TW202333750A - Rnai oligonucleotide conjugates - Google Patents

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

RNAi oligonucleotide conjugates

The present disclosure relates to oligonucleotides linked to lipid moieties that are useful for inhibiting target genes in various tissues. Specifically, the present disclosure relates to oligonucleotide-lipid conjugates, methods of making them, their chemical configurations, and the use of conjugated nucleic acids and oligonucleotides according to the descriptions provided herein. Methods of modulating (e.g., inhibiting or reducing) the expression of a target gene. The present disclosure also provides pharmaceutically acceptable compositions containing the conjugates of the present specification and methods of using the compositions to treat various diseases or conditions. Sequence Listing Electronic File Reference The contents of the sequence list electronic file (DICN_015_001TW_SeqList_ST26.xml; size 4,697,063 bits; creation date December 4, 2022) are incorporated by reference into this article in their entirety. [Related applications]

This application claims the rights and interests of U.S. Provisional Patent Application Serial No. 63/277,097 filed on November 8, 2021 and U.S. Provisional Patent Application Serial No. 63/340,291 filed on May 10, 2022, each of which is borrowed from They are incorporated herein by reference in their entirety.

Modulation of gene expression by modified nucleic acids shows great potential both as a research tool in the laboratory and as a therapeutic method in the clinic. Several types of oligonucleotide or nucleic acid-based therapies are already undergoing clinical research, including antisense oligonucleotide (ASO), short interfering RNA (siRNA), double-stranded nucleic acid (double- stranded nucleic acid (dsNA), aptamers, ribozymes, exon-skipping and splice-altering oligonucleotides, immunomodulatory oligonucleotides, mRNA, and CRISPR. Chemical modification of relevant molecules to allow functionality in various tissues, organs, and/or cell types is essential to overcome the challenges of oligonucleotide therapeutics, including improving nuclease stability, RNA binding affinity, and pharmacokinetics. plays a key role. Various chemical modification strategies for oligonucleotides, including modifications of sugars, nucleobases, and phosphodiester backbones, have been developed over the past three decades 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).

Dicer-processed RNAi technology utilizes short double-stranded RNA (dsRNA) with two nucleotide (nt) 3'-overhangs and approximately 21 base pairs in length for gene silencing. These dsRNA are generally called small interfering RNA (siRNA). siRNAs 12 to 22 nucleotides in length are active agents of RNAi. siRNA duplexes serve as guides for mRNA degradation. Once siRNA is incorporated into the RNA-induced silencing complex (RISC), the complex interacts with specific mRNAs and ultimately inhibits the mRNA signal. The sense strand or passenger strand of siRNA is typically cleaved by Argonaute 2 (Ago2) endonuclease at the 9th nucleotide downstream of the 5' end of the sense strand. 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 blockage of the targeted RNA.

However, the in vivo use of RNAi or siRNA molecules as pharmaceutical agents remains difficult due to obstacles encountered, such as low biological stability and unacceptable toxicity that may result from off-target effects. Various types of chemical modifications have been studied over the years to improve pharmacokinetics and overcome biological instability issues to improve the stability and specificity of RNAi duplexes. In some cases, chemical modification of siRNA has improved the serum stability of siRNA. However, RNAi activity is often lost, but careful placement of specific modified residues allows for enhanced siRNA biostability without loss of siRNA potency. Some of these modifications have reduced siRNA side effects, such as induction of immune responses in the recipient and inherent off-target effects, or even enhanced siRNA potency. Various chemically modified siRNAs have been studied, among them bridged nucleic acids (BNAs), such as 2',4'-methylene bridged nucleic acid 2',4'-BNA, also known as locked nucleic acids or LNAs. Some of these modified siRNAs show promising effects.

Therapeutic gene silencing mediated by RNAi oligonucleotide-based therapies including siRNA and double-stranded nucleic acid (dsNA) offers the potential to significantly expand the druggable target space and Offering the possibility to treat orphan diseases that are beyond the therapeutic reach of other drug modalities (e.g., antibodies and/or small molecules). RNAi oligonucleotide-based therapies that inhibit or reduce the expression of specific target genes in the liver have been developed and are currently in clinical use (Sehgal et al., (2013) Journal of Hepatology 59: 1354-59). There are still technical obstacles to the development and clinical use of RNAi oligonucleotides in extrahepatic cells, tissues, and organs. Therefore, there is a continuing need in the art to successfully develop novel and effective RNAi oligonucleotides to modulate the expression of target genes in extrahepatic cells, tissues, and/or organs. This is complicated by the variable nature of extrahepatic cell types and considerations regarding circulation patterns and cell membrane composition (such as receptor type).

Over the past decade, synthetic RNAi triggers such as double-stranded RNA have become popular tools in biological research, and large-scale basic and clinical development efforts recently culminated in the FDA approval of the first RNAi drug, ONPATTRO ^tm . Despite an emerging drug development pipeline and a large-scale excipient compendium of targeting ligands and delivery technologies, difficulties in delivering RNAi agents to specific populations of disease-relevant cells and/or tissues, particularly outside the liver, continue Limiting the potential of RNAi therapeutics. Over the past few years, repeated attempts to develop useful, active, and durable RNAi agents and constructs for use based on known liver delivery technologies have not convincingly demonstrated the intended effects outside the liver. Therefore, new dsRNAs with variant structures have been developed to overcome the limitations of this technical field.

The present disclosure is based, at least in part, on the discovery of lipid-conjugated RNAi oligonucleotides capable of inhibiting the expression of target genes in liver and extrahepatic tissues. As demonstrated herein, a lipid-conjugated RNAi oligonucleotide with a backbone loop at the 5' end of the oligonucleotide is shown to be more effective than a lipid-conjugated RNAi oligonucleotide with a backbone loop at the 3' end of the oligonucleotide. RNAi oligonucleotides have comparable efficacy in reducing the expression of target genes in several regions of the central nervous system. Furthermore, the presence of stable backbone loops (e.g., UACG) enhances target gene expression reduction compared to relatively less stable backbone loops (e.g., GAAA). In some aspects, the presence of _Tm -increasing nucleotides (e.g., locked nucleic acids) in the sense strand and/or truncation of the sense strand at the 3' end enhances the reduction in target gene expression.

It is further shown herein that lipid-conjugated RNAi oligonucleotides with "double overhangs" (e.g., an overhang of at least one nucleotide at each of the 5' and 3' ends of the antisense strand) can be used with respect to RNAi oligonucleotides with only one overhang (i.e., at the 3' end of the antisense strand) reduced CNS target gene expression to comparable levels. Truncation of up to three nucleotides is tolerated at the 3' end of the sense strand, while the introduction of _Tm -increasing nucleotides allows truncation of up to four nucleotides.

Lipid-conjugated RNAi oligonucleotides delivered to the eye have been shown to be effective in reducing ocular mRNA expression. Specifically, double-overhang RNAi oligonucleotides with lipids conjugated to the 5' nucleotide of the sense strand reduced expression of target genes in the optic nerve and retina.

Further shown herein are lipid-conjugated RNAi oligonucleotides capable of reducing target gene expression in hepatic macrophages. Specifically, an RNAi oligonucleotide having a blunt end (the blunt end includes the 3' end of the sense strand and the 5' end of the antisense strand) and an overhang of up to seven nucleotides at the 3' end of the antisense strand. Resulting in the reduction of genes expressed in macrophages. Furthermore, lipid-conjugated RNAi oligonucleotides with double overhangs, with or without _Tm -increasing nucleotides, similarly reduced expression of macrophage target genes. Macrophages constitute approximately 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 intoxication, steatosis, Infections (e.g. viral infections), inflammatory liver disease, fibrosis, hepatocellular carcinoma and cirrhosis.

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

Also shown herein are lipid-conjugated RNAi oligonucleotides with a blunt end or backbone loop at the 3' end of the oligonucleotide, which have a truncated sense strand (e.g., at the 3' of the antisense strand). Nucleotides with overhangs) and increased _Tm can reduce target gene expression in several tissues, including liver, skeletal muscle, fat, and adrenal gland.

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

In some aspects, the present disclosure provides a double-stranded oligonucleotide comprising an antisense strand of about 20 to 22 nucleotides in length and a sense strand of about 8 to 20 nucleotides in length, wherein the antisense strand and The sense strand forms a duplex region of approximately 8 to 20 base pairs, wherein the antisense strand contains a 5' to 3' direction, and wherein the antisense strand contains a 5' overhang of at least one nucleotide and at least one a 3' overhang of nucleotides, wherein the antisense strand comprises a region complementary to the 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 to 10 nucleotides. In some aspects, the 5' overhang is about 2 to 10 nucleotides. In some aspects, the 5' overhang is about 1 to 6 nucleotides. In some aspects, the 3' overhang is about 2 to 8 nucleotides. In some aspects, the 3' overhang is about 2 to 12 nucleotides. In some aspects, the 5' overhang is 2 nucleotides and the 3' overhang is about 3 to 7 nucleotides. In some aspects, the 3' overhang is 2 nucleotides and the 5' overhang is about 2 to 8 nucleotides. In some aspects, the 3' overhang is 6 to 8 nucleotides and the 5' overhang is about 2 to 4 nucleotides.

In any of the foregoing or related aspects, (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 7 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. In any of the foregoing or related aspects, (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.

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.

In any of the foregoing or related aspects, the lipid moiety is selected from: and .

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 some aspects, the hydrocarbon chain is a C22 hydrocarbon chain. In some aspects, the C22 hydrocarbon chain is represented by: .

In any of the foregoing or related aspects, the lipid moiety is conjugated to the 5' end nucleotide of the sense strand. In some aspects, the lipid moiety is conjugated to the 2' carbon of the ribose ring of the nucleotide.

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

In any of the foregoing or related aspects, the complementary region is completely complementary to the mRNA target sequence. In other aspects, the complementary region is partially complementary to the mRNA target sequence. In some aspects, the complementary region contains no more than four mismatches to the mRNA target sequence. In some aspects, the complementary region contains up to four mismatches to the mRNA target sequence.

In some aspects, the complementary region contains no more than four mismatches to the mRNA target sequence.

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 targeting sequence is a liver macrophage mRNA targeting 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.

In any of the preceding or related aspects, the oligonucleotide comprises at least one modified nucleotide. In some aspects, the modified nucleotide includes a 2'-modification. In some aspects, each of the nucleotides of the sense strand and the antisense strand includes a 2'-modification. In some aspects, the 2'-modification is selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy- Modification of 2'-fluoro-β-d-arabinose nucleic acid. In some aspects, the sense strand includes nucleotide positions numbered from 5' to 3', wherein each of positions 8 to 11 includes a 2'-fluoro modification. In some aspects, the sense strand comprises a 2'-fluoro modification at each of the nucleotides that form a base pair with nucleotides at positions 10 to 13 of the antisense strand, where positions are numbered from 5' to 3' . In some aspects, the antisense strand includes 22 nucleotides from 5' to 3' from positions 1 to 22, and wherein each of positions 2, 3, 4, 5, 7, 10, and 14 includes 2'- Fluorine modification. In some aspects, the residual nucleotides comprise a 2'-O-methyl modification, with the proviso that the nucleotide of the sense strand conjugated to the at least one lipid moiety does not comprise a 2'-O-methyl modification.

In any of the preceding 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 is (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 Contains phosphorothioate linkages with positions numbered 1 to 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 the positions range from 5' Toward 3' are numbered 1 to 22. In some aspects, the antisense strand contains phosphorothioate linkages between positions 13 and 14 and between positions 14 and 15. In some aspects, the sense strand is between positions 1 and 2, between the penultimate nucleotide and the third nucleotide from the 3' end, and between the penultimate nucleotide and the final nucleotide. Contains phosphorothioate linkages.

In any of the foregoing or related aspects, the antisense strand comprises a phosphorylated nucleotide at the 5' end, wherein the phosphorylated nucleotide is selected from the group consisting of 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 oxymethylphosphonate, vinylphosphonate, or malonylphosphonate. In some aspects, the phosphorylated nucleotide is 4'-O-monomethylphosphonate-2'-O-methyluridine.

In any of the foregoing or related aspects, the sense strand comprises at least one nucleotide that increases _Tm . In some aspects, the sense strand contains up to four nucleotides that increase _Tm . In some aspects, the _Tm -increasing nucleotide is a bicyclic nucleotide. In some aspects, the _Tm -increasing nucleotide is a locked nucleic acid.

In other aspects, the present 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 strand and the sense strand form 18 bases A duplex region of a base pair, wherein the antisense strand includes a direction from 5' to 3', wherein the antisense strand includes a 5' overhang and a 3' overhang, the 5' overhang includes at least two nucleotides and the the 3' overhang comprises at least two nucleotides, wherein the antisense strand comprises a region complementary to the mRNA target sequence, and wherein the sense strand comprises at least one lipid moiety conjugated to the 5' end nucleotide of the sense strand, And wherein each of the antisense strand and the sense strand includes at least one 2' modified nucleotide and at least one modified inter-nucleotide linkage.

In some aspects, the sense strand contains a 2'-fluoro modification at positions 8 to 11, numbered from 5' to 3'. In some aspects, the antisense strand contains 2'-fluoro modifications at positions 2 through 5, 7, 10, and 14, numbered from 5' to 3'. In some aspects, the residual nucleotides comprise a 2'-O-methyl modification, with the proviso 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 contains phosphorothioate linkages between nucleotides at positions 1 and 2, 16 and 17, and 17 and 18, numbered from 5' to 3'. In some aspects, the antisense strand comprises a phosphorothioate linkage between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22, numbered from 5' to 3' . In some aspects, the antisense strand contains phosphorylated uridine at position 1, numbered from 5' to 3'. In some aspects, the phosphorylated nucleotide is 4'-O-monomethylphosphonate-2'-O-methyluridine. In some aspects, the lipid moiety is a C16 hydrocarbon represented by: . In some aspects, the lipid moiety is a C22 hydrocarbon represented by: . 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 2 nucleotides and the 3' overhang is 2 nucleotides. In some aspects, the complementary region is completely complementary to the mRNA target sequence. In some aspects, the complementary region is partially complementary to the mRNA target sequence. In some aspects, the complementary region contains 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 a stellate cell mRNA target sequence.

In some aspects, the present 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 strand and the sense strand form 18 bases A duplex region of a base pair, wherein the antisense strand includes a direction from 5' to 3', wherein the antisense strand includes a 5' overhang and a 3' overhang, the 5' overhang includes two nucleotides and the 3' overhang 'The overhang contains two nucleotides, wherein the antisense strand contains a region complementary to the central nervous system mRNA target sequence, and wherein the sense strand contains at least one lipid moiety conjugated to the 5' end nucleotide of the sense strand , wherein the sense strand contains a 2'-fluoro modification at positions 8 to 11 and a 2'-O-methyl modification at positions 2 to 7 and 12 to 18, wherein the sense strand contains at positions 1 and 2, positions 16 and 17 and contains a phosphorothioate linkage between positions 17 and 18, wherein the antisense strand contains a 2'-fluoro modification at positions 2 to 5, 7, 10, and 14 and a 2'-fluoro modification at positions 1, 6, 8, 9, 11 to 13 and 15 to 22 contain a 2'-O-methyl modification, wherein the antisense strand contains a thio group between positions 1 and 2, positions 2 and 3, positions 3 and 4, positions 20 and 21, and positions 21 and 22 Phosphate linkage, and wherein the antisense strand contains 5'-terminal phosphorylated uridine.

In some aspects, the present 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 strand and the sense strand form 18 bases A duplex region of a base pair, wherein the antisense strand includes a direction from 5' to 3', wherein the antisense strand includes a 5' overhang and a 3' overhang, the 5' overhang includes two nucleotides and the 3' overhang 'The overhang comprises two nucleotides, wherein the antisense strand comprises a region complementary to the eye mRNA target sequence, and wherein the sense strand comprises at least one lipid moiety conjugated to the 5' end nucleotide of the sense strand, wherein The sense strand contains 2'-fluoro modifications at positions 8 to 11 and 2'-O-methyl modifications at positions 2 to 7 and 12 to 18, wherein the sense strand contains at positions 1 and 2, positions 16 and 17 and positions Contains a phosphorothioate linkage between 17 and 18, wherein the antisense strand contains 2'-fluoro modifications at positions 2 to 5, 7, 10 and 14 and at positions 1, 6, 8, 9, 11 to 13 and 15 to 22 contain a 2'-O-methyl modification, wherein the antisense strand contains a phosphorothioate between positions 1 and 2, positions 2 and 3, positions 3 and 4, positions 20 and 21, and positions 21 and 22 linked, and wherein the antisense strand contains 5'-terminal phosphorylated uridine.

In some aspects, the present 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 strand and the sense strand form 18 bases A duplex region of a base pair, wherein the antisense strand includes a direction from 5' to 3', wherein the antisense strand includes a 5' overhang and a 3' overhang, the 5' overhang includes two nucleotides and the 3' overhang 'The overhang contains two nucleotides, wherein the antisense strand contains a region complementary to the macrophage mRNA target sequence, and wherein the sense strand contains at least one lipid moiety conjugated to the 5' end nucleotide of the sense strand , wherein the sense strand contains a 2'-fluoro modification at positions 8 to 11 and a 2'-O-methyl modification at positions 2 to 7 and 12 to 18, wherein the sense strand contains at positions 1 and 2, positions 16 and 17 and contains a phosphorothioate linkage between positions 17 and 18, wherein the antisense strand contains a 2'-fluoro modification at positions 2 to 5, 7, 10, and 14 and a 2'-fluoro modification at positions 1, 6, 8, 9, 11 to 13 and 15 to 22 contain a 2'-O-methyl modification, wherein the antisense strand contains a thio group between positions 1 and 2, positions 2 and 3, positions 3 and 4, positions 20 and 21, and positions 21 and 22 Phosphate linkage, and wherein the antisense strand contains 5'-terminal phosphorylated uridine.

In other aspects, the present 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 strand and the sense strand form 17 bases A duplex region of a base pair, wherein the antisense strand comprises a 5' to 3' direction, wherein the antisense strand comprises a 5' overhang and a 3' overhang, the 5' overhang comprising at least three nucleotides and the the 3' overhang comprises at least two nucleotides, wherein the antisense strand comprises a region complementary to the mRNA target sequence, and wherein the sense strand comprises at least one lipid moiety conjugated to the 5' end nucleotide of the sense strand, And wherein each of the antisense strand and the sense strand includes at least one 2' modified nucleotide and at least one modified inter-nucleotide linkage.

In some aspects, the sense strand contains a 2'-fluoro modification at positions 8 to 11, numbered from 5' to 3'. In some aspects, the antisense strand contains 2'-fluoro modifications at positions 2 through 5, 7, 10, and 14, numbered from 5' to 3'. In some aspects, the residual nucleotides comprise a 2'-O-methyl modification, with the proviso 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 contains phosphorothioate linkages between nucleotides at positions 1 and 2, 15 and 16, and 16 and 17, numbered from 5' to 3'. In some aspects, the antisense strand comprises a phosphorothioate linkage between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22, numbered from 5' to 3' . In some aspects, the antisense strand contains phosphorylated uridine at position 1, numbered from 5' to 3'. In some aspects, the phosphorylated nucleotide is 4'-O-monomethylphosphonate-2'-O-methyluridine. In some aspects, the lipid moiety is a C16 hydrocarbon represented by: . 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 complementary region is completely complementary to the mRNA target sequence. In some aspects, the complementary region is partially complementary to the mRNA target sequence. In some aspects, the complementary region contains no more than four mismatches to the mRNA target sequence. In some aspects, the mRNA targeting sequence is a central nervous system (CNS) targeting sequence, optionally a neuronal mRNA targeting sequence.

In other aspects, the present 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 strand and the sense strand form 17 bases A duplex region of a base pair, wherein the antisense strand includes a direction from 5' to 3', wherein the antisense strand includes a 5' overhang and a 3' overhang, the 5' overhang includes three nucleotides and the 3' overhang 'The overhang contains two nucleotides, wherein the antisense strand contains a region complementary to the central nervous system mRNA target sequence, and wherein the sense strand contains at least one lipid moiety conjugated to the 5' end nucleotide of the sense strand , wherein the sense strand contains a 2'-fluoro modification at positions 8 to 11 and a 2'-O-methyl modification at positions 2 to 7 and 12 to 17, wherein the sense strand contains at positions 1 and 2, positions 15 and 16 and contains a phosphorothioate linkage between positions 16 and 17, wherein the antisense strand contains a 2'-fluoro modification at positions 2 to 5, 7, 10, and 14 and a 2'-fluoro modification at positions 1, 6, 8, 9, 11 to 13 and 15 to 22 contain a 2'-O-methyl modification, wherein the antisense strand contains a thio group between positions 1 and 2, positions 2 and 3, positions 3 and 4, positions 20 and 21, and positions 21 and 22 Phosphate linkage, and wherein the antisense strand contains 5'-terminal phosphorylated uridine.

In a further aspect, the present 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 16 bases A duplex region of a base pair, wherein the antisense strand comprises a 5' to 3' direction, wherein the antisense strand comprises a 5' overhang and a 3' overhang, the 5' overhang comprising at least four nucleotides and the the 3' overhang comprises at least two nucleotides, wherein the antisense strand comprises a region complementary to the mRNA target sequence, and wherein the sense strand comprises at least one lipid moiety conjugated to the 5' end nucleotide of the sense strand, wherein the sense strand includes up to five _Tm -increasing nucleotides, and wherein each of the antisense strand and the sense strand includes at least one 2' modified nucleotide and at least one modified inter-nucleotide linkage Union.

In some aspects, the sense strand contains a 2'-fluoro modification at positions 8 to 11, numbered from 5' to 3'. In some aspects, the antisense strand contains 2'-fluoro modifications at positions 2 through 5, 7, 10, and 14, numbered from 5' to 3'. In some aspects, the residual nucleotides comprise a 2'-O-methyl modification, with the proviso 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 contains phosphorothioate linkages between nucleotides at positions 1 and 2, 14 and 15, and 15 and 16, numbered from 5' to 3'. In some aspects, the antisense strand comprises a phosphorothioate linkage between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22, numbered from 5' to 3' . In some aspects, the antisense strand contains phosphorylated uridine at position 1, numbered from 5' to 3'. In some aspects, the phosphorylated nucleotide is 4'-O-monomethylphosphonate-2'-O-methyluridine. In some aspects, the lipid moiety is a C16 hydrocarbon represented by: . 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 contains at most 1 to 5, 1 to 4, 1 to 3, or 1 to 2 nucleotides that increase _Tm . In some aspects, the sense strand contains up to 1, 2, 3, 4, or 5 nucleotides that increase _Tm . In some aspects, the sense strand contains up to three nucleotides that increase _Tm . In some aspects, the sense strand contains _Tm -increasing nucleotides at positions 2, 15, and 16, numbered from 5' to 3'. In some aspects, the _Tm -increasing nucleotide is a bicyclic nucleotide, optionally a locked nucleic acid (LNA). In some aspects, the complementary region is completely complementary to the mRNA target sequence. In some aspects, the complementary region is partially complementary to the mRNA target sequence. In some aspects, the complementary region contains 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 a further aspect, the present 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 strand and the sense strand form 16 bases A duplex region of a base pair, wherein the antisense strand comprises a 5' to 3' direction, wherein the antisense strand comprises a 5' overhang and a 3' overhang, the 5' overhang comprising at least four nucleotides and the The 3' overhang includes at least two nucleotides, wherein the antisense strand includes a region complementary to the central nervous system mRNA target sequence, and wherein the sense strand includes at least one nucleotide conjugated to the 5' end of the sense strand A lipid moiety, wherein the sense strand contains _Tm -increasing nucleotides at positions 2, 15 and 16, wherein the sense strand contains a 2'-fluoro modification at positions 8 to 11 and 2 at positions 3 to 7 and 12 to 13 '-O-methyl modification, wherein the sense strand contains phosphorothioate linkages between positions 1 and 2, positions 14 and 15, and positions 15 and 16, wherein the antisense strand contains between positions 2 to 5, 7, 10 and 14 contain 2'-fluoro modifications and 2'-O-methyl modifications at positions 1, 6, 8, 9, 11 to 13 and 15 to 22, where the antisense strand is at positions 1 and 2, position 2 and 3, positions 3 and 4, positions 20 and 21, and positions 21 and 22 comprise a phosphorothioate linkage, and wherein the antisense strand comprises 5' phosphorylated uridine.

In yet a further aspect, the present 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 strand and the sense strand form 13 A duplex region of base pairs, wherein the antisense strand comprises a 5' to 3' direction, wherein the antisense strand comprises a 5' overhang and a 3' overhang, the 5' overhang comprising at least two nucleotides and the 3' overhang comprises at least seven nucleotides, wherein the antisense strand comprises a region complementary to the 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 includes up to three _Tm -increasing nucleotides, and wherein each of the antisense strand and the sense strand includes at least one 2' modified nucleotide and at least one modified inter-nucleotide linkage .

In some aspects, the sense strand contains a 2'-fluoro modification at positions 3 to 6, numbered from 5' to 3'. In some aspects, the antisense strand contains 2'-fluoro modifications at positions 2 through 5, 7, 10, and 14, numbered from 5' to 3'. In some aspects, the residual nucleotides comprise a 2'-O-methyl modification, with the proviso 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 contains phosphorothioate linkages between nucleotides at positions 1 and 2, 11 and 12, and 12 and 13, numbered from 5' to 3'. In some aspects, the antisense strand comprises a phosphorothioate linkage between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22, numbered from 5' to 3' . In some aspects, the antisense strand contains phosphorylated uridine at position 1, numbered from 5' to 3'. In some aspects, the phosphorylated nucleotide is 4'-O-monomethylphosphonate-2'-O-methyluridine. In some aspects, the lipid moiety is conjugated to nucleotide position 2 of the sense strand, numbered from 5' to 3'. In some aspects, the lipid moiety is a C22 hydrocarbon represented by: . 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 2 nucleotides and the 3' overhang is 7 nucleotides. In some aspects, the sense strand contains 1 to 3 or 1 to 2 nucleotides that increase the _Tm . In some aspects, the sense strand contains 1, 2, or 3 nucleotides that increase the _Tm . In some aspects, the sense strand contains _Tm -increasing nucleotides at (i) positions 1 and 10; or (ii) positions 1, 10, and 11, numbered from 5' to 3'. In some aspects, the _Tm -increasing nucleotide is a bicyclic nucleotide, optionally a locked nucleic acid (LNA). In some aspects, the complementary region is completely complementary to the mRNA target sequence. In some aspects, the complementary region is partially complementary to the mRNA target sequence. In some aspects, the complementary region contains no more than four mismatches to the mRNA target sequence. In some aspects, the mRNA targeting sequence is a liver targeting sequence. In some aspects, the mRNA targeting sequence is a hepatocyte targeting 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.

In yet a further aspect, the present 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 strand and the sense strand form 13 A duplex region of base pairs, wherein the antisense strand includes a direction from 5' to 3', wherein the antisense strand includes a 5' overhang and a 3' overhang, the 5' overhang includes two nucleotides and the The 3' overhang contains seven nucleotides, wherein the antisense strand contains a region complementary to the macrophage mRNA target sequence, and wherein the sense strand contains at least one lipid conjugated to the nucleotide at position 2 of the sense strand A portion wherein the sense strand contains nucleotides that increase _Tm at positions 1 and 11, wherein the sense strand contains a 2'-fluoro modification at positions 3 to 6 and a 2'-O at positions 7 to 10 and 12 to 13 -Methyl modification, wherein the sense strand contains phosphorothioate linkages between positions 1 and 2, positions 11 and 12, and positions 12 and 13, wherein the antisense strand is at positions 2 to 5, 7, 10, and 14 Contains a 2'-fluoro modification and a 2'-O-methyl modification at positions 1, 6, 8, 9, 11 to 13, and 15 to 22, wherein the antisense strand is at positions 1 and 2, positions 2 and 3, Phosphorothioate linkages are included between positions 3 and 4, positions 20 and 21, and positions 21 and 22, and wherein the antisense strand includes 5' phosphorylated uridine.

In some aspects, the present 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 strand and the sense strand form 13 bases A duplex region of a base pair, wherein the antisense strand includes a direction from 5' to 3', wherein the antisense strand includes a 5' overhang and a 3' overhang, the 5' overhang comprising two nucleotides and the 3' overhang 'The overhang contains seven nucleotides, wherein the antisense strand contains a region complementary to the macrophage mRNA target sequence, and wherein the sense strand contains at least one lipid moiety conjugated to the nucleotide at position 2 of the sense strand , wherein the sense strand contains _Tm -increasing nucleotides at positions 1, 10, and 11, wherein the sense strand contains a 2'-fluoro modification at positions 3 to 6 and 2'- at positions 7 to 19 and 12 to 13 O-methyl modification, wherein the sense strand contains phosphorothioate linkages between positions 1 and 2, positions 11 and 12, and positions 12 and 13, and wherein the antisense strand contains between positions 2 to 5, 7, 10, and 14 contains a 2'-fluoro modification and a 2'-O-methyl modification at positions 1, 6, 8, 9, 11 to 13 and 15 to 22, where the antisense strand is at positions 1 and 2, 2 and 3 , positions 3 and 4, positions 20 and 21, and positions 21 and 22 comprise phosphorothioate linkages, and wherein the antisense strand comprises 5'-terminal phosphorylated uridine.

In some aspects, the present 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 strand and the sense strand form 12 bases A duplex region of a base pair, wherein the antisense strand includes a direction from 5' to 3', wherein the antisense strand includes a 5' overhang and a 3' overhang, the 5' overhang includes at least two nucleotides and the the 3' overhang comprises at least seven nucleotides, wherein the antisense strand comprises a region complementary to the mRNA target sequence, and wherein the sense strand comprises at least one lipid moiety conjugated to the 5' end nucleotide of the sense strand, And wherein each of the antisense strand and the sense strand includes at least one 2' modified nucleotide and at least one modified inter-nucleotide linkage. In some aspects, the sense strand contains a 2'-fluoro modification at positions 3 to 6 or 4 to 7, numbered from 5' to 3'. In some aspects, the at least one modified internucleotide linkage is a phosphorothioate linkage. In some aspects, the sense strand contains phosphorothioate linkages between nucleotides at positions 1 and 2, 10 and 11, and 11 and 12, numbered from 5' to 3'. In some aspects, the antisense strand is a nucleoside between positions 1 and 2, 2 and 3, 3 and 4, 12 and 14, 14 and 15, 20 and 21, and 21 and 22, numbered from 5' to 3' The acid contains phosphorothioate linkages. 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 contains (i) 1 to 3 or 1 to 2 nucleotides that increase _Tm , or (ii) 1, 2, or 3 nucleotides that increase _Tm . In some aspects, the sense strand contains _Tm -increasing nucleotides at (i) positions 2, 10, and 11; or (ii) positions 2, 11, and 12, numbered from 5' to 3'. In some aspects, the _Tm -increasing nucleotide is a bicyclic nucleotide, optionally a locked nucleic acid (LNA).

