides a schematic of RNAi oligonucleotide-GalNAc conjugates targeting Aldh2 mRNA having the structures of Compounds 57 and 98-108.

[0229] FIG. 10D provides a graph measuring percent (%) of murine Aldh2 mRNA remaining in liver tissue of control mice administered PBS (group A) or mice administered Compounds 57 or 98-108 (respectively groups B-N).

[0230] FIG. 10E provides a schematic of RNAi oligonucleotide-lipid conjugates targeting PEC AM- 1 mRNA having the structures of Compounds 120 and 121 and CD68 mRNA having the structure of Compound 122.

[0231] FIGs. 10F-10G provide graphs measuring percent (%) of murine PEC AM mRNA (FIG. 10F) and CD68 mRNA (FIG. 10G) remaining in liver tissue of control mice administered PBS (group A) or mice administered Compounds 120-122 (respectively groups B-D) via subcutaneous injection.

[0232] FIGs. 10H-10I provide graphs measuring percent (%) of murine CD68 mRNA (FIG. 10H) and CD68 protein (FIG. 101) remaining in liver tissue of control mice administered PBS or mice administered Compound 122 at the indicated dose via subcutaneous injection, as measured by qPCR and immunohistochemistry (IHC) respectively.

[0233] FIG. 10J provides a representative image of liver tissue obtained from a PBS control mice as described in FIGs. 10H-10I that were imaged following CD68 immunostaining. Arrows indicate representative cells that morphologically present as hepatocytes or macrophages. Dark staining indicates CD68 protein expression.

[0234] FIG. 10K provides representative images of liver tissue obtained from mice described in FIGs. 10H-10I that were imaged following CD68 immunostaining. Dark staining indicates CD68 protein expression.

[0235] FIGs. 11A-11B provide schematics of RNAi oligonucleotide-lipid conjugates targeting CD68 mRNA having the structures of Compounds 58-97.

[0236] FIG. 12 provides a graph measuring percent (%) of murine CD68 mRNA remaining in liver tissue of control mice administered PBS or mice administered Compounds 58-97 (respectively groups B-AO) via subcutaneous injection.

[0237] FIG. 13 provides a schematic of RNAi oligonucleotide-lipid conjugates targeting Aldh2 mRNA having the structures of Compounds 109-117. FIGs. 14A-14D provide graphs measuring percent of murine Aldh2 mRNA remaining in liver (FIG. 14A), adipose (FIG. 14B), skeletal muscle (FIG. 14C), and adrenal (FIG. 14D) tissues in control mice administered PBS (group A) or mice administered Compounds 109-117 (respectively groups B-J) via subcutaneous injection.

[0238] FIG. 15 provides a schematic of RNAi oligonucleotide-lipid conjugates targeting STAT3 (Compounds 123 and 127), SLC25A1 (Compounds 124 and 128), HMGB1 (Compounds 125 and 129), and ALDH2 (Compounds 126 and 130).

[0239] FIGs. 16A-16D provide graphs measuring percent (%) of murine STAT3 mRNA remaining in liver tissue of mice administered PBS (group A) or compounds 123 or 127 (groups Bl and Cl respectively) (FIG. 16A); percent (%) of murine SLC25A1 mRNA remaining in liver tissue of mice administered PBS (group A) or compounds 124 or 128 (groups B2 and C2 respectively) (FIG. 16B); percent (%) of murine HMGB1 mRNA remaining in liver tissue of mice administered PBS (group A) or compounds 125 or 129 (groups B3 and C3 respectively) (FIG. 16C); and percent (%) of murine ALDH2 mRNA remaining in liver tissue of mice administered PBS (group A) or compounds 126 or 130 (groups B4 and C4 respectively) (FIG. 16D). Mice were administered RNAi oligonucleotidelipid conjugates via subcutaneous injection.

[0240] FIG. 17 provides a schematic of RNAi oligonucleotide-lipid conjugates targeting TUBB3 where the oligonucleotide comprises a P-4, P-6, or P-8 truncated sense strand (i.e. a 6, 8, and 10 nucleotide overhang of the antisense strand) (Compounds 131-133) or a p-4, p-6, or p-8 truncated sense strand comprising phosphorothioate linkages (Compounds 134-136).

[0241] FIGs. 18A-18F provide graphs measuring percent (%) of murine TUBB3 mRNA remaining in frontal cortex (FIG. 18A), hippocampus (FIG. 18B), cerebellum (FIG. 18C), lumbar dorsal root ganglion (DRG) (FIG. 18D), medulla (FIG. 18E), and lumbar spinal cord (SC) (FIG. 18F) of control mice (aCSF) or mice administered Compounds 131-136 (respectively Parent Cl 6, P-4, P-6, P-8, P-4 PS, P-6 PS, and P-8 PS) via lumbar intrathecal injection.

[0242] FIG. 19 provides a schematic of blunt-end RNAi oligonucleotide-lipid conjugates targeting TUBB3 where the oligonucleotide comprises a sense strand with different amounts of locked nucleic acids (LNA) (Compounds 137-140) or a P-6 truncated sense strand (i.e., an 8 nucleotide overhang of the antisense strand) comprising different amounts of LNAs (Compounds 141-145).

[0243] FIGs. 20A-20F provide graphs measuring percent (%) of murine TUBB3 mRNA remaining in frontal cortex (FIG. 20A), hippocampus (FIG. 20B), cerebellum (FIG. 20C), medulla (FIG. 20D), lumbar dorsal root ganglion (DRG) (FIG. 20E), and lumbar spinal cord (SC) (FIG. 20F) of control mice (aCSF) or mice administered Compounds 1 and 137-145 (respectively B-K) via lumbar intrathecal injection.

[0244] FIG. 21 provides a schematic of blunt-end RNAi oligonucleotide-lipid conjugates targeting TUBB3 where the oligonucleotide comprises a sense strand comprising different amounts of locked nucleic acids (LNA) (Compounds 138, 139, 140, 147, and 148).

[0245] FIGs. 22A-22F provide graphs measuring percent (%) of murine TUBB3 mRNA remaining in frontal cortex (FIG. 22A), hippocampus (FIG. 22B), medulla (FIG. 22C), lumbar dorsal root ganglion (DRG) (FIG. 22D), cerebellum (FIG. 22E), and lumbar spinal cord (SC) (FIG. 22F) of control mice (aCSF) or mice administered Compounds 1, 137, 146, 138, 139, 140, 147, and 148 (respectively B-I) via lumbar intrathecal injection.

[0246] FIG. 23 provides a schematic of blunt-end RNAi oligonucleotide-lipid conjugates targeting TUBB3 where the oligonucleotide comprises a P-4 truncated sense strand comprising different amounts of locked nucleic acids (LNA) (Compounds 149-154).

[0247] FIGs. 24A-24F provide graphs measuring percent (%) of murine TUBB3 mRNA remaining in frontal cortex (FIG. 24A), hippocampus (FIG. 24B), medulla (FIG. 24C), lumbar dorsal root ganglion (DRG) (FIG. 24D), cerebellum (FIG. 24E), and lumbar spinal cord (SC) (FIG. 24F) of control mice (aCSF) or mice administered Compounds 1, 137 and 149-154 (respectively B-I) via lumbar intrathecal injection.

[0248] FIG. 25 provides a schematic of blunt-end RNAi oligonucleotide-lipid conjugates targeting TUBB3 where the oligonucleotide comprises a P-8 truncated sense strand comprising different amounts of locked nucleic acids (LNA) (Compounds 155-160).

[0249] FIGs. 26A-26F provide graphs measuring percent (%) of murine TUBB3 mRNA remaining in frontal cortex (FIG. 26A), hippocampus (FIG. 26B), medulla (FIG. 26C), lumbar dorsal root ganglion (DRG) (FIG. 26D), cerebellum (FIG. 26E), and lumbar spinal cord (SC) (FIG. 26F) of control mice (aCSF) or mice administered Compounds 1, 137 and 155-160 (respectively B-I) via lumbar intrathecal injection.

[0250] FIG. 27 provides a schematic of blunt-end RNAi oligonucleotide-lipid conjugates targeting UGT8 where the oligonucleotide comprises a sense strand comprising different amounts of locked nucleic acids (LNA) (Compounds 162-165) or a P-6 truncated sense strand comprising different amounts of LNAs (Compounds 166-171).

[0251] FIGs. 28A-28F provide graphs measuring percent (%) of murine UGT8 mRNA remaining in frontal cortex (FIG. 28A), hippocampus (FIG. 28B), hypothalamus (FIG. 28C), cerebellum (FIG. 28D), medulla (FIG. 28E), and lumbar spinal cord (FIG. 26F) of control mice (aCSF) or mice administered Compounds 161-171 (respectively B-L) via lumbar intrathecal injection.

[0252] FIG. 29 provides a schematic of blunt-end RNAi oligonucleotide-lipid conjugates targeting GFAP where the oligonucleotide comprises a sense strand comprising different amounts of locked nucleic acids (LNA) (Compounds 173-176) or a P-6 truncated sense strand comprising different amounts of LNAs (Compounds 177-182).

[0253] FIGs. 30A-30F provide graphs measuring percent (%) of murine GFAP mRNA remaining in frontal cortex (FIG. 30A), hippocampus (FIG. 30B), cerebellum (FIG. 30C), medulla (FIG. 30D), hypothalamus (FIG. 30E), and lumbar spinal cord (FIG. 30F) of control mice (aCSF) or mice administered Compounds 172-182 (respectively B-L) via lumbar intrathecal injection.

[0254] FIGs. 31A-31D provide schematics of blunt-end RNAi oligonucleotide-lipid conjugates targeting TUBB3 where the oligonucleotide comprises different sense strand truncations including: no truncation (FIG. 31A; Compounds 137 and 183-190); a P-4 truncated sense strand (FIG. 31B; Compounds 149, and 191-194); a P-6 truncated sense strand (FIG. 31C; Compounds 141, 195, and 196); and, a P-8 truncated sense strand (FIG. 31D; Compound 197). Each compound comprises a C16 lipid conjugated to a different position of the sense strand as indicated in the schematics.

[0255] FIGs. 32A-32F provide graphs measuring percent (%) of murine TUBB3 mRNA remaining in frontal cortex (FIG. 32A), hippocampus (FIG. 32B), medulla (FIG. 32C), lumbar dorsal root ganglion (FIG. 32D), cerebellum (FIG. 32E), and lumbar spinal cord (FIG. 32F) of control mice (aCSF) or mice administered Compounds 137, 183-190, 149, 191-194, 141, and 195-197 via lumbar intrathecal injection.

[0256] FIGs. 33A-33C provide schematics of RNAi oligonucleotide-lipid conjugates targeting GFAP mRNA having the structures of Compounds 173, 176, 180, 200-205, and 207 -215.

[0257] FIGs. 34A-34D provide graphs measuring percent (%) of murine GFAP mRNA remaining in frontal cortex (FIG. 34A), hippocampus (FIG. 34B), medulla (FIG. 34C), and lumbar spinal cord (FIG. 34D) of control mice (aCSF) or mice administered compounds 173, 176, 180, 200-205, and 207 -215 (as identified in Table 20) via lumbar intrathecal injection.

[0258] FIGs. 35A- 35E provide schematics of RNAi oligonucleotide-lipid conjugates targeting TUBB3 mRNA having the structures of Compounds 137, 140, 144, 217-222, 224- 232, and 277-285. FIGs. 36A-36D provide graphs measuring percent (%) of murine TUBB3 mRNA remaining in frontal cortex (FIG. 34A), hippocampus (FIG. 34B), medulla (FIG. 34C), and lumbar spinal cord (FIG. 34D) of control mice (aCSF) or mice administered compounds 137, and 140, 144, 217-222, 224-232 (as identified in Table 21) via lumbar intrathecal injection.

[0259] FIG. 36E is a graph measuring (%) of murine TUBB3 mRNA remaining in frontal cortex hippocampus, hypothalamus, cerebellum, brain stem, and lumbar spinal cord of control mice (aCSF) or mice administered compounds 140, 277-278, 231, and 279-285 (respectively B-L) via lumbar intrathecal injection.

[0260] FIGs. 37A- 37B provide schematics of RNAi oligonucleotide-lipid conjugates targeting GFAP mRNA having the structures of Compounds 173, 176, and 233-245.

[0261] FIGs. 38A-38D provide graphs measuring percent (%) of murine GFAP mRNA remaining in frontal cortex (FIG. 38A), hippocampus (FIG. 38B), medulla (FIG. 38C), and lumbar spinal cord (FIG. 38D) of control mice (aCSF) or mice administered compounds 173, 176, and 233-245 (respectively B-P) via lumbar intrathecal injection.

[0262] FIGs. 39A- 39B provide schematics of RNAi oligonucleotide-lipid conjugates targeting GFAP mRNA having the structures of Compounds 173, 176, 200 and 246-255.

[0263] FIGs. 40A-40D provide graphs measuring percent (%) of murine GFAP mRNA remaining in frontal cortex (FIG. 40A), hippocampus (FIG. 40B), medulla (FIG. 40C), and lumbar spinal cord (FIG. 40D) of control mice (aCSF) or mice administered compounds 173, 176, 200 and 246-255 (respectively B-N) via lumbar intrathecal injection.

[0264] FIGs. 41 A- 41B provide schematics of RNAi oligonucleotide-lipid conjugates targeting TUBB3 mRNA having the structures of Compounds 137, 140, and 257-268.

[0265] FIGs. 42A-42D provide graphs measuring percent (%) of murine TUBB3 mRNA remaining in frontal cortex (FIG. 42A), hippocampus (FIG. 42B), medulla (FIG. 42C), and lumbar spinal cord (FIG. 42D) of control mice (aCSF) or mice administered compounds 137, 140, and 257-268 (respectively B-P) via lumbar intrathecal injection.

[0266] FIGs. 43 A- 43B provide schematics of RNAi oligonucleotide-lipid conjugates targeting TUBB3 mRNA having the structures of Compounds 137, 140, 217, and 269-276.

[0267] FIGs. 44A-44D provide graphs measuring percent (%) of murine TUBB3 mRNA remaining in frontal cortex (FIG. 44A), hippocampus (FIG. 44B), medulla (FIG. 44C), and lumbar spinal cord (FIG. 44D) of control mice (aCSF) or mice administered compounds 137, 140, 217, and 269-276 (respectively B-F and I-N) via lumbar intrathecal injection.

[0268] FIG. 45 provides schematics of RNAi oligonucleotide-lipid conjugates targeting GFAP mRNA having the structures of Compounds 286-292. FIGs. 46A-46D provide graphs measuring percent (%) of murine ALDH2 mRNA remaining in liver (FIG. 46A), quadricep (quad) muscle (FIG. 46B), heart muscle (FIG. 46C), and gonadal white adipose tissue (gWAT) (FIG. 46D) of control mice (PBS) or mice administered compounds 286-292 (respectively A-G) via subcutaneous injection.

[0269] FIG. 47 provides schematics of RNAi oligonucleotide-lipid conjugates targeting UGT8 mRNA having the structures of Compounds 162, 167, 293, and 294.

[0270] FIGs. 48A-48D provide graphs measuring percent (%) of murine UGT8 mRNA remaining in frontal cortex (FIG. 48A), hippocampus (FIG. 48B), brain stem (FIG. 48C), and lumbar spinal cord (FIG. 48D) of control mice (aCSF) or mice administered compounds 162, 167, 293, and 294 (respectively blunt, 5’ p-6, 3’ p-3, and 5’ p-6 & 3’ p-3) via lumbar intrathecal injection.

[0271] DETAILED DESCRIPTION

[0272] In some aspects, the disclosure provides oligonucleotide-lipid conjugates (e.g., RNAi oligonucleotide-lipid conjugates) that reduce expression of a target gene. In other aspects, the disclosure provides methods of treating a disease or disorder associated with expression of a target gene. 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 a target gene 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 a target gene.

[0273] In nucleic acid chemistry, many different artificial nucleic acids have been developed to alter the behavior of siRNAs under physiological conditions. In particular, phosphorothioate (PS), 2'-methoxy (2'-0Me), and 2'-fluoro nucleic acid have often been used to modify the siRNA its behavior, toxicity and thermostability. Recently a novel class of conformationally restricted artificial nucleic acids has been developed these are bridged nucleic acids (BNAs). The basic structure of the BNA is the bridged structure between 2'-0 and 4'-C which fixes the furanose ring to the N-type and 2',4'-BNA gives its nucleotide analog high nuclease resistance and affinity to complementary RNA. The concept of introducing a bulky group at the 2'- position of the furanose ring to achieve high nuclease resistance has little impact on sugar fluctuation, whereas in the BNAs, the bicyclic structure provides steric hindrance at the phosphodiester backbone and the reduction of entropic loss by the sugar fluctuation, and thus both high nuclease resistance and stabilization of the RNA duplex are realized. Many studies related to usage of 2',4'-BNA / locked nucleic acid (LNA) modifications in siRNAs have been conducted in vitro. Similarly, locked nucleic acids (LNA’s) are a class of nucleic acid analogues that display increased hybridization affinity towards complementary DNA and RNA sequences and that as such they are also useful in the design of RNAi trigger structures. Structural studies have shown LNA to be an RNA mimic, fitting seamlessly into an A-type duplex geometry. Several reports have revealed LNA as a most promising molecule for the development of oligonucleotide-based therapeutics.

[0274] According to the current disclosure the thermostability provided by LNA’s and BNA’s allow modifications of underlying RNAi structures. For example, significant truncations in the passenger / sense strand of a given trigger can be made and with the appropriate addition and use of LNA’s or BNA’s the gene knockdown activity of every shorter passenger strands can be rescued. The result is a LNS enhanced RNAi trigger that is lighter in weight with the equivalent activity in physiological systems. Similarly, phosphorothioate molecules can be used to control or lessen nuclease attack on a given RNAi trigger structure. In this sense the chemical modification of a given RNAi trigger can be a balancing act between those modifications such as LNA, BNA or Phosphorothioate (PS) placement to protect against nuclease degradation or loss of thermostability.

[0275] Lipid-Conjugated RNAi Oligonucleotides

[0276] The disclosure provides, inter alia, lipid-conjugated RNAi oligonucleotides (e.g., RNAi oligonucleotide-lipid conjugates) that reduce expression of a target gene. 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

[0277] In some embodiments, the lipid-conjugated RNAi oligonucleotide is targeted to a target sequence comprising a target mRNA. In some embodiments, the lipid-conjugated RNAi oligonucleotide is targeted to a target sequence within a target 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 double-stranded oligonucleotide) binds or anneals to a target sequence comprising a target mRNA, thereby reducing target gene expression. In some embodiments, the lipid-conjugated RNAi oligonucleotide is targeted to a target sequence comprising target mRNA for the purpose of reducing expression of a target gene in vivo. In some embodiments, the amount or extent of reduction of target gene expression by a lipid- conjugated RNAi oligonucleotide targeted to a specific 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 a lipid-conjugated RNAi oligonucleotide targeted to a specific 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.

[0278] 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 a target mRNA. In some embodiments, a portion or region of the sense strand of a double-stranded oligonucleotide described herein comprises a target sequence comprising a target mRNA.