In other aspects, the present 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 strand and the sense strand form 10 bases A duplex region of a base pair, wherein the antisense strand contains a direction from 5' to 3', wherein the antisense strand contains a 5' overhang and a 3' overhang, the 5' overhang contains one nucleotide and the 3' The overhang comprises eleven nucleotides, wherein the antisense strand comprises a region complementary to the mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to the 5' end nucleotide of the sense strand, and wherein The antisense strand and the sense strand each comprise at least one 2' modified nucleotide and at least one modified inter-nucleotide linkage. In some aspects, the at least one modified internucleotide linkage is a phosphorothioate linkage. In some aspects, the sense strand contains phosphorothioate linkages between nucleotides at positions 1 and 2, 8 and 9, and 9 and 10, numbered from 5' to 3'. In some aspects, the antisense strand is a nucleoside between positions 1 and 2, 2 and 3, 3 and 4, 12 and 14, 14 and 15, 20 and 21, and 21 and 22, numbered from 5' to 3' The acid contains phosphorothioate linkages. In some aspects, the oligonucleotide contains (i) 1 to 3 nucleotides that increase _Tm , or (ii) 1, 2, or 3 nucleotides that increase _Tm . In some aspects, the sense strand contains nucleotides at positions 2, 6, and 7 that increase the _Tm . In some aspects, the _Tm -increasing nucleotide is a bicyclic nucleotide, optionally a locked nucleic acid (LNA).

In any of the foregoing or related aspects, the antisense strand comprises 2'-fluoro modifications at positions 2 through 5, 7, 10 and 14, numbered from 5' to 3'. In some aspects, the residual nucleotides comprise a 2'-O-methyl modification, with the proviso that the nucleotide of the sense strand conjugated to the at least one lipid moiety does not comprise a 2'-O-methyl modification.

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

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

In other aspects, the present disclosure provides a method for treating an individual suffering from a disease, disorder, or condition associated with expression of a target mRNA, the method comprising administering to the individual a therapeutically effective amount of an oligonucleotide described herein. Glycosides or pharmaceutical compositions. In some aspects, the target mRNA is expressed in the central nervous system, optionally wherein the central nervous system includes the frontal cortex, hippocampus, medulla oblongata, cerebellum, lumbar dorsal root ganglia, and/or lumbar spinal cord. In some aspects, the target mRNA is expressed in neurons of the central nervous system. In some aspects, the target mRNA is expressed in macrophages. In some aspects, the macrophages are located in the liver. In some aspects, the target mRNA is expressed in the liver. In some aspects, the target mRNA is expressed in liver cells. In some aspects, the target mRNA is expressed in liver sinusoidal endothelial cells. In some aspects, the target mRNA is expressed in eye tissue. In some aspects, the target mRNA is expressed in central nervous system tissue, liver tissue, eye tissue, adipose tissue, muscle tissue, adrenal tissue, heart tissue, lung tissue, or any combination thereof.

In some aspects, the present disclosure provides a method for treating an individual suffering from a disease, disorder, or condition associated with expression of mRNA in the central nervous system, the method comprising administering to the individual a therapeutically effective amount of a therapeutic agent described herein. Oligonucleotides or pharmaceutical compositions, optionally wherein the central nervous system includes the frontal cortex, hippocampus, medulla oblongata, cerebellum, lumbar root ganglia and/or lumbar spinal cord. In some aspects, the central nervous system mRNA is neuronal mRNA.

In other aspects, the present disclosure provides a method for treating an individual suffering from a disease, disorder, or condition associated with expression of mRNA in the liver, the method comprising administering to the individual a therapeutically effective amount of an oligonucleotide described herein. Glycosides or pharmaceutical compositions.

In a further aspect, the present disclosure provides a method for treating an individual suffering from a disease, disorder or condition associated with expression of ocular mRNA, the method comprising administering to the individual a therapeutically effective amount of an oligonucleotide described herein Acid or pharmaceutical composition.

In yet a further aspect, the present disclosure provides a method for treating an individual suffering from a disease, disorder, or condition associated with expression of macrophage mRNA, the method comprising administering to the individual a therapeutically effective amount of a composition described herein Oligonucleotides or pharmaceutical compositions. In some aspects, the macrophage mRNA line is expressed in the liver.

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

In a further aspect, the present disclosure provides a method of reducing expression of a target mRNA in an individual, comprising administering to the individual 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 includes the frontal cortex, hippocampus, medulla oblongata, cerebellum, lumbar dorsal root ganglia, and/or lumbar spinal cord. In some aspects, the target mRNA is expressed in neurons 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 liver cells. In some aspects, the target mRNA is expressed in liver sinusoidal endothelial cells. In some aspects, the target mRNA is expressed in macrophages. In some aspects, the macrophages are located in the liver. In some aspects, the target mRNA is expressed in eye tissue.

In other aspects, the present disclosure provides a kit comprising an oligonucleotide as described herein, a pharmaceutically acceptable carrier if necessary, and a pharmaceutical preparation, the pharmaceutical preparation comprising administering a drug to a patient with a target mRNA. Instructions for administration to individuals with excessive manifestations of a disease, disease, or condition related to the disease, disease, or condition. In a further aspect, the present disclosure provides a kit comprising an oligonucleotide as described herein, a pharmaceutically acceptable carrier if necessary, and a pharmaceutical preparation, the pharmaceutical preparation comprising administering a drug to a patient with a target mRNA. A statement that administration to an individual has shown a reduction in the associated disease, disorder, or condition.

In some aspects, the present disclosure provides use of an oligonucleotide or pharmaceutical composition described herein for the manufacture of a medicament for the treatment of a disease, disorder, or condition associated with reduced expression of a target mRNA. In other aspects, the present disclosure provides use of an oligonucleotide or pharmaceutical composition described herein in the manufacture of a medicament for treating a disease, disorder, or condition associated with overexpression of a target mRNA. In a further aspect, the present disclosure provides an oligonucleotide or pharmaceutical composition described herein for use, or applicable for the treatment of a disease, disorder, or condition associated with the expression of a target mRNA.

In any of the foregoing or related aspects, the target mRNA is expressed in the central nervous system, neurons of the central nervous system, liver, macrophages, optionally macrophages of the liver, eye tissue, or any combination thereof , optionally wherein the central nervous system includes the frontal cortex, hippocampus, medulla oblongata, cerebellum, lumbar dorsal root ganglia and/or lumbar spinal cord.

In some aspects, the present disclosure provides a method of activating target-specific RNA interference (RNAi) in an organism, comprising administering to the organism an oligonucleotide described herein that is sufficient to An amount is administered that causes degradation of the target mRNA, thereby activating target-specific RNAi in the organism. In some aspects, the target mRNA specifies an amino acid sequence of a protein that is involved or expected to be involved in a disease or disorder in humans. In some aspects, the disease or condition is selected from the group consisting of viral infection, bacterial infection, parasitic infection, cancer, allergy, autoimmune disease, immunodeficiency, and immunosuppression.

In some aspects, the present disclosure provides a double-stranded oligonucleotide comprising an antisense strand of about 15 to 30 nucleotides in length and a sense strand of about 15 to 50 nucleotides in length, wherein the antisense strand and The sense strand forms a duplex region of approximately 15 to 30 base pairs, wherein the antisense strand includes a region complementary to the mRNA target sequence, and wherein the sense strand includes (i) a nucleotide conjugated to the sense strand At least one lipid portion and (ii) a backbone loop, wherein the backbone loop includes a nucleotide sequence represented by the formula: 5'-S1-L-S2-3', wherein S1 is complementary to S2, and wherein L forms a loop between S1 and S2, and the sense strand and the antisense strand each include a direction from 5' to 3', and the backbone loop is tied to the 5' end of the sense strand.

In any of the preceding or related aspects, the oligonucleotide comprises a blunt end. In some aspects, the blunt end includes the 3' end of the sense strand and the 5' end of the antisense strand.

In some aspects, the oligonucleotide contains an overhang of at least two nucleotides. In some aspects, the overhang includes the 5' end of the antisense strand.

In some aspects, the sense strand is about 28 to 38 nucleotides in length. In some aspects, the antisense strand is 22 nucleotides. In some aspects, the lipid moiety is conjugated to a nucleotide comprising the loop.

In any of the foregoing or related aspects, (i) The sense strand is 28 nucleotides, and the lipid part is conjugated to the nucleotide at position 4, and the positions are numbered from 5' to 3'; (ii) The sense strand is 30 nucleotides, and the lipid part is conjugated to the nucleotide at position 4, and the positions are numbered from 5' to 3'; (iii) The sense strand is 34 nucleotides, and the lipid moiety is conjugated to the nucleotide at position 6 or position 15, with the positions numbered from 5' to 3'; or (iv) The sense strand is 38 nucleotides long, and the lipid part is conjugated to the nucleotide at position 8, and the positions are numbered from 5' to 3'.

In any of the foregoing or related aspects, the lipid moiety is selected from: and .

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 preceding or related aspects, the lipid moiety is conjugated to the 2' carbon of the ribose ring of the nucleotide.

In any of the foregoing or related aspects, the complementary region is completely complementary to the mRNA target sequence. In other aspects, the complementary region is partially complementary to the mRNA target sequence. In some aspects, the complementary region contains no more than four mismatches to the mRNA target sequence.

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.

In any of the preceding or related aspects, the loop sequence is 5'-GAAA-3'. In some aspects, the loop sequence is 5'-UNCG-3', where N is any nucleotide. In some aspects, the loop sequence is 5'-UACG-3'.

In any of the preceding or related aspects, the oligonucleotide comprises at least one modified nucleotide. In some aspects, the modified nucleotide includes a 2'-modification. In some aspects, each of the nucleotides of the sense strand and the antisense strand includes a 2'-modification. In some aspects, the 2'-modification is selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy- Modification of 2'-fluoro-β-d-arabinose nucleic acid. In some aspects, the antisense strand includes 22 nucleotides from 5' to 3' from positions 1 to 22, and wherein each of positions 2, 3, 4, 5, 7, 10, and 14 includes 2'- Fluorine modification. in some aspects, (i) The sense strand is 38 nucleotides from positions 1 to 38 from 5' to 3', and each of positions 26 to 29 includes a 2'-fluorine modification; (ii) The sense strand is 34 nucleotides from positions 1 to 34 from 5' to 3', and each of positions 22 to 25 includes a 2'-fluorine modification; (iii) The sense strand is 30 nucleotides from 5' to 3' from positions 1 to 30, and each of positions 18 to 21 contains a 2'-fluoro modification; or (iv) The sense strand is 28 nucleotides from positions 1 to 28 from 5' to 3', and each of positions 18 to 21 includes a 2'-fluorine modification.

In any of the preceding or related aspects, it 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 is (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 Contains phosphorothioate linkages with positions numbered 1 to 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 the positions range from 5' Toward 3' are numbered 1 to 22. In some aspects, the sense strand contains a phosphorothioate bond between the penultimate nucleotide and the third nucleotide from the 3' end and between the penultimate nucleotide and the final nucleotide Union. in some aspects, (i) the sense strand is 38 nucleotides from positions 1 to 38 from 5' to 3', and wherein the sense strand contains a phosphorothioate linkage between positions 36 and 37 and 37 and 38; (ii) the sense strand is 34 nucleotides from 5' to 3' from positions 1 to 34, and wherein the sense strand contains a phosphorothioate linkage between positions 32 and 33 and 33 and 34; (iii) the sense strand is 30 nucleotides from 5' to 3' from positions 1 to 30, and wherein the sense strand contains a phosphorothioate linkage between positions 28 and 29 and 29 and 30; or (iv) The sense strand is 28 nucleotides from positions 1 to 28 from 5' to 3', and wherein the sense strand contains a phosphorothioate linkage between positions 26 and 27 and 27 and 28.

In any of the foregoing or related aspects, the antisense strand comprises a phosphorylated nucleotide at the 5' end, wherein the phosphorylated nucleotide is selected from the group consisting of 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 oxymethylphosphonate, vinylphosphonate, or malonylphosphonate. In some aspects, the phosphorylated nucleotide is 4'-O-monomethylphosphonate-2'-O-methyluridine.

In any of the foregoing or related aspects, the sense strand comprises at least one nucleotide that increases _Tm . In some aspects, the sense strand includes 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 nucleotides that increase _Tm . In some aspects, the sense strand contains up to 1, 2, 3, 4, 5, or 6 nucleotides that increase _Tm . In some aspects, the sense strand contains up to six nucleotides that increase _Tm . In some aspects, the _Tm -increasing nucleotide is a bicyclic nucleotide. In some aspects, the _Tm -increasing nucleotide is a locked nucleic acid.

In any of the foregoing or related aspects, S1 and S2 each comprise 1 to 6 nucleotides. In some aspects, S1 and S2 each comprise 4 nucleotides. In some aspects, S1 and S2 each comprise 2 nucleotides. In some aspects, S1 and S2 each comprise at least one nucleotide that increases _Tm . In some aspects, S1 and S2 are each 4 nucleotides, wherein 1 to 3 nucleotides of each S1 and S2 are nucleotides that increase the _Tm .

In any of the foregoing or related aspects, the residual nucleotide comprises a 2'-O-methyl modification, with the proviso 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 present disclosure provides a double-stranded oligonucleotide comprising an antisense strand of about 20 to 22 nucleotides in length and a sense strand of about 32 to 34 nucleotides in length, wherein the antisense strand and The sense strand forms a duplex region of approximately 20 to 22 base pairs and the oligonucleotide is blunt-ended, wherein the antisense strand contains a region complementary to the mRNA target sequence, and wherein the sense strand contains (i) the backbone A loop, wherein the backbone loop includes a nucleotide sequence represented by the formula: 5'-S1-L-S2-3', wherein S1 is complementary to S2, and wherein L forms a loop between S1 and S2, and (ii) at least one lipid moiety conjugated to a nucleotide of the loop, wherein the sense strand and antisense strand each comprise a 5' to 3' direction, and wherein the backbone loop is tethered to the sense strand The 5' end, and wherein each of the antisense strand and sense strand comprises at least one 2' modified nucleotide and at least one modified inter-nucleotide linkage.

In some aspects, the sense strand is 34 nucleotides and contains a 2'-fluoro modification at positions 22 to 25, numbered from 5' to 3'. In some aspects, the antisense strand is 22 nucleotides and contains 2'-fluoro modifications at positions 2 through 5, 7, 10, and 14, numbered from 5' to 3'. In some aspects, the residual nucleotides comprise a 2'-O-methyl modification, with the proviso 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 includes a phosphorothioate between nucleotides at positions 1 and 2, 2 and 3, 32 and 33, and 33 and 34, numbered from 5' to 3' Key link. In some aspects, the antisense strand is 22 nucleotides and is between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22, numbered from 5' to 3' Contains phosphorothioate linkages. In some aspects, the antisense strand contains phosphorylated uridine at position 1, numbered from 5' to 3'. In some aspects, the phosphorylated nucleotide is 4'-O-monomethylphosphonate-2'-O-methyluridine. In some aspects, the lipid moiety is a C16 hydrocarbon represented by: . 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 nucleotide at position 6 of the sense strand, numbered from 5' to 3'. In some aspects, S1 and S2 each comprise 1 to 6 nucleotides. In some aspects, S1 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 complementary region is completely complementary to the mRNA target sequence. In other aspects, the complementary region is partially complementary to the mRNA target sequence. In some aspects, the complementary region contains 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 present 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 strand and the sense strand form 22 bases a duplex region of a base pair and the oligonucleotide is blunt-ended, wherein the antisense strand comprises a region complementary to a central nervous system mRNA target sequence, wherein the sense strand comprises (i) a backbone loop, wherein the backbone The loop contains a nucleotide sequence represented by the formula: 5'-S1-L-S2-3', where S1 is complementary to S2, where S1 and S2 each contain 4 nucleotides, where L is between S1 and S2 and (ii) at least one lipid moiety conjugated to the nucleotides of the loop, wherein the sense strand and the antisense strand each comprise from 5' to 3 ' orientation, wherein the backbone loop is tethered to the 5' end of the sense strand, wherein the sense strand contains a 2'-fluoro modification at positions 22 to 25 and 2 at positions 1 to 5, 7 to 21, and 26 to 34 '-O-methyl modification, wherein the antisense strand contains 2'-fluoro modifications at positions 2 to 5, 7, 10, and 14 and 2 at positions 1, 6, 8, 9, 11 to 13, and 15 to 22 '-O-methyl modification, wherein the sense strand contains a phosphorothioate linkage between positions 1 and 2, 2 and 3, 32 and 33, and 33 and 34, and wherein the antisense strand is at positions 1 and 2 , 2 and 3, 3 and 4, 20 and 21, and 21 and 22 contain phosphorothioate linkages.

In some aspects, the present disclosure provides a double-stranded oligonucleotide comprising an antisense strand of about 20 to 22 nucleotides in length and a sense strand of about 26 to 28 nucleotides in length, wherein the antisense strand and The sense strand forms an asymmetric duplex region of approximately 20 to 22 base pairs, the asymmetric duplex region comprising a 3' overhang of at least 2 nucleotides of the antisense strand, wherein the antisense strand comprises A region complementary to an mRNA target sequence, wherein the sense strand includes: (i) a backbone loop, wherein the backbone loop includes a nucleotide sequence represented by the formula: 5'-S1-L-S2-3', wherein S1 is complementary to S2, and wherein L forms a loop between S1 and S2 and includes the sequence UNCG, and wherein S1 and S2 each include at least one nucleotide that increases the _Tm , and (ii) with the core of the loop At least one lipid moiety of nucleotide conjugation, wherein the sense strand includes a direction from 5' to 3', wherein the backbone loop is attached to the 5' end of the sense strand, and wherein each of the antisense strand and the sense strand They comprise at least one 2' modified nucleotide and at least one modified inter-nucleotide linkage.

In some aspects, the sense strand is 28 nucleotides and includes a 2'-fluoro modification at positions 18 to 21, numbered from 5' to 3'. In some aspects, the antisense strand is 22 nucleotides and contains 2'-fluoro modifications at positions 2 through 5, 7, 10, and 14, numbered from 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 includes a phosphorothioate linkage between nucleotides at positions 26 and 27 and 27 and 28, numbered from 5' to 3'. In some aspects, the antisense strand is 22 nucleotides and is between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22, numbered from 5' to 3' Contains phosphorothioate linkages. In some aspects, the 3' overhang is 2 nucleotides. In some aspects, the antisense strand contains phosphorylated uridine at position 1, numbered from 5' to 3'. In some aspects, the phosphorylated nucleotide is 4'-O-monomethylphosphonate-2'-O-methyluridine. In some aspects, the lipid moiety is a C16 hydrocarbon represented by: . 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 nucleotide at position 4 of the sense strand numbered from 5' to 3'. In some aspects, S1 and S2 each comprise 1 to 6 nucleotides. In some aspects, S1 and S2 are each 2 nucleotides. In some aspects, S1 and S2 each include a nucleotide that increases the _Tm . In 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 residual nucleotides comprise a 2'-O-methyl modification, with the proviso 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', where N is any nucleotide. In some aspects, L comprises the sequence 5'-UACG-3'. In some aspects, the complementary region is completely complementary to the mRNA target sequence. In some aspects, the complementary region is partially complementary to the mRNA target sequence. In some aspects, the complementary region contains 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 present 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 strand and the sense strand form 22 bases An asymmetric duplex region of a base pair, the asymmetric duplex region comprising a 2-nucleotide 3' end overhang of the antisense strand, wherein the antisense strand comprises a sequence complementary to the central nervous system mRNA target sequence A region, wherein the sense strand includes: (i) a backbone loop, wherein the backbone loop includes a nucleotide sequence represented by the formula: 5'-S1-L-S2-3', wherein S1 is complementary to S2, wherein S1 and S2 each comprise 2 nucleotides, wherein L forms a loop between S1 and S2 and comprises the sequence UNCG, and wherein S1 and S2 each comprise at least one nucleotide that increases _Tm , and (ii) with the At least one lipid portion of the nucleotide conjugate of the loop, wherein the sense strand contains a direction from 5' to 3', wherein the backbone loop is tied to the 5' end of the sense strand, and wherein the sense strand is at position 18 to 21 contains a 2'-fluoro modification and at positions 2 to 3, 5 to 7, 9 to 17, and 22 to 28 a 2'-O-methyl modification, wherein the antisense strand is at positions 2 to 5, 7, 10 and 14 contains a 2'-fluoro modification and a 2'-O-methyl modification at positions 1, 6, 8, 9, 11 to 13, and 15 to 22, where the sense strand is between positions 26 and 27 and 27 and 28 and wherein the antisense strand comprises a phosphorothioate linkage between positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22.

In some aspects, the present disclosure provides a double-stranded oligonucleotide comprising an antisense strand of about 20 to 22 nucleotides in length and a sense strand of about 32 to 34 nucleotides in length, wherein the antisense strand and The sense strand forms a duplex region of approximately 20 to 22 base pairs, and the oligonucleotide is blunt-ended, wherein the antisense strand contains a region complementary to the mRNA target sequence, wherein the sense strand contains: (i ) Backbone loop, wherein the backbone loop includes a nucleotide sequence represented by the formula: 5'-S1-L-S2-3', wherein S1 is complementary to S2, and wherein L is formed between S1 and S2 a loop, and wherein S1 and S2 each comprise at least one nucleotide that increases the _Tm , and (ii) at least one lipid moiety conjugated to the nucleotide of the loop, wherein the sense strand includes from 5' to 3' an orientation, wherein the backbone loop is attached to the 5' end of the sense strand, and wherein each of the antisense strand and the sense strand includes at least one 2' modified nucleotide and at least one modified nucleotide Inter-linkage.

In some aspects, the sense strand is 34 nucleotides and contains a 2'-fluoro modification at positions 22 to 25, numbered from 5' to 3'. In some aspects, the antisense strand is 22 nucleotides and contains 2'-fluoro modifications at positions 2 through 5, 7, 10, and 14, numbered from 5' to 3'. In some aspects, the at least one modified internucleotide linkage is a phosphorothioate linkage. In some aspects, the sense strand is 34 nucleotides and includes a phosphorothioate linkage between nucleotides at positions 32 and 33 and 33 and 34, numbered from 5' to 3'. In some aspects, the antisense strand is 22 nucleotides and is between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22, numbered from 5' to 3' Contains phosphorothioate linkages. In some aspects, the oligonucleotide includes 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 contains phosphorylated uridine at position 1, numbered from 5' to 3'. In some aspects, the phosphorylated nucleotide is 4'-O-monomethylphosphonate-2'-O-methyluridine. In some aspects, the lipid moiety is a C16 hydrocarbon represented by: . 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 nucleotide at position 6 of the sense strand, numbered from 5' to 3'. In some aspects, S1 and S2 each comprise 1 to 6 nucleotides. In some aspects, S1 and S2 are each 4 nucleotides. In some aspects, S1 and S2 each include a nucleotide that increases the _Tm . In 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 residual nucleotides comprise a 2'-O-methyl modification, with the proviso 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 complementary region is completely complementary to the mRNA target sequence. In some aspects, the complementary region is partially complementary to the mRNA target sequence. In some aspects, the complementary region contains 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 .

In some aspects, the present 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 strand and the sense strand form 22 bases a duplex region of a base pair, and the oligonucleotide is blunt-ended, wherein the antisense strand comprises a region complementary to a central nervous system mRNA target sequence, wherein the sense strand comprises: (i) a backbone loop, wherein The backbone loop includes a nucleotide sequence represented by the formula: 5'-S1-L-S2-3', where S1 is complementary to S2, where S1 and S2 each contain 4 nucleotides, and L is in S1 and S2 form a loop, wherein L includes 4 nucleotides, and wherein S1 and S2 each include at least one nucleotide that increases _Tm , and (ii) at least one nucleotide conjugated to the loop a lipid moiety, wherein the sense strand contains a 5' to 3' direction, wherein the backbone loop is tethered to the 5' end of the sense strand, wherein the sense strand contains a 2'-fluoro modification at positions 22 to 25 and at position 2 to 5, 7 to 11, 12 to 21 and 26 to 34 comprise a 2'-O-methyl modification, wherein the antisense strand comprises a 2'-fluoro modification at positions 2 to 5, 7, 10 and 14 and at position 1, 6, 8, 9, 11 to 13, and 15 to 22 contain a 2'-O-methyl modification, wherein the sense strand contains a phosphorothioate linkage between positions 32 and 33 and 33 and 34, and wherein The antisense strand contains phosphorothioate linkages between positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22.

In any of the foregoing or related aspects, the oligonucleotide is an endonuclease substrate. In a further aspect, the oligonucleotide reduces expression of the mRNA target sequence by a cell or cell population in vitro and/or in vivo.

In some aspects, the present 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.

In some aspects, the present disclosure provides a method for treating an individual suffering from a disease, disorder, or condition associated with expression of a target mRNA, the method comprising administering to the individual a therapeutically effective amount of an oligonucleotide described herein. Glycosides or pharmaceutical compositions.

In some aspects of the methods described herein, the target mRNA is expressed in the central nervous system, optionally neurons 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.

In some aspects, the present 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.

In some aspects, the present disclosure provides a method of reducing expression of a target mRNA in an individual, comprising administering to the individual 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 in neurons 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.

In some aspects, the present disclosure provides a kit comprising an oligonucleotide as described herein, an optional pharmaceutically acceptable carrier, and a pharmaceutical preparation, the pharmaceutical preparation comprising administering a drug to a patient with a target mRNA. Instructions for administration to an individual exhibiting a related disease, disorder, or condition.

In some aspects, the present disclosure provides use of an oligonucleotide or pharmaceutical composition described herein for the manufacture of a medicament for the treatment of a disease, disorder, or condition associated with the expression of a target mRNA.

In some aspects, the present disclosure provides any of the oligonucleotides or pharmaceutical compositions described herein for use in, or being applicable for, treating a disease, disorder, or condition associated with the expression of a target mRNA.

In some aspects of the methods described herein, the target mRNA is expressed in the central nervous system and/or neurons of the central nervous system. In some aspects, the target mRNA is expressed in eye tissue, optionally the retina and/or optic nerve.

In any of the preceding or related aspects of the methods described herein, the central nervous system includes the frontal cortex, hippocampus, cerebellum, brainstem, lumbar root ganglia, lumbar spinal cord, or combinations thereof.

In any of the foregoing or related aspects, the central nervous system includes the frontal cortex, hippocampus, cerebellum, brainstem, lumbar root ganglia, lumbar spinal cord, or combinations thereof.

In some aspects, the present disclosure provides a method of activating target-specific RNA interference (RNAi) in an organism, comprising administering to the organism a dsRNA oligonucleotide described herein, the oligonucleotide being An amount sufficient to cause degradation of the target mRNA is administered, thereby activating target-specific RNAi in the organism. In some aspects, the target mRNA specifies an amino acid sequence of a protein that is involved or expected to be involved in a disease or disorder in humans. In some aspects, the disease or condition is selected from the group consisting of viral infection, bacterial infection, parasitic infection, cancer, allergy, autoimmune disease, immunodeficiency, and immunosuppression.

In some aspects, the present disclosure provides a double-stranded oligonucleotide comprising an antisense strand of about 13 to 30 nucleotides in length and a sense strand of about 10 to 50 nucleotides in length, wherein the antisense strand and The sense strand is an individual strand that forms an asymmetric duplex region with an overhang of about 2 to 10 nucleotides at the 3' end of the antisense strand, where the duplex region is about 10 to 30 Nucleotides, wherein the antisense strand comprises a region complementary to the 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 present disclosure provides a double-stranded oligonucleotide comprising an antisense strand of about 13 to 30 nucleotides in length and a sense strand of about 10 to 50 nucleotides in length, wherein the antisense strand and The sense strand is an individual strand that forms an asymmetric duplex region with an overhang of about 2 to 12 nucleotides at the 3' end of the antisense strand, where the duplex region is about 10 to 30 Nucleotides, wherein the antisense strand comprises a region complementary to the 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 oligonucleotide contains a blunt end. In some aspects, the blunt end includes the 3' end of the sense strand and the 5' end of the antisense strand.

In a further aspect, the oligonucleotide comprises a backbone loop, wherein the backbone loop comprises a nucleotide sequence represented by the formula: 5'-S1-L-S2-3', wherein S1 is complementary to S2, and Wherein L forms a loop between S1 and S2, and the justice strand includes a direction from 5' to 3', and the backbone loop is tied to the 3' end of the justice strand.

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; (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.

in some aspects, (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.

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

in some aspects, (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.

In some aspects, the sense strand is 22 nucleotides numbered from positions 1 to 22 from 5' to 3'.

In some aspects, the lipid moiety is conjugated to a nucleotide of the sense strand that forms a base pair with the 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 the nucleotide at position 12 of the antisense strand. In some aspects, the lipid moiety is formed 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 sense strand and the antisense strand Base pair to nucleotide conjugation.

In a further aspect, the lipid moiety is conjugated to the nucleotide of the loop. In some aspects, the lipid moiety is conjugated to the 3' nucleotide on the sense strand. In some aspects, the lipid moiety is conjugated to the 5' nucleotide on the sense strand.

In any of the foregoing or related aspects, the lipid moiety is selected from: and .

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 some aspects, the hydrocarbon chain is a C22 hydrocarbon chain. In some aspects, the C22 hydrocarbon chain is represented by: .

In any of the preceding or related aspects, the lipid moiety is conjugated to the 2' carbon of the ribose ring of the nucleotide.

In any of the foregoing or related aspects, the complementary region is completely complementary to the mRNA target sequence. In some aspects, the complementary region is partially complementary to the mRNA target sequence. In some aspects, the complementary region contains no more than four mismatches to the mRNA target sequence.

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 the group consisting of frontal cortex, medulla oblongata, hippocampus, hypothalamus, cerebellum, lumbar spinal cord, lumbar dorsal root ganglia, and any combination thereof.

In any of the preceding or related aspects, the oligonucleotide comprises at least one modified nucleotide. In some aspects, the modified nucleotide includes a 2'-modification. In some aspects, each of the nucleotides of the sense strand and the antisense strand includes a 2'-modification. In some aspects, the 2'-modification is selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy- Modification of 2'-fluoro-β-d-arabinose nucleic acid. In some aspects, the antisense strand is 22 nucleotides numbered from 5' to 3' from positions 1 to 22, and wherein the sense strand is formed with nucleotides at positions 10 to 13 of the antisense strand Each of the nucleotides of the base pair contains a 2'-fluoro modification. In some aspects, the antisense strand is 22 nucleotides numbered from 5' to 3' from positions 1 to 22, and wherein the sense strand is at position 10, 11, 12, 13, or Each of the nucleotides forming a base pair of any combination thereof contains a 2'-fluoro modification. In some aspects, the antisense strand includes 22 nucleotides from 5' to 3' from positions 1 to 22, and wherein each of positions 2, 3, 4, 5, 7, 10, and 14 includes 2'- Fluorine modification. In some aspects, the residual nucleotides comprise a 2'-O-methyl modification, with the proviso that the nucleotide of the sense strand conjugated to the at least one lipid moiety does not comprise a 2'-O-methyl modification.

In any of the preceding 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 is (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 Contains phosphorothioate linkages with positions numbered 1 to 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 the positions range from 5' Toward 3' are numbered 1 to 22. In some aspects, the antisense strand contains phosphorothioate linkages between positions 13 and 14 and between positions 14 and 15. In some aspects, the antisense strand comprises phosphorothioate linkages 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 phosphorothioate linkages 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 is 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 Contains phosphorothioate linkages. In some aspects, the sense strand contains a phosphorothioate linkage between positions 1 and 2. In some aspects, the sense strand is between positions 1 and 2, between the penultimate nucleotide and the third nucleotide from the 3' end, and between the penultimate nucleotide and the final nucleotide. Contains phosphorothioate linkages.

In any of the foregoing or related aspects, the antisense strand comprises a phosphorylated nucleotide at the 5' end, wherein the phosphorylated nucleotide is selected from the group consisting of 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 oxymethylphosphonate, vinylphosphonate, or malonylphosphonate. In some aspects, the phosphorylated nucleotide is 4'-O-monomethylphosphonate-2'-O-methyluridine.