[0279] In some embodiments, the target mRNA is expressed in a tissue or cell of a subject. In some embodiments, the target mRNA is expressed in more than one tissue or cell of a subject, wherein the tissues or cells are different. In some embodiments, the target mRNA is differentially expressed in a tissue or cell. In some embodiments, the target mRNA is expressed throughout multiple tissue types and / or cell types. In some embodiments, the target mRNA is expressed in the central nervous system, liver tissue, ocular tissue, adipose tissue, adrenal tissue, or skeletal muscle tissue. In some embodiments, the target mRNA is expressed in the central nervous system, peripherial nervous system, liver tissue, ocular tissue, adipose tissue, adrenal tissue, heart tissue, lung tissue, skeletal muscle tissue, cardiac muscle tissue, smooth muscle tissue, or kidney tissue, or any combination thereof. In some embodiments, the target mRNA is expressed in the central nervous system. In some embodiments, the target mRNA is expressed in the peripherial nervous system. In some embodiments, the target mRNA is expressed in a region of the central nervous system. In some embodiments, a region of the central nervous system is selected from the eye, brain, cerebrum, cerebral cortex, frontal lobe, frontal cortex, parietal lobe, temporal lobe, occipital lobe, hippocampus, cerebellum, brain stem, dorsal root ganglion (DRG) or the spinal cord. In some embodiments, the target mRNA is expressed in a neuron. In some embodiments, the target mRNA is expressed in a glial cell. In some embodiments, the target mRNA is expressed in a neuron located in the central nervous system. In some embodiments, the target mRNA is expressed in liver tissue. In some embodiments, the target mRNA is expressed in a hepatocyte. In some embodiments, the target mRNA is expressed in a liver sinusoidal endothelial cell. In some embodiments, the target mRNA is expressed in a macrophage. In some embodiments, the target mRNA is expressed in a macrophage located in liver tissue. In some embodiments, the target mRNA is expressed in ocular tissue. In some embodiments, the target mRNA is expressed in the retina and / or optic nerve. In some embodiments, the target mRNA is expressed in adipose tissue. In some embodiments, the target mRNA is expressed in skeletal muscle tissue. In some embodiments, the target mRNA is expressed in adrenal tissue. In some embodiments, the target mRNA is expressed in heart tissue. In some embodiments, the target mRNA is expressed in lung tissue. In some embodiments, the target mRNA is expressed in the eye. In some embodiments, the target mRNA is expressed in the brain. In some embodiments, the target mRNA is expressed in the cerebrum. In some embodiments, the target mRNA is expressed in the cerebellum. In some embodiments, the target mRNA is expressed in the brain stem. In some embodiments, the target mRNA is expressed in the frontal lobe. In some embodiments, the target mRNA is expressed in the frontal cortex. In some embodiments, the target mRNA is expressed in the parietal lobe. In some embodiments, the target mRNA is expressed in the temporal lobe. In some embodiments, the target mRNA is expressed in the occipital lobe. In some embodiments, the target mRNA is expressed in the hippocampus. In some embodiments, the target mRNA is expressed in the DRG. In some embodiments, the target mRNA is expressed in the spinal cord.

[0280] RNAi Oligonucleotide Targeting Sequences

[0281] 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. 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. 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 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 within a 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 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 at least 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.

[0282] In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a targeting sequence that is fully complementary to a target sequence comprising a target mRNA. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a targeting sequence that is fully complementary to a target sequence within a 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 a target mRNA. In some embodiments, the targeting sequence comprises a region of contiguous nucleotides comprising the antisense strand.

[0283] In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a targeting sequence that is complementary to a contiguous sequence of nucleotides comprising a 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 a 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.

[0284] In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises 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 oligonucleotide comprises 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.

[0285] 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 a 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 a 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 a 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 a 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 a target mRNA and comprises 12 nucleotides, 13 nucleotides, 14 nucleotides, 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 a target mRNA and comprises 19 nucleotides of the antisense strand.

[0286] 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 a 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 a 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 a 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 a 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 a target mRNA and comprises 12 nucleotides, 13 nucleotides, 14 nucleotides, 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 a target mRNA and comprises 19 nucleotides of the antisense strand.

[0287] 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 a 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 a 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 a 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.

[0288] Types of Oligonucleotides

[0289] A variety of RNAi oligonucleotide types and / or structures are useful for reducing target gene expression in the methods herein. Any of the RNAi oligonucleotide types described herein or elsewhere are contemplated for use as a framework to incorporate a targeting sequence herein for the purposes of inhibiting or reducing corresponding target gene expression.

[0290] 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.

[0291] 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, the oligonucleotides herein interact with Dicer and are loaded into RISC. In some embodiments, upon endogenous Dicer processing, double-stranded nucleic acids of 19- 23 nucleotides in length capable of reducing expression of a 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 a 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; 9,193,753; 8,420,391; and, 8,552,171 all to Tuschl et al. Such patents also indicate a lack of activity with regard to double overhang constructs.

[0292] 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.

[0293] 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 etal. (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).

[0294] Antisense Strands

[0295] 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.”

[0296] 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 8 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 8 to about 40 (e.g., 8 to 40, 8 to 36, 8 to 32, 8 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, a lipid-conjugated RNAi oligonucleotide herein comprises an antisense strand of 12 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.

[0297] Sense Strands

[0298] 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 of at least about 10 nucleotides in length (e.g., at least 10, 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 in a range of about 10 to about 50 (e.g., 10 to 50, 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 38 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 12-21 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 10 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 11 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 12 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 13 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 14 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 15 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 16 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. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 37 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 38 nucleotides in length.

[0299] In some embodiments, a sense strand comprises a stem-loop structure at its 3' end. In some embodiments, a sense strand comprises a stem-loop structure at its 5' 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 1, 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 1 nucleotide 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 stem-loop 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.

[0300] 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.

[0301] In some embodiments, the tetraloop comprises the sequence 5’-GAAA-3’. In some embodiments, the tetraloop comprises the sequence 5’-UNCG-3’. In some embodiments, the tetraloop comprises the sequence 5’-UACG-3’. In some embodiments, the stem loop comprises the sequence 5’-GCAGCCGAAAGGCUGC-3’ (SEQ ID NO: 526).

[0302] 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.

[0303] 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.

[0304] 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). Duplex Length

[0305] In some embodiments, a duplex formed between a sense and antisense strand is at least 8 (e.g., at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16) nucleotides in length. 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 in the range of 10-30 nucleotides in length (e.g., 10 to 30, 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 10, 11, 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 10-18 base pairs 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 10 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand is 11 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand is 12 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand is 13 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand is 14 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand is 15 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand is 16 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.

[0306] Oligonucleotide Ends

[0307] 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).

[0308] 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

[0309] (I) to two (2) nucleotides in length. In some embodiments, the 3’-overhang is about two (2) to about twelve (12) 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

[0310] (I I) 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.

[0311] 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.

[0312] 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.

[0313] 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.

[0314] In some embodiments, the 5’ overhang is 2 nucleotides and the 3’ overhang is about 3-7 nucleotides. In some embodiments, the 5’ overhang is 2 nucleotides and the 3’ overhang is 3 nucleotides. In some embodiments, the 5’ overhang is 2 nucleotides and the 3’ overhang is 4 nucleotides. In some embodiments, the 5’ overhang is 2 nucleotides and the 3’ overhang is 5 nucleotides. In some embodiments, the 5’ overhang is 2 nucleotides and the 3’ overhang is 6 nucleotides. In some embodiments, the 5’ overhang is 2 nucleotides and the 3’ overhang is 7 nucleotides.

[0315] In some embodiments, the 3’ overhang is 6-8 nucleotides and the 5’ overhang is 2-4 nucleotides. In some embodiments, the 3’ overhang is 6 nucleotides and the 5’ overhang is 2 nucleotides. In some embodiments, the 3’ overhang is 6 nucleotides and the 5’ overhang is 3 nucleotides. In some embodiments, the 3’ overhang is 6 nucleotides and the 5’ overhang is 4 nucleotides. In some embodiments, the 3’ overhang is 7 nucleotides and the 5’ overhang is 2 nucleotides. In some embodiments, the 3’ overhang is 7 nucleotides and the 5’ overhang is 3 nucleotides. In some embodiments, the 3’ overhang is 7 nucleotides and the 5’ overhang is 4 nucleotides. In some embodiments, the 3’ overhang is 8 nucleotides and the 5’ overhang is 2 nucleotides. In some embodiments, the 3’ overhang is 8 nucleotides and the 5’ overhang is 3 nucleotides. In some embodiments, the 3’ overhang is 8 nucleotides and the 5’ overhang is 4 nucleotides. 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.

[0316] 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.

[0317] In some embodiments, an RNAi oligonucleotide conjugate disclosed herein comprises a stem-loop structure at the 5’ end of the sense strand and comprises an 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 5’ end of the sense strand comprises a stem-tetraloop structure and comprises two terminal overhang nucleotides at the 3’ end of the antisense strand.

[0318] In some embodiments, an RNAi oligonucleotide conjugate disclosed herein comprises a stem-loop structure at the 5’ end of the sense strand and comprises a blunt end at the 5’ end of the antisense strand.

[0319] In some embodiments, an RNAi oligonucleotide conjugate disclosed herein comprises an overhang of 1-8 nucleotides at the 5’ end of the sense strand and comprises an overhang of 1-8 nucleotides at the 5’ end of the antisense strand.

[0320] 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.

[0321] In some embodiments, the 5’ end and / or the 3 ’end of a sense or antisense strand has an inverted cap nucleotide.

[0322] In some embodiments, one or more (e.g., 2, 3, 4, 5, 6) modified intemucleotide linkages are provided between terminal nucleotides of the 3’ end or 5’ end of a sense and / or antisense strand. In some embodiments, modified intemucleotide linkages are provided between overhang nucleotides at the 3’ end or 5’ end of a sense and / or antisense strand.

[0323] In some embodiments, the sense strand is 18 nucleotides, and the antisense strand comprises a 5 ’overhang of 2 nucleotides, and a 3 ’overhang of 2 nucleotides. In some embodiments, the sense strand is 17 nucleotides, and the antisense strand comprises a 5’overhang of 3 nucleotides, and a 3’overhang of 2 nucleotides. In some embodiments, the sense strand is 16 nucleotides, and the antisense strand comprises a 5’overhang of 4 nucleotides, and a 3’overhang of 2 nucleotides. In some embodiments, the sense strand is 13 nucleotides, and the antisense strand comprises a 5’overhang of 2 nucleotides, and a 3’overhang of 7 nucleotides. In some embodiments, the sense strand is 12 nucleotides, and the antisense strand comprises a 5’overhang of 2 nucleotides, and a 3’overhang of 8 nucleotides. In some embodiments, the sense strand is 12 nucleotides, and the antisense strand comprises a 5’overhang of 3 nucleotides, and a 3’overhang of 7 nucleotides. In some embodiments, the sense strand is 10 nucleotides, and the antisense strand comprises a 5’overhang of 1 nucleotide, and a 3’overhang of 11 nucleotides.

[0324] In some embodiments, the sense strand is 18 nucleotides, the duplex region is 18 nucleotides, and the antisense strand comprises a 5’overhang of 2 nucleotides, and a 3’overhang of 2 nucleotides. In some embodiments, the sense strand is 17 nucleotides, the duplex region is 17 nucleotides, and the antisense strand comprises a 5’overhang of 3 nucleotides, and a 3’overhang of 2 nucleotides. In some embodiments, the sense strand is 16 nucleotides, the duplex region is 16 nucleotides, and the antisense strand comprises a 5’overhang of 4 nucleotides, and a 3’overhang of 2 nucleotides. In some embodiments, the sense strand is 13 nucleotides, the duplex region is 13 nucleotides, and the antisense strand comprises a 5’overhang of 2 nucleotides, and a 3’overhang of 7 nucleotides. In some embodiments, the sense strand is 12 nucleotides, the duplex region is 12 nucleotides, and the antisense strand comprises a 5’overhang of 2 nucleotides, and a 3’overhang of 8 nucleotides. In some embodiments, the sense strand is 12 nucleotides, the duplex region is 12 nucleotides, and the antisense strand comprises a 5’overhang of 3 nucleotides, and a 3’overhang of 7 nucleotides. In some embodiments, the sense strand is 10 nucleotides, the duplex region is 10 nucleotides, and the antisense strand comprises a 5’overhang of 1 nucleotide, and a 3’overhang of 11 nucleotides.

[0325] Oligonucleotide Modifications

[0326] 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. 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 internucleoside linkage, and at least one modified base.

[0327] 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).

[0328] Sugar Modifications

[0329] 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.

[0330] 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).

[0331] 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 ( / .< ., 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'-0Me, 2'-M0E, and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid).

[0332] 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.

[0333] 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 comprising nucleotides that are modified with 2'-F and 2'-0Me. In some embodiments, a lipid-conjugated RNAi oligonucleotide disclosed herein comprises a sense strand comprising nucleotides that are modified with 2'-F and 2'-OMe, provided that a nucleotide conjugated to a lipid moiety is not modified with 2’-F or 2’-0Me.

[0334] 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.

[0335] 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, one or more nucleotides forming a base pair with a nucleotide at one or more of positions 10-13 of the antisense 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.

[0336] In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein comprises an antisense strand 22 nucleotides in length, with positions 1-22 numbered 5’ to 3’, and a sense strand having a 2’ -fluoro modification at each of the nucleotides forming a base pair with nucleotides at one or more of positions 10, 11, 12, and 13 of the antisense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein comprises an antisense strand 22 nucleotides in length, with positions 1-22 numbered 5’ to 3’, and a sense strand having a 2’ -fluoro modification at each of the nucleotides forming a base pair with nucleotides at positions 10, 11, 12, 13, or any combination thereof, of the antisense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein comprises an antisense strand 22 nucleotides in length, with positions 1-22 numbered 5’ to 3’, and a sense strand having a 2’ -fluoro modification at each of the nucleotides forming a base pair with nucleotides at positions 10, 11, 12, and 13 of the antisense strand.

[0337] 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.

[0338] 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,

[0339] 17, 18, 19, 20, 21, and 22 of the antisense strand are modified with a 2’-OMe.

[0340] 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,

[0341] 18, 19, 20, 21, and 22 of the antisense strand are modified with a 2’-OMe.

[0342] In some embodiments, the sense strand comprises a 2’ -fluoro modification at positions 3-6 or 4-7, numbered 5’ to 3’. In some embodiments, the sense strand comprises a 2’-fluoro modification at positions 3-6. In some embodiments, the sense strand comprises a 2’-fluoro modification at positions 4-7.

[0343] 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.

[0344] 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).

[0345] 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).

[0346] 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).

[0347] 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).

[0348] 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

[0349] 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).

[0350] 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

[0351] 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).

[0352] 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).

[0353] 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-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).

[0354] 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.

[0355] 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.

[0356] 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).

[0357] 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'-0Me), 2'-O-methoxyethyl (2'-M0E), 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-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 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-methoxyethyl (2'-M0E), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-0-NMA), and 2'-deoxy- 2'-fluoro-P-d-arabinonucleic acid (2'-FANA).

[0358] 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.

[0359] 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, position 36, position 37 or position 38 modified with 2'-F.

[0360] 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.

[0361] 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, position 36, position 37 or position 38 modified with 2'-0Me.

[0362] 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).

[0363] 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, position 36, position 37 or position 38 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'-O- NMA), and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid (2'-FANA).

[0364] 5 ’-Terminal Phosphate 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”).

[0365] 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, CH2OCH2CH2Si (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.

[0366] 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:

[0367]

[0368] 4’-O-monomethylphosphonate-2’-O-methyluridine phosphorothioate

[0369] [MePhosphonate-4O-mUs, alternatively referred to as “MeMOP”]

[0370] Modified Internucleotide Linkage

[0371] 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 internucleotide 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.

[0372] 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.

[0373] 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.

[0374] In some embodiments, the oligonucleotide described herein has a phosphorothioate linkage between each of positions 1 and 2 of the sense strand, the third to last position and penultimate position of the sense strand, and the penultimate position and ultimate position of the sense strand.

[0375] In some embodiments, the oligonucleotide described herein has a phosphorothioate linkage between 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 13 and 14 of the antisense strand, positions 14 and 15 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand.

[0376] In some embodiments, the oligonucleotide described herein has a phosphorothioate linkage between 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 16 and 17 of the antisense strand, positions 17 and 18 of the antisense strand, positions 18 and 19 of the antisense strand, positions 19 and 20 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand.

[0377] In some embodiments, the oligonucleotide described herein has a phosphorothioate linkage between 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 13 and 14 of the antisense strand, positions 14 and 15 of the antisense strand, positions 16 and 17 of the antisense strand, positions 17 and 18 of the antisense strand, positions 18 and 19 of the antisense strand, positions 19 and 20 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand.

[0378] In some embodiments, the oligonucleotide comprises phosphorothioate linkages on the sense strand between nucleotides at positions 1 and 2, 8 and 9, and 9 and 10. In some embodiments, the oligonucleotide comprises phosphorothioate linkages on the antisense strand between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 12 and 14, 14 and 15, 20 and 21, and 21 and 22.

[0379] In some embodiments, the oligonucleotide described herein has a phosphorothioate linkage between 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 12 and 13 of the antisense strand, positions 13 and 14 of the antisense strand, positions 14 and 15 of the antisense strand, positions 15 and 16 of the antisense strand, positions 16 and 17 of the antisense strand, positions 17 and 18 of the antisense strand, positions 18 and 19 of the antisense strand, positions 19 and 20 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 comprises a nucleotide at position 14 of a 22 nucleotide antisense strand, wherein the nucleotide is flanked by phosphorothioate linkages (i.e. a phosphorothioate linkage between positions 13 and 14 and between positions 14 and 15). In some embodiments, the flanked nucleotide at position 14 is the ultimate nucleotide of a duplex between the antisense strand and sense strand. In some embodiments, the oligonucleotide comprises a sense and antisense strand, wherein the antisense strand comprises a flanked oligonucleotide at position 14 of a 22 nucleotide antisense strand (i.e. a phosphorothioate linkage between positions 13 and 14 and between positions 14 and 15), wherein the sense and antisense strand form a duplex and the antisense strand comprises an overhang, and wherein the nucleotide at position 14 is within the overhang.

[0380] 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- aminoethyl)glycine 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 intemucleotide linkage backbone of methylene morpholine rings linked through phosphorodiamidate groups.

[0381] Base Modifications

[0382] 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).

[0383] 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.

[0384] 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).

[0385] Tm-Incr easing Nucleotides

[0386] In some embodiments, the oligonucleotide described herein comprises at least one Tm- increasing nucleotide in the sense strand. In some embodiments, the oligonucleotide has one Tm-increasing nucleotide in the sense strand. In some embodiments, the oligonucleotide has up to two Tm-increasing nucleotides in the sense strand. In some embodiments, the oligonucleotide has up to three Tm-increasing nucleotides in the sense strand. In some embodiments, the oligonucleotide has up to four Tm-increasing nucleotides in the sense strand. In some embodiments, the oligonucleotide has up to five Tm-increasing nucleotides in the sense strand. In some embodiments, the oligonucleotide has up to six Tm-increasing nucleotides in the sense strand. In some embodiments, the oligonucleotide has up to seven Tm- increasing nucleotides in the sense strand. In some embodiments, the oligonucleotide has up to eight Tm-increasing nucleotides in the sense strand. In some embodiments, the oligonucleotide has up to nine Tm-increasing nucleotides in the sense strand. In some embodiments, the oligonucleotide has up to ten Tm-increasing nucleotides in the sense strand.