In any of the foregoing or related aspects, the sense strand comprises at least one nucleotide that increases _Tm . In some aspects, the sense strand contains up to nine nucleotides that increase the _Tm . In some aspects, the sense strand contains 1 to 3 nucleotides that increase the _Tm . In some aspects, S1 and S2 each comprise at least one nucleotide that increases _Tm . In some aspects, S1 and S2 are each 3 nucleotides, and each nucleotide is a nucleotide that increases the _Tm . In some aspects, the sense strand includes up to three _Tm -increasing nucleotide positions at nucleotide positions, with the proviso that the nucleotide positions are not in the backbone loop. In some aspects, the sense strand contains up to five nucleotides that increase _Tm .

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 to 20 from 5' to 3', and wherein the sense strand comprises a locked nucleic acid at the nucleotide at: (i) Position 2; (ii) Position 2 and Position 15; (iii) Position 2, Position 15 and Position 16; (iv) Position 2, Position 15, Position 16 and Position 18; or (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 to 16 from 5' to 3', and wherein the sense strand comprises a locked nucleic acid at the nucleotide 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.

In some aspects, the sense strand is 14 nucleotides in length, wherein the nucleotides are numbered 1 to 14 from 5' to 3', and wherein the sense strand comprises a locked nucleic acid at the nucleotide 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.

In some aspects, the sense strand is 12 nucleotides in length, wherein the nucleotides are numbered 1 to 12 from 5' to 3', and wherein the sense strand comprises a locked nucleic acid at the nucleotide 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.

In some aspects, the sense strand comprises a locked nucleic acid at a nucleotide that forms a base pair with a nucleotide of the antisense strand selected from: (i) Position 2; (ii) Position 3; (iii) position 5; (iv) Position 6; and (v) Any combination of (i) to (iv).

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

In some aspects, the present disclosure provides a pharmaceutical composition comprising any oligonucleotide described herein and a pharmaceutically acceptable carrier, delivery agent or excipient.

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

In some aspects, the present disclosure provides a method for treating an individual suffering from a disease, disorder, or condition associated with expression of a target mRNA, the method comprising administering to the individual a therapeutically effective amount of an oligonucleotide described herein. Glycosides or pharmaceutical compositions.

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

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 liver cells. In some aspects, the target mRNA is expressed in liver sinusoidal endothelial cells. In some aspects of the methods described herein, the target mRNA is expressed in macrophages 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 a stellate cell, a neuron, or an oligodendritic cell. In some aspects, the target mRNA is expressed in stellate cells, neurons, oligodendritic cells, or any combination thereof. In some aspects, the target mRNA is expressed in the frontal cortex, medulla oblongata, hippocampus, hypothalamus, cerebellum, lumbar spinal cord, lumbar dorsal root ganglia, or any combination thereof. In some aspects of the methods described herein, the target mRNA is expressed in eye 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.

In some aspects, the present disclosure provides a kit comprising an oligonucleotide as described herein, an optional pharmaceutically acceptable carrier, and a pharmaceutical preparation, the pharmaceutical preparation comprising administering a drug to a patient with a target mRNA. Instructions for administration to an individual exhibiting a related disease, disorder, or condition.

In some aspects, the present disclosure provides use of an oligonucleotide or pharmaceutical composition described herein for the manufacture of a medicament for the treatment of a disease, disorder, or condition associated with the expression of a target mRNA.

In some aspects, the present disclosure provides an oligonucleotide or pharmaceutical composition described herein for use in, or applicable for, the treatment of a disease, disorder, or condition associated with the expression of a target mRNA.

In any of the foregoing or related aspects, the target mRNA is expressed in the liver, optionally wherein the target mRNA is expressed in liver macrophages, liver hepatocytes, or liver sinusoidal endothelial cells. 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 group consisting of frontal cortex, medulla oblongata, hippocampus, hypothalamus, cerebellum, lumbar spinal cord, lumbar dorsal root ganglia, and any combination thereof.

In any of the foregoing or related aspects of the methods described herein, the disease or condition is selected from the group consisting of viral infection, bacterial infection, parasitic infection, cancer, allergy, autoimmune disease, immune deficiency and immunosuppression group.

In some aspects, the present disclosure provides a double-stranded oligonucleotide comprising: (i) a sense strand of 14 to 20 nucleotides in length, wherein the sense strand comprises at least one lipid moiety conjugated to the 5' end, and wherein the sense strand comprises at least one locked nucleic acid, and (ii) an antisense strand of 22 nucleotides in length, wherein the antisense strand contains a region complementary to the stellate cell target mRNA, and wherein the sense strand and antisense strand are separate strands forming an asymmetric duplex region having an overhang of about 2 to 8 nucleotides at the 3' end of the antisense strand, wherein the duplex region It is about 14 to 20 nucleotides in length.

In other aspects, the present disclosure provides a double-stranded oligonucleotide comprising: (i) a sense strand of 14 to 20 nucleotides in length, wherein the sense strand comprises at least one lipid moiety conjugated to the 5' end, and wherein the sense strand comprises at least one locked nucleic acid, and (ii) an antisense strand of 22 nucleotides in length, wherein the antisense strand contains a region complementary to the neuronal target mRNA, and wherein the sense strand and antisense strand are separate strands forming an asymmetric duplex region having an overhang of about 2 to 8 nucleotides at the 3' end of the antisense strand, wherein the duplex region It is about 14 to 20 nucleotides in length.

In a further aspect, the present disclosure provides a double-stranded oligonucleotide comprising: (i) a sense strand of 14 to 20 nucleotides in length, wherein the sense strand comprises at least one lipid moiety conjugated to the 5' end, and wherein the sense strand comprises at least one locked nucleic acid, and (ii) an antisense strand of 22 nucleotides in length, wherein the antisense strand contains a region complementary to the oligodendritic cell target mRNA, and wherein the sense strand and antisense strand are separate strands forming an asymmetric duplex region having an overhang of about 2 to 8 nucleotides at the 3' end of the antisense strand, wherein the duplex region It is about 14 to 20 nucleotides in length.

In any of the foregoing or related aspects, the sense strand is 14 nucleotides in length. In some aspects, the sense strand includes a locked nucleic acid at one or more of position 2, position 9, position 10, position 12, or position 13, numbered from 5' to 3'. In some aspects, the antisense strand is 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 contains a phosphorothioate linkage between positions 21 and 22.

In any of the foregoing or related aspects, the sense strand is 20 nucleotides in length. In some aspects, the sense strand includes a locked nucleic acid at one or more of position 2, position 15, or position 16, numbered from 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 .

In other aspects, the present disclosure provides a method of reducing the expression of target mRNA in stellate cells, comprising administering a double-stranded oligonucleotide, wherein the oligonucleotide comprises: (i) a sense strand, wherein the sense strand is 14 to 20 nucleotides in length, wherein the sense strand comprises at least one lipid moiety conjugated to the 5' end, 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 includes a region complementary to the target mRNA of the stellate cell, and wherein the sense strand and antisense strand are separate strands forming an asymmetric duplex region having an overhang of about 2 to 8 nucleotides at the 3' end of the antisense strand, wherein the duplex region is about 14 to 20 nucleotides, thereby reducing the expression of the target mRNA in the stellate cells. In some aspects, the oligonucleotide includes a blunt end comprising the 3' end of the sense strand and the 5' end of the antisense strand. In some aspects, the justice strand contains no more than 3 locked nucleic acids. In some aspects, the sense strand is 20 nucleotides in length, and wherein the reduction of the target mRNA in stellate cells is increased compared to the reduction in neurons. In some aspects, the target mRNA reduction is increased by at least 5%. In some aspects, the target mRNA reduction is increased by 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%, or at least 50% . In some aspects, the sense strand is 14 nucleotides in length, and wherein the reduction of the target mRNA in stellate cells is increased compared to the reduction in oligodendritic cells. In some aspects, the target mRNA reduction is increased by at least 5%. In some aspects, the target mRNA reduction is increased by 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%, or at least 50% . In some aspects, the sense strand is 20 nucleotides in length, and wherein the target mRNA is reduced to the same or similar levels in stellate cells and oligodendritic cells. In some aspects, the length of the sense strand is 14 nucleotides, wherein the target mRNA is reduced to the same or similar levels in stellate cells and in neurons, and wherein the target mRNA is reduced in stellate cells and neurons The decrease was increased compared to the decrease in oligodendritic cells. In some aspects, the target mRNA reduction is increased by at least 5%. In some aspects, the target mRNA reduction is increased by 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%, or at least 50% .

In other aspects, the present disclosure provides a method of reducing the expression of target mRNA in oligodendritic cells, comprising administering a double-stranded oligonucleotide, wherein the oligonucleotide comprises: (i) a sense strand, wherein the sense strand is 14 to 40 nucleotides in length, wherein the sense strand comprises at least one lipid moiety conjugated to the 5' end, 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 includes a region complementary to the target mRNA of the oligodendritic cell, and wherein the sense strand and antisense strand are separate strands forming an asymmetric duplex region having an overhang of about 2 to 8 nucleotides at the 3' end of the antisense strand, wherein the duplex region is about 14 to 20 nucleotides, thereby reducing the expression of the target mRNA in the oligodendritic cells. In some aspects, the oligonucleotide includes 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 is 36 nucleotides in length, and wherein the oligonucleotide includes a backbone loop. In some aspects, the target mRNA is reduced to the same or similar levels in stellate cells and oligodendritic cells, and wherein the reduction of the target mRNA in stellate cells and oligodendritic cells is compared to the reduction in neurons. system increases. In some aspects, the target mRNA reduction is increased by at least 5%. In some aspects, the target mRNA reduction is increased by 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%, or at least 50% .

In other aspects, the present disclosure provides a method of reducing expression of target mRNA in neurons, comprising administering a double-stranded oligonucleotide, wherein the oligonucleotide comprises: (i) a sense strand, wherein the sense strand is 14 to 20 nucleotides in length, wherein the sense strand comprises at least one lipid moiety conjugated to the 5' end, 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 includes a region complementary to the target mRNA of the neuron, and wherein the sense strand and antisense strand are separate strands forming an asymmetric duplex region having an overhang of about 2 to 8 nucleotides at the 3' end of the antisense strand, wherein the duplex region is about 14 to 20 nucleotides, thereby reducing the expression of the target mRNA in the neuron. In some aspects, the oligonucleotide includes a blunt end comprising the 3' end of the sense strand and the 5' end of the antisense strand. In some aspects, the justice strand contains no more than 5 locked nucleic acids. In some aspects, the sense strand is 14 nucleotides in length.

In some aspects, the present disclosure provides oligonucleotide-lipid conjugates (eg, RNAi oligonucleotide-lipid conjugates) that reduce expression of target genes. In other aspects, the present disclosure provides methods of treating diseases or conditions associated with expression of target genes. In other aspects, the present disclosure provides the use of lipid-conjugated RNAi oligonucleotides described herein, or pharmaceutically acceptable compositions thereof, to treat a disease or disorder associated with expression of a target gene (e.g., neurological disease and/or inappropriate gene expression). In other aspects, the present disclosure provides methods of using the lipid-conjugated RNAi oligonucleotides described herein in the manufacture of medicaments for treating diseases or conditions associated with expression of a target gene.

In nucleic acid chemistry, many different artificial nucleic acids have been developed to alter the behavior of siRNA under physiological conditions. In particular, phosphorothioate (PS), 2'-methoxy (2'-OMe), and 2'-fluoronucleic acid have been frequently used to modify the behavior, toxicity, and thermal stability of siRNA. A new class of conformationally constrained artificial nucleic acids has recently been developed, which are bridged nucleic acids (BNA). The basic structure of BNA is a bridge structure between 2'-O and 4'-C, which fixes the furanose ring into an N-type, and 2',4'-BNA gives high nucleic acid content to its nucleotide analogues. Enzyme resistance and affinity for complementary RNA. The concept of introducing large groups at the 2'-position of the furanose ring to achieve high nuclease resistance has a small impact on sugar fluctuations, while in BNA, the bicyclic structure provides steric hindrance in the phosphodiester backbone and reduces The entropy of sugar fluctuations is reduced, and thus both high nuclease resistance and RNA duplex stability are achieved. Many studies related to the use of 2',4'-BNA/locked nucleic acid (LNA) modifications in siRNA have been performed in vitro. Similarly, locked nucleic acids (LNAs) are a class of nucleic acid analogs that exhibit increased hybridization affinity to complementary DNA and RNA sequences, and thus they are also useful in designing RNAi trigger constructs. Structural studies indicate that LNA is an RNA mimetic that fits A-type duplex geometry seamlessly. Several reports have shown that LNA is the most promising molecule for developing oligonucleotide-based therapies.

According to the current revelation, the thermal stability provided by LNA and BNA allows modification of potential RNAi structures. For example, significant truncations can be made at the passenger/sense strand of a given trigger, and with appropriate addition and use of LNA or BNA, the gene knockdown activity of each shorter passenger strand can be rescued. The result is an LNS-enhanced RNAi trigger that is lighter in weight and equally active in physiological systems. Similarly, phosphorothioate molecules can be used to control or reduce nuclease attack on a given RNAi trigger substructure. In this sense, chemical modification of a given RNAi trigger can be a balancing act between placing modifications such as LNA, BNA, or phosphorothioate (PS) to protect it from nuclease degradation or loss of thermal stability . Lipid-conjugated RNAi oligonucleotides

In particular, the present disclosure provides lipid-conjugated RNAi oligonucleotides (eg, RNAi oligonucleotide-lipid conjugates) that reduce expression of target genes. In some embodiments, lipid-conjugated RNAi oligonucleotides provided by the present disclosure are targeted to mRNA encoding a target gene. The messenger RNA (mRNA) encoding the target gene and targeted by the lipid-conjugated RNAi oligonucleotides of the present disclosure is referred to herein as "target mRNA". mRNA target sequence

In some embodiments, lipid-conjugated RNAi oligonucleotides target target sequences comprising target mRNA. In some embodiments, lipid-conjugated RNAi oligonucleotides target target sequences within the target mRNA. In some embodiments, a lipid-conjugated RNAi oligonucleotide, or a portion, fragment, or strand thereof (e.g., an antisense or guide strand of a double-stranded oligonucleotide) is combined with a target sequence comprising a target mRNA. Bonding or adhesion, thereby reducing target gene expression. In some embodiments, lipid-conjugated RNAi oligonucleotides target target sequences comprising target mRNA for the purpose of reducing expression of the target gene in vivo. In some embodiments, the amount or degree of reduction in target gene expression by a lipid-conjugated RNAi oligonucleotide that targets a specific target sequence is related to the efficacy of the lipid-conjugated RNAi oligonucleotide. Union. In some embodiments, the amount or degree of reduction in target gene expression by a lipid-conjugated RNAi oligonucleotide that targets a specific target sequence is comparable to treatment with a lipid-conjugated RNAi oligonucleotide. Correlates the amount or degree of therapeutic benefit to an individual or patient suffering from a disease, disorder, or condition associated with expression of a target gene.

By examining the nucleotide sequences of mRNAs encoding target genes, including those from a variety of different species (e.g., humans, Malay monkeys, mice, and rats) and as a result of in vitro and in vivo tests, certain Certain systematic modifications of nucleotide sequences and those oligonucleotides are more suitable than others for reduction mediated by RNAi oligonucleotides and are therefore useful as oligos for targeting specific gene target sequences in other ways. part of a nucleotide. In some embodiments, the sense strand of a lipid-conjugated RNAi oligonucleotide described herein, or a portion or fragment thereof, comprises a target sequence similar to that comprising the target mRNA (e.g., having no more than 4 errors). match) or the same nucleotide sequence. In some embodiments, a portion or region of the sense strand of a double-stranded oligonucleotide described herein includes a target sequence that includes a target mRNA.

In some embodiments, the target mRNA is expressed in tissues or cells of the individual. In some embodiments, the target mRNA is expressed in more than one tissue or cell of an individual, wherein the tissues or cells are different. In some embodiments, the target mRNA is differentially expressed in tissues or cells of an individual. In some embodiments, the target mRNA is expressed across multiple tissue types and/or cell types. In some embodiments, the target mRNA is expressed in the central nervous system, liver tissue, eye tissue, adipose tissue, adrenal tissue or skeletal muscle tissue. In some embodiments, the target mRNA is expressed in the central nervous system, peripheral nervous system, liver tissue, eye tissue, adipose tissue, adrenal tissue, heart tissue, lung tissue, skeletal muscle tissue, cardiac 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 peripheral nervous system. In some embodiments, the target mRNA is expressed in the region of the central nervous system. In some embodiments, the region of the central nervous system is selected from the group consisting of eye, brain, cerebrum, cerebral cortex, frontal lobe, frontal cortex, parietal lobe, temporal lobe, occipital lobe, hippocampus, cerebellum, brainstem, dorsal root ganglion (DRG) or spinal cord. In some embodiments, the target mRNA is expressed in neurons. In some embodiments, the target mRNA is expressed in glial cells. In some embodiments, the target mRNA is expressed in neurons 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 liver cells. In some embodiments, the target mRNA is expressed in liver sinusoidal endothelial cells. In some embodiments, the target mRNA is expressed in macrophages. In some embodiments, the target mRNA is expressed in macrophages located in liver tissue. In some embodiments, the target mRNA is expressed in eye 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 cardiac 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 brain. In some embodiments, the target mRNA is expressed in the cerebellum. In some embodiments, the target mRNA is expressed in the brainstem. 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 a DRG. In some embodiments, the target mRNA is expressed in the spinal cord. RNAi oligonucleotide targeting sequence

In some embodiments, lipid-conjugated RNAi oligonucleotides provided by the present disclosure comprise targeting sequences. As used herein, the term "targeting sequence" refers to a nucleotide sequence having a region complementary to a specific nucleotide sequence comprising an mRNA. In some embodiments, lipid-conjugated RNAi oligonucleotides provided by the present disclosure comprise a gene-targeting sequence having a region complementary to a nucleotide sequence comprising a target sequence of a target mRNA.

The targeting sequence confers specificity to the lipid-conjugated RNAi oligonucleotide by bonding or adhering to the target sequence containing the target mRNA via complementary (Watson-Crick) base pairing. The ability to sexually target mRNA. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein (or strands thereof, e.g., the antisense strand or leader strand of a double-stranded oligonucleotide) comprise a targeting sequence having a complementary region that The complementary region bonds or adheres to the target sequence containing the target mRNA through complementary (Wasen-Click) base pairing. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein (or strands thereof, e.g., the antisense strand or leader strand of a double-stranded oligonucleotide) comprise a targeting sequence having a complementary region that The complementary region bonds or adheres to the target sequence within the target mRNA through complementary (Wasen-Click) base pairing. The targeting sequence generally has a suitable length and base content so that the lipid-conjugated RNAi oligonucleotide (or strand thereof) can bond or adhere to the specific target mRNA for the purpose of inhibiting the expression of the target gene. . In some embodiments, the length of 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 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 in length (eg, 12 to 30, 12 to 22, 15 to 25, 17 to 21, 18 to 27, 19 to 27, or 15 to 30) nucleotides. In some embodiments, the length of 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 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.

In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a targeting sequence that is completely complementary to a target sequence comprising a target mRNA. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a targeting sequence that is completely complementary to a target sequence within the target mRNA. In some embodiments, the targeting sequence is partially complementary to a target sequence comprising the target mRNA. In some embodiments, the targeting sequence is partially complementary to the target sequence within the target mRNA. In some embodiments, the targeting sequence includes a region of contiguous nucleotides that includes the antisense strand.

In some embodiments, lipid-conjugated RNAi oligonucleotides herein comprise a targeting sequence complementary to a contiguous sequence of nucleotides comprising the target mRNA, wherein the contiguous sequence of nucleotides is about 12 in length to about 30 nucleotides (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 oligonucleotide comprises a targeting sequence complementary to a contiguous sequence of nucleotides comprising the target mRNA, wherein the length of the contiguous sequence of nucleotides is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a targeting sequence complementary to a contiguous sequence of nucleotides comprising the target mRNA, wherein the contiguous sequence of nucleotides is 15 nucleotides in length acid. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a targeting sequence complementary to a contiguous sequence of nucleotides comprising the target mRNA, wherein the contiguous sequence of nucleotides is 19 nucleotides in length acid.

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

In some embodiments, the targeting sequence of the lipid-conjugated RNAi oligonucleotides herein is completely complementary (e.g., without mismatch) to the target sequence comprising the target mRNA and comprises the full length of the antisense strand . In some embodiments, the targeting sequence of the lipid-conjugated RNAi oligonucleotides herein is completely complementary (e.g., without mismatch) to the target sequence comprising the target mRNA and comprises the full length of the antisense strand part. In some embodiments, the targeting sequence of the lipid-conjugated RNAi oligonucleotides herein is completely complementary (e.g., no mismatch) to the target sequence comprising the target mRNA and includes 10 of the antisense strands. to 20 nucleotides. In some embodiments, the targeting sequence of the lipid-conjugated RNAi oligonucleotides herein is completely complementary (e.g., no mismatch) to the target sequence comprising the target mRNA and includes the antisense strand. to 19 nucleotides. In some embodiments, the targeting sequence of the lipid-conjugated RNAi oligonucleotides herein is completely complementary (e.g., no mismatch) to the target sequence comprising the target mRNA and includes the antisense strand. Nucleotides, 13 Nucleotides, 14 Nucleotides, 15 Nucleotides, 16 Nucleotides, 17 Nucleotides, 18 Nucleotides, 19 Nucleotides, 20 Nucleotides nucleotides, 21 nucleotides, or 22 nucleotides. In some embodiments, the targeting sequence of the lipid-conjugated RNAi oligonucleotides herein is completely complementary (e.g., no mismatch) to the target sequence comprising the target mRNA and includes the antisense strand. nucleotides.

In some embodiments, the targeting sequence of the lipid-conjugated RNAi oligonucleotides herein is partially complementary (e.g., with no more than 4 mismatches) to the target sequence comprising the target mRNA and includes anti- The total length of the stock. In some embodiments, the targeting sequence of the lipid-conjugated RNAi oligonucleotides herein is partially complementary (e.g., with no more than 4 mismatches) to the target sequence comprising the target mRNA and includes anti- Part of the full length of the stock. In some embodiments, the targeting sequence of the lipid-conjugated RNAi oligonucleotides herein is partially complementary (e.g., with no more than 4 mismatches) to the target sequence comprising the target mRNA and includes anti- 10 to 20 nucleotides of the strand. In some embodiments, the targeting sequence of the lipid-conjugated RNAi oligonucleotides herein is partially complementary (e.g., with no more than 4 mismatches) to the target sequence comprising the target mRNA and includes anti- 15 to 19 nucleotides of the strand. In some embodiments, the targeting sequence of the lipid-conjugated RNAi oligonucleotides herein is partially complementary (e.g., with no more than 4 mismatches) to the target sequence comprising the target mRNA and includes anti- 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides , 20 nucleotides, 21 nucleotides, or 22 nucleotides. In some embodiments, the targeting sequence of the lipid-conjugated RNAi oligonucleotides herein is partially complementary (e.g., with no more than 4 mismatches) to the target sequence comprising the target mRNA and includes anti- The 19 nucleotides of the righteous stock.

In some embodiments, lipid-conjugated RNAi oligonucleotides herein comprise a targeting sequence that has one or more base pairs (bp) mismatch with the corresponding target sequence comprising the 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 the target mRNA, with the proviso that The ability of the targeting sequence to bind or adhere 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 and the corresponding target sequence comprising the target mRNA comprise no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 bp of mismatch, limiting The condition is that the ability of the targeting sequence to bond or adhere to the target sequence and/or the ability of the lipid-conjugated RNAi oligonucleotide to inhibit or reduce the expression of the target gene is maintained under appropriate hybridization conditions. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a targeting sequence that has 1 mismatch with the corresponding target sequence. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a targeting sequence that has 2 mismatches with the corresponding target sequence. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a targeting sequence that has 3 mismatches with the corresponding target sequence. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a targeting sequence that has 4 mismatches with the corresponding target sequence. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a targeting sequence that has 5 mismatches with the corresponding target sequence. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a targeting sequence that has more than one mismatch (e.g., 2, 3, 4, 5 or more mismatches) with the corresponding target sequence, wherein at least 2 (eg, all) of the mismatches are located contiguously (eg, 2, 3, 4, 5 or more mismatches in a row), or wherein the mismatches are interspersed throughout the target anywhere in the sequence. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a targeting sequence that has 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 located contiguously (e.g., 2, 3, 4, 5 or more mismatches in succession), or where at least one or more non-mismatched bases The pairs are located between these mismatches, or a combination thereof. Type of oligonucleotide

Various RNAi oligonucleotide types and/or structures are useful in the methods herein to reduce target gene expression. Any of the types of RNAi oligonucleotides described herein or elsewhere are contemplated for use as a framework for targeting sequences incorporated herein for the purpose of inhibiting or reducing expression of the corresponding target gene.

In some embodiments, lipid-conjugated RNAi oligonucleotides herein inhibit target gene expression by engaging RNA interference (RNAi) pathways involving upstream or downstream endonucleases. For example, RNAi oligonucleotides have been developed with each strand having a size of about 19 to 25 nucleotides and having at least one 3' overhang of 1 to 5 nucleotides (see, eg, U.S. Patent No. 8,372,968). Longer oligonucleotides that are processed by Dicer to produce active RNAi products have also been developed (see, eg, US Pat. No. 8,883,996). Further work produces extended double-stranded oligonucleotides in which at least one end of at least one strand extends beyond the duplex targeting region, the extended double-stranded oligonucleotides comprising wherein one of the strands includes a thermodynamically The structure of a stable four-member loop structure (see, eg, US Patent Nos. 8,513,207 and 8,927,705, and International 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.

In some embodiments, the RNAi oligonucleotide conjugates herein engage the RNAi pathway downstream of an endonuclease (eg, endonuclease cleavage). In some embodiments, the RNAi oligonucleotides described herein are endonuclease substrates. In some embodiments, the RNAi oligonucleotides herein interact with endonucleases and are loaded into RISC. In some embodiments, after endogenous endonuclease processing, a double-stranded nucleic acid of 19 to 23 nucleotides in length is produced that reduces the expression of the target mRNA. In some embodiments, the lipid-conjugated RNAi oligonucleotide has an overhang at the 3' end of the sense strand (eg, is 1, 2, or 3 nucleotides in length). In some embodiments, lipid-conjugated RNAi oligonucleotides (e.g., siRNA conjugates) comprise a 21-nucleotide leader strand antisense to the target mRNA and a complementary passenger strand, where the two strands are bonded to form a 19 -bp duplex with 2 nucleotide overhang at either or both 3' ends. Longer oligonucleotide designs may also be considered, including oligonucleotides with a 23-nt leader strand and a 21-nt passenger strand, with the 3' end of the passenger strand on the right side of the molecule (the passenger strand). /5' end of the guide strand) has a blunt end, and there is a 3'-guide strand overhang of two nucleotides on the left side of the molecule (5' end of the passenger strand/3' end of the guide strand). In this type of molecule, there is a duplex region of 21 bp. See, for example, U.S. 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 regarding the double-slung construct.

In some embodiments, RNAi oligonucleotide conjugates disclosed herein comprise both having a length ranging from about 17 to 26 (eg, 17 to 26, 20 to 25, or 21 to 23) nucleotides. The righteous and antisense strands of acid. In some embodiments, lipid-conjugated RNAi oligonucleotides disclosed herein comprise a sense strand and an antisense strand both ranging in length from about 19 to 22 nucleotides. In some implementations, the sense strand and the antisense strand are of equal length. In some embodiments, lipid-conjugated RNAi oligonucleotides disclosed herein comprise a sense strand and an antisense strand, such that either the sense strand or the antisense strand, or both the sense strand and the antisense strand There is a 3' overhang. In some embodiments, for lipid-conjugated RNAi oligonucleotides having a sense strand and an antisense strand both ranging in length from about 21 to 23 nucleotides, the sense strand, antisense strand , or the length of the 3' overhang on both the sense and antisense strands is 1 or 2 nucleotides. In some embodiments, a lipid-conjugated RNAi oligonucleotide has a 22 nucleotide leader strand and a 20 nucleotide passenger strand, where on the right side of the molecule (the 3' end of the passenger strand/ There is a blunt end on the 5' end of the guide strand) and a 2-nucleotide 3'-guide overhang on the left side of the molecule (5' end of the passenger strand/3' end of the guide strand). In this type of molecule, there is a duplex region of 20 bp.

Other RNAi oligonucleotide designs for use with the compositions and methods herein include: 16-mer siRNA (see, e.g., Nucleic Acids in Chemistry and Biology , Blackburn (ed.), Royal Society of Chemistry, 2006), shRNA (e.g., have a backbone of 19 bp or less; see, e.g., Moore et al. (2010) Methods Mol. Biol. 629:141-58), blunt siRNA (e.g., have a length of 19 bp; see, e.g., Kraynack & Baker (2006) RNA 12:163-76), asymmetric siRNA (aiRNA; see e.g. Sun et al. (2008) Nat. Biotechnol. 26:1379-82), asymmetric shorter duplex siRNA (see e.g. Chang et al. (2009) Mol. Ther. 17:725-32), forked siRNA (see e.g. Hohjoh (2004) FEBS Lett. 557:193-98), and small internal segmented interfering RNA (siRNA; see e.g. Bramsen et al. (2007) Nucleic Acids Res. 35:5886-97). Further non-limiting examples of oligonucleotide structures that can be used in some embodiments to reduce or inhibit the expression of target genes are microRNA (miRNA), short hairpin RNA (shRNA), and short siRNA (see For example, Hamilton et al. (2002) EMBO J. 21:4671-79; see also, U.S. Patent Application Publication No. 2009/0099115). Antisense stocks

In some embodiments, the antisense strand of the lipid-conjugated RNAi oligonucleotide is referred to as the "lead strand." For example, antisense that engages the RNA-induced silencing complex (RISC) and binds to an Argonaute protein such as Ago2, or that engages or binds to one or more similar factors and directs silencing of the target gene stocks, so anti-negative stocks are called leading stocks. In some implementations, righteous stocks that are complementary to leading stocks are called "passenger stocks."