[0387] In some embodiments, the oligonucleotide has 1 to 2 Tm-increasing nucleotides in the sense strand. In some embodiments, the oligonucleotide has 1 to 3 Tm-increasing nucleotides in the sense strand. In some embodiments, the oligonucleotide has 1 to 4 Tm-increasing nucleotides in the sense strand. In some embodiments, the oligonucleotide has 1 to 5 Tm- increasing nucleotides in the sense strand.

[0388] In some embodiments, an oligonucleotide comprising a stem-loop comprises a Tm- increasing nucleotide in the stem. In some embodiments, an oligonucleotide comprising a stem-loop comprises Tm-increasing nucleotides in at least one base pair of the stem. In some embodiments, an oligonucleotide comprising a stem-loop comprises Tm-increasing nucleotides in one base pair of the stem. In some embodiments, an oligonucleotide comprising a stem-loop comprises Tm-increasing nucleotides in two base pairs of the stem. In some embodiments, an oligonucleotide comprising a stem-loop comprises Tm-increasing nucleotides in three base pairs of the stem. In some embodiments, an oligonucleotide comprising a stem-loop comprises Tm-increasing nucleotides in four base pairs of the stem. In some embodiments, an oligonucleotide comprising a stem-loop comprises Tm-increasing nucleotides in five base pairs of the stem. In some embodiments, an oligonucleotide comprising a stem-loop comprises Tm-increasing nucleotides in six base pairs of the stem.

[0389] Tm-increasing nucleotides include, but are not limited to, bicyclic nucleotides, tricyclic nucleotides, a G-clamp, and analogues thereof, hexitol nucleotides, or a modified nucleotide. In some embodiments, the Tm-increasing nucleotide is a bicyclic nucleotide. In some embodiments, the Tm-increasing nucleotide is a locked nucleic acid (LNA).

[0390] In some embodiments, the sense strand of the oligonucleotide comprises a Tm- increasing nucleotide at one or more of positions 2, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, and 19. In some embodiments, the sense strand of the oligonucleotide comprises a Tm-increasing nucleotide at position 2. In some embodiments, the sense strand of the oligonucleotide comprises a Tm-increasing nucleotide at position 9. In some embodiments, the sense strand of the oligonucleotide comprises a Tm-increasing nucleotide at position 10. In some embodiments, the sense strand of the oligonucleotide comprises a Tm-increasing nucleotide at position 11. In some embodiments, the sense strand of the oligonucleotide comprises a Tm- increasing nucleotide at position 12. In some embodiments, the sense strand of the oligonucleotide comprises a Tm-increasing nucleotide at position 14. In some embodiments, the sense strand of the oligonucleotide comprises a Tm-increasing nucleotide at position 15. In some embodiments, the sense strand of the oligonucleotide comprises a Tm-increasing nucleotide at position 16. In some embodiments, the sense strand of the oligonucleotide comprises a Tm-increasing nucleotide at position 18. In some embodiments, the sense strand of the oligonucleotide comprises a Tm-increasing nucleotide at position 19.

[0391] In some embodiments, a 10-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm-increasing nucleotide at one or more of positions 2, 6 and 7. In some embodiments, a 10-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm-increasing nucleotide at positions 2. In some embodiments, a 10-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm-increasing nucleotide at positions 2 and 6. In some embodiments, a 10-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm-increasing nucleotide at positions 2 and 7. In some embodiments, a 10- nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm-increasing nucleotide at 6 and 7. In some embodiments, a 10-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm-increasing nucleotide at positions 2, 6 and 7.

[0392] In some embodiments, a 12-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm-increasing nucleotide at one or more of positions 2, 7, 8, 10, and 11. In some embodiments, a 12-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm- increasing nucleotide at position 2. In some embodiments, a 12-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm- increasing nucleotide at position 2 and position 7. In some embodiments, a 12-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm- increasing nucleotide at position 2, position 7, and position 8. In some embodiments, a 12-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm- increasing nucleotide at position 2, position 7, position 8, and position 10. In some embodiments, a 12-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm- increasing nucleotide at position 2, position 7, positions, position 10, and position 11. In some embodiments, a 12-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm-increasing nucleotide at position 2, position 10, and position 11. In some embodiments, the sense strand comprises a Tm-increasing nucleotide at position 2, position 11, and position 12.

[0393] In some embodiments, a 14-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm-increasing nucleotide at one or more of positions 2, 9, 10, 12, and 13. In some embodiments, a 14-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm- increasing nucleotide at position 2. In some embodiments, a 14-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm- increasing nucleotide at position 2 and position 9. In some embodiments, a 14-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm- increasing nucleotide at position 2, position 9, and position

[0394] 10, In some embodiments, a 14-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm- increasing nucleotide at position 2, position 9, position 10, position 12, and position 13.

[0395] In some embodiments, a 16-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm-increasing nucleotide at one or more of positions 2, 11, 12, 14, and 15. In some embodiments, a 16-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm- increasing nucleotide at position 2. In some embodiments, a 16-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm- increasing nucleotide at position 2 and position 11. In some embodiments, a 16-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm- increasing nucleotide at position 2, position

[0396] 11, and position 12. In some embodiments, a 16-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm- increasing nucleotide at position 2, position 11, position

[0397] 12, and position 14. In some embodiments, a 16-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm- increasing nucleotide at position 2, position 11, position 12, position 14., and position 15.

[0398] In some embodiments, a 20-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm-increasing nucleotide at one or more of positions 2, 15, 16, 18, and 19. In some embodiments, a 20-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm-increasing nucleotide at position 2. In some embodiments, a 20-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm-increasing nucleotide at position 2 and position 15. In some embodiments, a 20-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm-increasing nucleotide at position 2, position

[0399] 15, and position 16. In some embodiments, a 20-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm-increasing nucleotide at position 2, position 15, position

[0400] 16, and position 18. In some embodiments, a 20-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm-increasing nucleotide at position 2, position 15, position 16, position 18, and position 19.

[0401] In some embodiments, the disclosure provides an RNAi oligonucleotide for reducing target gene expression by the RNAi pathway comprising a combination of one or more Tm- increasing nucleotides and one or more nucleotides (e.g., a modified nucleotide) having a lower binding affinity, wherein the duplex region comprising the RNAi oligonucleotide is maintained under physiological conditions and the ability of the RNAi oligonucleotide to inhibit or reduce target gene expression is maintained.

[0402] Bicyclic Nucleotides

[0403] Bicyclic nucleotides typically have a sugar moiety with a 4 to 7 membered ring (including but not limited to a furanosyl) comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicyclic structure. Such bicyclic nucleotides have various names including BNA's and LNA's for bicyclic nucleic acids and locked nucleic acids, respectively. The synthesis of bicyclic nucleotides and their incorporation into nucleic acid compounds has also been reported in the literature, including, for example, Singh et al., Chem. Commun., 1998, 4, 455-56; Koshkin et al., TETRAHEDRON, 1998, 54, 3607- 30; Wahlestedt et al., PROC. NATL. ACAD. SCI. U.S.A., 2000, 97, 5633-38; Kumar et al., BIOORG. MED. CHEM. LETT., 1998, 8, 2219-22; Singh et al., J. ORG. CHEM., 1998, 63, 10035- 039; U.S. Patent Nos. 7,427,672, 7,053,207, 6,794,499, 6,770,748, 6,268,490 and 6,794,499; and published U.S. applications 20040219565, 20040014959, 20030207841, 20040192918, 20030224377, 20040143114 and 20030082807; each of which is incorporated by reference herein, in its entirety.

[0404] In some embodiments, the Tm-increasing nucleotide is a bicyclic nucleotide that comprises a bicyclic sugar moiety. In certain embodiments, the bicyclic sugar moiety comprises a first ring of 4 to 7 members and a bridge forming a North-type sugar confirmation that connects any two atoms of the first ring of the sugar moiety to form a second ring. In certain embodiments, the bridge connects the 2'-carbon and the 4'-carbon of the first ring to form a second ring.

[0405] Typically, the bridge contains 2 to 8 atoms. In certain embodiments, the bridge contains 3 atoms. In certain embodiments, the bridge contains 4 atoms. In certain embodiments, the bridge contains 5 atoms. In certain embodiments, the bridge contains 6 atoms. In certain embodiments, the bridge contains 7 atoms. In certain embodiments, the bridge contains 8 atoms. In certain embodiments, the bridge contains more than 8 atoms.

[0406] In certain embodiments, the bicyclic sugar moiety is a substituted furanosyl comprising a bridge that connects the 2'-carbon and the 4'-carbon of the furanosyl to form the second ring. In certain embodiments, the bicyclic nucleotide has the structure of Formula I: Formula I wherein B is a nucleobase; wherein G is H, OH, NH2, Ci-Ce alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Ci-Ce alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl, substituted acyl, substituted amide, thiol, or substituted thio; wherein X is O, S, or NRi, wherein Ri is H, Ci-Ce alkyl, Ci-Ce alkoxy, benzene or pyrene; and wherein Wa and Wb are each independently, H, OH, a hydroxyl protecting group, a phosphorous moiety, or an internucleotide linking group attaching the nucleotide represented by Formula I to another nucleotide or to an oligonucleotide and wherein at least one of War or Wb is an internucleotide linking group attaching the nucleotide represented by Formula I to an oligonucleotide.

[0407] In certain embodiments of Formula I, G is H and X is NRi, wherein Ri is benzene or pyrene. In certain embodiments, of Formula I, G is H and X is S.

[0408] In certain embodiments of Formula I, G is H and X is O: Formula la

[0409] In certain embodiments of Formula I, G is H and X is NRi, wherein Ri is H, CH3, or

[0410] OCH3: Formula lb

[0411] In certain embodiments of Formula I, G is OH or NH2 and X is O.

[0412] In certain embodiments of Formula I, G is OH and X is O: Formula Ic

[0413] In certain embodiments of Formula I, G is NH2 and X is O: Formula Id

[0414] In certain embodiments, of Formula I, G is CH3 or CH2OCH3 and X is O. In certain embodiments, of Formula I, G is CH3 and X is O: Formula le

[0415] In certain embodiments, of Formula I, G is CH2OCH3 and X is O: Formula If

[0416] In certain embodiments, the bicyclic nucleotide has the structure of Formula II: Formula II wherein B is a nucleobase; wherein Qi is CH2 or O; wherein X is CH2, O, S, or NRi, wherein Ri is H, Ci-Ce alkyl, Ci-Ce alkoxy, benzene or pyrene; wherein if Qi is O, X is CH2; wherein if Qi is CH2, X is CH2, O, S, or NRi, wherein Ri is H, Ci-Ce alkyl, Ci-Ce alkoxy, benzene or pyrene; wherein Wa and Wb are each independently, H, OH, a hydroxyl protecting group, a phosphorous moiety, or an internucleotide linking group attaching the nucleotide represented by Formula II to another nucleotide or to an oligonucleotide and wherein at least one of Wa or

[0417] Wb is an intemucleotide linking group attaching the nucleotide represented by Formula II to an oligonucleotide.

[0418] In certain embodiments of Formula II, Qi is O and X is CH2: Formula Ila

[0419] In certain embodiments of Formula II, Qi is CH2 and X is O: Formula lib

[0420] In certain embodiments of Formula II, Qi is CH2 and X is NRi, wherein Ri is H, CH3 or OCH3: Formula lie

[0421] In certain embodiments of Formula II, Qi is CH2 and X is NH: Formula lid

[0422] In certain embodiments, the bicyclic nucleotide has the structure of Formula III: Formula III wherein B is a nucleobase; wherein Q2 is O or NRi, wherein Ri is H, Ci-Ce alkyl, Ci-Ce alkoxy, benzene or pyrene; wherein X is CH2, O, S, or NRi, wherein Ri is H, Ci-Ce alkyl, Ci-Ce alkoxy, benzene or pyrene; wherein if Q2 is O, X is NRi; wherein if Q2 is NRi, X is O or S; wherein Wa and Wb are each independently, H, OH, a hydroxyl protecting group, a phosphorous moiety, or an internucleotide linking group attaching the nucleotide represented by Formula III to another nucleotide or to an oligonucleotide and wherein at least one of Wa or Wb is an intemucleotide linking group attaching the nucleotide represented by Formula III to an oligonucleotide.

[0423] In certain embodiments of Formula III, Q2 is O and X is NRi. In certain embodiments of Formula III, Q2 is O and X is NRi, wherein Ri is Ci-Ce alkyl. In certain embodiments of Formula III, Q2 is O and X is NRi and Ri is H or CH3

[0424] In certain embodiments of Formula III, Q2 is O and X is NRi and Ri is CH3: Formula Illa

[0425] In certain embodiments of Formula III, Q2 is NRi and X is O. In certain embodiments of Formula III, Q2 is NRi, wherein Ri is Ci-Ce alkyl and X is O.

[0426] In certain embodiments of Formula III, Q2 is NCH3 and X is O: Formula Illb

[0427] In certain embodiments, the bicyclic nucleotide has the structure of Formula IV: Formula IV wherein B is a nucleobase; wherein Pi and P3 are CH2, P2 is CH2 or O and P4 is O; and wherein Wa and Wb are each independently, H, OH, a hydroxyl protecting group, a phosphorous moiety, or an internucleotide linking group attaching the nucleotide represented by Formula IV to another nucleotide or to an oligonucleotide and wherein at least one of Wa or Wb is an internucleotide linking group attaching the nucleotide represented by Formula IV to an oligonucleotide.

[0428] In certain embodiments of Formula IV, Pi, P2, and P3 are CH2, and P4 is O: Formula IVa

[0429] In certain embodiments of Formula IV, Pi and P3 are CH2, P2 is O and P4 is O: Formula IVb

[0430] In certain embodiments, the bicyclic nucleotide has the structure of Formula Va or Vb:

[0431] wherein B is a nucleobase; wherein rl, r2, r3, and r4 are each independently H, halogen, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl; substituted C2-C12 alkynyl; C1-C12 alkoxy; substituted C1-C12 alkoxy, OTi, STi, SOTi, SO2T1, NT1T2, N3, CN, C(=O)OTi, C(=O)NTIT2, C(=O)TI, O-C(=O)NTIT2, N(H)C(=NH)NTIT2, N(H)C(=O)NTIT2 or N(H)C(=S)NTIT2, wherein each of T1 and T2 is independently H, Ci- Ce alkyl, or substituted C1-C16 alkyl; or rl and r2 or r3 and r4 together are =C(r5)(r6), wherein r5 and r6 are each independently H, halogen, Ci-C 12 alkyl, or substituted C1-C12 alkyl; and wherein Wa and Wb are each independently, H, OH, a hydroxyl protecting group, a phosphorous moiety, or an internucleotide linking group attaching the nucleotide represented by Formula V to another nucleotide or to an oligonucleotide and wherein at least one of Wa or Wb is an internucleotide linking group attaching the nucleotide represented by Formula V to an oligonucleotide.

[0432] In certain embodiments, the bicyclic sugar moiety is a substituted furanosyl comprising a bridge that connects the 2'-carbon and the 4'-carbon of the furanosyl to form the second ring, wherein the bridge that connects the 2'-carbon and the 4'-carbon of the furanosyl includes, but is not limited to: a) 4'-CH2-O-N(R)-2' and 4'-CH2-N(R)-O-2', wherein R is H, C1-C12 alkyl, or a protecting group, including, for example, 4'-CH2-NH-O-2' (also known as BNANC), 4'-CH2-N(CH3)-O-2' (also known as BNANC[NMe]), (as described in U.S. Patent No. 7,427,672, which is hereby incorporated by reference in its entirety); b) 4'-CH2-2'; 4'-(CH2)2-2'; 4'-(CH2)3-2'; 4'-(CH2)-O-2' (also known as LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (also known as ENA); 4'-CH(CH3)-O-2' (also known as cEt); and 4'-CH(CH2OCH3)-O-2' (also known as cMOE), and analogs thereof (as described in U.S. Patent No. 7,399,845, which is hereby incorporated by reference in its entirety); c) 4'-C(CH3)(CH3)-O-2' and analogs thereof (as described in U. S. Patent No. 8,278,283, which is hereby incorporated by reference in its entirety); d) 4'-CH2-N(OCH3)-2' and analogs thereof (as described in U.S. Patent No.

[0433] 8.278.425, which is hereby incorporated by reference in its entirety); e) 4'-CH2-O-N(CH3)-2' and analogs thereof (as described in U.S. Patent Publication No. 2004 / 0171570, which is hereby incorporated by reference in its entirety); f) 4'-CH2-C(H)(CH3)-2' and analogs thereof (as described in Chattopadhyaya et al., J. ORG. CHEM., 2009, 74, 118-34, which is hereby incorporated by reference in its entirety); and g) 4'-CH2-C(=CH2)-2' and analogs thereof as described in U.S. Patent No.

[0434] 8.278.426, which is hereby incorporated by reference in its entirety).

[0435] In certain embodiments, the bicyclic nucleotide (BN) is one or more of the following: (a) methyleneoxy BN, (b) ethyleneoxy BN, (c) aminooxy BN; (d) oxyamino BN, (e) methyl(methyleneoxy) BN (also known as constrained ethyl or cET), (f) methylene-thio BN, (g) methylene amino BN, (h) methyl carbocyclic BN, and (i) propylene carbocyclic BN, as shown below.

[0436] ( )

[0437] In the bicyclic nucleotides of (a) to (i) above, B is a nucleobase, R2 is H or CH3 and Wa and Wb are each independently, H, OH, a hydroxyl protecting group, a phosphorous moiety, or an intemucleotide linking group attaching the bicyclic nucleotide to another nucleotide or to an oligonucleotide and wherein at least one of Wa or Wb is an intemucleotide linking group attaching the bicyclic nucleotide to an oligonucleotide.

[0438] In one embodiment of the oxyamino BN (d), R2 is CH3, as follows (also known as

[0439] BNANC[NMe]):

[0440] In certain embodiments, bicyclic sugar moieties and bicyclic nucleotides incorporating such bicyclic sugar moieties are further defined by isomeric configuration. In certain embodiments, the bicyclic sugar moiety or nucleotide is in the a-L configuration. In certain embodiments, the bicyclic sugar moiety or nucleotide is in the P-D configuration. For example, in certain embodiments, the bicyclic sugar moiety or nucleotide comprises a 2'0,4'-C- methylene bridge (2'-O-CH2-4') in the a-L configuration (a-L LNA). In certain embodiments, the bicyclic sugar moiety or nucleotide is in the R configuration. In certain embodiments, the bicyclic sugar moiety or nucleotide is in the S configuration. For example, in certain embodiments, the bicyclic sugar moiety or nucleotide comprises a 4'-CH(CH3)-O-2' bridge (i.e., cEt) in the S-configuration.