In some embodiments, lipid-conjugated RNAi oligonucleotides herein comprise 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. Antisense strands of up to 19, up to 17, up to 15 or up to 8 nucleotides in length). In some embodiments, lipid-conjugated RNAi oligonucleotides herein comprise at least about 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, to 22, at least 25, at least 27. An antisense strand of at least 30, at least 35 or at least 38 nucleotides in length). In some embodiments, the lengths included herein range from 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 some embodiments, lipid-conjugated RNAi oligonucleotides herein comprise antisense strands ranging from 15 to 30 nucleotides in length. In some embodiments, lipid-conjugated RNAi oligonucleotides herein comprise antisense strands ranging from 12 to 30 nucleotides in length. In some embodiments, the length of the antisense strand of any of the lipid-conjugated RNAi oligonucleotides disclosed herein is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides. In some embodiments, lipid-conjugated RNAi oligonucleotides comprise antisense strands ranging from 19 to 23 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises an antisense strand that is 19 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises an antisense strand that is 20 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises an antisense strand that is 21 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises an antisense strand that is 22 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises an antisense strand that is 23 nucleotides in length. justice unit

In some embodiments, lipid-conjugated RNAi oligonucleotides disclosed herein comprise 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) sense strand (or passenger strand). In some embodiments, lipid-conjugated RNAi oligonucleotides herein comprise 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. A sense strand of at least 36 or at least 38 nucleotides in length). In some embodiments, lipid-conjugated RNAi oligonucleotides herein comprise at least about 10 nucleotides in length (e.g., at least 10, at least 12, at least 15, at least 19, to 21, at least 25, at least 27. A sense strand of at least 30, at least 36 or at least 38 nucleotides in length). In some embodiments, lipid-conjugated RNAi oligonucleotides herein include a length ranging from 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) The justice strand of nucleotides. In some embodiments, lipid-conjugated RNAi oligonucleotides herein include a length ranging from 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 of the sense strand. In some embodiments, lipid-conjugated RNAi oligonucleotides herein comprise a sense strand ranging from 15 to 50 nucleotides in length. In some embodiments, lipid-conjugated RNAi oligonucleotides herein comprise a sense strand ranging from 18 to 38 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise lengths 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 The justice strand of nucleotides. In some embodiments, lipid-conjugated RNAi oligonucleotides herein comprise a sense strand ranging from 12 to 21 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a sense strand that is 10 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a sense strand that is 11 nucleotides in length. In some embodiments, lipid-conjugated RNAi oligonucleotides herein comprise a sense strand that is 12 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a sense strand that is 13 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a sense strand that is 14 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a sense strand that is 15 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a sense strand that is 16 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a sense strand that is 17 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a sense strand that is 18 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a sense strand that is 19 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a sense strand that is 20 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a sense strand that is 21 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a sense strand that is 22 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a sense strand that is 23 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a sense strand that is 24 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a sense strand that is 25 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a sense strand that is 26 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a sense strand that is 27 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a sense strand that is 28 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a sense strand that is 29 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a sense strand that is 30 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a sense strand that is 31 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a sense strand that is 32 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a sense strand that is 33 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a sense strand that is 34 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a sense strand that is 35 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a sense strand that is 36 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a sense strand that is 37 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a sense strand that is 38 nucleotides in length.

In some aspects, the justice strand contains a backbone loop structure at its 3' end. In some aspects, the justice strand contains a backbone loop structure at its 5' end. In some embodiments, the backbone loop is formed by intrastrand base pairing. In some aspects, the justice strand contains a backbone loop structure at its 5' end. In some embodiments, the backbone 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 backbone of the backbone loop includes a duplex that is 1 nucleotide in length. In some embodiments, the backbone of the backbone loop includes a duplex that is 2 nucleotides in length. In some embodiments, the backbone of the backbone loop includes a duplex that is 3 nucleotides in length. In some embodiments, the backbone of the backbone loop includes a duplex that is 4 nucleotides in length. In some embodiments, the backbone of the backbone loop includes a duplex that is 5 nucleotides in length. In some embodiments, the backbone of the backbone loop includes a duplex that is 6 nucleotides in length. In some embodiments, the backbone of the backbone loop includes a duplex that is 7 nucleotides in length. In some embodiments, the backbone of the backbone loop includes a duplex that is 8 nucleotides in length. In some embodiments, the backbone of the backbone loop includes a duplex that is 9 nucleotides in length. In some embodiments, the backbone of the backbone loop includes a duplex 10 nucleotides in length. In some embodiments, the backbone of the backbone loop includes a duplex 11 nucleotides in length. In some embodiments, the backbone of the backbone loop includes a duplex 12 nucleotides in length. In some embodiments, the backbone of the backbone loop includes a duplex 13 nucleotides in length. In some embodiments, the backbone of the backbone loop includes a duplex 14 nucleotides in length.

In some embodiments, the backbone loops provide lipid-conjugated RNAi oligonucleotides with protection against degradation (e.g., enzymatic degradation), promote or enhance targeting and/or delivery to target cells, tissues, or organs, Or both. For example, in some embodiments, the loop of the backbone loop provides nucleotides that include one or more modifications that promote, enhance, or increase targeting of a target mRNA (e.g., a target expressed in the CNS). target mRNA), inhibit target gene expression, and/or deliver to target cells, tissues, or organs (e.g., CNS), or combinations thereof. In some embodiments, the backbone loop itself or the modification(s) to the backbone loop do not substantially affect the inherent gene expression inhibition activity of the lipid-conjugated RNAi oligonucleotide, but promote and enhance , or increase stability (e.g., provide protection against degradation) and/or deliver the lipid-conjugated RNAi oligonucleotide to target cells, tissues, or organs (e.g., CNS). In certain embodiments, lipid-conjugated RNAi oligonucleotides herein comprise a sense strand comprising (e.g., at its 3' end) a backbone loop as shown below: S1-L-S2 , wherein S1 is complementary to S2, and wherein L is formed between S1 and S2 up to about 10 nucleotides in length (e.g., 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in length ) of a single-stranded loop. In some embodiments, loop (L) is 3 nucleotides in length. In some embodiments, loop (L) is 4 nucleotides in length.

In some aspects, the four-member loop includes the sequence 5'-GAAA-3'. In some aspects, the four-member loop includes the sequence 5'-UNCG-3'. In some aspects, the four-member loop includes the sequence 5'-UACG-3'. In some aspects, the backbone loop includes the sequence 5'-GCAGCCGAAAGGCUGC-3' (SEQ ID NO: 526).

In some aspects, the loop (L) having the backbone loop of structure S1-L-S2 as described above is a three-member loop. In some embodiments, the three-member loop includes ribonucleotides, deoxyribonucleotides, modified nucleotides, delivery ligands, and combinations thereof.

In some aspects, the loop (L) having the backbone loop of structure S1-L-S2 as described above is a four-member loop (eg, within a cutout four-member loop structure). In some embodiments, the four-member loop includes ribonucleotides, deoxyribonucleotides, modified nucleotides, delivery ligands, and combinations thereof.

In some aspects, the loop (L) having the backbone loop of structure S1-L-S2 as described above is a four-member loop as described in U.S. Patent No. 10,131,912 (e.g., in a notched four-member loop within the circle structure). duplex length

In some embodiments, the length of the duplex formed between the sense strand and the 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 some embodiments, the length of the duplex formed between the sense strand and the antisense strand is at least 12 (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21) nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand ranges from 12 to 30 nucleotides (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, the length of the duplex formed between the sense strand and the antisense strand ranges from 10 to 30 nucleotides (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, the length of the duplex formed between the sense strand and the antisense strand is 12, 13, 14, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the 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 some embodiments, the duplex formed between the sense strand and the antisense strand is 10 to 18 base pairs in length. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 15 to 30 base pairs. In some embodiments, the duplex formed between the sense strand and the antisense strand is 17 to 21 base pairs in length. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 10 base pairs. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 11 base pairs. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 12 base pairs. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 13 base pairs. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 14 base pairs. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 15 base pairs. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 16 base pairs. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 17 base pairs. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 18 base pairs. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 19 base pairs. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 20 base pairs. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 21 base pairs. In some implementations, the duplex formed between the sense strand and the antisense strand does not span the entire length of the sense strand and/or antisense strand. In some implementations, the double-stranded system between the sense strand and the antisense strand spans the entire length of the sense strand or the antisense strand. In some implementations, the double-stranded system between the sense strand and the antisense strand spans the entire length of both the sense strand and the antisense strand. oligonucleotide end

In some embodiments, lipid-conjugated RNAi oligonucleotides disclosed herein comprise a sense strand and an antisense strand, such that the sense strand or the antisense strand, or the sense strand and the antisense strand There is a 3' overhang. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein have a 5' end that is thermodynamically less stable than the other 5' end. In some embodiments, asymmetric lipid-conjugated RNAi oligonucleotides are provided that include a blunt end at the 3' end of the sense strand and an overhang at the 3' end of the antisense strand. In some embodiments, the 3' overhang on the antisense strand is 1 to 4 nucleotides in length (eg, 1, 2, 3, or 4 nucleotides in length).

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 has a length of approximately 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 approximately one (1) to two (2) nucleotides. 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 (11) nucleotides in length. In some embodiments, the 3' overhang is twelve (12) nucleotides in length. In some embodiments, the 3' overhang is thirteen (13) nucleotides in length. In some embodiments, the 3' overhang is fourteen (14) nucleotides in length. In some embodiments, the 3' overhang is fifteen (15) nucleotides in length. In some embodiments, the 3' overhang is sixteen (16) nucleotides in length. In some embodiments, the 3' overhang is seventeen (17) nucleotides in length. In some embodiments, the 3' overhang is eighteen (18) nucleotides in length. In some embodiments, the 3' overhang is nineteen (19) nucleotides in length. In some embodiments, the 3' overhang is twenty (20) nucleotides in length.

Typically, oligonucleotides used for RNAi have a two (2) nucleotide overhang on the 3' end of the antisense (leader) strand. However, other suspensions are also possible. In some embodiments, the overhang is a 3' overhang that includes 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 length. In some embodiments, the overhang is a 5' overhang comprising 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 length.

In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein the 5' end of either or both strands includes a 5'-overhang, the overhang comprising one or more nucleotides. In some embodiments, oligonucleotides herein include a sense strand and an antisense strand, wherein the sense strand includes a 5'-overhang, and the overhang includes one or more nucleotides. In some embodiments, the oligonucleotides herein include a sense strand and an antisense strand, wherein the antisense strand includes a 5'-overhang, and the overhang includes one or more nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein both the sense strand and the antisense strand comprise a 5'-overhang, the overhang comprising one or more nucleotides .

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 length of the 5' overhang is approximately 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 approximately one (1) to two (2) nucleotides. 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.

In some embodiments, the 5' overhang is 2 nucleotides and the 3' overhang is about 3 to 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.

In some embodiments, the 3' overhang is 6 to 8 nucleotides and the 5' overhang is 2 to 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 (eg, 2, 3, or 4) nucleotides at the 3' end or the 5' end of the sense strand and/or the antisense strand are modified. For example, in some embodiments, one or both nucleotides at the 3' end of the antisense strand are modified. In some embodiments, the last nucleotide at the 3' end of the antisense strand is modified, e.g., includes a 2' modification, e.g., 2'-O-methoxyethyl. In some embodiments, the last one or two nucleotides at the 3' end of the antisense strand are complementary to the target. In some embodiments, the last one or two nucleotides at the 3' end of the antisense strand are not complementary to the target.

In some embodiments, the RNAi oligonucleotide conjugates disclosed herein comprise a backbone loop structure at the 3' end of the sense strand and two overhanging nucleosides at the 3' end of the antisense strand. acid. In some embodiments, the RNAi oligonucleotide conjugates herein comprise a nicked four-member loop structure, wherein a backbone-four-member loop structure is included at the 3' end of the sense strand and at the antisense strand The 3' end of the strand contains two overhanging nucleotides.

In some embodiments, the RNAi oligonucleotide conjugates disclosed herein comprise a backbone loop structure at the 5' end of the sense strand and an overhang nucleotide at the 3' end of the antisense strand. In some embodiments, the RNAi oligonucleotide conjugates herein comprise a nicked four-member loop structure, wherein a backbone-four-member loop structure is included at the 5' end of the sense strand and at the antisense strand The 3' end of the strand contains two overhanging nucleotides.

In some embodiments, the RNAi oligonucleotide conjugates disclosed herein comprise a backbone loop structure at the 5' end of the sense strand and a blunt end at the 5' end of the antisense strand.

In some embodiments, the RNAi oligonucleotide conjugates disclosed herein comprise an overhang of 1 to 8 nucleotides at the 5' end of the sense strand and a 1 to 8 nucleotide overhang at the 5' end of the antisense strand. to an overhang of 8 nucleotides.

In some embodiments, the drape is selected from AA, GG, AG, and GA. In some embodiments, the drape is AA. In some implementations, the drape is AG. In some implementations, the drape is GA. In some embodiments, the overhanging nucleotides are GG. In some embodiments, one or both of the two terminal GG nucleotides of the antisense strand are not complementary to the target.

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

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

In some embodiments, the sense strand is 18 nucleotides and the antisense strand includes a 2 nucleotide 5' overhang and a 2 nucleotide 3' overhang. In some embodiments, the sense strand is 17 nucleotides and the antisense strand includes a 3' overhang of 3 nucleotides and a 3' overhang of 2 nucleotides. In some embodiments, the sense strand is 16 nucleotides and the antisense strand includes a 4 nucleotide 5' overhang and a 2 nucleotide 3' overhang. In some embodiments, the sense strand is 13 nucleotides and the antisense strand includes a 2 nucleotide 5' overhang and a 7 nucleotide 3' overhang. In some embodiments, the sense strand is 12 nucleotides and the antisense strand includes a 2 nucleotide 5' overhang and an 8 nucleotide 3' overhang. In some embodiments, the sense strand is 12 nucleotides and the antisense strand includes a 3' overhang of 3 nucleotides and a 3' overhang of 7 nucleotides. In some embodiments, the sense strand is 10 nucleotides and the antisense strand includes a 1 nucleotide 5' overhang and an 11 nucleotide 3' overhang.

In some embodiments, the sense strand is 18 nucleotides, the duplex region is 18 nucleotides, and the antisense strand includes a 5' overhang of 2 nucleotides and a 2 nucleotide 3' overhang. In some embodiments, the sense strand is 17 nucleotides, the duplex region is 17 nucleotides, and the antisense strand includes a 3 nucleotide 5' overhang and a 2 nucleotide 3' overhang. In some embodiments, the sense strand is 16 nucleotides, the duplex region is 16 nucleotides, and the antisense strand includes a 4 nucleotide 5' overhang and a 2 nucleotide 3' overhang. In some embodiments, the sense strand is 13 nucleotides, the duplex region is 13 nucleotides, and the antisense strand includes a 5' overhang of 2 nucleotides and a 7 nucleotide 3' overhang. In some embodiments, the sense strand is 12 nucleotides, the duplex region is 12 nucleotides, and the antisense strand includes a 5' overhang of 2 nucleotides and 8 nucleotides 3' overhang. In some embodiments, the sense strand is 12 nucleotides, the duplex region is 12 nucleotides, and the antisense strand includes a 3 nucleotide 5' overhang and a 7 nucleotide 3' overhang. In some embodiments, the sense strand is 10 nucleotides, the duplex region is 10 nucleotides, and the antisense strand includes a 5' overhang of 1 nucleotide and 11 nucleotides 3' overhang. Oligonucleotide modification

In some embodiments, RNAi oligonucleotide conjugates disclosed herein include one or more modifications. Oligonucleotides (e.g., RNAi oligonucleotides) can be modified in various ways to improve or control specificity, stability, delivery, bioavailability, resistance to nuclease degradation, immunogenicity, base-pairing properties, RNA distribution and cellular uptake and other characteristics 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 or any combination of the modifications described herein. For example, in some embodiments, an oligonucleotide described herein includes at least one modified sugar, a 5'-terminal phosphate group, at least one modified internucleoside linkage, and at least one modified the base.

The number of modifications on an oligonucleotide (eg, an RNAi oligonucleotide) and the location of those nucleotide modifications may affect the properties of the oligonucleotide. For example, oligonucleotides can be delivered in vivo by conjugating them to or including them in lipid nanoparticles (LNPs) or similar carriers. However, when the oligonucleotides are not protected by LNP or similar carriers, it may be advantageous to modify at least some of the nucleotides. Thus, in some embodiments, all or substantially all of the nucleotides of the oligonucleotide are modified. In some embodiments, more than half of the nucleotides are modified. In some embodiments, less than half of the nucleotides are modified. In some embodiments, the sugar 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 nucleotides conjugated to the lipid (e.g., the 5'-end nucleoside of the sense strand). acid). Modifications can be reversible or irreversible. In some embodiments, oligonucleotides as described herein possess sufficient properties to confer desirable properties (e.g., protection from enzymatic degradation, the ability to target a desired cell following in vivo administration, and/or thermodynamic stability ) number and type of modified nucleotides. Sugar modification

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

In some embodiments, the lipid-conjugated RNAi oligonucleotides described herein comprise at least about 1 (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, to 30, at least 35. At least 40, at least 45, at least 50, at least 55, at least 60 or more) modified nucleotides. In some embodiments, the sense strand of the lipid-conjugated RNAi oligonucleotide comprises at least about 1 (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) modified nucleotides. In some embodiments, the antisense strand of the lipid-conjugated RNAi oligonucleotide comprises at least about 1 (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, or more) modified Nucleotides.

In some embodiments, all nucleotides of the sense strand of the lipid-conjugated RNAi oligonucleotide are modified. In some embodiments, all nucleotides of the antisense strand of the lipid-conjugated RNAi oligonucleotide are modified. In some embodiments, all nucleotides of the lipid-conjugated RNAi oligonucleotide (ie, both the sense strand and the antisense strand) are modified. In some embodiments, the modified nucleotides include 2' modifications (e.g., 2'-F or 2'-OMe, 2'-MOE, and 2'-deoxy-2'-fluoro-β- d-arabinose nucleic acid).

In some embodiments, the present disclosure provides lipid-conjugated RNAi oligonucleotides with different modification patterns. In some embodiments, the modified lipid-conjugated RNAi oligonucleotide comprises a sense strand sequence having a modification pattern as shown in the Examples and Sequence Listing and modifications as shown in the Examples and Sequence Listing Pattern of antisense sequences.

In some embodiments, lipid-conjugated RNAi oligonucleotides disclosed herein comprise antisense strands having 2'-F modified nucleotides. In some embodiments, lipid-conjugated RNAi oligonucleotides disclosed herein comprise antisense strands comprising 2'-F and 2'-OMe modified nucleotides. In some embodiments, lipid-conjugated RNAi oligonucleotides disclosed herein comprise a sense strand having a 2'-F modified nucleotide. In some embodiments, lipid-conjugated RNAi oligonucleotides disclosed herein comprise a sense strand comprising 2'-F and 2'-OMe modified nucleotides. In some embodiments, lipid-conjugated RNAi oligonucleotides disclosed herein include a sense strand that includes 2'-F and 2'-OMe modified nucleotides, with the proviso that they are co-located with a lipid moiety. The conjugated nucleotides are not modified with 2'-F or 2'-OMe.

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

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 the nucleotide at one or more of positions 10 to 13 of the antisense strand are modified with a 2'-F group. In some embodiments, the sugar moiety of each nucleotide on the sense strand that is not modified with a 2'-F group or not conjugated to a lipid is modified with 2'-OMe. In some embodiments, the sugar moiety of each of nucleotides at positions 1 to 7 and 12 to 20 of the sense strand is modified with 2'-OMe.

In some embodiments, lipid-conjugated RNAi oligonucleotides provided herein comprise an antisense strand and a sense strand, the antisense strand being 22 cores numbered from 5' to 3' from positions 1 to 22 nucleotide, and the sense strand contains a 2'-fluorine modification on each of the nucleotides forming a base pair with the nucleotide at one or more of positions 10, 11, 12, and 13 of the antisense strand . In some embodiments, lipid-conjugated RNAi oligonucleotides provided herein comprise an antisense strand and a sense strand, the antisense strand being 22 cores numbered from 5' to 3' from positions 1 to 22 nucleotide, and the sense strand contains a 2'-fluoro modification at each of the nucleotides that form a base pair with the nucleotides at positions 10, 11, 12, 13, or any combination thereof, of the antisense strand. In some embodiments, lipid-conjugated RNAi oligonucleotides provided herein comprise an antisense strand and a sense strand, the antisense strand being 22 cores numbered from 5' to 3' from positions 1 to 22 nucleotide, and the sense strand contains a 2'-fluorine modification at each of the nucleotides forming a base pair with the nucleotides at positions 10, 11, 12, and 13 of the antisense strand.

In some embodiments, the sense strand includes at least one 2'-F modified nucleotide, wherein the remaining nucleotide that is not modified with a 2'-F group or conjugated to a lipid is 2'-modified. -OMe modification.

In some embodiments, the antisense strand has 7 nucleotides modified with 2'-F at the 2' position of the sugar moiety. In some embodiments, the sugar moieties at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand are modified with 2'-F. In some embodiments, the antisense strand has 14 nucleotides modified with 2'-OMe at the 2' position of the sugar moiety. In some embodiments, the sugar moieties at positions 6, 8, 9, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, and 22 of the antisense strand are modified with 2'-OMe Grooming.

In some embodiments, the sense strand has 4 nucleotides modified with 2'-F at the 2' position of the sugar moiety. In some embodiments, the sugar moieties at positions 2, 3, 8, 9, 10, and 11 of the sense strand are modified with 2'-F. In some embodiments, the sense strand has 15 nucleotides modified with 2'-OMe at the 2' position of the sugar moiety. In some embodiments, the sugar moieties at positions 6, 8, 9, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21 and 22 of the sense strand are modified with 2'-OMe .

In some embodiments, the sense strand contains a 2'-fluoro modification at positions 3 to 6 or 4 to 7, numbered from 5' to 3'. In some embodiments, the sense strand contains a 2'-fluoro modification at positions 3 to 6. In some embodiments, the sense strand contains a 2'-fluoro modification at positions 4 to 7.

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

In some embodiments, lipid-conjugated RNAi oligonucleotides provided herein comprise an antisense strand having a 2' -F modified sugar moiety, and the sugar moiety of each of the remaining nucleotides of the antisense strand is modified by a modification selected from the group consisting of: 2'-O-propargyl, 2'-O- Propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxy Ethyl (2'-MOE), 2'-O-[2-(methylamino)-2-side-oxyethyl] (2'-O-NMA), and 2'-deoxy-2' -Fluoro-β-d-arabinose nucleic acid (2'-FANA).

In some embodiments, lipid-conjugated RNAi oligonucleotides provided herein comprise an antisense strand at positions 2, 3, 4, 5, 7, 10, 14, 16 and Each of the nucleotides of 19 has a sugar moiety modified by 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand is modified by a modification selected from the group consisting of: 2' -O-propargyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2' -OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-side oxyethyl](2'-O- NMA), and 2'-deoxy-2'-fluoro-β-d-arabinose nucleic acid (2'-FANA).

In some embodiments, lipid-conjugated RNAi oligonucleotides provided herein comprise an antisense strand having a specific nucleotide at each of positions 1, 2, 5, and 14 of the antisense strand. 2'-F modified sugar moiety, and the sugar moiety of each of the remaining nucleotides of the antisense strand is modified by a modification selected from the group consisting of: 2'-O-propargyl, 2'- O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methyl Oxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-side oxyethyl] (2'-O-NMA), and 2'-deoxy- 2'-Fluoro-β-d-arabinose nucleic acid (2'-FANA).

In some embodiments, lipid-conjugated RNAi oligonucleotides provided herein comprise an antisense strand between nucleotides at positions 1, 2, 3, 5, 7, and 14 of the antisense strand. Each has a sugar moiety modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand is modified with a modification selected from the group consisting of: 2'-O-propargyl , 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2' -O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-side oxyethyl] (2'-O-NMA), and 2' -Deoxy-2'-fluoro-β-d-arabinose nucleic acid (2'-FANA).

In some embodiments, lipid-conjugated RNAi oligonucleotides provided herein comprise an antisense strand between nucleotides at positions 1, 2, 3, 5, 10, and 14 of the antisense strand. Each has a sugar moiety modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand is modified with a modification selected from the group consisting of: 2'-O-propargyl , 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2' -O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-side oxyethyl] (2'-O-NMA), and 2' -Deoxy-2'-fluoro-β-d-arabinose nucleic acid (2'-FANA).

In some embodiments, lipid-conjugated RNAi oligonucleotides provided herein comprise an antisense strand between nucleotides at positions 2, 3, 5, 7, 10, and 14 of the antisense strand. Each has a sugar moiety modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand is modified with a modification selected from the group consisting of: 2'-O-propargyl , 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2' -O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-side oxyethyl] (2'-O-NMA), and 2' -Deoxy-2'-fluoro-β-d-arabinose nucleic acid (2'-FANA).

In some embodiments, lipid-conjugated RNAi oligonucleotides provided herein comprise an antisense strand at positions 2, 3, 4, 5, 7, 10, 14, 16 and Each of the nucleotides of 19 has a sugar moiety modified by 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand is modified by a modification selected from the group consisting of: 2' -O-propargyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2' -OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-side oxyethyl](2'-O- NMA), and 2'-deoxy-2'-fluoro-β-d-arabinose nucleic acid (2'-FANA).

In some embodiments, lipid-conjugated RNAi oligonucleotides provided herein comprise an antisense strand with nucleosides at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand. Each of the acids has a sugar moiety modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand is modified with a modification selected from the group consisting of: 2'-O-yne Propyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-pentoxyethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinose nucleic acid (2'-FANA).

In some embodiments, lipid-conjugated RNAi oligonucleotides provided herein comprise antisense strands at position 1, position 2, position 3, position 4, position 5, position 6, position 7, and 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 have modified by 2'-F Sugar part.

In some embodiments, lipid-conjugated RNAi oligonucleotides provided herein comprise antisense strands at position 1, position 2, position 3, position 4, position 5, position 6, position 7, and 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 have 2'-OMe modified Sugar part.

In some embodiments, lipid-conjugated RNAi oligonucleotides provided herein comprise antisense strands 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 have been selected from the following: Modified sugar moiety of the group: 2'-O-propargyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA ), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2- Pendant oxyethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinose nucleic acid (2'-FANA).

In some embodiments, lipid-conjugated RNAi oligonucleotides provided herein comprise a sense strand having a 2'-F modified sugar moiety at positions 8 to 11. In some embodiments, lipid-conjugated RNAi oligonucleotides provided herein comprise a sense strand having a 2'-F modification at positions 3, 5, 8, 10, 12, 13, 15, and 17 The sugar part. In some embodiments, lipid-conjugated RNAi oligonucleotides provided herein comprise a sense strand having a 2'OMe modified sugar moiety at positions 1 to 7 and 12 to 17 or 12 to 20. In some embodiments, lipid-conjugated RNAi oligonucleotides provided herein comprise a sense strand having a 2'OMe modified sugar moiety at positions 2 to 7 and 12 to 17 or 12 to 20. In some embodiments, lipid-conjugated RNAi oligonucleotides provided herein comprise a sense strand having a 2'OMe modified sugar moiety at positions 1 to 6 and 12 to 17 or 12 to 20. In some embodiments, lipid-conjugated RNAi oligonucleotides provided herein comprise a sense strand having at positions 1, 2, 4, 6, 7, 9, 11, 14, 16, and 18 to 20 2'OMe modified sugar moiety. In some embodiments, lipid-conjugated RNAi oligonucleotides provided herein comprise a sense strand with each of nucleotides at positions 1 to 7 and 12 to 17 or 12-20 of the sense strand A sugar moiety having a modification selected from the group consisting of: 2'-O-propargyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2' -Aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methyl (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinose nucleic acid (2'-FANA). In some embodiments, lipid-conjugated RNAi oligonucleotides provided herein comprise a sense strand with each of nucleotides at positions 2 to 7 and 12 to 17 or 12-20 of the sense strand A sugar moiety having a modification selected from the group consisting of: 2'-O-propargyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2' -Aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methyl (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinose nucleic acid (2'-FANA). In some embodiments, lipid-conjugated RNAi oligonucleotides provided herein comprise a sense strand with each of nucleotides at positions 1 to 6 and 12 to 17 or 12 to 20 of the sense strand A sugar moiety having a modification selected from the group consisting of: 2'-O-propargyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2' -Aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methyl (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinose nucleic acid (2'-FANA). In some embodiments, lipid-conjugated RNAi oligonucleotides provided herein comprise a sense strand at positions 1, 2, 4, 6, 7, 9, 11, 14, 16 and Each of nucleotides 18 to 20 has a sugar moiety modified by a modification selected from the group consisting of: 2'-O-propargyl, 2'-O-propylamino, 2'-amine base, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-side-oxyethyl](2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinose Nucleic acid (2'-FANA).

In some embodiments, lipid-conjugated RNAi oligonucleotides provided herein comprise sense strands 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 have a 2'-F modified sugar moiety.

In some embodiments, lipid-conjugated RNAi oligonucleotides provided herein comprise a sense strand 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 have a 2'-F modified sugar moiety.

In some embodiments, lipid-conjugated RNAi oligonucleotides provided herein comprise sense strands 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 have a 2'-OMe modified sugar moiety.

In some embodiments, lipid-conjugated RNAi oligonucleotides provided herein comprise sense strands 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 have a 2'-OMe modified sugar moiety.

In some embodiments, lipid-conjugated RNAi oligonucleotides provided herein comprise a sense strand 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 has a modified sugar moiety selected from the group consisting of : 2'-O-propargyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-side oxyethyl](2' -O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinose nucleic acid (2'-FANA).

In some embodiments, lipid-conjugated RNAi oligonucleotides provided herein comprise a sense strand 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 have a group selected from the following Modified sugar part: 2'-O-propargyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2 '-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-side oxy Ethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinose nucleic acid (2'-FANA). 5'-terminal phosphate

In some embodiments, the lipid-conjugated RNAi oligonucleotides described herein comprise a 5'-terminal phosphate. In some embodiments, the 5'-terminal phosphate group of the lipid-conjugated RNAi oligonucleotide enhances the interaction with Ago2. However, oligonucleotides containing 5'-phosphate groups may be susceptible to degradation by phosphatases or other enzymes, which may limit their in vivo bioavailability. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise analogs of the 5' phosphate that are resistant to such degradation. In some embodiments, the phosphate analog is oxymethylphosphonate, vinylphosphonate, or malonylphosphonate, or a combination thereof. In some embodiments, the 5' end of the lipid-conjugated RNAi oligonucleotide strand is attached to a chemical moiety that mimics the electrostatic and steric properties of the natural 5'-phosphate group ("phosphate mimetic"). mimic)").

In some embodiments, the lipid-conjugated RNAi oligonucleotides herein have a phosphate analog at the 4'-carbon position of the sugar (referred to as a "4'-phosphate analog). ”). See, for example, International Patent Application Publication No. WO 2018/045317. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a 4'-phosphate analog at the 5'-terminal nucleotide. In some embodiments, the phosphate analog is an oxymethylphosphonate, wherein the oxygen atom of the oxymethyl is bonded to the sugar moiety (eg, at the 4' carbon) or the like. In other embodiments, the 4'-phosphate analog is a thiomethylphosphonate or an aminomethylphosphonate, in which the sulfur atom of the thiomethyl group or the nitrogen atom of the aminomethyl group is with a sugar. Partial 4'-carbon bond or the like. In some embodiments, the 4'-phosphate analog is oxymethylphosphonate. In some embodiments, the oxymethylphosphonate is represented by the formula -O-CH ₂ -PO(OH) ₂ , -O-CH ₂ -PO(OR) ₂ or -O-CH2-POOH(R) represents, where R is independently selected from H, CH ₃ , alkyl, CH ₂ CH ₂ CN, CH ₂ OCOC(CH ₃ ) ₃ , CH ₂ OCH ₂ CH ₂ Si (CH ₃ ) 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 embodiments, R is CH3. In some embodiments, the 4'-phosphate analog is 5'-methoxyphosphonate-4'-oxy. In some embodiments, the 4'-phosphate analog is 4'-oxymethylphosphonate.

In some embodiments, lipid-conjugated RNAi oligonucleotides provided herein comprise a sense strand comprising a 4'-phosphate analog at the 5'-terminal nucleotide, wherein the 5'-terminal nucleotide Contains the following structures: 4'-O-Monomethylphosphonate-2'-O-methyluridine phosphorothioate [MePhosphonate-4O-mUs, alternatively referred to as "MeMOP"] Modified internucleotide linkage

In some embodiments, lipid-conjugated RNAi oligonucleotides herein comprise modified internucleotide linkages. In some embodiments, the phosphate modification or substitution results in an oligonucleotide comprising at least about 1 (eg, at least 1, at least 2, at least 3, or at least 5) modified internucleotide linkages. In some embodiments, any 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 inter-nucleotide linkages. In some embodiments, any of the oligonucleotides disclosed herein comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified internucleotide linkages Union.