[0441] Tricyclic Nucleotides

[0442] In some embodiments, the Tm-increasing nucleotide is a tricyclic nucleotide. The synthesis of tricyclic nucleotides and their incorporation into nucleic acid compounds has also been reported in the literature, including, for example, Steffens et al., J. AM. CHEM. SOC. 1997;119: 11548-549; Steffens et al., J. ORG. CHEM. 1999;121(14):3249-55; Renneberg et al., J. AM. CHEM. SOC. 2002;124:5993-6002; Ittig et al., NUCLEIC ACIDS RES. 2004;32(l):346-53; Scheidegger et al., CHEMISTRY 2006;12:8014-23; Ivanova et al., OLIGONUCLEOTIDES 2007;17:54-65; each of which is each hereby incorporated by reference in its entirety.

[0443] In certain embodiments, the tricyclic nucleotide is a tricyclo nucleotide (also called tri cyclo DNA) in which the 3 '-carbon and 5 '-carbon centers are connected by an ethylene that is fused to a cyclopropane ring, as discussed for example in Leumann CJ, BIOORG. MED. CHEM. 2002;10:841-54 and published U.S. Applications 2015 / 0259681 and 2018 / 0162897, which are each hereby incorporated by reference. In certain embodiments, the tricyclic nucleotide comprises a substituted furanosyl ring comprising a bridge that connects the 2'-carbon and the 4'-carbon of the furanosyl to form a second ring, and a third fused ring resulting from a group connecting the 5 '-carbon to the methylene group of the bridge that connects the 2'-carbon and the 4'-carbon of the furanosyl, as discussed, for example, in published U.S. Application 2015 / 0112055, which is hereby incorporated by reference.

[0444] Other Tm-increasing nucleotides

[0445] In addition to bicyclic and tricyclic nucleotides, other Tm-increasing nucleotides can be used in the RNAi oligonucleotides described herein. For example, in certain embodiments, the Tm-increasing nucleotide is a G-clamp, guanidine G-clamp or analogue thereof (Wilds et al., CHEM, 2002; 114: 123 and Wilds et al., CHIM ACTA 2003; 114: 123), a hexitol nucleotide (Herdewijn, CHEM. BIODIVERSITY 2010;7: 1-59), or a modified nucleotide. The modified nucleotide can have a modified nucleobase, as described herein, including for example, 5- bromo-uracil, 5-iodo-uracil, 5-propynyl-modified pyrimidines, or 2-amino adenine (also called 2,6-diaminopurine) (Deleavey et al., CHEM. & BIOL. 2012;19:937-54) or 2-thio uridine, 5 Me- thio uridine, and pseudo uridine. The modified nucleotide can also have a modified sugar moiety, as described for example, in U.S. Patent No. 8,975,389, which is hereby incorporated by reference, or as described herein, except that the Tm-increasing nucleotide is not modified at the 2'-carbon of the sugar moiety with a 2'-F or a 2'-OMe.

[0446] In certain embodiments, the Tm-increasing nucleotide is a bicyclic nucleotide. In certain embodiments, the Tm-increasing nucleotide is a tricyclic nucleotide. In certain embodiments, the Tm-increasing nucleotide a G-clamp, guanidine G-clamp or analogue thereof. In certain embodiments, the Tm-increasing nucleotide is a hexitol nucleotide. In certain embodiments, the Tm-increasing nucleotide is a bicyclic or tricyclic nucleotide. In certain embodiments, the Tm- increasing nucleotide is a bicyclic nucleotide, a tricyclic nucleotide, or a G-clamp, guanidine G-clamp or analogue thereof. In certain embodiments, the Tm-increasing nucleotide is a bicyclic nucleotide, a tricyclic nucleotide, a G-clamp, guanidine G-clamp or analogue thereof, or a hexitol nucleotide.

[0447] In certain embodiments, the Tm-increasing nucleotide increases the Tmof the nucleic acid inhibitor molecule by at least 2 °C per incorporation. In certain embodiments, the Tm- increasing nucleotide increases the Tm of nucleic acid inhibitor molecule by at least 3 °C per incorporation. In certain embodiments, the Tm-increasing nucleotide increases the Tm of nucleic acid inhibitor molecule by at least 4 °C per incorporation. In certain embodiments, the Tm-increasing nucleotide increases the Tm of nucleic acid inhibitor molecule by at least 5 °C per incorporation.

[0448] Targeting Ligands

[0449] 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). 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 liver. 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 eye.

[0450] 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). 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.

[0451] 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.

[0452] In some embodiments, an oligonucleotide of the 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.

[0453] 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.

[0454] In some embodiments, the tetraloop is any combination of adenine and guanine nucleotides. In some embodiments, the tetraloop is any combination of adenine, guanine, cytosine, and uridine nucleotides.

[0455] 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):

[0456] 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):

[0457]

[0458] 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:

[0459] 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:

[0460]

[0461] 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.

[0462]

[0463] 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.

[0464]

[0465] 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.

[0466] 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.

[0467] Lipid Conjugates

[0468] 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.

[0469] 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 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

[0470] 29, position 30, position 31, position 32, position 33, position 34, position 35, position 36, position 37 or position 38 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 4 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 8 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 28 of the sense strand. In some embodiments, the lipid moiety is conjugated to position 38 of the sense strand.

[0471] In some embodiments, the lipid moiety is conjugated to a nucleotide of the sense strand that forms a base pair with a nucleotide at position 20, position 19, position 18, position 17, position 16, position 15, position 14, position 13, or position 12 of the antisense strand. In some embodiments, the lipid moiety is conjugated to a nucleotide of the sense strand that forms a base pair with a nucleotide at position 16, position 14, or position 12 of the antisense strand. In some embodiments, the lipid moiety is conjugated to a nucleotide of the sense strand that forms a base pair with a nucleotide at position 20 of the antisense strand. In some embodiments, the lipid moiety is conjugated to a nucleotide of the sense strand that forms a base pair with a nucleotide at position 19 of the antisense strand. In some embodiments, the lipid moiety is conjugated to a nucleotide of the sense strand that forms a base pair with a nucleotide at position 18 of the antisense strand. In some embodiments, the lipid moiety is conjugated to a nucleotide of the sense strand that forms a base pair with a nucleotide at position 17 of the antisense strand. In some embodiments, the lipid moiety is conjugated to a nucleotide of the sense strand that forms abase pair with a nucleotide at position 16 of the antisense strand. In some embodiments, the lipid moiety is conjugated to a nucleotide of the sense strand that forms a base pair with a nucleotide at position 15 of the antisense strand. In some embodiments, the lipid moiety is conjugated to a nucleotide of the sense strand that forms a base pair with a nucleotide at position 14 of the antisense strand. In some embodiments, the lipid moiety is conjugated to a nucleotide of the sense strand that forms a base pair with a nucleotide at position 13 of the antisense strand. In some embodiments, the lipid moiety is conjugated to a nucleotide of the sense strand that forms a base pair with a nucleotide at position 12 of the antisense strand.

[0472] 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.

[0473] 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.

[0474] In some embodiments, the lipid moiety is a C 16 hydrocarbon chain.

[0475] In some embodiments, the lipid moiety is conjugated to the oligonucleotide via a linker.

[0476] In some embodiments, a nucleotide of the lipid-conjugated oligonucleotide is represented by formula Il-b or II-c:

[0477] II-c or a pharmaceutically acceptable salt thereof, wherein:

[0478] 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)-, -

[0479] R4is hydrogen, RA, or a suitable amine protection group; and

[0480] 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 -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR-, or -P(S)OR.

[0481] In some embodiments of the lipid-conjugated RNAi oligonucleotide, R5is selected from

[0482]

[0483] In certain embodiments of the lipid-conjugated RNAi oligonucleotide,

[0484] R5is selected from

[0485]

[0486] In some embodiments, R5is

[0487] In some embodiments, a nucleotide of the lipid-conjugated RNAi oligonucleotide is represented by formula Il-Ib or II-Ic:

[0488] II-Ic or a pharmaceutically acceptable salt thereof; wherein

[0489] B is a nucleobase or hydrogen; m is 1-50;

[0490] X1is -O-, or -S-;

[0491] __ . , 1

[0492] Y is hydrogen,

[0493] R3is hydrogen, or a suitable protecting group;

[0494] X2is O, or S;

[0495] X3is -O-, -S-, or a covalent bond;

[0496] Y1is a linking group attaching to the 2'- or 3 '-terminal of a nucleoside, a nucleotide, or an oligonucleotide;

[0497] Y2is hydrogen, a phosphoramidite analogue, an internucleotide linking group attaching to the 5 '-terminal of a nucleoside, a nucleotide, or an oligonucleotide, or a linking group attaching to a solid support;

[0498] 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 - 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.

[0499] In some embodiments, the lipid is 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.

[0500] 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.

[0501] Exemplary Oligonucleotides

[0502] 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. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a nucleotide conjugated with a 22-carbon (C22) lipid. In some embodiments, the oligonucleotide of the lipid-conjugated RNAi oligonucleotide is conjugated to a C16 lipid as shown in:

[0503] In some embodiments, the oligonucleotide of the lipid-conjugated RNAi oligonucleotide is conjugated to a C22 lipid as shown in:

[0504] 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 5’ end of the antisense strand comprises an overhang. In some embodiments, the 5’ and 3’ ends of the antisense strand each comprise an overhang.

[0505] 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.

[0506] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’- [mXs][mXs][mX][mX][mX][ademX-L][mX][mX][mX][mX][mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [fX] [fX] [fX] [fX] [mX] [mX] [mX] [mX] [ mX] [mX] [mXs] [mXs] [mX] -3 ’ Hybridized to:

[0507] 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, and [ademX-L] = Lipid attached to a nucleotide.

[0508] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’- [+X][mX][mX][ademX-

[0509] L] [mX] [mX] [mX] [+X] [mX] [mX] [mX] [mX] [mX]

[0510] [mX] [mX] [mX] [mX] [fX] [fX] [fX] [fX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’ Hybridized to:

[0511] Antisense Strand: 5’ - [MePhosphonate-4O- mXs] [fXs] [fXs] [fX] [fX] [mX] [fX] [mX] [mX] [fX]

[0512] [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-L] = Lipid attached to a nucleotide, [+X] = a Tm-increasing nucleotide, optionally an LNA, and [+Xs] = a Tm- increasing nucleotide with a phosphorothioate linkage to the neighboring nucleotide, optionally an LNA.

[0513] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’- [+X][mX][mX][mX][mX][ademX- L] [mX] [mX] [mX] [mX] [mX] [+X] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [fX] [fX] [fX] [fX] [mX] [mX] [mX] [mX] [mX] [mX] [ mXs][mXs][mX] -3’ Hybridized to:

[0514] Antisense Strand: 5’ - [MePhosphonate-4O- mXs] [fXs] [fXs] [fX] [fX] [mX] [fX] [mX] [mX] [fX]

[0515] [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-L] = Lipid attached to a nucleotide, [+X] = a Tm-increasing nucleotide, optionally an LNA, and [+Xs] = a Tm- increasing nucleotide with a phosphorothioate linkage to the neighboring nucleotide, optionally an LNA.

[0516] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’- [ademX-Ls] [mX][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX] [mX][mX][mX][mXs][mXs][mX] -3’ Hybridized to:

[0517] Antisense Strand: 5’ - [MePhosphonate-4O- mXs] [fXs] [fXs] [fX] [fX] [mX] [fX] [mX] [mX] [fX]

[0518] [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, and [ademX-Ls] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide.

[0519] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of

[0520] Sense Strand: 5’- [ademX-Ls] [mX][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX] [mX][mX][mXs][mXs][mX] -3’ Hybridized to:

[0521] Antisense Strand: 5’ - [MePhosphonate-4O- mXs] [fXs] [fXs] [fX] [fX] [mX] [fX] [mX] [mX] [fX]

[0522] [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, and [ademX-Ls] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide.

[0523] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’- [ademX-Ls] [+X][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX][mX] [mXs][+Xs][+X] -3’ Hybridized to:

[0524] Antisense Strand: 5’ - [MePhosphonate-4O- mXs] [fXs] [fXs] [fX] [fX] [mX] [fX] [mX] [mX] [fX]

[0525] [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, [+X] = a Tm- increasing nucleotide, optionally an LNA, and [+Xs] = a Tm-increasing nucleotide with a phosphorothioate linkage to the neighboring nucleotide, optionally an LNA or BNA.

[0526] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of

[0527] Sense Strand: 5’- [ademX-Ls] [mX][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX] [mX][mX][mX][mXs][mXs][mX] -3’

[0528] Hybridized to:

[0529] Antisense Strand: 5’ - [MePhosphonate-4O- mXs] [fXs] [fXs] [fX] [fX] [mX] [fX] [mX] [mX] [fX]

[0530] [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, and [ademX-Ls] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide.

[0531] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’- [ademX-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:

[0532] 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, and [ademX-Ls] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide.

[0533] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of

[0534] Sense Strand: 5’- [+Xs] [ademX-L] [fX][fX][fX][fX] [mX][mX][mX][mX] [+Xs][mXs][mX]- 3’

[0535] Hybridized to:

[0536] 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-L] = Lipid attached to a nucleotide, [+X] = a Tm-increasing nucleotide, optionally an LNA, and [+Xs] = a Tm- increasing nucleotide with a phosphorothioate linkage to the neighboring nucleotide, optionally an LNA.

[0537] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of

[0538] Sense Strand: 5’- [+Xs] [ademX-L] [fX][fX][fX][fX] [mX][mX][mX][+X] [+Xs][mXs][mX]- 3’

[0539] 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’-( -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-L] = Lipid attached to a nucleotide, [+X] = a Tm-increasing nucleotide, optionally an LNA, and [+Xs] = a Tm- increasing nucleotide with a phosphorothioate linkage to the neighboring nucleotide, optionally an LNA.

[0540] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’- [ademX-

[0541] Ls] [mX] [mX] [mX] [mX] [mX] [mX] [fX] [fX] [fX] [fX] [mX] [mX] [mX] [mX][mX][mX][mXs][mXs][mX]- 3’ Hybridized to:

[0542] 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.

[0543] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of

[0544] Sense Strand: 5’- [ademX-Ls] [mX][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX] [mX][mX][mX][mXs][mXs][mX]- 3’

[0545] Hybridized to:

[0546] 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.

[0547] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of

[0548] Sense Strand: 5’- [mXs][mXs][mX][mX][mX][ademX-C16][mX][mX][mX][mX][mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [fX] [fX] [fX] [fX] [mX] [mX] [mX] [mX] [ mX] [mX] [mXs] [mXs] [mX] -3 ’

[0549] Hybridized to:

[0550] 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, and [ademX-C16] = C16 Lipid attached to a nucleotide.

[0551] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’- [+X][mX][mX][ademX-

[0552] C 16] [mX] [mX] [mX] [+X] [mX] [mX] [mX] [mX] [mX]

[0553] [mX] [mX] [mX] [mX] [fX] [fX] [fX] [fX] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] -3 ’ Hybridized to:

[0554] 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, and [ademX-C16] = C16 Lipid attached to a nucleotide, [+X] = a Tm-increasing nucleotide, optionally an LNA.

[0555] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’- [+X][mX][mX][mX][mX][ademX- C 16] [mX] [mX] [mX] [mX] [mX] [+X] [mX]

[0556] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [fX] [fX] [fX] [fX] [mX] [mX] [mX] [mX] [mX] [mX] [ mXs][mXs][mX] -3’

[0557] Hybridized to:

[0558] Antisense Strand: 5’ - [MePhosphonate-4O- mXs] [fXs] [fXs] [fX] [fX] [mX] [fX] [mX] [mX] [fX]

[0559] [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-C16] = C16 Lipid attached to a nucleotide, [+X] = a Tm-increasing nucleotide, optionally an LNA.

[0560] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of

[0561] Sense Strand: 5’- [ademX-C16s][mX][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX] [mX][mX][mX][mXs][mXs][mX] -3’

[0562] Hybridized to:

[0563] Antisense Strand: 5’ - [MePhosphonate-4O- mXs] [fXs] [fXs] [fX] [fX] [mX] [fX] [mX] [mX] [fX]

[0564] [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-C16s] = C16 Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide.

[0565] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of

[0566] Sense Strand: 5’- [ademX-C16s][mX][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX] [mX][mX][mXs][mXs][mX] -3’

[0567] Hybridized to:

[0568] Antisense Strand: 5’ - [MePhosphonate-4O- mXs] [fXs] [fXs] [fX] [fX] [mX] [fX] [mX] [mX] [fX]

[0569] [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-C16s] = C16 Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide.

[0570] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of

[0571] Sense Strand: 5’- [ademX-C16s][+X][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX][mX] [mXs][+Xs][+X] -3’

[0572] Hybridized to:

[0573] Antisense Strand: 5’ - [MePhosphonate-4O- mXs] [fXs] [fXs] [fX] [fX] [mX] [fX] [mX] [mX] [fX]

[0574] [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-C16s] = C16 lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide, [+X] = a Tm-increasing nucleotide, optionally an LNA, and [+Xs] = a Tm-increasing nucleotide with a phosphorothioate linkage to the neighboring nucleotide, optionally an LNA or BNA. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of

[0575] Sense Strand: 5’- [ademX-C22s][mX][mX][mX][mX][mX][rnX][fX][fX][fX][fX][rnX] [mX][mX][mX][mXs][mXs][mX] -3’

[0576] Hybridized to:

[0577] Antisense Strand: 5’ - [MePhosphonate-4O- mXs] [fXs] [fXs] [fX] [fX] [mX] [fX] [mX] [mX] [fX]

[0578] [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-C22s] = C22 Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide.

[0579] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’- [ademX-

[0580] C22s] [mX] [mX] [mX] [mX] [mX] [mX] [fX] [fX] [fX] [fX] [mX] [mX]

[0581] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [ mX][mX][mX][mX][mX][mX]- 3’

[0582] Hybridized to:

[0583] 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-C22s] = C22 Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide.

[0584] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’- [+Xs][ademX-

[0585] C22] [fX] [fX] [fX] [fX] [mX] [mX] [mX] [mX] [+Xs] [mXs] [mX] - 3 ’

[0586] Hybridized to:

[0587] 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-C22] = C22 Lipid attached to a nucleotide, [+X] = a Tm-increasing nucleotide, optionally an LNA, and [+Xs] = a Tm-increasing nucleotide with a phosphorothioate linkage to the neighboring nucleotide, optionally an LNA.

[0588] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’- [+Xs][ademX- C22] [fX] [fX] [fX] [fX] [mX] [mX] [mX] [+X] [+Xs] [mXs] [mX] - 3 ’ Hybridized to:

[0589] 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-C22] = C22 Lipid attached to a nucleotide, [+X] = a Tm-increasing nucleotide, optionally an LNA, and [+Xs] = a Tm-increasing nucleotide with a phosphorothioate linkage to the neighboring nucleotide, optionally an LNA.

[0590] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’- [ademX-

[0591] C22s] [mX] [mX] [mX] [mX] [mX] [mX] [fX] [fX] [fX] [fX] [mX] [mX]

[0592] [mX] [mX] [mX] [mX] [mXs] [mXs] [mX] - 3 ’

[0593] Hybridized to:

[0594] 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, [fX] =2’- fluoro modified nucleotide with phosphodiester linkages to neighboring nucleotides, [MePhosphonate-4O-mX] = 4’-O- monomethylphosphonate-2’-O-m ethyl modified nucleotide, [ademX-C22s] = C22 Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide.