The modified inter-nucleotide linkage can be a phosphorodithioate linkage, a phosphorothioate linkage, a phosphotriester linkage, a thionoalkylphosphonate linkage, or a thioalkyl group. Phosphonate triester linkage, phosphoamide linkage, phosphonate linkage or borate phosphate linkage. In some embodiments, at least one modified internucleotide linkage of any of the oligonucleotides disclosed herein is linked to a phosphorothioate linkage.

In some embodiments, the lipid-conjugated RNAi oligonucleotides provided herein are at positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, and There is a phosphorothioate linkage between one or more of positions 3 and 4 of the strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand. In some embodiments, the oligonucleotides described herein are at positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, and positions 20 and 2 of the antisense strand. 21, and the antisense strand has a phosphorothioate linkage between each of positions 21 and 22. In some embodiments, the oligonucleotides described herein are at 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 3 of the antisense strand. 4. There is a phosphorothioate bond between positions 20 and 21 of the antisense strand and positions 21 and 22 of the antisense strand. In some embodiments, the oligonucleotides described herein are at 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, There is a phosphorothioate linkage between each of positions 2 and 3 of the antisense strand, positions 3 and 4 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand.

In some embodiments, the oligonucleotides described herein are at positions 1 and 2 of the sense strand, the third to last and penultimate positions of the sense strand, and the penultimate position of the sense strand. There is a phosphorothioate linkage between each of the and final positions.

In some embodiments, the oligonucleotides described herein are at 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 3 of the antisense strand. 4. There are phosphorothioate bonds between 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.

In some embodiments, the oligonucleotides described herein are at 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 3 of the antisense strand. 4. Antisense stocks are at positions 16 and 17, antisense stocks are at positions 17 and 18, antisense stocks are at positions 18 and 19, antisense stocks are at positions 19 and 20, antisense stocks are at positions 20 and 21, and antisense stocks The strand has a phosphorothioate linkage between positions 21 and 22.

In some embodiments, the oligonucleotides described herein are at 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 3 of the antisense strand. 4. The positions 13 and 14 of the anti-anti stock, the positions 14 and 15 of the anti-anti stock, the positions 16 and 17 of the anti-anti stock, the positions 17 and 18 of the anti-anti stock, the positions 18 and 19 of the anti-anti stock, the anti-anti stock There are phosphorothioate linkages between 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 aspects, the oligonucleotide comprises a phosphorothioate linkage between nucleotides at positions 1 and 2, 8 and 9, and 9 and 10 on the sense strand. In some embodiments, the oligonucleotide has 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 on the antisense strand Contains phosphorothioate linkages.

In some embodiments, the oligonucleotides described herein are at 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 3 of the antisense strand. 4. The positions 12 and 13 of the anti-anti-shares, the positions 13 and 14 of the anti-sense stocks, the positions 14 and 15 of the anti-sense stocks, the positions 15 and 16 of the anti-sense stocks, the positions 16 and 17 of the anti-sense stocks, the anti-sense stocks There is a phosphorothioate bond between 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 Union. In some embodiments, the oligonucleotide includes a nucleotide at position 14 of the 22-nucleotide antisense strand, wherein the nucleotide is flanked by a phosphorothioate linkage (i.e., at Phosphorothioate linkages between positions 13 and 14 and between positions 14 and 15). In some embodiments, the flanked nucleotide at position 14 is the final nucleotide of the duplex between the antisense and sense strands. In some embodiments, the oligonucleotide includes a sense strand and an antisense strand, wherein the antisense strand includes a flanked nucleotide at position 14 of the 22-nucleotide antisense strand (i.e., at position 14 phosphorothioate linkage between 13 and 14 and between positions 14 and 15), wherein the sense strand and antisense strand form a duplex and the antisense strand includes an overhang, and wherein the nucleoside at position 14 The acid is tied into this overhang.

In some embodiments, the oligonucleotide conjugates described herein comprise peptide nucleic acid (PNA). PNA is an oligonucleotide mimetic in which the sugar-phosphate backbone is replaced by a pseudopeptide skeleton composed of N-(2-aminoethyl)glycine units. The nucleobase is connected to this backbone through a two-atom carboxymethyl spacer. In some embodiments, oligonucleotide conjugates described herein comprise N-morpholino oligomers (PMOs), which comprise subunits linked through phosphorodiamidate groups. The backbone of the inter-nucleotide linkage of the methyl N-morpholine ring. base modification

In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise one or more modified nucleobases. In some embodiments, a modified nucleobase (also referred to herein as a base analog) is attached to the 1' position of the sugar moiety of the nucleotide. In some embodiments, the modified nucleobase is a nitrogen-containing base. In some embodiments, the modified nucleobase does not contain nitrogen atoms. See, for example, US Patent Application Publication No. 2008/0274462. In some embodiments, modified nucleobases comprise universal bases. In some embodiments, the modified nucleotide contains no nucleobases (missing bases).

In some embodiments, the universal base is located at the 1' position of the nucleotide sugar moiety of the modified nucleotide, or at the equivalent position of the heterocyclic moiety to which the nucleotide sugar moiety is substituted, when present in the double In a duplex, the heterocyclic moiety can be positioned relative to more than one type of base without substantially changing the structure of the duplex. In some embodiments, a single-stranded nucleic acid containing universal bases forms a duplex with the target nucleic acid compared to a reference single-stranded nucleic acid (e.g., an oligonucleotide) that is completely complementary to the target nucleic acid. The strand has a lower _Tm than the duplex formed with the complementary nucleic acid. In some embodiments, the single-stranded nucleic acid containing the universal base forms a duplex with the target nucleic acid when compared to a reference single-stranded nucleic acid in which the universal base has been replaced with a base, resulting in a single mismatch. The duplex has a higher _Tm than the duplex formed with the nucleic acid containing the mismatch.

Non-limiting examples of common bonded nucleotides include, but are not limited to, inosine, 1-β-D-ribofuranosyl-5-nitroindole, and/or 1-β-D-ribofuranosyl -3-Nitropyrrole (see U.S. Patent Application Publication No. 2007/0254362; Van Aerschot et al. (1995) Nucleic Acids Res. 23:4363-70; Loakes et al. (1995) Nucleic Acids Res. 23: 2361-66; and Loakes & Brown (1994) Nucleic Acids Res. 22:4039-43). Nucleotides that increase T _m

In some embodiments, oligonucleotides described herein comprise at least one _Tm- increasing nucleotide in the sense strand. In some embodiments, the oligonucleotide has a _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 on the sense strand. In some embodiments, the oligonucleotide has up to four _Tm- increasing nucleotides on the sense strand. In some embodiments, the oligonucleotide has up to five _Tm- increasing nucleotides on the sense strand. In some embodiments, the oligonucleotide has up to six _Tm- increasing nucleotides on 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 on the sense strand. In some embodiments, the oligonucleotide has up to ten _Tm- increasing nucleotides on the sense strand.

In some embodiments, the oligonucleotide has 1 to 2 _Tm -increasing nucleotides on the sense strand. In some embodiments, the oligonucleotide has 1 to 3 _Tm -increasing nucleotides on the sense strand. In some embodiments, the oligonucleotide has 1 to 4 _Tm -increasing nucleotides on the sense strand. In some embodiments, the oligonucleotide has 1 to 5 _Tm -increasing nucleotides on the sense strand.

In some embodiments, oligonucleotides comprising backbone loops include _Tm- increasing nucleotides in the backbone. In some embodiments, an oligonucleotide comprising a backbone loop includes a _Tm -increasing nucleotide in at least one base pair of the backbone. In some embodiments, an oligonucleotide comprising a backbone loop includes a _Tm -increasing nucleotide in one base pair of the backbone. In some embodiments, an oligonucleotide comprising a backbone loop contains _Tm -increasing nucleotides in two base pairs of the backbone. In some embodiments, an oligonucleotide comprising a backbone loop contains _Tm -increasing nucleotides within three base pairs of the backbone. In some embodiments, an oligonucleotide comprising a backbone loop includes _Tm -increasing nucleotides within four base pairs of the backbone. In some embodiments, an oligonucleotide comprising a backbone loop contains _Tm -increasing nucleotides within five base pairs of the backbone. In some embodiments, an oligonucleotide comprising a backbone loop contains _Tm -increasing nucleotides within six base pairs of the backbone.

Nucleotides that increase _Tm include, but are not limited to, bicyclic nucleotides, tricyclic nucleotides, G-clamps and their analogs, hexitol nucleotides, or modified nucleotides. In some embodiments, the _Tm -increasing nucleotide is a bicyclic nucleotide. In some embodiments, the _Tm -increasing nucleotide is a locked nucleic acid (LNA).

In some embodiments, the sense strand of the oligonucleotide includes an increased T _m at one of more than one of positions 2, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, and 19 of nucleotides. In some embodiments, the sense strand of the oligonucleotide includes a _Tm- increasing nucleotide at position 2. In some embodiments, the sense strand of the oligonucleotide includes a _Tm- increasing nucleotide at position 9. In some embodiments, the sense strand of the oligonucleotide includes a _Tm- increasing nucleotide at position 10. In some embodiments, the sense strand of the oligonucleotide includes a _Tm- increasing nucleotide at position 11. In some embodiments, the sense strand of the oligonucleotide includes a _Tm- increasing nucleotide at position 12. In some embodiments, the sense strand of the oligonucleotide includes a _Tm- increasing nucleotide at position 14. In some embodiments, the sense strand of the oligonucleotide includes a _Tm- increasing nucleotide at position 15. In some embodiments, the sense strand of the oligonucleotide includes a _Tm- increasing nucleotide at position 16. In some embodiments, the sense strand of the oligonucleotide includes a _Tm- increasing nucleotide at position 18. In some embodiments, the sense strand of the oligonucleotide includes a _Tm- increasing nucleotide at position 19.

In some embodiments, the 10-nucleotide sense strand, numbered from 5' to 3', contains a _Tm -increasing nucleotide at one or more of positions 2, 6, and 7. In some embodiments, the 10-nucleotide sense strand, numbered from 5' to 3', contains a _Tm -increasing nucleotide at position 2. In some embodiments, the 10-nucleotide sense strand, numbered from 5' to 3', contains _Tm -increasing nucleotides at positions 2 and 6. In some embodiments, the 10-nucleotide sense strand, numbered from 5' to 3', contains _Tm -increasing nucleotides at positions 2 and 7. In some embodiments, the 10-nucleotide sense strand, numbered from 5' to 3', includes nucleotides at 6 and 7 that increase the _Tm . In some embodiments, the 10-nucleotide sense strand, numbered from 5' to 3', contains _Tm -increasing nucleotides at positions 2, 6, and 7.

In some embodiments, the 12-nucleotide sense strand, with nucleotides numbered from 5' to 3', includes a _Tm -increasing nucleotide at one or more of positions 2, 7, 8, 10, and 11 . In some embodiments, the 12-nucleotide sense strand, numbered from 5' to 3', contains a _Tm -increasing nucleotide at position 2. In some embodiments, the 12-nucleotide sense strand, numbered from 5' to 3', contains _Tm -increasing nucleotides at position 2 and position 7. In some embodiments, the 12-nucleotide sense strand, numbered from 5' to 3', contains _Tm -increasing nucleotides at position 2, position 7, and position 8. In some embodiments, the 12-nucleotide sense strand, numbered from 5' to 3', contains _Tm -increasing nucleotides at position 2, position 7, position 8, and position 10. In some embodiments, the 12-nucleotide sense strand, with nucleotides numbered from 5' to 3', contains _Tm -increasing nucleotides at position 2, position 7, position 8, position 10, and position 11. In some embodiments, the 12-nucleotide sense strand, numbered from 5' to 3', contains _Tm -increasing nucleotides at position 2, position 10, and position 11. In some embodiments, the sense strand contains _Tm -increasing nucleotides at position 2, position 11, and position 12.

In some embodiments, the 14-nucleotide sense strand, nucleotides numbered from 5' to 3', includes a _Tm -increasing nucleotide at one or more of positions 2, 9, 10, 12, and 13 . In some embodiments, the 14-nucleotide sense strand, numbered from 5' to 3', contains a _Tm -increasing nucleotide at position 2. In some embodiments, the 14-nucleotide sense strand, numbered from 5' to 3', contains _Tm -increasing nucleotides at position 2 and position 9. In some embodiments, the 14-nucleotide sense strand, numbered from 5' to 3', contains _Tm -increasing nucleotides at position 2, position 9, and position 10. In some embodiments, the 14-nucleotide sense strand, numbered from 5' to 3', contains _Tm -increasing nucleotides at position 2, position 9, position 10, position 12, and position 13.

In some embodiments, the 16-nucleotide sense strand, with nucleotides numbered from 5' to 3', includes a _Tm -increasing nucleotide at one or more of positions 2, 11, 12, 14, and 15 . In some embodiments, the 16-nucleotide sense strand, numbered from 5' to 3', contains a _Tm -increasing nucleotide at position 2. In some embodiments, the 16-nucleotide sense strand, numbered from 5' to 3', contains _Tm -increasing nucleotides at position 2 and position 11. In some embodiments, the 16-nucleotide sense strand, numbered from 5' to 3', contains _Tm -increasing nucleotides at position 2, position 11, and position 12. In some embodiments, the 16-nucleotide sense strand, numbered from 5' to 3', contains _Tm -increasing nucleotides at position 2, position 11, position 12, and position 14. In some embodiments, the 16-nucleotide sense strand, numbered from 5' to 3', contains _Tm -increasing nucleotides at position 2, position 11, position 12, position 14, and position 15.

In some embodiments, the 20-nucleotide sense strand, with nucleotides numbered from 5' to 3', includes a _Tm -increasing nucleotide at one or more of positions 2, 15, 16, 18, and 19 . In some embodiments, the 20-nucleotide sense strand, numbered from 5' to 3', includes a _Tm -increasing nucleotide at position 2. In some embodiments, the 20-nucleotide sense strand, numbered from 5' to 3', contains _Tm -increasing nucleotides at position 2 and position 15. In some embodiments, the 20-nucleotide sense strand, numbered from 5' to 3', contains _Tm -increasing nucleotides at position 2, position 15, and position 16. In some embodiments, the 20-nucleotide sense strand, numbered from 5' to 3', contains _Tm -increasing nucleotides at position 2, position 15, position 16, and position 18. In some embodiments, the 20-nucleotide sense strand, numbered from 5' to 3', contains _Tm -increasing nucleotides at position 2, position 15, position 16, position 18, and position 19.

In some embodiments, the present disclosure provides an RNAi oligonucleotide for reducing target gene expression through the RNAi pathway, which includes one or more nucleotides that increase T _m and one or more nucleotides with lower A combination of binding affinity nucleotides (e.g., modified nucleotides) in which the duplex region comprising the RNAi oligonucleotide is maintained under physiological conditions and the RNAi oligonucleotide inhibits or The ability to reduce target gene expression is maintained. bicyclic nucleotide

Bicyclic nucleotides typically have a sugar moiety (including but not limited to a furanosyl group) with a 4- to 7-membered ring that contains a bridge that connects two atoms of the 4- to 7-membered ring to form a second ring, thereby creating Double ring structure. Such bicyclic nucleotides go by various names, including BNA and LNA 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; and published U.S. Application Nos. 20040219565, 20040014959, 20030207841, 20040192918, 20030224377, 20040143114, and 20030082807; each of which is incorporated herein by reference in its entirety. .

In some embodiments, the _Tm -increasing nucleotide is a bicyclic nucleotide comprising a bicyclic sugar moiety. In some embodiments, the bicyclic sugar moiety includes a first ring of 4 to 7 members and a bridge forming a North-type sugar 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.

Typically, the bridge contains 2 to 8 atoms. In some embodiments, the bridge contains 3 atoms. In some embodiments, the bridge contains 4 atoms. In some embodiments, the bridge contains 5 atoms. In some embodiments, the bridge contains 6 atoms. In some embodiments, the bridge contains 7 atoms. In some embodiments, the bridge contains 8 atoms. In some embodiments, the bridge contains more than 8 atoms.

In certain embodiments, the bicyclic sugar moiety is a substituted furanosyl group containing a bridge connecting the 2'-carbon and 4'-carbon of the furanosyl group to form a second ring. In certain embodiments, the bicyclic nucleotide has the structure of Formula I: where B is a nucleobase; where G is H, OH, NH ₂ , C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, substituted C ₁ -C ₆ alkyl group, substituted C ₂ -C ₆ alkenyl, substituted C ₂ -C ₆ alkynyl, hydroxyl, substituted hydroxyl, substituted amide, thiol or substituted thio; where X is O, S or NR ₁ , wherein R ₁ is H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, benzene or pyrene; and wherein W _a and W _b are each independently H, OH, A hydroxyl protecting group, phosphorus-containing moiety, or internucleotide linking group attaches the nucleotide represented by Formula I to another nucleotide or oligonucleotide, and wherein at least one of W _a or W _b One is an internucleotide linking group that attaches the nucleotide represented by Formula I to the oligonucleotide.

In certain embodiments of Formula I, G is H and X is NR ₁ , wherein R ₁ is benzene or pyrene. In certain embodiments of Formula I, G is H and X is S.

In certain implementations of Formula I, G is H and X is O:

In certain embodiments of Formula I, G is H and X is NR ₁ , wherein R ₁ is H, CH ₃ or OCH ₃ :

In certain embodiments of Formula I, G is OH or _NH2 and X is O.

In certain embodiments of Formula I, G is OH and X is O:

In certain embodiments of Formula I, G is NH _and X is O:

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:

In certain embodiments of Formula I, G is CH ₂ OCH ₃ and X is O:

In certain embodiments, the bicyclic nucleotide has the structure of Formula II: where B is a nucleobase; where Q ₁ is CH ₂ or O; where X is CH ₂ , O, S or NR ₁ , where R ₁ is H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy base, benzene or pyrene; where if Q ₁ is O, then X is CH ₂ ; where if Q ₁ is CH ₂ , then X is CH ₂ , O, S or NR ₁ , where R ₁ is H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, benzene or pyrene; wherein W _a and W _b are each independently H, OH, hydroxyl protecting group, phosphorus-containing moiety or the nucleotide represented by formula II is attached An internucleotide linking group attached to another nucleotide or oligonucleotide, and wherein at least one of W _a or W _b attaches the nucleotide represented by Formula II to the oligonucleotide Acidic internucleotide linking group.

In certain embodiments of Formula II, Q ₁ is O and X is CH ₂ :

In certain embodiments of Formula II, _Q1 is _CH2 and X is O:

In certain embodiments of Formula II, Q ₁ is CH ₂ and X is NR ₁ , wherein R ₁ is H, CH ₃ or OCH ₃ :

In certain embodiments of Formula II, _Q1 is _CH2 and X is NH:

In certain embodiments, the bicyclic nucleotide has the structure of Formula III: where B is a nucleobase; where Q ₂ is O or NR ₁ , where R ₁ is H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, benzene or pyrene; where X is CH ₂ , O , S or NR ₁ , where R ₁ is H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, benzene or pyrene; where if Q ₂ is O, then X is NR ₁ ; where if Q _{2 is} NR _{1 ,} then acid or an internucleotide linking group of an oligonucleotide, and wherein at least one of W _a or W _b is an internucleotide linking group that attaches the nucleotide represented by Formula III to the oligonucleotide. linking group.

In certain embodiments of Formula III, _Q2 is O and X is _NR1 . In certain embodiments of Formula III, Q ₂ is O and X is NR ₁ , wherein R ₁ is C ₁ -C ₆ alkyl. In certain embodiments of Formula III, _Q2 is O and X is _NR1 , and _R1 is H or _CH3 .

In certain embodiments of Formula III, _Q2 is O and X is _NR1 , and _R1 is _CH3 :

In certain embodiments of Formula III, _Q2 is _NR1 and X is O. In certain embodiments of Formula III, Q ₂ is NR ₁ , wherein R ₁ is C ₁ -C ₆ alkyl and X is O.

In certain embodiments of Formula III, _Q2 is _NCH3 and X is O:

In certain embodiments, the bicyclic nucleotide has the structure of Formula IV: wherein B is a nucleobase; wherein P ₁ and P ₃ are CH ₂ , P ₂ is CH ₂ or O and P ₄ is O; and wherein W _a and W _b are each independently H, OH, hydroxyl protecting group, containing a phosphorus moiety or an internucleotide linking group that attaches the nucleotide represented by Formula IV to another nucleotide or oligonucleotide, and wherein at least one of W _a or W _b is the The nucleotide represented by Formula IV is attached to the internucleotide linking group of the oligonucleotide.

In certain embodiments of Formula IV, P ₁ , P ₂ and P ₃ are CH ₂ and P ₄ is O:

In certain embodiments of Formula IV, P ₁ and P ₃ are CH ₂ , P ₂ is O and P ₄ is O:

In certain embodiments, the bicyclic nucleotide has the structure of formula Va or Vb: wherein B is a nucleobase; wherein r1, r2, r3 and r4 are each independently H, halogen, C ₁ -C ₁₂ alkyl, substituted C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, Substituted C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl; substituted C ₂ -C ₁₂ alkynyl; C ₁ -C ₁₂ alkoxy; substituted C ₁ -C ₁₂ alkoxy , OT ₁ , ST ₁ , SOT ₁ , SO ₂ T ₁ , NT ₁ T ₂ , N3, CN, C(=O)OT ₁ , C(=O)NT ₁ T ₂ , C(=O)T ₁ , OC(=O)NT ₁ T2, N(H)C(=NH)NT ₁ T ₂ , N(H)C(=O)NT ₁ T ₂ or N(H)C(=S)NT ₁ T ₂ , wherein each of T1 and T2 is independently H, C ₁ -C ₆ alkyl, or substituted C ₁ -C ₁₆ alkyl; or r1 and r2 or r3 and r4 together are =C(r5)(r6 ), where r5 and r6 are each independently H, halogen, C ₁ -C ₁₂ alkyl or substituted C ₁ -C ₁₂ alkyl; and wherein W _a and W _b are each independently H, OH, hydroxyl protection group, phosphorus-containing moiety, or internucleotide linking group that attaches the nucleotide represented by Formula V to another nucleotide or oligonucleotide, and wherein at least one of W _a or W _b is the internucleotide linking group that attaches the nucleotide represented by Formula V to the oligonucleotide.

In certain embodiments, the bicyclic sugar moiety is a substituted furanosyl group containing a bridge connecting the 2'-carbon and 4'-carbon of the furanosyl group to form a second ring in which the furanose group is attached. The bridge between the 2'-carbon and the 4'-carbon of the sugar group includes, but is not limited to: a) 4'-CH ₂ -ON(R)-2' and 4'-CH ₂ -N(R)-O-2 ', where R is H, C ₁ -C ₁₂ alkyl or protecting group, including, for example, 4'-CH ₂ -NH-O-2' (also known as BNA ^NC ), 4'-CH ₂ -N (CH ₃ )-O-2' (also known as BNA ^NC [NMe]) (as described in U.S. Patent No. 7,427,672, which is incorporated by reference in its entirety); b) 4'-CH ₂ -2';4'-(CH ₂ ) ₂ -2';4'-(CH ₂ ) ₃ -2';4'-(CH ₂ )-O-2' (also known as LNA); 4'- (CH ₂ )-S-2';4'-(CH ₂ ) ₂ -O-2' (also called ENA); 4'-CH(CH ₃ )-O-2' (also called cEt); and 4'-CH(CH ₂ OCH ₃ )-O-2' (also known as cMOE) and analogs thereof (as described in U.S. Patent No. 7,399,845, which is incorporated herein by reference in its entirety); c) 4'-C(CH ₃ )(CH ₃ )-O-2' and analogs thereof (as described in U.S. Patent No. 8,278,283, which is incorporated herein by reference in its entirety); d) 4 '-CH ₂ -N(OCH ₃ )-2' and analogs thereof (as described in U.S. Patent No. 8,278,425, which is incorporated herein by reference in its entirety); e) 4'-CH ₂ -ON (CH ₃ )-2' and analogs thereof (as described in U.S. Patent Publication No. 2004/0171570, which is incorporated herein by reference in its entirety); f) 4'-CH ₂ -C(H )(CH ₃ )-2' and analogs thereof (as described in Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-34, which is incorporated herein by reference in its entirety); and g) 4'- _CH2 -C(= _CH2 )-2' and analogs thereof (as described in U.S. Patent No. 8,278,426, which is incorporated herein by reference in its entirety).

In certain embodiments, the bicyclic nucleotide (BN) is one or more of the following: (a) methyleneoxy BN, (b) ethyloxy BN, (c) amine Oxy BN; (d) Oxyamine BN, (e) Methyl (methyleneoxy) BN (also known as constrained ethyl or cET), (f) Methylene-thio BN, ( g) methyleneamine BN, (h) methyl carbocyclic BN and (i) propylene carbocyclic BN, as shown below.

In the bicyclic nucleotides (a) to (i) above, B is a nucleobase, R ₂ is H or CH ₃ and wherein W _a and W _b are each independently H, OH, hydroxyl protecting group, phosphorus-containing moiety or an internucleotide linking group that attaches the bicyclic nucleotide to another nucleotide or oligonucleotide, and wherein at least one of W _a or W _b is the bicyclic nucleotide attached to An internucleotide linking group attached to an oligonucleotide.

In one embodiment of oxyamine BN(d), R ₂ is CH ₃ as follows (also referred to as BNA ^NC [NMe]): .

In certain embodiments, bicyclic sugar moieties and bicyclic nucleotides incorporating such bicyclic sugar moieties are further defined by isomeric configurations. In certain embodiments, the bicyclic sugar moiety or nucleotide adopts the alpha-L configuration. In certain embodiments, the bicyclic sugar moiety or nucleotide adopts the β-D configuration. For example, in certain embodiments, the bicyclic sugar moiety or nucleotide includes a 2'O,4'-C-methylene bridge (2'-O-CH ₂ -4' in an α-L configuration )(α-L LNA). In certain embodiments, the bicyclic sugar moiety or nucleotide adopts the R configuration. In certain embodiments, the bicyclic sugar moiety or nucleotide adopts the S configuration. For example, in certain embodiments, the bicyclic sugar moiety or nucleotide includes a 4'-CH( _CH3 )-O-2' bridge in the S configuration (ie, cEt). tricyclic nucleotides

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(1): 346-53; Scheidegger et al., Chemistry 2006; 12: 8014-23; Ivanova et al., Oligonucleotides 2007; 17: 54-65; each incorporated by reference in its entirety. incorporated herein.

In certain embodiments, the tricyclic nucleotide is a cyclic nucleotide (also known as cyclic DNA), in which the 3'-carbon and 5'-carbon centers are fused to a cyclopropane ring. Ethyl linkages are as described, for example, in Leumann CJ, Bioorg. Med. Chem. 2002; 10: 841-54 and published U.S. Application Nos. 2015/0259681 and 2018/0162897, each of which is incorporated by reference. into this article. In certain embodiments, the tricyclic nucleotide comprises a substituted furanose ring comprising a bridge connecting the 2'-carbon and 4'-carbon of the furanose group to form a second ring, and a third fused ring resulting from the methylene group connecting the 5'-carbon to the bridge connecting the 2'-carbon and 4'-carbon of the furanosyl group, as disclosed in, for example, U.S. Application No. No. 2015/0112055, which is incorporated herein by reference. Other nucleotides that increase _Tm

In addition to bicyclic and tricyclic nucleotides, other _Tm -increasing nucleotides may also be used in the RNAi oligonucleotides described herein. For example, in certain embodiments, the _Tm -increasing nucleotide is a G-clamp, a guanidine G-clamp, or an analog thereof (Wilds et al., Chem, 2002; 114: 123 and Wilds et al., Chim Acta 2003; 114: 123), hexitol nucleotides (Herdewijn, Chem. Biodiversity 2010; 7: 1-59) or modified nucleotides. The modified nucleotide may have a modified nucleobase, as described herein, including, for example, 5-bromo-uracil, 5-iodo-uracil, 5-propynyl-modified pyrimidine, or 2 -Aminoadenine (also known as 2,6-diaminopurine) (Deleavey et al., Chem. & Biol. 2012; 19: 937-54) or 2-thiouridine, 5-Me-thio Uridine and pseudouridine. The modified nucleotide may also have a modified sugar moiety, as described, for example, in U.S. Patent No. 8,975,389, which is incorporated herein by reference, or as described herein, in addition to the _Tm -increasing nucleotide The 2'-carbon in the sugar moiety is not modified with 2'-F or 2'-OMe.

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 is a G-clamp, a guanidine G-clamp, or the like. 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, a guanidine G-clamp, or the like. In certain embodiments, the _Tm -increasing nucleotide is a bicyclic nucleotide, a tricyclic nucleotide, a G-clamp, a guanidine G-clamp or an analog thereof, or a hexitol nucleotide.

In certain embodiments, each incorporation of the _Tm -increasing nucleotide increases the _Tm of the nucleic acid inhibitor molecule by at least 2°C. In certain embodiments, each incorporation of the _Tm -increasing nucleotide increases the _Tm of the nucleic acid inhibitor molecule by at least 3°C. In certain embodiments, each incorporation of the _Tm -increasing nucleotide increases the _Tm of the nucleic acid inhibitor molecule by at least 4°C. In certain embodiments, each incorporation of the _Tm -increasing nucleotide increases the _Tm of the nucleic acid inhibitor molecule by at least 5°C. Targeting ligand

In some embodiments, it is desirable to target the oligonucleotides of the present disclosure (eg, 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 present disclosure (eg, 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 present disclosure (eg, lipid-conjugated RNAi oligonucleotides) to one or more cells or tissues of the eye.

Such a strategy may help avoid undesirable effects in other organs or avoid excessive loss of the oligonucleotide to cells, tissues, or organs that do not benefit from the oligonucleotide. Accordingly, in some embodiments, lipid-conjugated RNAi oligonucleotides disclosed herein are modified to promote targeting and/or delivery to a specific tissue, cell, or organ (e.g., to promote targeting to a specific tissue, cell, or organ). CNS delivers this conjugate). In some embodiments, lipid-conjugated RNAi oligonucleotides comprise at least one (e.g., 1, 2, 3, 4, 5, 6, or more nucleotides) and one or more targeting ligands Conjugated nucleotides.

In some embodiments, one or more (eg, 1, 2, 3, 4, 5, or 6) nucleotides of the lipid-conjugated RNAi oligonucleotides disclosed herein are each associated with a separate Targeting ligand conjugation. In some embodiments, one nucleotide of the lipid-conjugated RNAi oligonucleotides herein is conjugated to a separate targeting ligand. In some embodiments, 2 to 4 nucleotides of the lipid-conjugated RNAi oligonucleotides herein are each conjugated to a separate targeting ligand. In some embodiments, the targeting ligand is conjugated to 2 to 4 nucleotides at either end of the sense or antisense strand (e.g., the targeting ligand is conjugated to 5 of the sense or antisense strand). The 2 to 4 nucleotide overhang or extension on the ' or 3' end is conjugated) such that the targeting ligand resembles the bristles of a toothbrush and the lipid-conjugated RNAi oligonucleotide resembles a toothbrush. For example, a lipid-conjugated RNAi oligonucleotide can comprise a backbone loop at the 5' or 3' end of the sense strand and 1, 2, 3, or 4 nucleotides of the loop of the backbone can be individually combined with Targeting ligand conjugation. In some embodiments, the lipid-conjugated RNAi oligonucleotides provided by the present disclosure can include a backbone loop at the 3' end of the sense strand, wherein the loop of the backbone loop includes a three-member loop or A four-membered loop, and the 3 or 4 nucleotides respectively containing the three-membered loop or the four-membered loop are individually conjugated to the targeting ligand.

GalNAc is a high-affinity ligand for ASGPR, which is mainly expressed on the sinusoidal surface of hepatocytes and is involved in the binding, internalization, and subsequent clearance of circulating glycoproteins containing terminal galactose or GalNAc residues. sialoglycoprotein) plays a major role. Conjugation (indirect or direct) of the GalNAc moiety to the oligonucleotides of the present disclosure can be used to target these oligonucleotides to ASGPR expressed on cells. In some embodiments, oligonucleotides of the present disclosure are conjugated to at least one or more GalNAc moieties, wherein the GalNAc moiety targets the oligonucleotide for expression on human liver cells (e.g., human hepatocytes) The ASGPR. In some embodiments, the GalNAc portion targets the oligonucleotide to the liver.

In some embodiments, oligonucleotides of the present disclosure are conjugated directly or indirectly to 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 typically is conjugated to 3 or 4 unit price GalNAc partially conjugated). In some embodiments, the oligonucleotide is conjugated to one or more divalent GalNAc, trivalent GalNAc, or tetravalent GalNAc moieties.

In some embodiments, one or more (eg, 1, 2, 3, 4, 5, or 6) of the oligonucleotides are each conjugated to a GalNAc moiety. In some embodiments, 2 to 4 nucleotides of the four-member loop are each conjugated to a separate GalNAc. In some embodiments, 1 to 3 nucleotides of the three-member loop are each conjugated to a separate GalNAc. In some embodiments, the targeting ligand is conjugated to 2 to 4 nucleotides at either end of the sense or antisense strand (e.g., the ligand is conjugated to the 5' or 5' end of the sense or antisense strand). The 2 to 4 nucleotide overhang or extension on the 3' end is conjugated) such that the GalNAc portion resembles the bristles of a toothbrush and the oligonucleotide resembles a toothbrush. In some embodiments, the GalNAc moiety is conjugated to the nucleotide of the sense strand. For example, four (4) GalNAc moieties may be conjugated to the nucleotides of the four-member loop of the sense strand, with each GalNAc moiety conjugated to 1 nucleotide.

In some embodiments, the four-member loop is any combination of adenine and guanine nucleotides. In some embodiments, the four-member loop is any combination of adenine, guanine, cytosine, and uridine nucleotides.

In some embodiments, the four-member loop (L) has a monovalent GalNAc moiety attached to any one or more guanine nucleotides of the four-member loop via any linker described herein, as follows Shown (X = heteroatom):

In some embodiments, the four-member loop (L) has a monovalent GalNAc attached to any one or more adenine nucleotides of the four-member loop via any linker described herein, as follows Indicates (X=heteroatom):

In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a monovalent GalNAc attached to a guanine nucleotide, referred to as [ademG-GalNAc] or 2'-aminodiethoxy Methanol-guanine-GalNAc, as shown below:

In some embodiments, lipid-conjugated RNAi oligonucleotides herein comprise a monovalent GalNAc attached to an adenine nucleotide, referred to as [ademA-GalNAc] or 2'-aminodiethoxy Methanol-Adenine-GalNAc, as shown below:

An example of such a conjugation comprising a loop from 5' to 3' of the nucleotide sequence GAAA is shown below (L=linker, X=heteroatom). Such a ring may appear, for example, at positions 27 to 30 of the righteous strand. In this chemical formula, is used to describe the point of attachment to the oligonucleotide strand.

Appropriate methods or chemistry (eg, click chemistry) can be used to attach the targeting ligand to the nucleotide. In some embodiments, targeting ligands are conjugated to nucleotides using click linkers. In some embodiments, an acetal-based linker is used to conjugate a targeting ligand to a nucleotide of any of the oligonucleotides described herein. Acetal-based linkers are disclosed, for example, in International Patent Application Publication No. WO 2016/100401. In some embodiments, the linker is an unstable linker. However, in other embodiments, the linker is stable. Shown below is an example of a loop containing the 5' to 3' nucleotide GAAA, where the GalNAc moiety is attached to the nucleotides of the loop using an acetal linker. Such a ring may appear, for example, at positions 27 to 30 of the righteous strand. In this chemical formula, is the attachment point to the oligonucleotide strand. or

As described above, a variety of suitable methods or chemical synthesis techniques (eg, click chemistry) can be used to attach targeting ligands to nucleotides. In some embodiments, targeting ligands are conjugated to nucleotides using click linkers. In some embodiments, an acetal-based linker is used to conjugate a targeting ligand to a nucleotide of any of the oligonucleotides described herein. Acetal-based linkers are disclosed, for example, in International Patent Application Publication No. WO 2016/100401. In some embodiments, the linker is an unstable linker. However, in other embodiments, the linker is a stable linker.

In some embodiments, a duplex extension (e.g., having up to 3, 4, 5, or 6 bp) is provided between the targeting ligand (e.g., GalNAc moiety) and the lipid-conjugated RNAi oligonucleotide. length). In some embodiments, the lipid-conjugated RNAi oligonucleotides herein do not have GalNAc conjugated thereto. lipid conjugate

In some embodiments, any of the lipid moieties described herein is conjugated to the nucleotides of the sense strand of the oligonucleotide. In some embodiments, the lipid moiety is conjugated to a terminal position of the oligonucleotide. In some embodiments, the lipid moiety is conjugated to the 5' end nucleotide of the sense strand. In some embodiments, the lipid moiety is conjugated to the 3' end nucleotide of the sense strand.

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 at each end of the sense strand. In some embodiments, the lipid moiety is conjugated to one or more internal positions on the sense strand. In some embodiments, the lipid moiety is with 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 are conjugate. In some embodiments, the lipid moiety is conjugated to position 1 on the sense strand. In some embodiments, the lipid moiety is conjugated to position 2 on the sense strand. In some embodiments, the lipid moiety is conjugated to position 4 on the sense strand. In some embodiments, the lipid moiety is conjugated to position 6 on the sense strand. In some embodiments, the lipid moiety is conjugated to position 8 on the sense strand. In some embodiments, the lipid moiety is conjugated to position 15 on the sense strand. In some embodiments, the lipid moiety is conjugated to position 28 on the sense strand. In some embodiments, the lipid moiety is conjugated to position 38 on the sense strand.

In some embodiments, the lipid moiety is the core of position 20, position 19, position 18, position 17, position 16, position 15, position 14, position 13, or position 12 of the sense strand and the antisense strand Nucleotide conjugation of nucleotides forming base pairs. 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 the 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 the 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 the 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 the 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 a base pair with the 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 the 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 the 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 the 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 the nucleotide at position 12 of the antisense strand.

In some embodiments, lipid-conjugated RNAi oligonucleotides described herein comprise at least one nucleotide conjugated to 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 adenine nucleotides. In some embodiments, one or more lipid moieties are conjugated to guanine nucleotides. In some embodiments, one or more lipid moieties are conjugated to cytosine nucleotides. In some embodiments, one or more lipid moieties are conjugated to thymine nucleotides. In some embodiments, one or more lipid moieties are conjugated to uracil nucleotides.

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 linear. In some embodiments, the lipid moiety is a C8-C30 hydrocarbon chain. In some embodiments, the lipid moiety is C8:0, C10:0, C11:0, C12:0, C14:0, C16:0, C17:0, C18:0, C18:1, C18:2 , C22:5, C22:0, C24:0, C26:0, C22:6, C24:1, diyl group C16:0 or diyl group C18:1. In some embodiments, the lipid moiety is a C16 hydrocarbon chain.

In some embodiments, the lipid moiety is conjugated to the oligonucleotide via a linker. In some embodiments, the nucleotide of the lipid-conjugated oligonucleotide is represented by Formula II-b or II-c: Or a pharmaceutically acceptable salt thereof, wherein: L ¹ is a covalent bond, a monovalent or divalent saturated or unsaturated linear or branched C _1-50 hydrocarbon chain, wherein the hydrocarbon chain has 0 to 10 methylene The base units are independently -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)- , -S(O) ₂ -, -P(O)OR-, -P(S)OR-or Replacement; R ⁴ is hydrogen, R ^A or a suitable amine protecting group; and R ⁵ is adamantyl, or a saturated or unsaturated linear or branched C _1-50 hydrocarbon chain, wherein the hydrocarbon chain has 0 to The 10 methylene units 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 substitution.

In some embodiments of the lipid-conjugated RNAi oligonucleotide, ^R5 is selected from and .

In certain embodiments of the lipid-conjugated RNAi oligonucleotide, ^R5 is selected from and .

In some implementations, R ⁵ is .

In some implementations, R ⁵ is .

In some implementations, R ⁵ is .

In some embodiments, the nucleotide of the lipid-conjugated RNAi oligonucleotide is represented by Formula II-Ib or II-Ic: or a pharmaceutically acceptable salt thereof; wherein B is a nucleobase or hydrogen; m is 1 to 50; X ¹ is -O- or -S-; Y is hydrogen, or ; R ³ is hydrogen or ^a suitable protecting group; ^{X 2} is O or S; A linking group at the 2'- or 3'-end of a nucleotide; Y ² is hydrogen, a phosphoramidite analog, a linking group attached to the 5'-end of a nucleoside, nucleotide, or oligonucleotide , or a linking group attached to a solid support; R ⁵ is an adamantyl group, or a saturated or unsaturated linear or branched C _1-50 hydrocarbon chain, wherein the hydrocarbon chain has 0 to 10 methylene groups Units are independently -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O ) ₂ -, -P(O)OR- or -P(S)OR-replacement; and R is hydrogen, a suitable protecting group, or an optionally substituted group selected from the following: C _1-6 lipid Group, phenyl, 4 to 7 membered saturated or partially unsaturated heterocyclic ring with 1 to 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and 5- to 6-membered heteroaryl rings of sulfur heteroatoms.

In some embodiments, the lipid is or .

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 includes backbone loops. In some embodiments, the backbone loop is shown as S1-L-S2, where S1 is complementary to S2, and where L forms a loop between S1 and S2. In some embodiments, the ligand is conjugated to any of the nucleotides of the loop of the backbone loop. In some embodiments, the ligand is conjugated to any of the nucleotides of the backbone of the backbone loop. In some embodiments, the ligand is conjugated to the first nucleotide from 5' to 3' of the loop. In some embodiments, the ligand is conjugated to the second nucleotide from 5' to 3' of the loop. In some embodiments, the ligand is conjugated to the third nucleotide from 5' to 3' of the loop. In some embodiments, the ligand is conjugated to the fourth nucleotide from 5' to 3' of the loop. In some embodiments, the ligand is conjugated to one, two, three, or four nucleotides in the loop. In some embodiments, the ligand is conjugated to three nucleotides in the backbone loop.

In some embodiments, the backbone loop is 16 nucleotides in length. In some embodiments, the ligand is conjugated to the third nucleotide from 5' to 3' of the backbone loop. In some embodiments, the ligand is conjugated to the eighth nucleotide from 5' to 3' of the backbone loop. In some embodiments, the ligand is conjugated to the ninth nucleotide from 5' to 3' of the backbone loop. In some embodiments, the ligand is conjugated to the tenth nucleotide from 5' to 3' of the backbone loop. Exemplary oligonucleotides

In some embodiments, lipid-conjugated RNAi oligonucleotides comprise nucleotides conjugated to fatty acids. 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 oligonucleotides comprise nucleotides conjugated to lipids. 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 to a 16-carbon (C16) lipid. In some embodiments, the C16 lipid contains at least one double bond. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a nucleotide conjugated to a 22-carbon (C22) lipid.

In some embodiments, the oligonucleotide of the lipid-conjugated RNAi oligonucleotide is conjugated to a C16 lipid as follows: .

In some embodiments, the oligonucleotide of the lipid-conjugated RNAi oligonucleotide is conjugated to a C22 lipid as follows: .

In some implementations, the 3' end of the positive strand is blunt. In some embodiments, the 5' end of the antisense strand is blunt. In some embodiments, the 3' end of the antisense strand includes an overhang. In some embodiments, the 5' end of the antisense strand includes an overhang. In some embodiments, the 5' and 3' ends of the antisense strand each include an overhang.

In some embodiments, the lipid-conjugated RNAi oligonucleotide contains one or more 2' modifications. In some embodiments, the 2' modification is selected from 2'-fluoro or 2'-methyl.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression comprise the following modification pattern 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' Cross to: Antisense: 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' where [mXs]=the phosphorothioate linkage to the adjacent nucleotide 2'- O - Methyl-modified nucleotide, [fXs] = 2'-fluoro-modified nucleotide with phosphorothioate linkage to adjacent nucleotide, [mX] = phosphate with adjacent nucleotide 2'- O -methyl modified nucleotide with diester linkage, [fX] = 2'-fluoro modified nucleotide with phosphodiester linkage with adjacent nucleotide, [MePhosphonate- 4O-mX] = nucleotide modified with 4'-O-monomethylphosphonate-2'-O-methyl, and [ademX-L] = lipid attached to nucleotide.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression comprise the following modification pattern sense strand: 5'-[+X][mX][mX][ademX-L ][mX][mX][mX] [+X][mX][mX][mX][mX][mX][mX][mX][mX][mX][fX][fX][fX] [fX][mX][mX][mX][mX][mXs][mXs][mX]-3' Cross to: Antisense: 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' where [mXs]=a 2'- O -methyl modified nucleotide having a phosphorothioate linkage with the adjacent nucleotide, [fXs]=with the adjacent nucleotide Nucleotide 2'-fluoro modified nucleotide with phosphorothioate linkage, [mX] = 2'- O -methyl modified core with phosphodiester linkage to adjacent nucleotide Urinic acid, [fX] = 2'-fluorine modified nucleotide with phosphodiester linkage to adjacent nucleotide, [MePhosphonate-4O-mX] = 4'-O-monomethylphosphonic acid Ester-2'-O-methyl modified nucleotide, [ademX-L] = lipid attached to nucleotide, [+X] = nucleotide that increases _Tm , optionally LNA, and [+ Xs] = _Tm -increasing nucleotide with phosphorothioate linkage to adjacent nucleotide, optionally LNA.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression include the following modification pattern 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' Hybridize to: antisense Stock: 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' where [mXs] = phosphorothioate linkage to adjacent nucleotide 2' -O -Methyl modified nucleotide, [fXs] = 2'-fluoro modified nucleotide having phosphorothioate linkage with adjacent nucleotide, [mX] = with adjacent nucleotide 2'- O -methyl modified nucleotide having a phosphodiester linkage, [fX] = 2'-fluoro modified nucleotide having a phosphodiester linkage to the adjacent nucleotide, [ MePhosphonate-4O-mX]=Nucleotide modified with 4'-O-monomethylphosphonate-2'-O-methyl, [ademX-L]=Lipid attached to nucleotide, [+X ] = _Tm -increasing nucleotide, optionally LNA, and [+Xs] = _Tm -increasing nucleotide having a phosphorothioate linkage to an adjacent nucleotide, optionally LNA.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression comprise the following modification pattern sense strand: 5'-[ademX-Ls][mX][mX][mX] [mX][mX][mX] [fX][fX][fX][fX][mX][mX][mX][mX][mXs][mXs][mX]-3' Hybridize to: antisense Stock: 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' where [mXs] = phosphorothioate linkage to adjacent nucleotide 2' -O -Methyl modified nucleotide, [fXs] = 2'-fluoro modified nucleotide having phosphorothioate linkage with adjacent nucleotide, [mX] = with adjacent nucleotide A 2'- O -methyl modified nucleotide having a phosphodiester linkage, [fX] = a 2'-fluoro modified nucleotide having a phosphodiester linkage to an adjacent nucleotide, [ MePhosphonate-4O-mX] = 4'-O-monomethylphosphonate-2'-O-methyl modified nucleotide, and [ademX-Ls] = lipid attached to adjacent nucleotide Nucleotides with phosphorothioate linkages.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression comprise the following modification pattern sense strand: 5'-[ademX-Ls][mX][mX][mX] [mX][mX][mX] [fX][fX][fX][fX][mX][mX][mX][mXs][mXs][mX]-3' Cross 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' where [mXs]=the phosphorothioate linkage to the adjacent nucleotide 2'- O - Methyl-modified nucleotides, [fXs] = 2'-fluorine-modified nucleotides with phosphorothioate linkages to adjacent nucleotides, [mX] = 2'-fluorine-modified nucleotides with phosphorothioate linkages to adjacent nucleotides 2'- O -methyl modified nucleotide with ester linkage, [fX] = 2'-fluoro modified nucleotide with phosphodiester linkage with adjacent nucleotide, [MePhosphonate-4O -mX]=Nucleotide modified with 4'-O-monomethylphosphonate-2'-O-methyl, and [ademX-Ls]=lipid attached to an adjacent nucleotide with a thio group Phosphate linked nucleotides.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression comprise the following modification pattern sense strand: 5'-[ademX-Ls][+X][mX][mX ][mX][mX][mX] [fX][fX][fX][fX][mX][mX][mXs][+Xs][+X]-3' Cross 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' where [mXs]=the phosphorothioate linkage to the adjacent nucleotide 2'- O - Methyl-modified nucleotides, [fXs] = 2'-fluorine-modified nucleotides with phosphorothioate linkages to adjacent nucleotides, [mX] = 2'-fluorine-modified nucleotides with phosphorothioate linkages to adjacent nucleotides 2'- O -methyl modified nucleotide with ester linkage, [fX] = 2'-fluoro modified nucleotide with phosphodiester linkage with adjacent nucleotide, [MePhosphonate-4O -mX]=Nucleotide modified with 4'-O-monomethylphosphonate-2'-O-methyl, [ademX-Ls]=lipid attached to adjacent nucleotide with phosphorothioate Nucleotides with ester linkages, [+X] = Nucleotides that increase _Tm , optionally LNA, and [+Xs] = Nucleotides that increase _Tm with phosphorothioate linkages to adjacent nucleotides Nucleotides, LNA or BNA as appropriate.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression comprise the following modification pattern sense strand: 5'-[ademX-Ls][mX][mX][mX] [mX][mX][mX] [fX][fX][fX][fX][mX][mX][mX][mX][mXs][mXs][mX]-3' Hybridize to: antisense Stock: 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' where [mXs] = phosphorothioate linkage to adjacent nucleotide 2' -O -Methyl modified nucleotide, [fXs] = 2'-fluoro modified nucleotide having phosphorothioate linkage with adjacent nucleotide, [mX] = with adjacent nucleotide A 2'- O -methyl modified nucleotide having a phosphodiester linkage, [fX] = a 2'-fluoro modified nucleotide having a phosphodiester linkage to an adjacent nucleotide, [ MePhosphonate-4O-mX] = 4'-O-monomethylphosphonate-2'-O-methyl modified nucleotide, and [ademX-Ls] = lipid attached to adjacent nucleotide Nucleotides with phosphorothioate linkages.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression comprise the following modification pattern 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' hybrid To: Antisense: 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' where [mXs] = has phosphorothioate linkage to adjacent nucleotide The 2'- O -methyl modified nucleotide, [fXs] = the 2'-fluoro modified nucleotide having a phosphorothioate linkage with the adjacent nucleotide, [mX] = the corresponding 2'- O -methyl modified nucleotide with phosphodiester linkage to adjacent nucleotide, [fX] = 2'-fluorine modified core with phosphodiester linkage to adjacent nucleotide nucleotide, [MePhosphonate-4O-mX] = 4'-O-monomethylphosphonate-2'-O-methyl modified nucleotide, and [ademX-Ls] = lipid attached to the phase Nucleotides with adjacent nucleotides having phosphorothioate linkages.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression comprise the following modification pattern sense strand: 5'-[+Xs][ademX-L][fX][fX ][fX][fX][mX] [mX][mX][mX][+Xs][mXs][mX]-3' Cross to: Antisense: 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' where [mXs]=a 2'- O -methyl modified nucleotide with a phosphorothioate linkage to the adjacent nucleotide, [fXs ] = 2'-fluoro modified nucleotide with phosphorothioate linkage to adjacent nucleotide, [mX] = 2'- O - with phosphodiester linkage to adjacent nucleotide Methyl-modified nucleotides, [fX] = 2'-fluorine-modified nucleotides with phosphodiester linkages to adjacent nucleotides, [MePhosphonate-4O-mX] = 4'-O- Monomethylphosphonate-2'-O-methyl modified nucleotide, [ademX-L] = lipid attached to nucleotide, [+X] = nucleotide increasing T _m , optional LNA, and [+Xs] = _Tm -increasing nucleotide with a phosphorothioate linkage to the adjacent nucleotide, optionally LNA.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression comprise the following modification pattern sense strand: 5'-[+Xs][ademX-L][fX][fX ][fX][fX][mX] [mX][mX][+X][+Xs][mXs][mX]-3' Cross to: Antisense: 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' where [mXs]=a 2'- O -methyl modified nucleotide with a phosphorothioate linkage to the adjacent nucleotide, [ fXs] = 2'-fluorine modified nucleotide with phosphorothioate linkage to adjacent nucleotide, [mX] = 2'- O modified nucleotide with phosphodiester linkage to adjacent nucleotide - Methyl modified nucleotide, [fX] = 2'-fluoro modified nucleotide with phosphodiester linkage to adjacent nucleotide, [MePhosphonate-4O-mX] = 4'-O - Monomethylphosphonate-2'-O-methyl modified nucleotide, [ademX-L] = lipid attached to nucleotide, [+X] = nucleotide that increases _Tm , as needed LNA, and [ ₊

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression comprise the following modification pattern sense strand: 5'-[ademX-Ls][mX][mX][mX] [mX][mX][mX] [fX][fX][fX][fX][mX][mX][mX][mX][mX][mX][mXs][mXs][mX]-3 ' Cross to: Antisense: 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' where [mXs] = has phosphorothioate with adjacent nucleotide Linked 2'- O -methyl modified nucleotides, [fXs]= 2'-fluoro modified nucleotides having phosphorothioate linkages to adjacent nucleotides, [mX]= 2'- O -methyl modified nucleotide with phosphodiester linkage to adjacent nucleotide, [fX] = 2'-fluoro modified nucleotide with phosphodiester linkage to adjacent nucleotide Nucleotide, [MePhosphonate-4O-mX] = Nucleotide modified with 4'-O-monomethylphosphonate-2'-O-methyl, [ademX-Ls] = Lipid attached to Nucleotides in which adjacent nucleotides have phosphorothioate linkages.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression comprise the following modification pattern sense strand: 5'-[ademX-Ls][mX][mX][mX] [mX][mX] [mX][fX][fX][fX][fX][mX][mX][mX][mX][mXs][mXs][mX]-3' Hybridize to: antisense Stock: 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' where [mXs] = phosphorothioate linkage to adjacent nucleotide 2' -O -Methyl modified nucleotide, [fXs] = 2'-fluoro modified nucleotide having phosphorothioate linkage with adjacent nucleotide, [mX] = with adjacent nucleotide 2'- O -methyl modified nucleotide having a phosphodiester linkage, [fX] = 2'-fluoro modified nucleotide having a phosphodiester linkage to the adjacent nucleotide, [ MePhosphonate-4O-mX]=Nucleotide modified with 4'-O-monomethylphosphonate-2'-O-methyl, [ademX-Ls]=lipid attached to adjacent nucleotide Phosphorothioate linked nucleotides.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression comprise the following modification pattern 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' Cross to: Antisense: 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' where [mXs]=the phosphorothioate linkage to the adjacent nucleotide 2'- O - Methyl-modified nucleotide, [fXs] = 2'-fluoro-modified nucleotide with phosphorothioate linkage to adjacent nucleotide, [mX] = phosphate with adjacent nucleotide 2'- O -methyl modified nucleotide with diester linkage, [fX] = 2'-fluoro modified nucleotide with phosphodiester linkage with adjacent nucleotide, [MePhosphonate- 4O-mX] = nucleotide modified with 4'-O-monomethylphosphonate-2'-O-methyl, and [ademX-C16] = nucleotide to which a C16 lipid is attached.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression comprise the following modification pattern sense strand: 5'-[+X][mX][mX][ademX-C16 ][mX][mX] [mX][+X][mX][mX][mX][mX][mX][mX][mX][mX][mX][fX][fX][fX] [fX][mX][mX][mX][mX][mXs][mXs][mX]-3' Cross to: Antisense: 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' where [mXs]=a 2'- O -methyl modified nucleotide having a phosphorothioate linkage with the adjacent nucleotide, [fXs]=with the adjacent nucleotide Nucleotide 2'-fluoro modified nucleotide with phosphorothioate linkage, [mX] = 2'- O -methyl modified core with phosphodiester linkage to adjacent nucleotide Urinic acid, [fX] = 2'-fluorine modified nucleotide with phosphodiester linkage to adjacent nucleotide, [MePhosphonate-4O-mX] = 4'-O-monomethylphosphonic acid Ester-2'-O-methyl modified nucleotide, and [ademX-C16] = nucleotide to which C16 lipid is attached, [+X] = nucleotide to increase T _m , optional LNA .