[0595] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of

[0596] Sense Strand: 5’- [ademX-C22s][mX][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX] [mX][mX][mX][mXs][mXs][mX]- 3’

[0597] Hybridized to:

[0598] 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-C22s] = C22 Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide.

[0599] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’-

[0600] [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: Antisense Strand: 5’ - [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX][mX] [fX] [mX] [mX] [mX] [fX] [mX] [mX / mXs] [mX / mXs] [mX / mXs] [mX / mXs] [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, [fX] =2’- fluoro modified nucleotide with phosphodiester linkages to neighboring nucleotides, [MePhosphonate-4O-mX] = 4’-O- monomethylphosphonate-2’-O-m ethyl modified nucleotide, [mX / mXs]= the position is either a 2’-O-methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides or a 2’-O-methyl modified nucleotide with phosphorothioate linkages to neighboring nucleotides, and [ademX-L] = Lipid attached to a nucleotide.

[0601] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’-

[0602] [+Xs] [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:

[0603] Antisense Strand: 5’ - [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX][mX] [fX] [mX] [mX] [mXs] [fXs] [mX / mXs] [mX / mXs] [mX / mXs] [mX / mXs] [mX / mXs] [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, [mX / mXs]= the position is either a 2’-O-methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides or a 2’-O-methyl modified nucleotide with phosphorothioate linkages to neighboring nucleotides, and [ademX-L] = Lipid attached to a nucleotide.

[0604] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’-

[0605] [+Xs] [fX] [fX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [m X] [mX] [ademX-L] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX]-3 ’ Hybridized to:

[0606] Antisense Strand: 5’ - [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX][mX] [fX] [mX] [mX / mXs] [mXs] [fXs ] [mX / mXs] [mX / mXs] [mX / mXs] [mX / mXs] [mX / mXs] [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, [mX / mXs]= the position is either a 2’-( -methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides or a 2’-( -methyl modified nucleotide with phosphorothioate linkages to neighboring nucleotides, and [ademX-L] = Lipid attached to a nucleotide.

[0607] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of

[0608] Sense Strand: 5’- [ademXs-L][mX / +X][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX] [mX] [mX] [mX / +X] [mX / +X] [mX] [mXs / +Xs] [mXs / +Xs] [mX]-3 ’ Hybridized to:

[0609] 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, [mX / +X]= the position is either a 2’-( -methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides or a Tm-increasing nucleotide with phosphodiester linkages to neighboring nucleotides, optionally an LNA, and [ademXs-C16] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of

[0610] Sense Strand: 5’- [ademXs-L] [fX / +X][fX][fX][fX][rnX][rnX][rnX][rnX / +X][mX / +X][rnX] [mXs / +Xs] [mXs / +Xs] [mX]-3 ’ Hybridized to:

[0611] Antisense Strand: 5’ - [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX] [mX] [fX] [mX] [mX] [mXs] [fXs] [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, [mX / +X]= the position is either a 2’-( -methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides or a Tm-increasing nucleotide, [mXs / +Xs]= the position is either a 2’-( -methyl modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide or a Tm-increasing nucleotide with phosphorothioate linkage to neighboring nucleotide, optionally an LNA, [fXs / +Xs]= the position is either a 2’- fluoro modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide or a Tm-increasing nucleotide with phosphorothioate linkage to neighboring nucleotide, optionally an LNA and [ademXs-L] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide.

[0612] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of

[0613] Sense Strand: 5’- [ademXs-L] [mX / +X][mX][fX][fX][fX][fX][mX][mX][mX][mX / +X] [mX / +X] [mX] [mXs / +Xs] [mXs / +Xs] [mX]-3 ’ Hybridized to:

[0614] 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, [mX / +X]= the position is either a 2’-( -methyl modified nucleotide with phosphodiester linkages to neighboring nucleotides or a Tm-increasing nucleotide, [mXs / +Xs]= the position is either a 2’-( -methyl modified nucleotide with a phosphorothioate linkage to the neighboring nucleotide or a Tm-increasing nucleotide with phosphorothioate linkage to neighboring nucleotide, optionally an LNA, and [ademXs-L] = Lipid attached to a nucleotide with a phosphorothioate linkage to the neighboring nucleotide.

[0615] In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’- [ademXs-L] [mX / +X][mX][fX][mX][mX][mX][mX / +X] [mX / +X] [mX] [mXs / +Xs] [mXs / +Xs] [mX]-3 ’ Hybridized to:

[0616] Antisense Strand: 5’ - [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX] [mX] [fX] [mX] [mX] [mXs] [fXs] [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...

Claims

CLAIMS1. A double-stranded oligonucleotide comprising an antisense strand of about 20-22 nucleotides in length and a sense strand of about 8-20 nucleotides in length, wherein the antisense and sense strands form a duplex region of about 8-20 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang of at least one nucleotide and a 3’ overhang of at least one nucleotide, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, and wherein the sense strand comprises at least one lipid moiety conjugated to a nucleotide on the sense strand.

2. The oligonucleotide of claim 1, wherein the 5’ overhang is about 1-10 nucleotides.

3. The oligonucleotide of claim 1, wherein the 5’overhang is about 2-10 nucleotides.

4. The oligonucleotide of claim 1, wherein the 5’ overhang is about 1-6 nucleotides.

5. The oligonucleotide of any one of claims 1-4, wherein the 3’ overhang is about 2-12 nucleotides.

6. The oligonucleotide of any one of claims 1-4, wherein the 3 ’overhang is about 2-8 nucleotides.

7. The oligonucleotide of any one of claims 1-5, wherein the 5’overhang is 2 nucleotides and the 3’overhang is about 3-7 nucleotides.

8. The oligonucleotide of any one of claims 1-5, wherein the 3’overhang is 2 nucleotides, and the 5’overhang is about 2-8 nucleotides.

9. The oligonucleotide of any one of claims 1-5, wherein the 3’overhang is 6-8 nucleotides and the 5’overhang is 2-4 nucleotides.

10. The oligonucleotide of any one of claims 1-6, wherein:(i) the sense strand is 18 nucleotides, the duplex region is 18 nucleotides, the 5’overhang is 2 nucleotides and the 3’overhang is 2 nucleotides;(ii) the sense strand is 17 nucleotides, the duplex region is 17 nucleotides, the 5’overhang is 3 nucleotides and the 3’overhang is 2 nucleotides;(iii) the sense strand is 16 nucleotides, the duplex region is 16 nucleotides, the 5’overhang is 4 nucleotides and the 3’overhang is 2 nucleotides; or(iv) the sense strand is 13 nucleotides, the duplex region is 13 nucleotides, the 5’ overhang is 2 nucleotides and the 3’overhang is 7 nucleotides.

11. The oligonucleotide of any one of claims 1-6, wherein:(i) the sense strand is 12 nucleotides, the duplex region is 12 nucleotides, the 5’overhang is 2 nucleotides, and the 3’overhang is 8 nucleotides;(ii) the sense strand is 12 nucleotides, the duplex region is 12 nucleotides, the 5’overhang is 3 nucleotides, and the 3’overhang is 7 nucleotides; or(iii) the sense strand is 10 nucleotides, the duplex region is 10 nucleotides, the 5’overhang is 1 nucleotide, and the 3’overhang is 11 nucleotides.

12. The oligonucleotide of any one of claims 1-6, wherein the sense strand is 13 nucleotides, the duplex region is 13 nucleotides, the 5’ overhang is 2 nucleotides, and the 3’ overhang is 7 nucleotides.

13. The oligonucleotide of any one of claims 1-6, wherein the sense strand is 12 nucleotides, the duplex region is 12 nucleotides, the 5’overhang is 2 nucleotides, and the 3’overhang is 8 nucleotides.

14. The oligonucleotide of any one of claims 1-6, wherein the sense strand is 12 nucleotides, the duplex region is 12 nucleotides, the 5’overhang is 3 nucleotides, and the 3’overhang is 7 nucleotides.

15. The oligonucleotide of any one of claims 1-14, wherein the lipid moiety is selected from:

16. The oligonucleotide of any one of claims 1-14, wherein the lipid moiety is a hydrocarbon chain.

17. The oligonucleotide of claim 16, wherein the hydrocarbon chain is a C8-C30 hydrocarbon chain.

18. The oligonucleotide of claim 17, wherein the hydrocarbon chain is a C16 hydrocarbon chain.

19. The oligonucleotide of claim 18, wherein the C16 hydrocarbon chain is represented by20. The oligonucleotide of claim 17, wherein the hydrocarbon chain is a C22 hydrocarbon chain.

21. The oligonucleotide of claim 20, wherein the C22 hydrocarbon chain is represented by22. The oligonucleotide of any one of claims 1-21, wherein the lipid moiety is conjugated to the 5 ’terminal nucleotide of the sense strand.

23. The oligonucleotide of any one of claims 1-22, wherein the antisense strand is 22 nucleotides, wherein positions are numbered 1-22 from 5’ to 3’.

24. The oligonucleotide of claim 23, wherein the nucleotide conjugated to the lipid moiety forms a base pair with a nucleotide at position 16, 14, or 12 of the antisense strand, wherein positions are numbered 5’ to 3’.

25. The oligonucleotide of claim 23, wherein the nucleotide conjugated to the lipid moiety forms a base pair with a nucleotide at position 14 of the antisense strand, wherein positions are numbered 5’ to 3’.

26. The oligonucleotide of any one of claims 1-25, wherein the oligonucleotide comprises at least one modified nucleotide.

27. The oligonucleotide of claim 26, wherein the modified nucleotide comprises a 2'- modification.

28. The oligonucleotide of claim 27, wherein each of the nucleotides of the sense strand and the antisense strand comprise a 2'-modification.

29. The oligonucleotide of claim 27 or 28, 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.

30. The oligonucleotide of any one of claims 27-29, wherein the sense strand comprises nucleotide positions numbered 5’ to 3’, wherein each of positions 8-11 comprise a 2'-fluoro modification.

31. The oligonucleotide of any one of claims 27-29, wherein the sense strand comprises a 2’ -fluoro modification at each of nucleotides forming a base pair with nucleotides at positions 10-13 of the antisense strand, wherein positions are numbered 5’ to 3’.

32. The oligonucleotide of any one of claims 27-31, 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.

33. The oligonucleotide of any one of claims 30-32, wherein the remaining nucleotides comprise a 2'-O-methyl modification, provided that the nucleotide of the sense strand conjugated to the at least one lipid moiety does not comprise a 2’-O-methyl modification.

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

35. The oligonucleotide of claim 34, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.

36. The oligonucleotide of claim 35, wherein the antisense strand comprises a phosphorothioate linkage (i) between positions 1 and 2, and between positions 2 and 3; or (ii)317between 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’.

37. The oligonucleotide of claim 35 or 36 , 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'.

38. The oligonucleotide of claim 37, wherein the antisense strand comprises a phosphorothioate linkage between positions 13 and 14 and between positions 14 and 15.

39. The oligonucleotide of any one of claims 35-38, wherein the sense strand comprises a phosphorothioate linkage between positions 1 and 2, between the penultimate nucleotide and third nucleotide from the 3’ end, and between the penultimate nucleotide and ultimate nucleotide.

40. The oligonucleotide of any one of claims 1-39, wherein the sense strand comprises at least one Tm-increasing nucleotide.

41. The oligonucleotide of claim 40, wherein the sense strand comprises up to four Tm- increasing nucleotides.

42. The oligonucleotide of claim 40 or 41, wherein the Tm-increasing nucleotide is a bicyclic nucleotide.

43. The oligonucleotide of claim 40 or 41, wherein the Tm-increasing nucleotide is a locked nucleic acid.

44. A double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 18 nucleotides in length, wherein the antisense and sense strands form a duplex region of 18 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising at least two nucleotides and a 3’ overhang comprising at least two nucleotides, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, wherein the sense318strand comprises at least one lipid moiety conjugated to a 5’ terminal nucleotide of the sense strand, and wherein each of the antisense and sense strands comprise at least one 2’ -modified nucleotide and at least one modified internucleotide linkage.

45. The oligonucleotide of claim 44, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.

46. The oligonucleotide of claim 45, wherein the sense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 16 and 17, and 17 and 18, numbered 5’ to 3’.

47. The oligonucleotide of claim 45 or 46, wherein the antisense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22, numbered 5’ to 3’.

48. The oligonucleotide of any one of claims 44-47, wherein the 5’overhang is 2 nucleotides and the 3 ’overhang is 2 nucleotides.

49. The oligonucleotide of any one of claims 1-48, wherein the mRNA target sequence is a central nervous system (CNS) target sequence, optionally a neuronal mRNA target sequence or an ocular mRNA target sequence.

50. The oligonucleotide of claim 49, wherein the CNS target sequence is an astrocyte mRNA target sequence.

51. The oligonucleotide of any one of claims 44-50, wherein the mRNA target sequence is a liver mRNA target sequence, optionally a liver macrophage mRNA target sequence, a liver hepatocyte mRNA target sequence, or a liver sinusoidal endothelial cell mRNA target sequence.

52. A double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 17 nucleotides in length, wherein the antisense and sense strands form a duplex region of 17 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising at least three319nucleotides and a 3’ overhang comprising at least two nucleotides, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to a 5’ terminal nucleotide of the sense strand, and wherein each of the antisense and sense strands comprise at least one 2’ -modified nucleotide and at least one modified internucleotide linkage.

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

54. The oligonucleotide of claim 53, wherein the sense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 15 and 16, and 16 and 17, numbered 5’ to 3’.

55. The oligonucleotide of claim 53 or 54, wherein the antisense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22, numbered 5’ to 3’.

56. The oligonucleotide of any one of claims 52-55, wherein the 5’overhang is 3 nucleotides and the 3 ’overhang is 2 nucleotides.

57. A double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 16 nucleotides in length, wherein the antisense and sense strands form a duplex region of 16 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising at least four nucleotides and a 3’ overhang comprising at least two nucleotides, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to a 5’ terminal nucleotide of the sense strand, wherein the sense strand comprises up to five Tm-increasing nucleotides, and wherein each of the antisense and sense strands comprise at least one 2’ -modified nucleotide and at least one modified internucleotide linkage.

58. The oligonucleotide of any one of claims 44-57, wherein the sense strand comprises a 2’ -fluoro modification at positions 8-11, numbered 5’ to 3’.32059. The oligonucleotide of claim 58, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.

60. The oligonucleotide of claim 59, wherein the sense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 14 and 15, and 15 and 16, numbered 5’ to 3’.

61. The oligonucleotide of claim 59 or 60, wherein the antisense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22, numbered 5’ to 3’.

62. The oligonucleotide of any one of claims 57-61, wherein the 5 ’overhang is 4 nucleotides and the 3 ’overhang is 2 nucleotides.

63. The oligonucleotide of any one of claims 57-62, comprising:(i) 1-5, 1-4, 1-3, or 1-2 Tm-increasing nucleotides;(ii) 1, 2, 3, 4 or 5 Tm-increasing nucleotides; or(iii) up to three Tm-increasing nucleotides.

64. The oligonucleotide of claim 63, comprising two Tm-increasing nucleotides.

65. The oligonucleotide of claim 63, comprising three Tm-increasing nucleotides.

66. The oligonucleotide of any one of claims 57-65, wherein the sense strand comprises a Tm-increasing nucleotide at positions 2, 15 and 16, numbered 5’ to 3’.

67. The oligonucleotide of any one of claims 57-66, wherein the Tm-increasing nucleotide is a bicyclic nucleotide, optionally, a locked nucleic acid (LNA).

68. The oligonucleotide of any one of claims 44-65, wherein the mRNA target sequence is a central nervous system (CNS) target sequence, optionally a neuronal mRNA target sequence or an ocular mRNA target sequence.32169. A double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 13 nucleotides in length, wherein the antisense and sense strands form a duplex region of 13 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising at least two nucleotides and a 3’ overhang comprising at least seven nucleotides, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to an internal nucleotide of the sense strand, wherein the sense strand comprises up to three Tm-increasing nucleotides, and wherein each of the antisense and sense strands comprise at least one 2’ -modified nucleotide and at least one modified internucleotide linkage.

70. The oligonucleotide of claim 69, wherein the sense strand comprises a 2’ -fluoro modification at positions 3-6, numbered 5’ to 3’.

71. The oligonucleotide of claim 69 or 70, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.

72. The oligonucleotide of claim 71, wherein the sense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 11 and 12, and 12 and 13, numbered 5’ to 3’.

73. The oligonucleotide of claim 71 or 72, wherein the antisense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22, numbered 5’ to 3’.

74. The oligonucleotide of any one of claims 69-73, wherein the lipid moiety is conjugated at a nucleotide at position 2 of the sense strand, numbered 5’ to 3’.

75. The oligonucleotide of any one of claims 69-74, wherein the 5’overhang is 2 nucleotides and the 3 ’overhang is 7 nucleotides.

76. The oligonucleotide of any one of claims 69-75, comprising:(i) 1-3 or 1-2 Tm-increasing nucleotides, or322(ii) 1, 2 or 3 Tm-increasing nucleotides.

77. The oligonucleotide of any one of claims 69-76, wherein the sense strand comprises a Tm-increasing nucleotide at (i) positions 1 and 10, or (ii) positions 1, 10 and 11, numbered 5’ to 3’.

78. The oligonucleotide of any one of claims 69-77, wherein the Tm-increasing nucleotide is a bicyclic nucleotide, optionally a locked nucleic acid (LNA).

79. A double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 12 nucleotides in length, wherein the antisense and sense strands form a duplex region of 12 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising at least two nucleotides and a 3’ overhang comprising at least seven nucleotides, wherein the antisense strand comprises a region of complementarity to an mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to a 5 ’terminal nucleotide of the sense strand, and wherein each of the antisense and sense strands comprise at least one 2’- modified nucleotide and at least one modified internucleotide linkage.

80. The oligonucleotide of claim 79, wherein the sense strand comprises a 2’ -fluoro modification at positions 3-6 or 4-7, numbered 5’ to 3’.

81. The oligonucleotide of any one of claims 79-80, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.

82. The oligonucleotide of claim 81, wherein the sense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 10 and 11, and 11 and 12, numbered 5’ to 3’.

83. The oligonucleotide of claim 81 or 82, wherein the antisense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 12 and 14, 14 and 15, 20 and 21, and 21 and 22, numbered 5’ to 3’.32384. The oligonucleotide of any one of claims 79-83, wherein the 5’overhang is 2 nucleotides and the 3 ’overhang is 8 nucleotides.

85. The oligonucleotide of any one of claims 79-83, wherein the 5’overhang is 3 nucleotides and the 3 ’overhang is 7 nucleotides.

86. The oligonucleotide of any one of claims 79-85, comprising:(i) 1-3 or 1-2 Tm-increasing nucleotides, or(ii) 1, 2 or 3 Tm-increasing nucleotides.

87. The oligonucleotide of any one of claims 79-86, wherein the sense strand comprises a Tm-increasing nucleotide at (i) positions 2, 10 and 11, or (ii) positions 2, 11 and 12, numbered 5’ to 3’.

88. The oligonucleotide of any one of claims 79-87, wherein the Tm-increasing nucleotide is a bicyclic nucleotide, optionally a locked nucleic acid (LNA).

89. A double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 10 nucleotides in length, wherein the antisense and sense strands form a duplex region of 10 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising one nucleotide and a 3’ overhang comprising eleven nucleotides, wherein the antisense strand comprises a region of complementarity to an mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to a 5 ’terminal nucleotide of the sense strand, and wherein each of the antisense and sense strands comprise at least one 2’ -modified nucleotide and at least one modified internucleotide linkage.

90. The oligonucleotide of claim 89, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.

91. The oligonucleotide of claim 90, wherein the sense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 8 and 9, and 9 and 10, numbered 5’ to 3’.32492. The oligonucleotide of claim 90 or 91, wherein the antisense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 12 and 14, 14 and 15, 20 and 21, and 21 and 22, numbered 5’ to 3’.

93. The oligonucleotide of any one of claims 89-92, comprising:(i) 1-3 Tm-increasing nucleotides, or(ii) 1, 2 or 3 Tm-increasing nucleotides.

94. The oligonucleotide of any one of claims 89-93, wherein the sense strand comprises a Tm-increasing nucleotide at positions 2, 6 and 7.

95. The oligonucleotide of any one of claims 89-94, wherein the Tm-increasing nucleotide is a bicyclic nucleotide, optionally a locked nucleic acid (LNA).

96. The oligonucleotide of any one of claims 44-95, wherein the antisense strand comprises a 2’-fluoro modification at positions 2-5, 7, 10 and 14, numbered 5’ to 3’.

97. The oligonucleotide of any one of claims 58-68, 70-78, 80-88 and 96, wherein the remaining nucleotides comprise a 2'-O-methyl modification, provided that the nucleotide of the sense strand conjugated to the at least one lipid moiety does not comprise a 2’-O-methyl modification.

98. The oligonucleotide of any one of claims 44-68 and 79-98, wherein the lipid moiety is a C16 hydrocarbon represented by:

99. The oligonucleotide of any one of claims 44-51, 69-78, and 96-97, wherein the lipid moiety is a C22 hydrocarbon represented by:

100. The oligonucleotide of any one of claims 1-99, wherein the lipid moiety is conjugated to the 2’ carbon of the ribose ring of the nucleotide.325101. The oligonucleotide of any one of claims 1-100, wherein the antisense strand comprises a phosphorylated nucleotide at the 5’ terminus, wherein the phosphorylated nucleotide is selected from uridine and adenosine.

102. The oligonucleotide of claim 101, wherein the phosphorylated nucleotide is uridine.

103. 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.

104. The oligonucleotide of claim 103, wherein the phosphate analog is oxymethyl phosphonate, vinyl phosphonate or malonyl phosphonate.

105. The oligonucleotide of any one of claims 102-104, wherein the phosphorylated nucleotide is 4’-O-monomethylphosphonate-2’-O-methyl uridine.

106. The oligonucleotide of any one of claims 1-105, wherein the region of complementarity is fully complementary to the mRNA target sequence.

107. The oligonucleotide of any one of claims 1-105, wherein the region of complementarity is partially complementary to the mRNA target sequence.

108. The oligonucleotide of claim 107, wherein the region of complementarity comprises no more than four mismatches to the mRNA target sequence.

109. The oligonucleotide of any one of claims 69-108, wherein the mRNA target sequence is a liver mRNA target sequence, optionally a hepatocyte mRNA target sequence, a liver macrophage mRNA target sequence, or a liver sinusoidal endothelial cell mRNA target sequence.

110. The oligonucleotide of any one of claims 1-109, wherein the oligonucleotide is a Dicer substrate.326111. The oligonucleotide of any one of claims 1-110, wherein the oligonucleotide reduces expression of the mRNA target sequence in a cell or population of cells in vitro and / or in vivo.

112. A pharmaceutical composition comprising the oligonucleotide of any one of claims 1- 111, and a pharmaceutically acceptable carrier, delivery agent or excipient.

113. A pharmaceutical composition comprising the oligonucleotide of any one of claims 44-51, 68, and 96-111, and a pharmaceutically acceptable carrier, delivery agent or excipient.

114. A pharmaceutical composition comprising the oligonucleotide of any one of claims 52-56, 68, and 96-111, and a pharmaceutically acceptable carrier, delivery agent or excipient.

115. A pharmaceutical composition comprising the oligonucleotide of any one of claims 57-68 and 96-111, and a pharmaceutically acceptable carrier, delivery agent or excipient.

116. A pharmaceutical composition comprising the oligonucleotide of any one of claims 69-78, and 96-111, and a pharmaceutically acceptable carrier, delivery agent or excipient.

117. A pharmaceutical composition comprising the oligonucleotide of any one of claims 79-88, and 96-111, and a pharmaceutically acceptable carrier, delivery agent or excipient.

118. A pharmaceutical composition comprising the oligonucleotide of any one of claims 89-111, and a pharmaceutically acceptable carrier, delivery agent or excipient.

119. A method for treating a subject having a disease, disorder or condition associated with expression of a target mRNA, the method comprising administering to the subject a therapeutically effective amount of the oligonucleotide of any one of claims 1-111, or the pharmaceutical composition of any one of claims 112-118.

120. A method of reducing expression of a target mRNA in a subject, comprising administering to the subject the oligonucleotide of any one of claims 1-111, or the pharmaceutical composition of any one of claims 112-118.327121. The method of claim 119 or 120, wherein the target mRNA is expressed in the central nervous system, optionally wherein the central nervous system comprises the frontal cortex, hippocampus, medulla, cerebellum, lumbar dorsal root ganglion, and / or lumbar spinal cord.

122. The method of claim 121, wherein the target mRNA is expressed in a neuron of the central nervous system.

123. The method of claim 119 or 120, wherein the target mRNA is expressed in a liver, optionally a hepatocyte, a liver macrophage, or a liver sinusoidal endothelial cell.

124. The method of claim 123, wherein the target mRNA is expressed in a macrophage of the liver.

125. The method of claim 119 or 120, wherein the target mRNA is expressed in ocular tissue.

126. The method of claim 119 or 120, wherein the target mRNA is expressed in a tissue of the central nervous system, liver tissue, ocular tissue, adipose tissue, muscle tissue, adrenal tissue, cardiac tissue, lung tissue, or any combination thereof.

127. A method for treating a subject having a disease, disorder or condition associated with expression of an mRNA of the central nervous system, an mRNA of the liver, or an ocular mRNA, the method comprising administering to the subject a therapeutically effective amount of the oligonucleotide of any one of claims 44-51, 68, and 96-111 or the pharmaceutical composition of claim 113, optionally wherein the central nervous system comprises the frontal cortex, hippocampus, medulla, cerebellum, lumbar dorsal root ganglion, and / or lumbar spinal cord.

128. The method of claim 127, wherein the mRNA of the central nervous system is a neuronal mRNA.

129. A method for treating a subject having a disease, disorder or condition associated with expression of an mRNA of the central nervous system, the method comprising administering to the subject a therapeutically effective amount of the oligonucleotide of any one of claims32852-68 and 96-11, or the pharmaceutical composition of claim 114 or 115, optionally wherein the central nervous system comprises the frontal cortex, hippocampus, medulla, cerebellum, lumbar dorsal root ganglion, and / or lumbar spinal cord.

130. The method of claim 129, wherein the mRNA of the central nervous system is a neuronal mRNA.

131. A method for treating a subject having a disease, disorder or condition associated with expression of a macrophage mRNA, the method comprising administering to the subject a therapeutically effective amount of the oligonucleotide of any one of claims 69-78 and 96-111 or the pharmaceutical composition of claim 116.

132. The method of claim 131, wherein the macrophage mRNA is expressed in the liver.

133. A method of delivering an oligonucleotide to a cell or population of cells in the central nervous system, liver tissue or ocular tissue, the method comprising administering the pharmaceutical composition of any one of claims 112-118.

134. A method for reducing expression of a target mRNA expressed in the central nervous system of a subject, a target mRNA expressed in the liver of a subject, or a target mRNA expressed in ocular tissue of a subject, comprising administering to the subject the oligonucleotide of any one of claims 44-51, 68 and 96-111 or the pharmaceutical composition of claim 113, optionally wherein the central nervous system comprises the frontal cortex, hippocampus, medulla, cerebellum, lumbar dorsal root ganglion, and / or lumbar spinal cord.

135. The method of claim 134, wherein the target mRNA is expressed in neurons of the central nervous system.

136. A method for reducing expression of a target mRNA expressed in the central nervous system of a subject, comprising administering to the subject a therapeutically effective amount of the oligonucleotide of any one of claims 52-68 and 96-111 or the pharmaceutical composition of claim 114 or 115, optionally wherein the central nervous system comprises the frontal cortex, hippocampus, medulla, cerebellum, lumbar dorsal root ganglion, and / or lumbar spinal cord.329137. The method of claim 136, wherein the target mRNA is expressed in neurons of the central nervous system.

138. A method for reducing expression of a target mRNA expressed in a macrophage of a subject, comprising administering to the subject a therapeutically effective amount of the oligonucleotide of any one of claims 69-78 and 96-111 or the pharmaceutical composition of claim 116.

139. The method of claim 138, wherein the macrophage is in the liver of the subject.

140. A method for reducing expression of a target mRNA expressed in the central nervous system of a subject, comprising administering to the subject a therapeutically effective amount of the oligonucleotide of any one of claims 79-88 and 96-111 or the pharmaceutical composition of claim 117, optionally wherein the central nervous system comprises the frontal cortex, hippocampus, medulla, cerebellum, lumbar dorsal root ganglion, and / or lumbar spinal cord.

141. A method for reducing expression of a target mRNA expressed in the central nervous system of a subject, comprising administering to the subject a therapeutically effective amount of the oligonucleotide of any one of claims 89-111 or the pharmaceutical composition of claim 118, optionally wherein the central nervous system comprises the frontal cortex, hippocampus, medulla, cerebellum, lumbar dorsal root ganglion, and / or lumbar spinal cord.

142. A kit comprising the oligonucleotide of any one of claims 1-111, an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administration to a subject having a disease, disorder or condition associated with the overexpression of a target mRNA.

143. A kit comprising the oligonucleotide of any one of claims 1-111, an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administration to a subject having a disease, disorder or condition associated with the reduction in expression of a target mRNA.330144. Use of the oligonucleotide of any one of claims 1-111, or the pharmaceutical composition of any one of claims 112-118, in the manufacture of a medicament for the treatment of a disease, disorder, or condition associated with the reduction in the expression of a target mRNA.

145. Use of the oligonucleotide of any one of claims 1-111, or the pharmaceutical composition of any one of claims 112-118, in the manufacture of a medicament for the treatment of a disease, disorder, or condition associated with the overexpression of a target mRNA.

146. The oligonucleotide of any one of claims 1-111, or the pharmaceutical composition of any one of claims 112-118, for use, or adaptable for use, in the treatment of a disease, disorder, or condition associated with expression of a target mRNA.

147. The kit of claim 142 or 143, the use of claim 144 or 145, or the oligonucleotide of claim 146, wherein the target mRNA is expressed in the central nervous system, a neuron of the central system, a macrophage, optionally a macrophage in the liver, ocular tissue, or any combination thereof, optionally wherein the central nervous system comprises the frontal cortex, hippocampus, medulla, cerebellum, lumbar dorsal root ganglion, and / or lumbar spinal cord.

148. A method of activating target-specific RNA interference (RNAi) in an organism comprising administering to said organism the oligonucleotide of claims 1, 44, 52, 57 or 69, said oligonucleotide being administered in an amount sufficient for degradation of the target mRNA to occur, thereby activating target-specific RNAi in the organism.

149. A method of activating target-specific RNA interference (RNAi) in an organism comprising administering to said organism the oligonucleotide of claims 1, 44, 52, 57, 69, 79 or 89, said oligonucleotide being administered in an amount sufficient for degradation of the target mRNA to occur, thereby activating target-specific RNAi in the organism.

150. The method of claim 148 or 149, wherein the target mRNA specifies the amino acid sequence of a protein involved or predicted to be involved in a human disease or disorder.331151. The method of claim 150, wherein the disease or disorder is selected from the group consisting of viral infections, bacterial infections, parasitic infections, cancers, allergies, autoimmune diseases, immunodeficiencies, and immunosuppression.

152. A double-stranded oligonucleotide comprising an antisense strand of about 15-30 nucleotides in length and a sense strand of about 15-50 nucleotides in length, wherein the antisense and sense strands form a duplex region of about 15-30 base pairs, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, wherein the sense strand comprises (i) at least one lipid moiety conjugated to a nucleotide of the sense strand, and (ii) a stem-loop, 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, and wherein the sense and antisense strands each comprise an orientation of 5’ to 3’, and wherein the stem-loop is at the 5’ end of the sense strand.

153. The oligonucleotide of claim 152, comprising a blunt end.

154. The oligonucleotide of claim 153, wherein the blunt end comprises the 3’ end of the sense strand and the 5’ end of the antisense strand.

155. The oligonucleotide of claim 152, comprising an overhang of at least two nucleotides.

156. The oligonucleotide of claim 155, wherein the overhang comprises the 5’end of the antisense strand.

157. The oligonucleotide of any one of claims 152-156, wherein the sense strand is about 28-38 nucleotides.

158. The oligonucleotide of any one of claims 152-157, wherein the antisense strand is 22 nucleotides.

159. The oligonucleotide of any one of claims 152-158, wherein the lipid moiety is conjugated to a nucleotide comprising the loop.332160. The oligonucleotide of any one of claims 152-159, wherein:(i) the sense strand is 28 nucleotides, and the lipid moiety is conjugated to a nucleotide at position 4, positions numbered 5’ to 3’;(ii) the sense strand is 30 nucleotides, and the lipid moiety is conjugated to a nucleotide at position 4, positions numbered 5’ to 3’;(iii) the sense strand is 34 nucleotides, and the lipid moiety is conjugated to a nucleotide at position 6 or position 15, positions numbered 5’ to 3’; or(iv) the sense strand is 38 nucleotides, and the lipid moiety is conjugated to a nucleotide at position 8, positions numbered 5’ to 3’.

161. The oligonucleotide of any one of claims 152-160, wherein the lipid moiety is selected from:333162. The oligonucleotide of any one of claims 152-160, wherein the lipid moiety is a hydrocarbon chain.

163. The oligonucleotide of claim 162, wherein the hydrocarbon chain is a C8-C30 hydrocarbon chain.

164. The oligonucleotide of claim 163, wherein the hydrocarbon chain is a C16 hydrocarbon chain.

165. The oligonucleotide of claim 164, wherein the C16 hydrocarbon chain is represented by166. The oligonucleotide of any one of claims 152-165, wherein the mRNA target sequence is a central nervous system (CNS) target sequence, optionally a neuronal mRNA target sequence or an ocular mRNA target sequence.

167. The oligonucleotide of any one of claims 152-165, wherein the mRNA target sequence is an ocular mRNA target sequence.334168. The oligonucleotide of any one of claims 152-167, wherein the loop sequence is 5’- GAAA-3’.

169. The oligonucleotide of any one of claims 152-167, wherein the loop sequence is 5’- UNCG-3’, wherein N is any nucleotide.

170. The oligonucleotide of claim 169, wherein the loop sequence is 5’-UACG-3’.

171. The oligonucleotide of any one of claims 152-170, wherein the oligonucleotide comprises at least one modified nucleotide.

172. The oligonucleotide of claim 171, wherein the modified nucleotide comprises a 2'- modification.

173. The oligonucleotide of claim 171, wherein each of the nucleotides of the sense strand and the antisense strand comprise a 2'-modification.

174. The oligonucleotide of claim 172 or 173, 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.

175. The oligonucleotide of any one of claims 172-174, 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.

176. The oligonucleotide of any one of claims 172-175, wherein:(i) the sense strand is 38 nucleotides with positions 1-38 from 5’ to 3’, and wherein each of positions 26-29 comprise a 2’ -fluoro modification;(ii) the sense strand is 34 nucleotides with positions 1-34 from 5’ to 3’, and wherein each of positions 22-25 comprise a 2’ -fluoro modification;(iii) the sense strand is 30 nucleotides with positions 1-30 from 5’ to 3’, and wherein each of positions 18-21 comprise a 2’ -fluoro modification; or335(iv) the sense strand is 28 nucleotides with positions 1-28 from 5’ to 3’, and wherein each of positions 18-21 comprise a 2’ -fluoro modification.

177. The oligonucleotide of any one of claims 152-176, wherein the oligonucleotide comprises at least one modified intemucleotide linkage.

178. The oligonucleotide of claim 177, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.

179. The oligonucleotide of claim 178, 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’.

180. The oligonucleotide of claim 177 or 178 , 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'.

181. The oligonucleotide of any one of claims 177-180, wherein the sense strand comprises a phosphorothioate linkage between the penultimate nucleotide and third nucleotide from the 3’ end, and between the penultimate nucleotide and ultimate nucleotide.

182. The oligonucleotide of any one of claims 177-180, wherein(i) the sense strand is 38 nucleotides with positions 1-38 from 5’ to 3’, and wherein the sense strand comprises a phosphorothioate linkage between positions 36 and 37, and 37 and 38;(ii) the sense strand is 34 nucleotides with positions 1-34 from 5’ to 3’, and wherein the sense strand comprises a phosphorothioate linkage between positions 32 and 33, and 33 and 34;(iii) the sense strand is 30 nucleotides with positions 1-30 from 5’ to 3’, and wherein the sense strand comprises a phosphorothioate linkage between positions 28 and 29, and 29 and 30; or336(iv) the sense strand is 28 nucleotides with positions 1-28 from 5’ to 3’, and wherein the sense strand comprises a phosphorothioate linkage between positions 26 and 27, and 27 and 28.

183. The oligonucleotide of any one of claims 152-182, wherein the sense strand comprises at least one Tm-increasing nucleotide.

184. The oligonucleotide of claim 183, wherein the sense strand comprises up to six Tm- increasing nucleotides.

185. The oligonucleotide of claim 183 or 184, wherein the Tm-increasing nucleotide is a bicyclic nucleotide or a locked nucleic acid.

186. The oligonucleotide of any one of claims 152-185, wherein SI and S2 each comprise 1-6 nucleotides.

187. The oligonucleotide of claim 186, wherein SI and S2 each comprise 4 nucleotides.

188. The oligonucleotide of claim 186, wherein SI and S2 each comprise 2 nucleotides.

189. The oligonucleotide of any one of claims 182-188, wherein SI and S2 each comprise at least one Tm-increasing nucleotide.

190. The oligonucleotide of claim 189, wherein SI and S2 are each 4 nucleotides, wherein 1-3 nucleotides of each SI and S2 are Tm-increasing nucleotides.

191. The oligonucleotide of any one of claims 175-190, wherein the remaining nucleotides comprise a 2'-O-methyl modification, provided that the nucleotide of the sense strand conjugated to the at least one lipid moiety does not comprise a 2’-O-methyl modification.