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression include the following modification pattern sense strand: 5'-[+X][mX][mX][mX][ mX][ademX-C16] [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' Hybridize to: antisense Stock: 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' where [mXs] = phosphorothioate linkage to adjacent nucleotide 2' -O -Methyl modified nucleotide, [fXs] = 2'-fluoro modified nucleotide having phosphorothioate linkage with adjacent nucleotide, [mX] = with adjacent nucleotide 2'- O -methyl modified nucleotide having a phosphodiester linkage, [fX] = 2'-fluoro modified nucleotide having a phosphodiester linkage to the adjacent nucleotide, [ MePhosphonate-4O-mX]=Nucleotide modified with 4'-O-monomethylphosphonate-2'-O-methyl, [ademX-C16]=Nucleotide to which C16 lipid is attached, [+X]=Nucleotide to increase T _m , LNA if necessary.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression comprise the following modification pattern sense strand: 5'-[ademX-C16s][mX][mX][mX] [mX][mX] [mX][fX][fX][fX][fX][mX][mX][mX][mX][mXs][mXs][mX]-3' Hybridize to: antisense Stock: 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' where [mXs] = phosphorothioate linkage to adjacent nucleotide 2' -O -Methyl modified nucleotide, [fXs] = 2'-fluoro modified nucleotide having phosphorothioate linkage with adjacent nucleotide, [mX] = with adjacent nucleotide 2'- O -methyl modified nucleotide having a phosphodiester linkage, [fX] = 2'-fluoro modified nucleotide having a phosphodiester linkage to the adjacent nucleotide, [ MePhosphonate-4O-mX]=Nucleotide modified with 4'-O-monomethylphosphonate-2'-O-methyl, [ademX-C16s]=C16 lipid attached to adjacent nucleotide Nucleotides with phosphorothioate linkages.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression comprise the following modification pattern sense strand: 5'-[ademX-C16s][mX][mX][mX] [mX][mX] [mX][fX][fX][fX][fX][mX][mX][mX][mXs][mXs][mX]-3' Cross 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' where [mXs]=the phosphorothioate linkage to the adjacent nucleotide 2'- O - Methyl-modified nucleotides, [fXs] = 2'-fluorine-modified nucleotides with phosphorothioate linkages to adjacent nucleotides, [mX] = 2'-fluorine-modified nucleotides with phosphorothioate linkages to adjacent nucleotides 2'- O -methyl modified nucleotide with ester linkage, [fX] = 2'-fluoro modified nucleotide with phosphodiester linkage with adjacent nucleotide, [MePhosphonate-4O -mX]=Nucleotide modified with 4'-O-monomethylphosphonate-2'-O-methyl, [ademX-C16s]=C16 lipid attached to an adjacent nucleotide with a thio group Phosphate linked nucleotides.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression comprise the following modification pattern sense strand: 5'-[ademX-C16s][+X][mX][mX ][mX][mX] [mX][fX][fX][fX][fX][mX][mX][mXs][+Xs][+X]-3' Cross 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' where [mXs]=the phosphorothioate linkage to the adjacent nucleotide 2'- O - Methyl-modified nucleotides, [fXs] = 2'-fluorine-modified nucleotides with phosphorothioate linkages to adjacent nucleotides, [mX] = 2'-fluorine-modified nucleotides with phosphorothioate linkages to adjacent nucleotides 2'- O -methyl modified nucleotide with ester linkage, [fX] = 2'-fluoro modified nucleotide with phosphodiester linkage with adjacent nucleotide, [MePhosphonate-4O -mX]=Nucleotide modified with 4'-O-monomethylphosphonate-2'-O-methyl, [ademX-C16s]=C16 lipid attached to an adjacent nucleotide with a thio group Phosphate linked nucleotides, [+X] = nucleotides that increase T _m , optionally LNA, and [+Xs] = increased T _m with phosphorothioate linkages to adjacent nucleotides The nucleotide is LNA or BNA as needed.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression comprise the following modification pattern sense strand: 5'-[ademX-C22s][mX][mX][mX] [mX][mX] [mX][fX][fX][fX][fX][mX][mX][mX][mX][mXs][mXs][mX]-3' Hybridize to: antisense Stock: 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' where [mXs] = phosphorothioate linkage to adjacent nucleotide 2' -O -Methyl modified nucleotide, [fXs] = 2'-fluoro modified nucleotide having phosphorothioate linkage with adjacent nucleotide, [mX] = with adjacent nucleotide 2'- O -methyl modified nucleotide having a phosphodiester linkage, [fX] = 2'-fluoro modified nucleotide having a phosphodiester linkage to the adjacent nucleotide, [ MePhosphonate-4O-mX]=Nucleotide modified with 4'-O-monomethylphosphonate-2'-O-methyl, [ademX-C22s]=C22 lipid lipid attached to adjacent nucleoside Acid nucleotides with phosphorothioate linkages.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression comprise the following modification pattern sense strand: 5'-[ademX-C22s][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' hybrid To: Antisense: 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' where [mXs] = has phosphorothioate linkage to adjacent nucleotide The 2'- O -methyl modified nucleotide, [fXs] = the 2'-fluoro modified nucleotide having a phosphorothioate linkage with the adjacent nucleotide, [mX] = the corresponding 2'- O -methyl modified nucleotide with phosphodiester linkage to adjacent nucleotide, [fX] = 2'-fluorine modified core with phosphodiester linkage to adjacent nucleotide Glycoside, [MePhosphonate-4O-mX] = 4'-O-monomethylphosphonate-2'-O-methyl modified nucleotide, [ademX-C22s] = C22 lipid attached to the phase Nucleotides with adjacent nucleotides having phosphorothioate linkages.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression include the following modification pattern sense strand: 5'-[+Xs][ademX-C22][fX][fX ][fX][fX][mX] [mX][mX][mX][+Xs][mXs][mX]-3' Cross to: Antisense: 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' where [mXs]=a 2'- O -methyl modified nucleotide with a phosphorothioate linkage to the adjacent nucleotide, [fXs ] = 2'-fluoro modified nucleotide with phosphorothioate linkage to adjacent nucleotide, [mX] = 2'- O - with phosphodiester linkage to adjacent nucleotide Methyl-modified nucleotides, [fX] = 2'-fluorine-modified nucleotides with phosphodiester linkages to adjacent nucleotides, [MePhosphonate-4O-mX] = 4'-O- Monomethylphosphonate-2'-O-methyl modified nucleotide, [ademX-C22] = nucleotide to which C22 lipid is attached, [+X] = nucleotide that increases T _m , LNA, optionally, and [+Xs] = _Tm- increasing nucleotide with phosphorothioate linkage to adjacent nucleotide, LNA, optionally.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression comprise the following modification pattern sense strand: 5'-[+Xs][ademX-C22][fX][fX ][fX][fX][mX] [mX][mX][+X][+Xs][mXs][mX]-3' Cross to: Antisense: 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' where [mXs]=a 2'- O -methyl modified nucleotide with a phosphorothioate linkage to the adjacent nucleotide, [ fXs] = 2'-fluorine modified nucleotide with phosphorothioate linkage to adjacent nucleotide, [mX] = 2'- O modified nucleotide with phosphodiester linkage to adjacent nucleotide - Methyl modified nucleotide, [fX] = 2'-fluoro modified nucleotide with phosphodiester linkage to adjacent nucleotide, [MePhosphonate-4O-mX] = 4'-O - Monomethylphosphonate-2'-O-methyl modified nucleotide, [ademX-C22] = nucleotide to which C22 lipid is attached, [+X] = nucleotide that increases T _m , optionally LNA, and [+Xs] = a _Tm -increasing nucleotide with a phosphorothioate linkage to an adjacent nucleotide, optionally LNA.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression comprise the following modification pattern sense strand: 5'-[ademX-C22s][mX][mX][mX] [mX][mX] [mX][fX][fX][fX][fX][mX][mX][mX][mX][mX][mX][mXs][mXs][mX]-3 ' Cross to: Antisense: 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' where [mXs] = has phosphorothioate with adjacent nucleotide Linked 2'- O -methyl modified nucleotides, [fXs]= 2'-fluoro modified nucleotides having phosphorothioate linkages to adjacent nucleotides, [mX]= 2'- O -methyl modified nucleotide with phosphodiester linkage to adjacent nucleotide, [fX] = 2'-fluoro modified nucleotide with phosphodiester linkage to adjacent nucleotide Nucleotide, [MePhosphonate-4O-mX]=Nucleotide modified with 4'-O-monomethylphosphonate-2'-O-methyl, [ademX-C22s]=C22 lipid attached to A nucleotide that has a phosphorothioate linkage to an adjacent nucleotide.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression comprise the following modification pattern sense strand: 5'-[ademX-C22s][mX][mX][mX] [mX][mX] [mX][fX][fX][fX][fX][mX][mX][mX][mX][mXs][mXs][mX]-3' Hybridize to: antisense Stock: 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' where [mXs] = phosphorothioate linkage to adjacent nucleotide 2' -O -Methyl modified nucleotide, [fXs] = 2'-fluoro modified nucleotide having phosphorothioate linkage with adjacent nucleotide, [mX] = with adjacent nucleotide A 2'- O -methyl modified nucleotide having a phosphodiester linkage, [fX] = a 2'-fluoro modified nucleotide having a phosphodiester linkage to an adjacent nucleotide, [ MePhosphonate-4O-mX]=Nucleotide modified with 4'-O-monomethylphosphonate-2'-O-methyl, [ademX-C22s]=C22 lipid attached to adjacent nucleotide Nucleotides with phosphorothioate linkages.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression comprise the following modification pattern sense strand: 5'-[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' Cross to: Antisense: 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' where [mXs] = phosphorothioate linkage to adjacent nucleotide 2' -O -Methyl modified nucleotide, [fXs] = 2'-fluoro modified nucleotide having phosphorothioate linkage with adjacent nucleotide, [mX] = with adjacent nucleotide 2'- O -methyl modified nucleotide having a phosphodiester linkage, [fX] = 2'-fluoro modified nucleotide having a phosphodiester linkage to the adjacent nucleotide, [ MePhosphonate-4O-mX]=Nucleotide modified with 4'-O-monomethylphosphonate-2'-O-methyl, [mX/mXs]=This position has a phosphate with the adjacent nucleotide A 2'- O -methyl modified nucleotide with a diester linkage or a 2'- O -methyl modified nucleotide with a phosphorothioate linkage to an adjacent nucleotide, and [ademX- L] = lipid attached to nucleotide.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression include the following modification pattern sense strand: 5'-[+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' Cross to: Antisense: 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' where [mXs]=the phosphorothioate linkage to the adjacent nucleotide 2'- O - Methyl-modified nucleotides, [fXs] = 2'-fluorine-modified nucleotides with phosphorothioate linkages to adjacent nucleotides, [mX] = 2'-fluorine-modified nucleotides with phosphorothioate linkages to adjacent nucleotides 2'- O -methyl modified nucleotide with ester linkage, [fX] = 2'-fluoro modified nucleotide with phosphodiester linkage with adjacent nucleotide, [MePhosphonate-4O -mX]=Nucleotide modified with 4'-O-monomethylphosphonate-2'-O-methyl, [mX/mXs]=This position has a phosphodiester bond with the adjacent nucleotide A 2'- O -methyl-modified nucleotide or a 2'- O -methyl-modified nucleotide having a phosphorothioate linkage to an adjacent nucleotide, and [ademX-L]= Lipids are attached to nucleotides.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression include the following modification pattern sense strand: 5'-[+Xs][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' Cross to: Antisense: 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' where [mXs]= 2'- O -methyl modified nucleotide with phosphorothioate linkage to adjacent nucleotide Nucleotide, [fXs] = 2'-fluorine modified nucleotide with phosphorothioate linkage to adjacent nucleotide, [mX] = phosphodiester linkage to adjacent nucleotide 2'- O -methyl modified nucleotide, [fX]= 2'-fluoro modified nucleotide having a phosphodiester linkage with the adjacent nucleotide, [MePhosphonate-4O-mX]= Nucleotide modified with 4'-O-monomethylphosphonate-2'-O-methyl, [mX/mXs] = the position is a phosphodiester linkage with the adjacent nucleotide 2 ' -O -methyl modified nucleotide or 2'- O -methyl modified nucleotide having a phosphorothioate linkage to an adjacent nucleotide, and [ademX-L]=lipid attached to Nucleotides.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression comprise the following modification pattern 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' Cross to: Antisense: 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' where [mXs] = 2'- O -methyl modified nucleotide with phosphorothioate linkage to adjacent nucleotide, [fXs] = phosphorothioate linkage to adjacent nucleotide 2'-fluoro modified nucleotide, [mX] = 2'- O -methyl modified nucleotide having a phosphodiester linkage with the adjacent nucleotide, [fX] = with the adjacent core The nucleotide has a phosphodiester linkage and is modified with 2'-fluorine, [MePhosphonate-4O-mX] = modified with 4'-O-monomethylphosphonate-2'-O-methyl Nucleotide, [mX / + The Tm-increasing nucleotide of an _ester linkage, optionally LNA, and [ademXs-C16] = lipid is attached to a nucleotide with a phosphorothioate linkage to the adjacent nucleotide.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression comprise the following modification pattern sense strand: 5'-[ademXs-L][fX/+X][fX] [fX][fX][mX] [mX][mX][mX/+X][mX/+X][mX][mXs/+Xs][mXs/+Xs][mX]-3' Cross to : Antisense: 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' where [mXs]= has a phosphorothioate linkage to the adjacent nucleotide 2'- O -methyl modified nucleotide, [fXs] = 2'-fluoro modified nucleotide with phosphorothioate linkage to adjacent nucleotide, [mX] = adjacent nucleotide Nucleotides are 2'- O -methyl modified nucleotides with phosphodiester linkages, [fX] = 2'-fluoro modified nucleosides with phosphodiester linkages to adjacent nucleotides Acid, [MePhosphonate-4O-mX]=Nucleotide modified with 4'-O-monomethylphosphonate-2'-O-methyl, [mX/+X]=This position is with the adjacent nuclei A 2'- O -methyl modified nucleotide with a phosphodiester linkage or a nucleotide that increases _Tm , [mXs/+Xs] = this position has a thio group with the adjacent nucleotide. Phosphate-linked 2'- O -methyl-modified nucleotides or _Tm -increasing nucleotides with phosphorothioate linkages to adjacent nucleotides, optionally LNA, [fXs/+ Xs] = This position is a 2'-fluoro modified nucleotide with a phosphorothioate linkage to an adjacent nucleotide or a _Tm -increasing core with a phosphorothioate linkage to an adjacent nucleotide The nucleotide, optionally LNA, and [ademXs-L]=lipid are attached to the nucleotide with a phosphorothioate linkage to the adjacent nucleotide.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression comprise the following modification pattern 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' Cross to: Antisense: 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' where [mXs]= has sulfur with adjacent nucleotide 2'- O -methyl modified nucleotide with phosphorothioate linkage, [fXs] = 2'-fluoro modified nucleotide with phosphorothioate linkage to adjacent nucleotide, [ mX] = 2'- O -methyl modified nucleotide having a phosphodiester linkage with the adjacent nucleotide, [fX] = 2' having a phosphodiester linkage with the adjacent nucleotide -Fluorine modified nucleotide, [MePhosphonate-4O-mX]=Nucleotide modified with 4'-O-monomethylphosphonate-2'-O-methyl, [mX/+X]=the The position is a 2'- O -methyl modified nucleotide with a phosphodiester linkage to the adjacent nucleotide or a nucleotide that increases _Tm , [mXs/+Xs] = the position is with the adjacent nucleotide A 2'- O -methyl modified nucleotide with a phosphorothioate linkage or a _Tm -increasing nucleotide with a phosphorothioate linkage to an adjacent nucleotide, as appropriate LNA, and [ademXs-L]=lipid attached to a nucleotide with a phosphorothioate linkage to the adjacent nucleotide.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression comprise the following modification pattern sense strand: 5'-[ademXs-L][mX/+X][mX] [fX][mX] [mX][mX][mX/+X][mX/+X][mX][mXs/+Xs][mXs/+Xs][mX]-3' Hybridize to: antisense Stock: 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' where [mXs] = phosphorothioate linkage to adjacent nucleotide 2' -O -Methyl modified nucleotide, [fXs] = 2'-fluoro modified nucleotide having phosphorothioate linkage with adjacent nucleotide, [mX] = with adjacent nucleotide A 2'- O -methyl modified nucleotide having a phosphodiester linkage, [fX] = a 2'-fluoro modified nucleotide having a phosphodiester linkage to an adjacent nucleotide, [ MePhosphonate-4O-mX]=Nucleotide modified with 4'-O-monomethylphosphonate-2'-O-methyl, [mX/+X]=This position is shared by adjacent nucleotides Phosphodiester-linked 2'- O -methyl modified nucleotides or nucleotides that increase T _m , [mXs/+Xs] = this position has a phosphorothioate bond with the adjacent nucleotide Coupled with 2'- O -methyl modified nucleotides or _Tm -increasing nucleotides with phosphorothioate linkages to adjacent nucleotides, optionally LNA, [fXs/+Xs]= The position is a 2'-fluoro modified nucleotide having a phosphorothioate linkage to an adjacent nucleotide or a _Tm -increasing nucleotide having a phosphorothioate linkage to an adjacent nucleotide, Optionally LNA, and [ademXs-L]=lipid are attached to the nucleotide with a phosphorothioate linkage to the adjacent nucleotide.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression comprise the following modification pattern sense strand: 5'-[+Xs][ademX-L][fX][fX ][fX][fX][mX] [mX][mX][mX][+Xs][mXs][mX]-3' Cross to: Antisense: 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' where [mXs]=a 2'- O -methyl modified nucleotide with a phosphorothioate linkage to the adjacent nucleotide, [fXs ] = 2'-fluoro modified nucleotide with phosphorothioate linkage to adjacent nucleotide, [mX] = 2'- O - with phosphodiester linkage to adjacent nucleotide Methyl-modified nucleotides, [fX] = 2'-fluorine-modified nucleotides with phosphodiester linkages to adjacent nucleotides, [MePhosphonate-4O-mX] = 4'-O- Monomethylphosphonate-2'-O-methyl modified nucleotide, [+X] = nucleotide that increases T _m , [+Xs] = has a phosphorothioate bond with adjacent nucleotides linked to a _Tm -increasing nucleotide, and [ademXs-L] = lipid attached to a nucleotide with a phosphorothioate linkage to an adjacent nucleotide, wherein the antisense strand contains 2 nucleotides 5' overhang and a 3' overhang of 7 nucleotides.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression comprise the following modification pattern sense strand: 5'-[+Xs][ademX-L][fX][fX ][fX][fX][mX] [mX][mX][+X][+Xs][mXs][mX]-3' Cross to: Antisense: 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' where [mXs]=a 2'- O -methyl modified nucleotide with a phosphorothioate linkage to the adjacent nucleotide, [ fXs] = 2'-fluorine modified nucleotide with phosphorothioate linkage to adjacent nucleotide, [mX] = 2'- O modified nucleotide with phosphodiester linkage to adjacent nucleotide - Methyl modified nucleotide, [fX] = 2'-fluoro modified nucleotide with phosphodiester linkage to adjacent nucleotide, [MePhosphonate-4O-mX] = 4'-O - Monomethylphosphonate-2'-O-methyl modified nucleotide, [+X] = nucleotide that increases T _m , [+Xs] = phosphorothioate with adjacent nucleotide Linked _Tm -increasing nucleotides, and [ademXs-L] = lipid attached to a nucleotide with a phosphorothioate linkage to an adjacent nucleotide, wherein the antisense strand contains 2 nucleosides A 5' overhang of acid and a 3' overhang of 7 nucleotides.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression comprise the following modification pattern sense strand: 5'-[ademXs-L][+X][fX][fX ][fX][mX][mX] [mX][+X][+Xs][mXs][mX]-3' Cross to: Antisense: 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' where [mXs]= 2'- O -methyl modified nucleotide with phosphorothioate linkage to adjacent nucleotide, [fXs]= 2'-Fluorine modified nucleotide with phosphorothioate linkage to adjacent nucleotide, [mX] = 2'- O -methyl group with phosphodiester linkage to adjacent nucleotide Modified nucleotides, [fX] = 2'-fluoro modified nucleotides with phosphodiester linkages to adjacent nucleotides, [MePhosphonate-4O-mX] = 4'-O-monomethyl Phosphonate-2'-O-methyl modified nucleotides, [+X] = nucleotides that increase _Tm , [+Xs] = phosphorothioate linkages to adjacent nucleotides Nucleotide that increases _Tm , and [ademXs-L]=lipid attached to a nucleotide with a phosphorothioate linkage to an adjacent nucleotide, wherein the antisense strand contains 5 of 2 nucleotides ' overhang and a 3-of-8 ' overhang.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression comprise the following modification pattern sense strand: 5'-[ademXs-L][+X][fX][fX ][fX][fX][mX] [mX][mX][+Xs][+Xs][mX]-3' Cross to: Antisense: 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' where [mXs]= 2'- O -methyl modified nucleotide with phosphorothioate linkage to adjacent nucleotide, [fXs]= 2'-Fluorine modified nucleotide with phosphorothioate linkage to adjacent nucleotide, [mX] = 2'- O -methyl group with phosphodiester linkage to adjacent nucleotide Modified nucleotides, [fX] = 2'-fluoro modified nucleotides with phosphodiester linkages to adjacent nucleotides, [MePhosphonate-4O-mX] = 4'-O-monomethyl Phosphonate-2'-O-methyl modified nucleotides, [+X] = nucleotides that increase _Tm , [+Xs] = phosphorothioate linkages to adjacent nucleotides Nucleotide that increases _Tm , and [ademXs-L]=lipid attached to a nucleotide with a phosphorothioate linkage to an adjacent nucleotide, wherein the antisense strand contains 5 of 3 nucleotides ' overhang and a 3-of-7 ' overhang.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing target gene expression comprise the following modification pattern sense strand: 5'-[ademXs-L][+X][mX][mX ][mX][+X][+X] [mXs][mXs][mX]-3' Cross to: Antisense: 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' where [mXs] = 2'- O -methyl modified nucleotide with phosphorothioate linkage to adjacent nucleotide, [fXs] = and adjacent nucleotide 2'-fluoro-modified nucleotides with phosphorothioate linkages, [mX] = 2'- O -methyl-modified nucleotides with phosphodiester linkages to adjacent nucleotides, [fX] = 2'-fluorine modified nucleotide with phosphodiester linkage to adjacent nucleotide, [MePhosphonate-4O-mX] = 4'-O-monomethylphosphonate-2 '-O-methyl modified nucleotide, [+X] = nucleotide that increases _Tm , and [ademXs-L] = lipid attached to a nucleotide with a phosphorothioate linkage to the adjacent nucleotide nucleotides, wherein the antisense strand contains a 5' overhang of 1 nucleotide and a 3' overhang of 11 nucleotides.

In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of Compounds 2-14 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in Compound 15 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of Compounds 16-18 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of Compounds 19-38 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of Compounds 39-40, 45, and 48-49 described herein . In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of Compounds 41 to 44 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 46-47 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in compounds 57 and any of 98-108 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 120-122 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 59-64 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 65-69 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of Compounds 70-71 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of Compounds 72-74 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of Compounds 75-76 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of Compounds 77-79 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in compound 58 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 80-81 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 82-84 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 85-86 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 87-89 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of Compounds 90-91 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 92-94 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in compound 95 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 96-97 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of Compounds 109-117 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 123 to 126 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as set forth in any of 127 to 130 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 131 to 136 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 137-140 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 141 to 145 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 146-148 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 149-154 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 155-160 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in compound 161 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 162 to 165 and 171 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 166-170 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in compound 172 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 173 to 176 and 182 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 177-181 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as set forth in any one of 183 to 190 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 191 to 194 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 195-197 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 200-201 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise any of compounds 202 to 205, 207 to 210, and 211 to 215 described herein. Modification mode. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 217-218 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 219-222 and 224-232 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as set forth in any of 277 to 278 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 279-285 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 198-199 and 233 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 234-236 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 237-239 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 240-242 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 243-245 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 246-251 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 252-255 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in compound 256 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 257-259 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 260-262 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of Compounds 152 and 264-264 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 266-268 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 269-272 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 273-276 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 286 and 292 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 287-291 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of compounds 293-294 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise a modification pattern as shown in any of Compounds 2 to 15, 46, and 47 described herein. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise compounds 137 to 140, 146, 148, 162 to 165, 173 to 176, 183 to Modification patterns shown in any one of 190, 200, 217, 247, 270, 277, 278, 286 and 292. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing the expression of a target gene comprise compounds 16, 19 to 28, 30 to 38, 41 to 44, 65 to 79, as described herein. 80 to 97, 127 to 130, 202 to 205, 207 to 210, 212 to 215, 219 to 221, 224 to 227, 229 to 232, 237 to 239, 243 to 245, 252 to 255, 260 to 262, 266 to Modification patterns shown in any one of 268, 274 to 276, 280 to 285, 287 to 291, and 293 to 294. In some embodiments, lipid-conjugated RNAi oligonucleotides for reducing expression of a target gene comprise compounds 40, 45, 49, 59 to 64, 98, 99 to 117, 131 to 136, 141 to 145, 149 to 160, 166 to 170, 177 to 181, 191 to 197, 201, 211, 218, 228, 234 to 236, 240 to 242, 248 to 251, 257 to 259, 264, 265, Modification mode shown in any one of 272 to 273 and 279. In some or any of the preceding embodiments, reference to compound numbering refers to the modification pattern (eg, phosphorothioate linkage, 2' modification, conjugation) rather than the nucleotide sequence. Provides general methods for nucleic acids and their analogs

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

In certain embodiments, methods for synthesizing provided nucleic acids comprise (a) attaching a nucleoside or analog thereof to a solid support via a covalent linkage; (b) nucleoside phosphoramidite or an analog thereof coupled to a reactive hydroxyl group on the nucleoside or analog thereof of step (a) to form an internucleotide bond therebetween, wherein any uncoupled nucleoside or analog thereof on the solid support The system is capped with a capping reagent; (c) the internucleotide bond is oxidized with an oxidizing agent; and (d) steps (b) to (c) are repeated with subsequent nucleoside phosphoramidites or analogs thereof to Forming a nucleic acid or an analog thereof, wherein at least the nucleoside or analog thereof of step (a), the nucleoside phosphoramidite or analog thereof of step (b) or the subsequent nucleoside phosphoramidite of step (d) or At least one of the analogs thereof includes a lipid conjugate moiety described herein. Typically, the coupling, capping/oxidation, and optional deprotection steps are repeated until the oligonucleotide reaches the desired length and/or sequence, after which it is cleaved from the solid support. In certain embodiments, oligonucleotides are prepared that include 1 to 3 nucleic acids or analogs thereof that comprise lipid conjugate units on a four-membered loop.

In Scheme A below, while specific protecting groups, leaving groups, and transformation conditions are shown, one of ordinary skill in the art will understand that other protecting groups, leaving groups, and transformation conditions are also suitable and are contemplated. . Certain reactive functional groups that require additional protecting group strategies as contemplated in Scheme A (eg, -N(H)-, -OH, etc.) are also contemplated and understood by those of ordinary skill in the art. Such groups and transformations are described in detail in March 's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure , MB Smith and J. March, ^5th Edition, John Wiley & Sons, 2001, Comprehensive Organic Transformations , (RC Larock, 2 ^nd Edition, John Wiley & Sons, 1999) and Protecting Groups in Organic Synthesis , (TW Greene and PGM Wuts, ^3rd edition, John Wiley & Sons, 1999), each of which is incorporated herein by reference in its entirety. middle.

In certain embodiments, the nucleic acids of the present disclosure and their analogs are generally prepared according to Scheme A, Scheme A1 and Scheme B shown below: Scheme A: Ligand-conjugated oligonucleotides of the present disclosure Synthesis Procedure A1: Synthesis of lipid-conjugated oligonucleotides of the present disclosure

As illustrated in Scheme A and Scheme A1 above, a nucleic acid of Formula I-1 or an analog thereof is conjugated with one or more ligands/lipophilic compounds to form a conjugate comprising one or more ligands/lipids A compound of formula I or Ia. Typically, the conjugation is by techniques known in the art in series or parallel by a nucleic acid of Formula I-1 or I-1a or an analog thereof with one or more adamantyl and/or lipophilic compounds ( For example, esterification or amidation reaction between fatty acids). Nucleic acids of Formula I or Ia or analogs thereof can then be deprotected to form compounds of Formula I-2 or I-2a and protected with a suitable hydroxy protecting group (eg, DMTr) to form compounds of Formula I-3 or I -3a compound. In one aspect, a nucleic acid-ligand conjugate of Formula I-3 or I-3a can be covalently attached to a solid support (e.g., via a succinic acid linking group) to form a form comprising one or more adamantane Solid support nucleic acid-ligand conjugates of formula I-4 or I-4a or analogs thereof of base and/or lipid conjugates. In another aspect, the nucleic acid-ligand conjugate of Formula I-3 or I-3a can form a reagent with P(III) (e.g., 2-cyanoethyl N , N -di-isopropyl chloride Phosphoramidite) reaction to form a nucleic acid of formula I-5 or I-5a or an analog thereof containing a P(III) group. Nucleic acid-ligand conjugates of Formula I-5 or I-5a or analogs thereof can then be subjected to oligopolymerization conditions using known and commonly used procedures to prepare oligonucleotides in the art. . For example, a compound of formula I-5 or I-5a is coupled to a solid supported nucleic acid-ligand conjugate bearing a 5'-hydroxyl group or the like. Further steps may include one or more deprotection, coupling, phosphite oxidation, and/or cleavage from the solid support to provide oligonucleotides of various nucleotide lengths including one or A plurality of lipid conjugate nucleotide units represented by compounds of formula II-1 or II-Ia. B, E, L, ligand, LC, n, PG ¹ , PG ² , PG ⁴ , R ¹ , R ² , R ³ , X, X ¹ , X ² , X ³ , and Z are as above Definitions and as described in this article. Process B: Synthesis of lipid conjugates after oligonucleotides of the present disclosure

As illustrated in Scheme B above, a nucleic acid of Formula I-1 or an analog thereof can be deprotected to form a compound of Formula I-6 and protected with a suitable hydroxy protecting group (eg, DMTr) to form a compound of Formula I- 7, and react with a P(III)-forming reagent (such as 2-cyanoethyl N , N -di-isopropyl phosphoramidite chloride) to form a formula I- containing a P(III) group 8 nucleic acids or analogs thereof. Next, the nucleic acid of Formula I-8 or analogs thereof can be subjected to oligomeric forming conditions using known and commonly used procedures to prepare oligonucleotides known in the art. For example, a compound of formula I-8 is coupled to a solid supported nucleic acid bearing a 5'-hydroxyl group or an analog thereof. Further steps may include one or more deprotection, coupling, phosphite oxidation, and/or cleavage from the solid support to provide oligonucleotides of various nucleotide lengths represented by the compounds of Formula II-2. acid. The oligonucleotide of Formula II-2 can then be conjugated with one or more ligands (e.g., adamantyl, or a lipophilic compound (e.g., fatty acid)) to form a conjugate containing one or more ligands. A compound of formula II-1. Typically, conjugation is by esterification or amide between a nucleic acid of formula II-2 or an analog thereof and one or more adamantyl groups or fatty acids in a series or parallel manner by techniques known in the art. chemical reaction to proceed. B, E, L, ligand, LC, n, PG ¹ , PG ² , PG ⁴ , R ¹ , R ² , R ³ , X, X ¹ , X ² , X ³ , and Z are as above Definitions and as described in this article.

In certain embodiments, the nucleic acids of the present disclosure and their analogs are prepared according to Scheme C and Scheme D shown below: Scheme C: Synthesis of lipid-conjugated oligonucleotides of the present disclosure

As illustrated in Scheme C above, a nucleic acid of formula C1 or an analog thereof is protected to form a compound of formula C2. The nucleic acid of formula C2 or analog thereof is then alkylated (eg, via Pummerer rearrangement using DMSO and acetic acid) to form the monothioacetal compound of formula C3. Next, the nucleic acid of formula C3 or an analog thereof is coupled with C4 under appropriate conditions (eg, mild oxidative conditions) to form a nucleic acid of formula C5 or an analog thereof. The nucleic acid of formula C5 or analog thereof can then be deprotected to form a compound of formula C6 and combined with a ligand of formula C7 (adamantyl or lipophilic compound (e.g., fatty acid)) under appropriate amide forming conditions (e.g., , HATU, DIPEA) to form a nucleic acid-ligand conjugate of Formula Ib or an analog thereof comprising the lipid conjugate of the present disclosure. The nucleic acid-ligand conjugate of Formula Ib or analog thereof can then be deprotected to form a compound of Formula C8 and protected with a suitable hydroxy protecting group (eg, DMTr) to form a compound of Formula C9. In one aspect, a nucleic acid of Formula C9, or an analog thereof, can be covalently attached to a solid support (e.g., via a succinic acid linking group) to form a ligand conjugate (adamantyl or Lipid moiety) solid support nucleic acid-ligand conjugate of formula C10 or analog thereof. In another aspect, nucleic acid-ligand conjugates of Formula C9 or analogs thereof can be formed with P(III) reagents (e.g., 2-cyanoethyl N , N -di-isopropyl chlorophosphoramidite ) reaction to form a nucleic acid-ligand conjugate of formula C11 containing a P(III) group or an analog thereof. The nucleic acid-ligand conjugate of formula C11 or analog thereof can then be subjected to oligomeric forming conditions using known and commonly used procedures to prepare oligonucleotides known in the art. For example, a compound of formula C11 is coupled to a solid supported nucleic acid-ligand conjugate bearing a 5'-hydroxyl group or the like. Further steps may include one or more deprotection, coupling, phosphite oxidation, and/or cleavage from the solid support to provide oligonucleotides of various nucleotide lengths including one or more an adamantyl and/or lipid conjugate nucleotide unit represented by a compound of formula II-b-3. B, E, L ² , PG ¹ , PG 2 , PG ³ , PG ⁴ , R ¹ , R ² , R ³ , R ⁴ , ^{R 5 ,} X ^{1 , X 2 ,} X ³ , V, W, and Z Each is as defined above and described herein. Process D: Oligonucleotides of the present disclosure followed by synthesis of lipid conjugates

B, E, L ² , PG ¹ , PG 2 , PG ³ , PG ⁴ , R ¹ , R ² , R ³ , R ⁴ , ^{R 5 ,} X ^{1 , X 2 ,} X ³ , V, W, and Z Each is as defined above and described herein. As illustrated in Scheme D above, a nucleic acid of Formula C5 or an analog thereof can be selectively deprotected to form a compound of Formula D1 and protected with a suitable hydroxy protecting group (e.g., DMTr) to form a compound of Formula D2 The compound is reacted with a P(III)-forming reagent (eg, 2-cyanoethyl N , N -di-isopropyl phosphoramidite chloride) to form a nucleic acid of formula D3 or an analog thereof. Next, the nucleic acid of Formula D3 or an analog thereof can be subjected to oligomeric forming conditions using known and commonly used procedures to prepare oligonucleotides known in the art. For example, a compound of formula D3 is coupled to a solid supported nucleic acid bearing a 5'-hydroxyl group or an analog thereof. Further steps may include one or more deprotection, coupling, phosphite oxidation, and/or cleavage from the solid support to provide oligonucleotides of various nucleotide lengths represented by the compound of Formula D4. Oligonucleotides of formula D4 can then be deprotected to form compounds of formula D5 and combined with hydrophobic ligands (eg, adamantyl or lipophilic moieties) to form compounds of formula C7 (eg, adamantyl or fatty acids) Compounds are coupled under appropriate amide-forming conditions (e.g., HATU, DIPEA) to form oligonucleotides of Formula II-b-3 comprising conjugates of the ligands of the present disclosure (e.g., adamantyl or fatty acids) .

One of ordinary skill in the art will understand that various functional groups (such as aliphatic groups, alcohols, carboxylic acids, esters, amides, aldehydes, halogens, and nitriles) present in the nucleic acids of the present disclosure or their analogs can be Interconversion by techniques well known in the art, including but not limited to reduction, oxidation, esterification, hydrolysis, partial oxidation, partial reduction, halogenation, dehydration, partial hydration, and hydration. See, e.g., " March 's Advanced Organic Chemistry ", ( ^5th Ed., Ed.: Smith, MB and March, J., John Wiley & Sons, New York: 2001), each of which is incorporated by reference in its entirety and incorporated into this article. Such interconversion may require one or more of the aforementioned techniques, and certain methods for synthesizing the nucleic acids provided by this disclosure are described in the examples below.

In some embodiments, the present disclosure provides a method of preparing an oligonucleotide comprising one or more lipid conjugates, or a pharmaceutically acceptable salt thereof, the lipid conjugate unit being composed of Formula II-a-1 Represents: , the method includes the following steps: (a) providing the nucleic acid of formula I-5a or an analog thereof: or a salt thereof, and (b) oligomerize the compound of formula I-5a to form a compound of formula II-1a, wherein B, E, L, LC, n, PG ⁴ , R ¹ , R ² , R ³ Each of , X, X ¹ , X ² , X ³ , E, and Z is as defined above and described herein.

In step (b) above, oligopolymerization refers to oligopolymerization-forming conditions using known and commonly used procedures to prepare oligonucleotides in the art. For example, a compound of formula I-5a is coupled to a solid supported nucleic acid bearing a 5'-hydroxyl group or an analog thereof. Further steps may include one or more deprotection, coupling, phosphite oxidation, and cleavage from the solid support to provide various cores represented by compounds of Formula II-la comprising the lipid conjugates of the present disclosure. Oligonucleotide length.