192. A double-stranded oligonucleotide comprising an antisense strand of about 20-22 nucleotides in length and a sense strand of about 32-34 nucleotides in length, wherein the antisense and sense strands form a duplex region of about 20-22 base pairs and the oligonucleotide is blunt ended, wherein the antisense strand comprises a region of337complementarity to a mRNA target sequence, wherein the sense strand comprises (i) a stemloop, wherein the stem-loop comprises a nucleotide sequence represented by the formula: 5’- S1-L-S2-3’, wherein SI is complementary to S2, and wherein L forms a loop between SI and S2, and (ii) at least one lipid moiety conjugated to a nucleotide of the loop, wherein the sense and antisense strands each comprise an orientation of 5’ to 3’, wherein the stem-loop is at the 5’ end of the sense strand, and wherein each of the antisense and sense strands comprise at least one 2’ -modified nucleotide and at least one modified internucleotide linkage.

193. The oligonucleotide of claim 192, wherein the sense strand is 34 nucleotides and comprises a 2’-fluoro modification at positions 22-25, numbered 5’ to 3’.

194. The oligonucleotide of any one of claims 192-193, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.

195. The oligonucleotide of claim 194, wherein the sense strand is 34 nucleotides and comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 2 and 3, 32 and 33, and 33 and 34, numbered 5’ to 3’.

196. The oligonucleotide of claim 194 or 195, wherein the antisense strand is 22 nucleotides and comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22, numbered 5’ to 3’.

197. The oligonucleotide of any one of claims 192-196, wherein the lipid moiety is conjugated to a nucleotide at position 6 of the sense strand, numbered 5’ to 3’.

198. The oligonucleotide of any one of claims 192-197, wherein SI and S2 each comprise 1-6 nucleotides.

199. The oligonucleotide of claim 198, wherein SI and S2 are each 4 nucleotides.

200. The oligonucleotide of any one of claims 192-199, wherein L is 4 nucleotides.

201. The oligonucleotide of claim 200, wherein L comprises the sequence 5’-GAAA-3’.338202. A double-stranded oligonucleotide comprising an antisense strand of about 20-22 nucleotides in length and a sense strand of about 26-28 nucleotides in length, wherein the antisense and sense strands form an asymmetric duplex region of about 20-22 base pairs comprising a 3’ terminal overhang of at least 2 nucleotides of the antisense strand, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, wherein the sense strand comprises: (i) a stem-loop, wherein the stem-loop comprises a nucleotide sequence represented by the formula: 5’-Sl-L-S2-3’, wherein SI is complementary to S2, wherein L forms a loop between SI and S2 and comprises the sequence UNCG, and wherein SI and S2 each comprise at least one Tm-increasing nucleotide, and (ii) at least one lipid moiety conjugated to a nucleotide of the loop, wherein the sense strand comprises an orientation of 5’ to 3’, wherein the stem-loop is at the 5’ end of the sense strand, and wherein each of the antisense and sense strands comprise at least one 2’- modified nucleotide and at least one modified internucleotide linkage.

203. The oligonucleotide of claim 202, wherein the sense strand is 28 nucleotides and comprises a 2’ -fluoro modification at positions 18-21, numbered 5’ to 3’.

204. The oligonucleotide of any one of claims 202-203, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.

205. The oligonucleotide of claim 204, wherein the sense strand is 28 nucleotides and comprises phosphorothioate linkages between nucleotides at positions 26 and 27, and 27 and 28, numbered 5’ to 3’.

206. The oligonucleotide of claim 204 or 205, wherein the antisense strand is 22 nucleotides and comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22, numbered 5’ to 3’.

207. The oligonucleotide of any one of claims 202-206, wherein the 3 ’terminal overhang is 2 nucleotides.

208. The oligonucleotide of any one of claims 202-207, wherein the lipid moiety is conjugated to a nucleotide at position 4 of the sense strand, numbered 5’ to 3’.339209. The oligonucleotide of any one of claims 202-208, wherein SI and S2 each comprise 1-6 nucleotides.

210. The oligonucleotide of claim 209, wherein SI and S2 are each 2 nucleotides.

211. The oligonucleotide of claim 210, wherein SI and S2 each comprise one Tm- increasing nucleotide.

212. The oligonucleotide of any one of claims 202-211, wherein the Tm-increasing nucleotide is a bicyclic nucleotide.

213. The oligonucleotide of any one of claims 202-211, wherein the Tm-increasing nucleotide is a locked nucleic acid (LNA).

214. The oligonucleotide of any one of claims 202-213, wherein L is 4 nucleotides.

215. The oligonucleotide of claim 214, wherein L comprises the sequence 5’-UNCG-3’, wherein N is any nucleotide.

216. The oligonucleotide of claim 215, wherein L comprises the sequence 5’-UACG-3.

217. A double-stranded oligonucleotide comprising an antisense strand of about 20-22 nucleotides in length and a sense strand of about 32-34 nucleotides in length, wherein the antisense and sense strands form a duplex region of about 20-22 base pairs, and the oligonucleotide is blunt ended, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, wherein the sense strand comprises: (i) a stemloop, wherein the stem-loop comprises a nucleotide sequence represented by the formula: 5’- S1-L-S2-3’, wherein SI is complementary to S2, wherein L forms a loop between SI and S2, and wherein SI and S2 each comprise at least one Tm-increasing nucleotide, and (ii) at least one lipid moiety conjugated to a nucleotide of the loop, wherein the sense strand comprises an orientation of 5’ to 3’, wherein the stem-loop is at the 5’ end of the sense strand, and wherein each of the antisense and sense strands comprise at least one 2’ -modified nucleotide and at least one modified internucleotide linkage.340218. The oligonucleotide of claim 217, wherein the sense strand is 34 nucleotides and comprises a 2’-fluoro modification at positions 22-25, numbered 5’ to 3’.

219. The oligonucleotide of any one of claims 217-218, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.

220. The oligonucleotide of claim 219, wherein the sense strand is 34 nucleotides and comprises phosphorothioate linkages between nucleotides at positions 32 and 33, and 33 and 34, numbered 5’ to 3’.

221. The oligonucleotide of claim 219 or 220, wherein the antisense strand is 22 nucleotides and comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22, numbered 5’ to 3’.

222. The oligonucleotide of any one of claims 217-221, wherein oligonucleotide comprises a blunt end comprising the 5’ end of the antisense strand and the 3’ end of the sense strand.

223. The oligonucleotide of any one of claims 217-222, wherein the lipid moiety is conjugated to a nucleotide at position 6 of the sense strand, numbered 5’ to 3’.

224. The oligonucleotide of any one of claims 217-223, wherein SI and S2 each comprise 1-6 nucleotides.

225. The oligonucleotide of claim 224, wherein SI and S2 are each 4 nucleotides.

226. The oligonucleotide of claim 225, wherein SI and S2 each comprise one Tm- increasing nucleotide.

227. The oligonucleotide of any one of claims 217-226, wherein the Tm-increasing nucleotide is a bicyclic nucleotide.

228. The oligonucleotide of any one of claims 217-226, wherein the Tm-increasing nucleotide is a locked nucleic acid (LNA).341229. The oligonucleotide of any one of claims 217-228, wherein L is 4 nucleotides.

230. The oligonucleotide of claim 229, wherein L comprises the sequence 5’-GAAA-3’.

231. The oligonucleotide of any one of claims 192-230, wherein the antisense strand is 22 nucleotides and comprises a 2’-fluoro modification at positions 2-5, 7, 10 and 14, numbered 5’ to 3’.

232. The oligonucleotide of any one of claims 193-201, 203-216 and 218-231, wherein the remaining nucleotides comprise a 2'-O-methyl modification, provided that the nucleotide of the sense strand conjugated to the at least one lipid moiety does not comprise a 2’-O-methyl modification.

233. The oligonucleotide of any one of claims 192-232, wherein the lipid moiety is a C16 hydrocarbon represented by:

234. The oligonucleotide of any one of claims 152-233, wherein the antisense strand comprises a phosphorylated nucleotide at the 5’ terminus, wherein the phosphorylated nucleotide is selected from uridine and adenosine.

235. The oligonucleotide of claim 234, wherein the phosphorylated nucleotide is uridine.

236. The oligonucleotide of any one of claims 152-235, wherein the 4'-carbon of the sugar of the 5 '-nucleotide of the antisense strand comprises a phosphate analog.

237. The oligonucleotide of claim 236, wherein the phosphate analog is oxymethyl phosphonate, vinyl phosphonate or malonyl phosphonate.

238. The oligonucleotide of any one of claims 235-237, wherein the phosphorylated nucleotide is 4’-O-monomethylphosphonate-2’-O-methyl uridine.342239. The oligonucleotide of any one of claims 152-238, wherein the lipid moiety is conjugated to the 2’ carbon of the ribose ring of the nucleotide.

240. The oligonucleotide of any one of claims 152-239, wherein the region of complementarity is fully complementary to the mRNA target sequence.

241. The oligonucleotide of any one of claims 152-239, wherein the region of complementarity is partially complementary to the mRNA target sequence.

242. The oligonucleotide of claim 241, wherein the region of complementarity comprises no more than four mismatches to the mRNA target sequence.

243. The oligonucleotide of any one of claims 152-242, wherein the mRNA target sequence is (i) a central nervous system (CNS) mRNA target sequence, optionally a neuronal mRNA target sequence or an ocular mRNA target sequence; or (ii) an ocular tissue mRNA target sequence.

244. The oligonucleotide of any one of claims 152-242, wherein the target mRNA is expressed in a tissue of the central nervous system, liver tissue, ocular tissue, adipose tissue, muscle tissue, adrenal tissue, cardiac tissue, lung tissue, or any combination thereof.

245. The oligonucleotide of any one of claims 152-244, wherein the oligonucleotide is a Dicer substrate.

246. The oligonucleotide of any one of claims 152-245, wherein the oligonucleotide reduces expression of the mRNA target sequence in a cell or population of cells in vitro and / or in vivo.

247. A pharmaceutical composition comprising the oligonucleotide of any one of claims 152-246, and a pharmaceutically acceptable carrier, delivery agent or excipient.

248. A method for treating a subject having a disease, disorder or condition associated with expression of a target mRNA, the method comprising administering to the subject a343therapeutically effective amount of the oligonucleotide of any one of claims 152-246, or the pharmaceutical composition of claim 247.

249. The method of claim 248, wherein the target mRNA is expressed in the central nervous system, optionally a neuron of the central nervous system.

250. The method of claim 248, wherein the target mRNA is expressed in ocular tissue, optionally the optic nerve and / or retina.

251. A method of delivering an oligonucleotide to a cell or population of cells in the central nervous system or ocular tissue, the method comprising administering the pharmaceutical composition of claim 247.

252. A method of reducing expression of a target mRNA in a subject, comprising administering to the subject the oligonucleotide of any one of claims 152-246, or the pharmaceutical composition of claim 247.

253. The method of claim 252, wherein the target mRNA is expressed in the central nervous system, optionally a neuron of the central nervous system.

254. The method of claim 253, wherein the target mRNA is expressed in ocular tissue, optionally the optic nerve and / or retina.

255. A kit comprising the oligonucleotide of any one of claims 152-246, 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 a target mRNA.

256. Use of the oligonucleotide of any one of claims 152-246, or the pharmaceutical composition of claim 247, in the manufacture of a medicament for the treatment of a disease, disorder, or condition associated with expression of a target mRNA.344257. The oligonucleotide of any one of claims 152-246, or the pharmaceutical composition of claim 122, for use, or adaptable for use, in the treatment of a disease, disorder, or condition associated with expression of a target mRNA.

258. The kit of claim 255, the use of claim 256, or the oligonucleotide of claim 257, wherein the target mRNA is expressed in the central nervous system and / or a neuron of the central nervous system.

259. The kit of claim 255, the use of claim 256, or the oligonucleotide of claim 257, wherein the target mRNA is expressed in ocular tissue, optionally the retina and / or optic nerve.

260. The method of any one of claims 249, 251, and 253, wherein the central nervous system comprises the frontal cortex, the hippocampus, the cerebellum, the brainstem, lumbar dorsal root ganglion, the lumbar spinal cord, or combinations thereof.

261. The kit, use or oligonucleotide of claim 258, wherein the central nervous system comprises the frontal cortex, the hippocampus, the cerebellum, the brainstem, lumbar dorsal root ganglion, the lumbar spinal cord, or combinations thereof.

262. A method of activating target-specific RNA interference (RNAi) in an organism comprising administering to said organism the dsRNA oligonucleotide of claims 152, 192, 202 or 217, said oligonucleotide being administered in an amount sufficient for degradation of the target mRNA to occur, thereby activating target-specific RNAi in the organism.

263. The method of claim 262, wherein the target mRNA specifies the amino acid sequence of a protein involved or predicted to be involved in a human disease or disorder.

264. The method of claim 263, wherein the disease or disorder is selected from the group consisting of viral infections, bacterial infections, parasitic infections, cancers, allergies, autoimmune diseases, immunodeficiencies, and immunosuppression.

265. A double-stranded oligonucleotide comprising an antisense strand of about 13-30 nucleotides in length and a sense strand of about 10-50 nucleotides in length, wherein the345antisense and sense strands are separate strands which form an asymmetric duplex region having an overhang of about 2-10 nucleotides at the 3’ terminus of the antisense strand, wherein the duplex region is about 10-30 nucleotides, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, and wherein the sense strand comprises at least one lipid moiety conjugated to a nucleotide on the sense strand.

266. A double-stranded oligonucleotide comprising an antisense strand of about 13-30 nucleotides in length and a sense strand of about 10-50 nucleotides in length, wherein the antisense and sense strands are separate strands which form an asymmetric duplex region having an overhang of about 2-12 nucleotides at the 3’ terminus of the antisense strand, wherein the duplex region is about 10-30 nucleotides, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, and wherein the sense strand comprises at least one lipid moiety conjugated to a nucleotide on the sense strand.

267. The oligonucleotide of claim 265 or 266, comprising a blunt end.

268. The oligonucleotide of claim 267, wherein the blunt end comprises the 3’ end of the sense strand and the 5’ end of the antisense strand.

269. The oligonucleotide of claim 265 or 266, comprising a stem loop, 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, and wherein the sense strand comprises an orientation of 5’ to 3’, and wherein the stem-loop is at the 3’ end of the sense strand.

270. The oligonucleotide of any one of claims 267-268, wherein:(i) the sense strand is 19 nucleotides, the duplex region is 19 nucleotides, and the overhang is 3 nucleotides;(ii) the sense strand is 18 nucleotides, the duplex region is 18 nucleotides, and the overhang is 4 nucleotides;(iii) the sense strand is 17 nucleotides, the duplex region is 17 nucleotides, and the overhang is 5 nucleotides;(iv) the sense strand is 16 nucleotides, the duplex region is 16 nucleotides, and the overhang is 6 nucleotides; or(v) the sense strand is 15 nucleotides, the duplex region is 15 nucleotides, and the overhang is 7 nucleotides.

271. The oligonucleotide of any one of claims 267-268, wherein:(i) the sense strand is 19 nucleotides, the duplex region is 19 nucleotides, and the overhang is 3 nucleotides;(ii) the sense strand is 18 nucleotides, the duplex region is 18 nucleotides, and the overhang is 4 nucleotides;(iii) the sense strand is 17 nucleotides, the duplex region is 17 nucleotides, and the overhang is 5 nucleotides;(iv) the sense strand is 16 nucleotides, the duplex region is 16 nucleotides, and the overhang is 6 nucleotides;(v) the sense strand is 15 nucleotides, the duplex region is 15 nucleotides, and the overhang is 7 nucleotides;(vi) the sense strand is 14 nucleotides, the duplex region is 14 nucleotides, and the overhang is 8 nucleotides;(vii) the sense strand is 13 nucleotides, the duplex region is 13 nucleotides, and the overhang is 8 nucleotides; or(viii) the sense strand is 12 nucleotides, the duplex region is 12 nucleotides, and the overhang is 10 nucleotides.

272. The method of any one of claims 267-268, wherein the sense strand is 10 nucleotides, the duplex region is 10 nucleotides, and the overhang is 12 nucleotides.

273. The oligonucleotide of claim 269, wherein:(i) the sense strand is 32 nucleotides, the duplex region is 16 nucleotides, and the overhang is 6 nucleotides;(ii) the sense strand is 30 nucleotides, the duplex region is 14 nucleotides, and the overhang is 8 nucleotides; or(iii) the sense strand is 28 nucleotides, the duplex region is 12 nucleotides, and the overhang is 10 nucleotides.

274. The oligonucleotide of any one of claims 265-273, wherein the antisense strand is 22 nucleotides, with positions 1-22 numbered 5’ to 3’.

275. The oligonucleotide of any one of claims 270-274, wherein the lipid moiety is conjugated to a nucleotide of the sense strand that forms a base pair with a nucleotide at position 14 of the antisense strand.

276. The oligonucleotide of any one of claims 270-274, wherein the lipid moiety is conjugated to a nucleotide of the sense strand that forms a base pair with a nucleotide at position 12 of the antisense strand.

277. The oligonucleotide of any one of claims 270-274, wherein the lipid moiety is conjugated to a nucleotide of the sense strand that forms a base pair with a nucleotide at position 20, position 19, position 18, position 17, position 16, position 15, position 14, position 13, or position 12 of the antisense strand.

278. The oligonucleotide of claim 269 or 274, wherein the lipid moiety is conjugated to a nucleotide in the loop.

279. The oligonucleotide of any one of claims 265-278, wherein the lipid moiety is conjugated to the 3 ’terminal nucleotide on the sense strand.

280. The oligonucleotide of any one of claims 265-278, wherein the lipid moiety is conjugated to the 5’ terminal nucleotide on the sense strand.

281. The oligonucleotide of any one of claims 265-280, wherein the lipid moiety is selected from:348282. The oligonucleotide of any one of claims 265-280, wherein the lipid moiety is a hydrocarbon chain.

283. The oligonucleotide of claim 282, wherein the hydrocarbon chain is a C8-C30 hydrocarbon chain.349284. The oligonucleotide of claim 283, wherein the hydrocarbon chain is a C16 hydrocarbon chain.

285. The oligonucleotide of claim 284, wherein the C16 hydrocarbon chain is represented by286. The oligonucleotide of claim 283, wherein the hydrocarbon chain is a C22 hydrocarbon chain.

287. The oligonucleotide of claim 286, wherein the C22 hydrocarbon chain is represented by288. The oligonucleotide of any one of claims 265-287, wherein the lipid moiety is conjugated to the 2’ carbon of the ribose ring of the nucleotide.

289. The oligonucleotide of any one of claims 265-288, wherein the region of complementarity is fully complementary to the mRNA target sequence.

290. The oligonucleotide of any one of claims 265-288, wherein the region of complementarity is partially complementary to the mRNA target sequence.

291. The oligonucleotide of claim 290, wherein the region of complementarity comprises no more than four mismatches to the mRNA target sequence.

292. The oligonucleotide of any one of claims 265-291, wherein the mRNA target sequence is a liver mRNA target sequence, optionally a liver macrophage mRNA target sequence, a liver hepatocyte mRNA target sequence, or a liver sinusoidal endothelial cell mRNA target sequence.350293. The oligonucleotide of any one of claims 265-291, wherein the mRNA target sequence is an ocular mRNA target sequence.

294. The oligonucleotide of any one of claims 265-291, wherein the mRNA target sequence is expressed in liver tissue, skeletal muscle tissue, adipose tissue and / or adrenal tissue.

295. The oligonucleotide of any one of claims 265-291, wherein the mRNA target sequence is expressed in at least one tissue of the central nervous system.

296. The oligonucleotide of claim 295, wherein the at least one tissue of the central nervous system is selected from frontal cortex, medulla, hippocampus, hypothalamus, cerebellum, lumbar spinal cord, lumbar dorsal root ganglion, and any combination thereof.

297. The oligonucleotide of any one of claims 265-296, wherein the oligonucleotide comprises at least one modified nucleotide.

298. The oligonucleotide of claim 297, wherein the modified nucleotide comprises a 2'- modification.

299. The oligonucleotide of claim 298, wherein each of the nucleotides of the sense strand and the antisense strand comprise a 2'-modification.

300. The oligonucleotide of claim 297 or 298, 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.

301. The oligonucleotide of any one of claims 298-300, wherein the antisense strand is 22 nucleotides with positions 1-22 numbered 5’ to 3’, and wherein the sense strand comprises a 2’ -fluoro modification at each of nucleotides forming a base pair with nucleotides at positions 10-13 of the antisense strand.

302. The oligonucleotide of any one of claims 298-300, wherein the antisense strand is 22 nucleotides with positions 1-22 numbered 5’ to 3’, and wherein the sense strand comprises a3512’ -fluoro modification at each of nucleotides forming a base pair with nucleotides at positions 10, 11, 12, 13, or any combination thereof, of the antisense strand.

303. The oligonucleotide of any one of claims 298-301, 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.

304. The oligonucleotide of any one of claims 301-303, wherein the remaining nucleotides comprise a 2'-O-methyl modification, provided that the nucleotide of the sense strand conjugated to the at least one lipid moiety does not comprise a 2’-O-methyl modification.

305. The oligonucleotide of any one of claims 265-304, wherein the oligonucleotide comprises at least one modified intemucleotide linkage.

306. The oligonucleotide of claim 305, wherein the at least one modified intemucleotide linkage is a phosphorothioate linkage.

307. The oligonucleotide of claim 306, 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’.

308. The oligonucleotide of claim 306 or 307, 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'.

309. The oligonucleotide of claim 308, wherein the antisense strand comprises a phosphorothioate linkage between positions 13 and 14 and between positions 14 and 15.

310. The oligonucleotide of claim 308, wherein the antisense strand comprises a phosphorothioate linkage between positions 16 and 17, between positions 17 and 18, between positions 18 and 19, and between positions 19 and 20.352311. The oligonucleotide of claim 308, wherein the antisense strand comprises a phosphorothioate linkage between positions 13 and 14, between positions 14 and 15, between positions 15 and 16, between positions 16 and 17, between positions 17 and 18, between positions 18 and 19, and between positions 19 and 20.

312. The oligonucleotide of claim 308, wherein the antisense strand comprises a phosphorothioate linkage between positions 12 and 13, between positions 13 and 14, between positions 14 and 15, between positions 15 and 16, between positions 16 and 17, between positions 17 and 18, between positions 18 and 19, and between positions 19 and 20.

313. The oligonucleotide of any one of claims 306-312, wherein the sense strand comprises a phosphorothioate linkage between positions 1 and 2.

314. The oligonucleotide of any one of claims 306-312, wherein the sense strand comprises a phosphorothioate linkage between positions 1 and 2, between the penultimate nucleotide and third nucleotide from the 3’ end, and between the penultimate nucleotide and ultimate nucleotide.

315. The oligonucleotide of any one of claims 265-314, wherein the antisense strand comprises a phosphorylated nucleotide at the 5' terminus, wherein the phosphorylated nucleotide is selected from uridine and adenosine.

316. The oligonucleotide of claim 315, wherein the phosphorylated nucleotide is uridine.

317. The oligonucleotide of any one of claims 265-316, wherein the 4'-carbon of the sugar of the 5 '-nucleotide of the antisense strand comprises a phosphate analog.

318. The oligonucleotide of claim 317, wherein the phosphate analog is oxymethyl phosphonate, vinyl phosphonate or malonyl phosphonate.

319. The oligonucleotide of any one of claims 316-318, wherein the phosphorylated nucleotide is 4’-O-monomethylphosphonate-2’-O-methyl uridine.353320. The oligonucleotide of any one of claims 265-319, wherein the sense strand comprises at least one Tm-increasing nucleotide.

321. The oligonucleotide of claim 320, wherein the sense strand comprises up to nine Tm- increasing nucleotides.

322. The oligonucleotide of claim 321, wherein the sense strand comprises 1-3 Tm- increasing nucleotides.

323. The oligonucleotide of any one of claims 269 and 279-322, wherein SI and S2 each comprise at least one Tm-increasing nucleotide.

324. The oligonucleotide of claim 323, wherein SI and S2 are each 3 nucleotides, and each nucleotide is a Tm-increasing nucleotide.

325. The oligonucleotide of claim 323 or 324, wherein the sense strand comprises up to three Tm-increasing nucleotides at nucleotide positions, provided the nucleotide positions are not in the stem-loop.

326. The oligonucleotide of any one of claims 267-268, and 270-322, wherein the sense strand comprises up to five Tm-increasing nucleotides.

327. The oligonucleotide of any one of claims 320-326, wherein the Tm-increasing nucleotide is a bicyclic nucleotide.

328. The oligonucleotide of any one of claims 320-326, wherein the Tm-increasing nucleotide is a locked nucleic acid.

329. The oligonucleotide of claim 328, wherein the sense strand is 20 nucleotides in length, wherein the nucleotides are numbered 1-20 5’ to 3’, and wherein the sense strand comprises a locked nucleic acid at a nucleotide located at:(i) position 2;(ii) position 2 and position 15;(iii) position 2, position 15, and position 16;354(iv) position 2, position 15, position 16, and position 18; or,(v) position 2, position 15, position 16, position 18, and position 19.

330. The oligonucleotide of claim 328, wherein the sense strand is 16 nucleotides in length, wherein the nucleotides are numbered 1-16 5’ to 3’, and wherein the sense strand comprises a locked nucleic acid at a nucleotide located at:(i) position 2;(ii) position 2 and position 11;(iii) position 2, position 11, and position 12;(iv) position 2, position 11, position 12, and position 14; or,(v) position 2, position 11, position 12, position 14, and position 15.

331. The oligonucleotide of claim 328, wherein the sense strand is 14 nucleotides in length, wherein the nucleotides are numbered 1-14 5’ to 3, and wherein the sense strand comprises a locked nucleic acid at a nucleotide located at:(i) position 2;(ii) position 2 and position 9;(iii) position 2, position 9, and position 10; or,(iv) position 2, position 9, position 10, position 12, and position 13.

332. The oligonucleotide of claim 328, wherein the sense strand is 12 nucleotides in length, wherein the nucleotides are numbered 1-12 5’ to 3’, and wherein the sense strand comprises a locked nucleic acid at a nucleotide located at:(i) position 2;(ii) position 2 and position 7;(iii) position 2, position 7, and position 8;(iv) position 2, position 7, position 8, and position 10;(v) position 2, position 7, position 8, position 10, and position 11.

333. The oligonucleotide of any one of claims 265-332, wherein the oligonucleotide is a Dicer substrate.355334. The oligonucleotide of any one of claims 265-333, wherein the oligonucleotide reduces expression of the mRNA target sequence in a cell or population of cells in vitro and / or in vivo.

335. A pharmaceutical composition comprising the oligonucleotide of any one of claims 265-334, and a pharmaceutically acceptable carrier, delivery agent or excipient.

336. A method of delivering an oligonucleotide to a cell or population of cells in the central nervous system, liver tissue, muscle tissue, adipose tissue, adrenal tissue and / or ocular tissue, the method comprising administering the pharmaceutical composition of claim 69.

337. A method for treating a subject having a disease, disorder or condition associated with expression of a target mRNA, the method comprising administering to the subject a therapeutically effective amount of the oligonucleotide of any one of claims 265-334, or the pharmaceutical composition of claim 335.

338. A method of reducing expression of a target mRNA in a subject, comprising administering to the subject the oligonucleotide of any one of claims 265-334, or the pharmaceutical composition of claim 335.

339. The method of claim 337 or 338, wherein the target mRNA is expressed in the liver, optionally a macrophage of the liver.

340. The method of claim 337 or 338, wherein the target mRNA is expressed in at least one cell type of the central nervous system.

341. The method of claim 340, wherein the at least one cell type of the central nervous system is an astrocyte, a neuron, or an oligodendrocyte.

342. The method of claim 337 or 338, wherein the target mRNA is expressed in an astrocyte, a neuron, an oligodendrocyte, or any combination thereof.356343. The method of any one of claims 340-342, wherein the target mRNA is expressed in the frontal cortex, medulla, hippocampus, hypothalamus, cerebellum, lumbar spinal cord, lumbar dorsal root ganglion, or any combination thereof.

344. The method of claim 337 or 338, wherein the target mRNA is expressed in ocular tissue, optionally the retina or optic nerve, liver tissue, skeletal muscle tissue, adipose tissue and / or adrenal tissue.

345. A kit comprising the oligonucleotide of any one of claims 265-334, 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 a target mRNA.

346. Use of the oligonucleotide of any one of claims 265-334, or the pharmaceutical composition of claim 335 in the manufacture of a medicament for the treatment of a disease, disorder, or condition associated with expression of a target mRNA.

347. The oligonucleotide of any one of claims 265-334, or the pharmaceutical composition of claim 335, for use, or adaptable for use, in the treatment of a disease, disorder, or condition associated with expression of a target mRNA.

348. The method of claim 347, wherein the target mRNA is expressed in at least one cell type of the central nervous system.

349. The method of claim 348, wherein the at least one cell type of the central nervous system is an astrocyte, a neuron, or an oligodendrocyte, optionally wherein the target mRNA is expressed in an astrocyte, a neuron, an oligodendrocyte, or any combination thereof.

350. The method of any one of claims 347-349, wherein the target mRNA is expressed in the frontal cortex, medulla, hippocampus, hypothalamus, cerebellum, lumbar spinal cord, lumbar dorsal root ganglion, or any combination thereof.357351. The kit of claim 345, the use of claim 346, or the oligonucleotide of claim 347, wherein the target mRNA is expressed in the liver, optionally wherein the target mRNA is expressed in a liver macrophage, a liver hepatocyte, or a liver sinusoidal endothelial cell.

352. The kit of claim 345, the use of claim 346, or the oligonucleotide of claim 347, wherein the target mRNA is expressed in ocular tissue, optionally the retina or optic nerve.

353. The kit of claim 345, the use of claim 346, or the oligonucleotide of claim 347, wherein the target mRNA is expressed in liver tissue, skeletal muscle tissue, adipose tissue and / or adrenal tissue.

354. The kit of claim 345, the use of claim 346, or the oligonucleotide of claim 347, wherein the target mRNA is expressed in at least one tissue of the central nervous system.

355. The kit, use or oligonucleotide of claim 354, wherein the at least one tissue of the central nervous system is selected from the frontal cortex, medulla, hippocampus, hypothalamus, cerebellum, lumbar spinal cord, lumbar dorsal root ganglion, and any combinations thereof.

356. The method of claim 337, wherein the disease or disorder is selected from the group consisting of viral infections, bacterial infections, parasitic infections, cancers, allergies, autoimmune diseases, immunodeficiencies, and immunosuppression.

357. A double-stranded oligonucleotide comprising:(i) a sense strand 14-20 nucleotides in length, wherein the sense strand comprises at least one lipid moiety conjugated to the 5’ terminus, and wherein the sense strand comprises at least one locked nucleic acid, and(ii) an antisense strand 22 nucleotides in length, wherein the antisense strand comprises a region of complementarity to an astrocyte target mRNA, and wherein the sense and antisense strand are separate strands which form an asymmetric duplex region having an overhang of about 2-8 nucleotides at the 3’ terminus of the antisense strand, wherein the duplex region is about 14-20 nucleotides.

358. A double-stranded oligonucleotide comprising:358(i) a sense strand 14-20 nucleotides in length, wherein the sense strand comprises at least one lipid moiety conjugated to the 5’ terminus, and wherein the sense strand comprises at least one locked nucleic acid, and(ii) an antisense strand 22 nucleotides in length, wherein the antisense strand comprises a region of complementarity to a neuron target mRNA, and wherein the sense and antisense strand are separate strands which form an asymmetric duplex region having an overhang of about 2-8 nucleotides at the 3’ terminus of the antisense strand, wherein the duplex region is about 14-20 nucleotides.

359. A double-stranded oligonucleotide comprising:(i) a sense strand 14-20 nucleotides in length, wherein the sense strand comprises at least one lipid moiety conjugated to the 5’ terminus, and wherein the sense strand comprises at least one locked nucleic acid, and(ii) an antisense strand 22 nucleotides in length, wherein the antisense strand comprises a region of complementarity to an oligodendrocyte target mRNA, and wherein the sense and antisense strand are separate strands which form an asymmetric duplex region having an overhang of about 2-8 nucleotides at the 3’ terminus of the antisense strand, wherein the duplex region is about 14-20 nucleotides.

360. The oligonucleotide of any one of claims 357-359, wherein the sense strand is 14 nucleotides in length.

361. The oligonucleotide of claim 360, wherein the sense strand comprises a locked nucleic acid at one or more of position 2, position 9, position 10, position 12, or position 13 with positions numbered 5’ to 3’.

362. The oligonucleotide of claim 360 or 361, wherein the antisense strand comprises a phosphorothioate linkage between positions 1 and 2, between positions 2 and 3, between positions 3 and 4, between positions 13 and 14, between positions 14 and 15, between positions 20 and 21, and between positions 21 and 22.

363. The oligonucleotide of any one of claims 357-359 wherein the sense strand is 20 nucleotides in length.359364. The oligonucleotide of claim 363, wherein the sense strand comprises a locked nucleic acid at one or more of position 2, position 15, or position 16 with positions numbered 5’ to 3’.

365. The oligonucleotide of claim 363 or 364, wherein the antisense strand comprises a phosphorothioate linkage between positions 1 and 2, between positions 2 and 3, between positions 3 and 4, between positions 20 and 21, and between positions 21 and 22.

366. A method of reducing expression of a target mRNA in an astrocyte, comprising administering a double-stranded oligonucleotide wherein the oligonucleotide comprises:(i) a sense strand, wherein the sense strand is 14-20 nucleotides in length, wherein the sense strand comprises at least one lipid moiety conjugated to the 5’ terminus, and wherein the sense strand comprises at least one locked nucleic acid, and(ii) an antisense strand, wherein the antisense strand is 22 nucleotides in length, and wherein the antisense strand comprises a region of complementarity to a target mRNA in the astrocyte, and wherein the sense and antisense strand are separate strands which form an asymmetric duplex region having an overhang of about 2-8 nucleotides at the 3’ terminus of the antisense strand, wherein the duplex region is about 14-20 nucleotides, thereby reducing expression of the target mRNA in the astrocyte.

367. The method of claim 366, wherein the oligonucleotide comprises a blunt end comprising the 3’ end of the sense strand and the 5’ end of the antisense strand.

368. The method of any one of claims 366-367, wherein the sense strand comprises no more than 3 locked nucleic acids.

369. The method of any one of claims 366-368, wherein the sense strand is 20 nucleotides in length, and wherein reduction of the target mRNA in an astrocyte is increased compared to reduction in a neuron, optionally wherein reduction of the target mRNA is increased by at least 5%.

370. The method of any one of claims 366-368, wherein the sense strand is 14 nucleotides in length, and wherein reduction of the target mRNA in an astrocyte is increased compared to360reduction in an oligodendrocyte, optionally wherein reduction of the target mRNA is increased by at least 5%.

371. The method of any one of claims 366-368, wherein the sense strand is 20 nucleotides in length, and wherein the target mRNA is reduced in an astrocyte and an oligodendrocyte to the same or similar level.

372. The method of any one of claims 366-368, wherein the sense strand is 14 nucleotides in length, wherein the target mRNA is reduced in an astrocyte and in a neuron to the same or similar level, and wherein reduction of the target mRNA in an astrocyte and in a neuron is increased compared to reduction in an oligodendrocyte, optionally wherein reduction of the target mRNA is increased by at least 5%.

373. A method of reducing expression of a target mRNA in an oligodendrocyte, comprising administering a double-stranded oligonucleotide wherein the oligonucleotide comprises:(i) a sense strand, wherein the sense strand is 14-40 nucleotides in length, wherein the sense strand comprises at least one lipid moiety conjugated to the 5’ terminus, and wherein the sense strand comprises at least one locked nucleic acid, and(ii) an antisense strand, wherein the antisense strand is 22 nucleotides in length, and wherein the antisense strand comprises a region of complementarity to a target mRNA in the oligodendrocyte, and wherein the sense and antisense strand are separate strands which form an asymmetric duplex region having an overhang of about 2-8 nucleotides at the 3’ terminus of the antisense strand, wherein the duplex region is about 14-20 nucleotides, thereby reducing expression of the target mRNA in the oligodendrocyte.

374. The method of claim 373, wherein the oligonucleotide comprises a blunt end comprising the 3’ end of the sense strand and the 5’ end of the antisense strand.

375. The method of claim 374, wherein the sense strand is 20 nucleotides in length.

376. The method of claim 373, wherein the sense strand in 36 nucleotides in length, and wherein the oligonucleotide comprises a stem-loop.361377. The method of claim 376, wherein the target mRNA is reduced in an astrocyte and the oligodendrocyte to the same or similar level, and wherein reduction of the target mRNA is increased in the astrocyte and the oligodendrocyte compared to reduction in a neuron, optionally wherein reduction of the target mRNA is increased by at least 5%.

378. A method of reducing expression of a target mRNA in a neuron, comprising administering a double-stranded oligonucleotide wherein the oligonucleotide comprises:(i) a sense strand, wherein the sense strand is 14-20 nucleotides in length, wherein the sense strand comprises at least one lipid moiety conjugated to the 5’ terminus, and wherein the sense strand comprises at least one locked nucleic acid, and(ii) an antisense strand, wherein the antisense strand is 22 nucleotides in length, and wherein the antisense strand comprises a region of complementarity to a target mRNA in the neuron, and wherein the sense and antisense strand are separate strands which form an asymmetric duplex region having an overhang of about 2-8 nucleotides at the 3’ terminus of the antisense strand, wherein the duplex region is about 14-20 nucleotides, thereby reducing expression of the target mRNA in the neuron.

379. The method of claim 378, wherein the oligonucleotide comprises a blunt end comprising the 3’ end of the sense strand and the 5’ end of the antisense strand.

380. The method of claim 378 or 379, wherein the sense strand comprises no more than 5 locked nucleic acids.

381. The method of any one of claims 378-380, wherein the sense strand is 14 nucleotides in length.362

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