In some embodiments, the present disclosure provides a method of preparing an oligonucleotide comprising one or more lipid conjugates, further comprising preparing a nucleic acid of Formula I-5a or an analog thereof: or a salt thereof, the preparation comprising the following steps: (a) providing a nucleic acid of formula Ia or an analog thereof: or a salt thereof, (b) deprotecting the nucleic acid of formula Ia or an analog thereof to form a compound of formula I-2a: or a salt thereof, (c) protecting the nucleic acid of formula I-2 or an analog thereof to form a compound of formula I-3a: or a salt thereof, and (d) treating the nucleic acid of formula I-3a or an analog thereof with a P(III) forming reagent to form a nucleic acid of formula I-5a or an analog thereof, wherein B, E, L, LC Each of , n, PG ⁴ , R ¹ , R ² , R ³ , X, X ¹ , X ² , X ³ , E, and Z is as defined above and described herein.

In step (b) above, PG ¹ and PG ² of the compound of formula Ia comprise silyl ether or cyclic silylene derivative that can be removed under acidic conditions or with fluoride anions. Examples of reagents that provide fluoride anions to remove silicon-based protecting groups include hydrofluoric acid, pyridine hydrogen fluoride, triethylamine trihydrofuride, tetrakis- N -butylammonium fluoride, and the like.

In step (c) above, the compound of formula I-2a is protected with a suitable hydroxyl protecting group. In certain embodiments, the protecting group PG ⁴ used to protect the 5'-hydroxyl group of the compound of Formula I-2a includes acid-labile protecting groups such as trityl, 4-methoxytrityl, 4,4'-dimethoxytrityl, 4,4',4"-trimethoxytrityl, 9-phenyl-xanthen-9-yl ), 9-(p-phenylmethyl)-𠮿-9-yl, phenyl𠮿yl (pixyl), 2,7-dimethylphenyl𠮿yl, etc. In some embodiments, acid-labile protection The base system is suitable for deprotection using, for example, dichloroacetic acid or trichloroacetic acid during both solution and solid phase synthesis of acid-sensitive nucleic acids or their analogues.

In the above step (d), the compound of formula I-3a is treated with a P(III) forming reagent to obtain the compound of formula I-5a. In the context of this disclosure, a P(III)-forming reagent is a phosphorus reagent that reacts to form a phosphorus(III) compound. In some embodiments, the P(III)-forming reagent is 2-cyanoethyl N , N -diisopropyl phosphoramidite chloride or 2-cyanoethyl dichlorophosphate. In certain embodiments, the P(III)-forming reagent is 2-cyanoethyl N , N -diisopropyl phosphoramidite chloride. One of ordinary skill will recognize that the leaving group in the P(III)-forming reagent is displaced from ^X1 of the compound of formula I-3a in the presence or absence of a suitable base. Such suitable bases are well known in the art and include organic and inorganic bases. In certain embodiments, basic tertiary amines such as triethylamine or diisopropylethylamine are used. In other embodiments, the above step (d) is performed using N , N -dimethylphosphoramic dichloride as the P(V) forming reagent.

In some embodiments, the present disclosure provides a method of preparing an oligonucleotide comprising one or more lipid conjugates, further comprising preparing a nucleic acid-lipid conjugate of Formula Ia or an analog thereof: or a salt thereof, the preparation includes the following steps: (a) providing a nucleic acid of formula I-1 or an analog thereof: or a salt thereof, and (b) conjugate one or more lipophilic compounds to a nucleic acid of Formula I-1 or an analog thereof to form a nucleic acid of Formula Ia or an analog thereof comprising one or more lipid conjugates , where: each of B, E, L, LC, n, PG ¹ , PG ² , R1, R ² , X, X ¹ , and Z is as defined above and described herein.

In the above step (b), the nucleic acid of Formula I-1a or its analog is conjugated with one or more lipophilic compounds to form the compound of Formula Ia including one or more lipid conjugates of the present disclosure. Typically, conjugation is carried out by an esterification or amidation reaction between a nucleic acid of formula I-la or an analog thereof and one or more fatty acids in a series or parallel manner by techniques known in the art. . In certain embodiments, conjugation is performed under suitable amide-forming conditions to provide a compound of Formula I that includes a plurality of lipid conjugates. Suitable amide forming conditions may include the use of amide coupling agents known in the art, such as, but not limited to, HATU, PyBOP, DCC, DIC, EDC, HBTU, HCTU, PyAOP, PyBrOP, BOP, BOP-Cl, DEPBT, T3P, TATU, TBTU, TNTU, TOTU, TPTU, TSTU, or TDBTU. Alternatively, conjugation of lipophilic compounds can be accomplished by any of the cross-coupling techniques described in Table A herein.

In some embodiments, the present disclosure provides a method of preparing an oligonucleotide comprising one or more lipid conjugates, or a pharmaceutically acceptable salt thereof, the lipid conjugate unit being represented by Formula II-1 : , the method includes the following steps: (a) providing an oligonucleotide of formula II-2: or a salt thereof, and (b) conjugating one or more lipophilic compounds to an oligonucleotide of Formula II-2 to form an oligonucleotide of Formula II-1 comprising one or more lipid conjugates . In the above step (b), the oligonucleotide of formula II-2 is conjugated with one or more lipophilic compounds to form the oligonucleotide of formula II-1 including one or more lipid conjugates of the present disclosure. Nucleotides. Typically, conjugation is performed by esterification or amidation reactions between an oligonucleotide of formula II-2 and one or more fatty acids in a series or parallel manner by techniques known in the art. In certain embodiments, conjugation is performed under suitable amide-forming conditions to obtain an oligonucleotide of Formula II-1 comprising a plurality of lipid conjugates. Suitable amide forming conditions may include the use of amide coupling agents known in the art, such as, but not limited to, HATU, PyBOP, DCC, DIC, EDC, HBTU, HCTU, PyAOP, PyBrOP, BOP, BOP-Cl, DEPBT, T3P, TATU, TBTU, TNTU, TOTU, TPTU, TSTU, or TDBTU. Alternatively, conjugation of lipophilic compounds can be accomplished by any of the cross-coupling techniques described in Table A herein.

In some embodiments, the present disclosure provides a preparation comprising Formula II-2: A method for making an oligonucleotide of the unit represented or a pharmaceutically acceptable salt thereof, the method comprising the following steps: (a) providing a nucleic acid of formula I-8 or an analog thereof: or a salt thereof, and (b) oligomerizing the compound of formula I-8 to form a compound of formula II-2.

In step (b) above, oligopolymerization refers to oligopolymerization-forming conditions using known and commonly used procedures to prepare oligonucleotides in the art. For example, a compound of formula I-8 is coupled to a solid supported nucleic acid bearing a 5'-hydroxyl group or an analog thereof. Further steps may include one or more deprotection, coupling, phosphite oxidation, and cleavage from the solid support to provide oligonucleotides of various nucleotide lengths represented by compounds of Formula II-2.

In some embodiments, the present disclosure provides a method of preparing a nucleic acid or an analog thereof comprising one or more lipid conjugates, further comprising preparing a nucleic acid of Formula I-8 or an analog thereof: or a salt thereof, the method includes the following steps: (a) providing a nucleic acid of formula I-1 or an analog thereof: or a salt thereof, (b) deprotecting the nucleic acid of formula I-1 or its analog to form a compound of formula I-6: or a salt thereof, (b) protecting the nucleic acid of formula I-6 or an analog thereof to form a compound of formula I-7: or a salt thereof, and (d) treating the nucleic acid of Formula I-7 or an analog thereof with a P(III) forming reagent to form a nucleic acid of Formula I-8 or an analog thereof. In step (b) above, PG ¹ and PG ² of the compound of formula I-1 comprise silyl ether or cyclic silylene derivatives that can be removed under acidic conditions or with fluoride anions. Examples of reagents that provide fluoride anions to remove silicon-based protecting groups include hydrofluoric acid, pyridine hydrogen fluoride, triethylamine trihydrofuride, tetrakis- N -butylammonium fluoride, and the like.

In step (c) above, the compound of formula I-6 is protected with a suitable hydroxyl protecting group. In certain embodiments, the protecting group PG ⁴ used to protect the 5'-hydroxyl group of the compound of Formula I-6 includes acid-labile protecting groups such as trityl, 4-methoxytrityl, 4,4'-Dimethoxytrityl, 4,4',4"-Trimethoxytrityl, 9-phenyl-𠮿-9-yl, 9-(p-phenylmethyl)- 𠮿-9-yl, phenyl 𠮿yl, 2,7-dimethylphenyl 𠮿yl, etc. In some embodiments, the acid-labile protecting group is suitable for use in acid-sensitive nucleic acids or their analogs. Deprotection is performed during both solution phase synthesis and solid phase synthesis using, for example, dichloroacetic acid or trichloroacetic acid.

In the above step (d), the compound of formula I-7 is treated with a P(III) forming reagent to obtain the compound of formula I-8. In the context of this disclosure, a P(III)-forming reagent is a phosphorus reagent that reacts to form a phosphorus(III) compound. In some embodiments, the P(III)-forming reagent is 2-cyanoethyl N , N -diisopropyl phosphoramidite chloride or 2-cyanoethyl dichlorophosphate. In certain embodiments, the P(III)-forming reagent is 2-cyanoethyl N , N -diisopropyl phosphoramidite chloride. One of ordinary skill will recognize that the leaving group in the P(III)-forming reagent is displaced from ^X1 of the compound of formula I-7 in the presence or absence of a suitable base. Such suitable bases are well known in the art and include organic and inorganic bases. In certain embodiments, basic tertiary amines such as triethylamine or diisopropylethylamine are used. In other embodiments, the above step (d) is performed using N , N -dimethylphosphoramidite dichloride as the P(V) forming reagent.

In some embodiments, the present disclosure provides a method of preparing an oligonucleotide-ligand conjugate comprising one or more adamantyl and/or lipid moieties, or a pharmaceutically acceptable salt thereof, the lipid co- The yoke unit is represented by formula II-b-3: , the method includes the following steps: (a) providing a nucleic acid-ligand conjugate of formula C11 or an analog thereof: or a salt thereof, and (b) oligomerize the compound of formula C11 to form a compound of formula II-b-3. In step (b) above, oligopolymerization refers to oligopolymerization-forming conditions using known and commonly used procedures to prepare oligonucleotides in the art. For example, a compound of formula C11 is coupled to a solid supported nucleic acid bearing a 5'-hydroxyl group or an analog thereof. Further steps may include one or more deprotection, coupling, phosphite oxidation, and cleavage from the solid support to provide oligos of various nucleotide lengths with one or more nucleic acid-ligand conjugate units. Nucleotide-ligand conjugates, wherein each unit is represented by a compound of Formula II-b-3 of the present disclosure containing an adamantyl or lipid moiety.

In some embodiments, the method of preparing an oligonucleotide of formula II-b-3 comprising one or more lipid conjugates further comprises preparing a nucleic acid-lipid conjugate of formula C11 or an analog thereof: or a salt thereof, the preparation comprising the following steps: (a) providing a nucleic acid of formula Ib or an analog thereof: or a salt thereof, (b) deprotecting the nucleic acid-ligand conjugate of formula Ib or an analog thereof to form a compound of formula C8: or a salt thereof, (c) protecting the nucleic acid-ligand conjugate of formula C8 or an analog thereof to form a compound of formula C9: or a salt thereof, and (d) treating the nucleic acid-ligand conjugate of Formula C9 or an analog thereof with a P(III) forming reagent to form a nucleic acid of Formula C11 or an analog thereof. In step (b) above, PG ¹ and PG ² of the compound of formula Ib comprise silyl ethers or cyclic silylene derivatives that can be removed under acidic conditions or with fluoride anions. Examples of reagents that provide fluoride anions to remove silicon-based protecting groups include hydrofluoric acid, pyridine hydrogen fluoride, triethylamine trihydrofuride, tetrakis- N -butylammonium fluoride, and the like.

In step (c) above, the compound of formula C8 is protected with a suitable hydroxyl protecting group. In certain embodiments, the protecting group PG ⁴ used to protect the 5'-hydroxyl group of the compound of Formula C8 includes acid-labile protecting groups such as trityl, 4-methoxytrityl, 4, 4'-dimethoxytrityl, 4,4',4"-trimethoxytrityl, 9-phenyl-𠮿-9-yl, 9-(p-phenylmethyl)-𠮿- 9-yl, phenyl, 2,7-dimethylphenyl, etc. In some embodiments, the acid-labile protecting group is suitable for use in the solution phase of acid-sensitive nucleic acids or their analogs. Deprotection is performed using, for example, dichloroacetic acid or trichloroacetic acid during both synthesis and solid phase synthesis.

In the above step (d), the compound of formula C9 is treated with a P(III) forming reagent to obtain the compound of formula C11. In the context of this disclosure, a P(III)-forming reagent is a phosphorus reagent that reacts to form a phosphorus(III) compound. In some embodiments, the P(III)-forming reagent is 2-cyanoethyl N , N -diisopropyl phosphoramidite chloride or 2-cyanoethyl dichlorophosphate. In certain embodiments, the P(III)-forming reagent is 2-cyanoethyl N , N -diisopropyl phosphoramidite chloride. One of ordinary skill will recognize that the leaving group in the P(III)-forming reagent is displaced from ^X1 of the compound of formula C9 in the presence or absence of a suitable base. Such suitable bases are well known in the art and include organic and inorganic bases. In certain embodiments, basic tertiary amines such as triethylamine or diisopropylethylamine are used. In other embodiments, the above step (d) is performed using N , N -dimethylphosphoramidite dichloride as the P(V) forming reagent.

In some embodiments, the present disclosure provides a method for preparing an oligonucleotide-ligand conjugate of Formula II-b-3, the oligonucleotide-ligand conjugate comprising one or more oligonucleotide-ligand conjugates each comprising One or more nucleic acid-ligand conjugate units of an adamantyl or lipid moiety, the method further comprising preparing a nucleic acid-ligand conjugate of formula Ib or an analog thereof: or a salt thereof, the preparation comprising the following steps: (a) providing a nucleic acid-ligand conjugate of formula C6 or an analog thereof: or a salt thereof, and (b) conjugate a lipophilic compound to a nucleic acid of formula C6 or an analog thereof to form a nucleic acid-ligand conjugate of formula Ib comprising one or more adamantyl and/or lipid conjugates Yoke or the like. In step (b) above, conjugation is carried out under suitable amide-forming conditions to obtain compounds of formula Ib containing adamantyl and/or lipid conjugates. Suitable amide forming conditions may include the use of amide coupling agents known in the art, such as, but not limited to, HATU, PyBOP, DCC, DIC, EDC, HBTU, HCTU, PyAOP, PyBrOP, BOP, BOP-Cl, DEPBT, T3P, TATU, TBTU, TNTU, TOTU, TPTU, TSTU, or TDBTU. In certain embodiments, the amide forming conditions include HATU, and DIPEA or TEA.

In certain embodiments, the nucleic acid-ligand conjugate of Formula C6 or analog thereof is provided in salt form (e.g., fumarate) and converted to the free base (e.g., prior to performing the conjugation step). , using sodium bicarbonate).

In some embodiments, the present disclosure provides a method of preparing an oligonucleotide-ligand conjugate of Formula II-b-3, the oligonucleotide-ligand conjugate comprising one or more nucleic acids- Ligand conjugate unit, the method further comprises preparing a nucleic acid-ligand conjugate of formula C6 or an analog thereof: or a salt thereof, the preparation comprising the following steps: (a) providing a nucleic acid of formula C1 or an analog thereof: or a salt thereof, and (b) protect the nucleic acid of formula C1 or an analog thereof to form a compound of formula C2: or a salt thereof, (c) alkylating the nucleic acid of formula C2 or an analog thereof to form a compound of formula C3: or a salt thereof, (d) using the nucleic acid of formula C3 or its analogue with a compound of formula C4: Or its salt is substituted to form a compound of formula C5: or a salt thereof, (e) deprotecting the nucleic acid of formula C5 or an analog thereof to form a nucleic acid-ligand conjugate of formula C6 or an analog thereof. In step (b) above, the PG ¹ and PG ² groups of formula C2 are taken together with their intervening atoms to form a cyclic diol protecting group, such as a cyclic acetal or ketal. Such groups include methylene, ethylene, benzylene, isopropylene, cyclohexylene, and cyclopentylene, silicone derivatives such as di-tertiary butylsilylene and 1, 1,3,3-tetraisopropyldisiloxanylidene (1,1,3,3-tetraisopropylidisiloxanylidene), cyclic carbonate, cyclic boronic acid ester, and cyclic adenosine monophosphate-based (also That is, the cyclic monophosphate derivative of cAMP). In certain embodiments, the cyclic diol protecting group is a 1,1,3,3-tetraisopropyl disiloxane group, which is formed from the diol of formula C1 and 1,3 under basic conditions. - Preparation by reaction of dichloro-1,1,3,3-tetraisopropyldisiloxane.

In step (c) above, the nucleic acid of formula C2 or analog thereof is alkylated with a mixture of DMSO and acetic anhydride under acidic conditions. In certain embodiments, when -V-H is a hydroxyl group, the mixture of DMSO and acetic anhydride undergoes Pumerel rearrangement in the presence of acetic acid to form (methylthio)methyl acetate in situ, and then combines with formula C2 The hydroxyl group of a nucleic acid or analog thereof is reacted to provide a monothioacetal functionalized fragment nucleic acid of formula C3 or an analog thereof.

In the above step (d), the nucleic acid of Formula C4 or its analogue is used to replace the thiomethyl group of the nucleic acid of Formula C3 or its analogue to obtain the nucleic acid of Formula C4 or its analogue. In certain embodiments, substitution occurs under mild oxidative and/or acidic conditions. In some embodiments, V is oxygen. In some embodiments, mild oxidizing reagents include elemental iodine and hydrogen peroxide, urea hydrogen peroxide complex, silver nitrate/silver sulfate, sodium bromate, ammonium peroxodisulfate, tetrabutyl peroxodisulfate tetrabutylammonium peroxydisulfate, Oxone®, Chloramine T, Selectfluor®, Selectfluor® II, sodium hypochlorite, or potassium iodate/sodium periodate. In some embodiments, mild oxidizing reagents include N-iodosuccinimide, N-bromosuccinimide, N-chlorosuccinimide, 1,3-diiodo-5,5-dimethyl Hydantoin (1,3-diiodo-5,5-dimethylhydantion), pyridinium tribromide (pyridinium tribromide), iodine chloride or its complex, etc. Typical acids used under mild oxidizing conditions include sulfuric acid, p-toluenesulfonic acid, trifluoromethanesulfonic acid, methanesulfonic acid, and trifluoroacetic acid. In certain embodiments, the mild oxidizing agent includes a mixture of N-succinimide and triflate.

In the above step (e), PG ³ and optionally R ⁴ (when R ⁴ is a suitable amine protecting group) of the nucleic acid-ligand conjugate of formula C5 or its analog are removed to obtain the formula C6 Nucleic acid-ligand conjugate or analog thereof or salt thereof. In some embodiments, PG ³ and/or R ⁴ comprise carbamate derivatives, which are removable under acidic or basic conditions. In certain embodiments, the protecting group (e.g., both PG ³ and R ⁴ or independently PG ³ or R ⁴ ) of the nucleic acid-ligand conjugate of Formula C5 or analog thereof is generated by acid hydrolysis. Remove. It will be understood that upon acid hydrolysis of the protecting group of the nucleic acid-ligand conjugate of formula C5 or analog thereof, a salt thereof of formula C6 is formed. For example, when the acid-labile protecting group of a nucleic acid-ligand conjugate of formula C5 or an analog thereof is removed by treatment with an acid, such as hydrochloric acid, the resulting amine compound will form its hydrochloride salt. One of ordinary skill in the art will recognize that a wide variety of acids can be used to remove acid-labile amine protecting groups, and thus contemplates a wide variety of salts of the nucleic acid of formula C6 or analogs thereof form.

In other embodiments, the protecting group (eg, both PG ³ and R ⁴ or independently PG ³ or R ⁴ ) of the nucleic acid of Formula C5 or analog thereof is removed by alkaline hydrolysis. For example, Fmoc and trifluoroacetyl protecting groups can be removed by treatment with a base. One of ordinary skill in the art will recognize that a wide variety of bases can be used to remove base-labile amine protecting groups. In some embodiments, the base is piperidine. In some embodiments, the base is 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). In certain embodiments, the nucleic acid-ligand conjugate of Formula C5 or analog thereof is deprotected under basic conditions and then treated with an acid to form a salt of Formula C6. In certain embodiments, the acid is fumarate and the salt of formula C6 is fumarate.

In some embodiments, the present disclosure provides a method of preparing an oligonucleotide-ligand conjugate or a pharmaceutically acceptable salt thereof comprising one or more nucleic acid-ligand conjugates. The body conjugated body unit is represented by formula II-b-3: , the method includes the following steps: (a) providing an oligonucleotide of formula D5: or a salt thereof, and (b) conjugating one or more adamantyl or lipophilic compounds to an oligonucleotide of Formula D5 to form Formula II-b comprising one or more nucleic acid ligand conjugate units -3 oligonucleotide-ligand conjugate. In the above step (b), the conjugation is carried out under suitable amide formation conditions to obtain a compound of formula D5 containing an adamantyl group or a lipid conjugate. Suitable amide forming conditions may include the use of amide coupling agents known in the art, such as, but not limited to, HATU, PyBOP, DCC, DIC, EDC, HBTU, HCTU, PyAOP, PyBrOP, BOP, BOP-Cl, DEPBT, T3P, TATU, TBTU, TNTU, TOTU, TPTU, TSTU, or TDBTU. In certain embodiments, the amide forming conditions include HATU, and DIPEA or TEA.

In some embodiments, the present disclosure provides a preparation comprising Formula D5: A method for producing an oligonucleotide-ligand conjugate of the unit represented or a salt thereof, the method comprising the following steps: (a) providing a nucleic acid-ligand conjugate of formula D4 or an analog thereof: or a salt thereof, and (b) deprotecting the compound of formula D4 to form a compound of formula D5. In the above step (b), PG ³ and optionally R ⁴ (when R ⁴ is a suitable amine protecting group) of the oligonucleotide of formula D4 are removed to obtain the oligonucleotide-ligand of formula D5. conjugate or its salt. In some embodiments, PG ³ and/or R ⁴ comprise urethane derivatives that are removed under acidic or basic conditions. In certain embodiments, the protecting group (e.g., both PG ³ and R ⁴ or independently PG ³ or R ⁴ ) of the oligonucleotide-ligand conjugate of Formula D4 is removed by acid hydrolysis. remove. It is understood that upon acid hydrolysis of the protecting group of the oligonucleotide-ligand conjugate of formula D4, its salt of formula D5 is formed. For example, when an acid-labile protecting group of an oligonucleotide of formula D4 is removed by treatment with an acid, such as hydrochloric acid, the resulting amine compound will form its hydrochloride salt. One of ordinary skill in the art will recognize that a wide variety of acids can be used to remove acid-labile amine protecting groups and thus contemplate nucleic acid-ligand conjugate units of formula D5 or the like thereof of various salt forms.

In other embodiments, the protecting group (e.g., both PG ³ and R ⁴ or independently PG ³ or R ⁴ ) of the oligonucleotide-ligand conjugate of Formula D4 is removed by alkaline hydrolysis . For example, Fmoc and trifluoroacetyl protecting groups can be removed by treatment with a base. One of ordinary skill in the art will recognize that a wide variety of bases can be used to remove base-labile amine protecting groups. In some embodiments, the base is piperidine. In some embodiments, the base is 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).

In some embodiments, the present disclosure provides methods for preparing oligonucleotide-ligand conjugates comprising one or more nucleic acid-ligand conjugate units having one or more adamantyl and/or lipid moieties. Or its pharmaceutically acceptable salt method, the conjugate unit is represented by formula D4: , the method includes the following steps: (a) providing a nucleic acid of formula D3 or an analog thereof: or a salt thereof, and (b) oligomerizes the compound of formula D3 to form a compound of formula D4. In step (b) above, oligopolymerization refers to oligopolymerization-forming conditions using known and commonly used procedures to prepare oligonucleotides in the art. For example, a nucleic acid of formula D3 or an analog thereof is coupled to a solid supported nucleic acid bearing a 5'-hydroxyl group or an analog thereof. Further steps may include one or more deprotection, coupling, phosphite oxidation, and cleavage from the solid support to provide the adamantyl or lipid conjugate-containing lipid conjugate of the present disclosure of Formula D4. The compounds represent oligonucleotides of various nucleotide lengths.

In some embodiments, the present disclosure provides a method of preparing a nucleic acid or an analog thereof comprising one or more lipid conjugates, further comprising preparing a nucleic acid of Formula D3 or an analog thereof: or a salt thereof, the preparation comprising the following steps: (a) providing a nucleic acid of formula C5 or an analog thereof: or a salt thereof, (b) deprotecting the nucleic acid of formula C5 or an analog thereof to form a compound of formula D1: or a salt thereof, (c) protecting the nucleic acid of formula D1 or an analog thereof to form a nucleic acid of formula D2 or an analog thereof. or a salt thereof, and (d) treating the nucleic acid of formula D2 or an analog thereof with a P(III) forming reagent to form a nucleic acid of formula D3 or an analog thereof. In the above step (b), PG ¹ and PG ² of the nucleic acid of formula C5 or its analog include silyl ether or cyclic silylene derivatives that can be removed under acidic conditions or with fluoride anions. Examples of reagents that provide fluoride anions to remove silicon-based protecting groups include hydrofluoric acid, pyridine hydrogen fluoride, triethylamine trihydrofuride, tetrakis- N -butylammonium fluoride, and the like.

In step (c) above, the nucleic acid of formula D1 or analog thereof is protected with a suitable hydroxyl protecting group. In certain embodiments, the protecting group PG ⁴ used to protect the 5'-hydroxyl group of the compound of Formula D1 includes an acid-labile protecting group such as trityl, 4-methoxytrityl, 4, 4'-dimethoxytrityl, 4,4',4"-trimethoxytrityl, 9-phenyl-𠮿-9-yl, 9-(p-phenylmethyl)-𠮿- 9-yl, phenyl, 2,7-dimethylphenyl, etc. In some embodiments, the acid-labile protecting group is suitable for use in the solution phase of acid-sensitive nucleic acids or their analogs. Deprotection is performed using, for example, dichloroacetic acid or trichloroacetic acid during both synthesis and solid phase synthesis.

In the above step (d), the nucleic acid of formula D2 or its analog is treated with a P(III) forming reagent to obtain the compound of formula D3. In the context of this disclosure, a P(III)-forming reagent is a phosphorus reagent that reacts to form a phosphorus(III) compound. In some embodiments, the P(III)-forming reagent is 2-cyanoethyl N , N -diisopropyl phosphoramidite chloride or 2-cyanoethyl dichlorophosphate. In certain embodiments, the P(III)-forming reagent is 2-cyanoethyl N , N -diisopropyl phosphoramidite chloride. One of ordinary skill will recognize that the leaving group in the P(III)-forming reagent is displaced from ^X1 of the compound of formula D2 in the presence or absence of a suitable base. Such suitable bases are well known in the art and include organic and inorganic bases. In certain embodiments, basic tertiary amines such as triethylamine or diisopropylethylamine are used. In other embodiments, the above step (d) is performed using N , N -dimethylphosphoramidite dichloride as the P(V) forming reagent. Preparations

Various formulations have been developed to facilitate the use of oligonucleotides. For example, oligonucleotides (e.g., lipid-conjugated RNAi oligonucleotides) can be delivered to an individual or cellular environment using formulations that minimize degradation, facilitate delivery and/or uptake, or that The oligonucleotide provides another beneficial property. In some embodiments, provided herein are compositions comprising oligonucleotides (e.g., lipid-conjugated RNAi oligonucleotides) that reduce target mRNA (e.g., expressed in neurons of the CNS). target mRNA). In some embodiments, provided herein are compositions comprising oligonucleotides (e.g., lipid-conjugated RNAi oligonucleotides) that reduce targets expressed in one or more tissues or cells of an individual Expression of mRNA. Such compositions may be suitably formulated so that when administered to an individual (either into the immediate environment of a target cell or systemically), a sufficient portion of the oligonucleotide enters the cell to reduce the target gene Performance. Any of a variety of suitable oligonucleotide formulations may be used to deliver oligonucleotides for reducing target gene expression as disclosed herein. In some embodiments, oligonucleotides are formulated in buffer solutions such as phosphate buffered saline solution, liposomes, microstructures, and shells.

In some embodiments, the formulations herein include excipients. In some embodiments, excipients impart to the composition increased stability, increased absorption, increased solubility, and/or therapeutic enhancement of the active ingredients. In some embodiments, the excipient is a buffer (eg, sodium citrate, sodium phosphate, tris base, or sodium hydroxide) or vehicle (eg, buffered solution, paraffin oil, dimethyl sulfoxide, or mineral oil). In some embodiments, the oligonucleotide is freeze-dried to extend its shelf life and then made into solution prior to use (eg, administration to an individual). Accordingly, the excipient in a composition comprising any of the oligonucleotides described herein may be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrole dextran) or collapse temperature modifier (such as dextran, Ficoll™ or gelatin). Likewise, the oligonucleotides herein may be provided in their free acid form.

In some embodiments, pharmaceutical compositions are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intramuscular, intraperitoneal, intradermal, subcutaneous, intrathecal), oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. give. In some embodiments, pharmaceutical compositions are formulated for delivery to the central nervous system (eg, intrathecal, epidural). In some embodiments, pharmaceutical compositions are formulated for delivery to the eye (e.g., ophthalmic, intraocular, subconjunctival, intravitreal, retrobulbar, intracameral) .

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (when water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof. In many cases it will be preferable to include isotonic agents such as sugars, polyols (such as mannitol, sorbitol), sodium chloride in the composition. Sterile injectable solutions can be prepared by incorporating the oligonucleotide in the required amount and, when necessary, one or a combination of the components enumerated above in a solvent of choice, followed by filtered sterilization.

In some embodiments, the compositions may contain at least about 0.1% or more of a therapeutic agent (e.g., a lipid-conjugated RNAi oligonucleotide herein), although the percentage of active ingredient(s) may be between Between about 1% and about 80% or more by weight or volume of the total composition. One of ordinary skill in the art in preparing such pharmaceutical formulations should take into account factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations, and accordingly, various dosages and treatments The plan can be whatever you want. Structural modification

Because nucleic acids are polymers of subunits or compounds, many of the modifications described below occur at repeated positions within the nucleic acid (eg, modifications of bases or phosphate moieties or non-bridging oxygens of the phosphate moiety). In some cases, modification will occur at all subject positions in the nucleic acid, but this will not occur in many cases, and indeed in most cases. For example, modifications may occur only at the 3' or 5' end positions, may occur only at the internal unpaired regions, may occur only at the end regions, such as at the terminal nucleotides of the strand or at the last 2, 3, 4, 5 or a position on 10 nucleotides. In some embodiments, modifications will occur at all subject positions in the nucleic acid. Modifications can occur in double-stranded regions, single-stranded regions, or both. Modification can occur only in the double-stranded region of the RNA agent or only in the single-stranded region of the RNA agent. (For example, phosphorothioate modifications at non-bridging oxygen positions can occur only at one or two ends, can occur only at the end region or at the terminal nucleotides of the strand or the last 2, 3, 4, 5 or 10 cores. Positions on the nucleotides may occur in double-stranded and single-stranded regions, particularly at the ends. The 5' end(s) may be phosphorylated.

Many studies in the art have shown that modified oligonucleotides and oligonucleotide analogs may be less susceptible to internalization than their natural counterparts. Therefore, many previously available RNAi trigger molecules are not active enough for practical therapeutic, research or diagnostic purposes.

Modifications to enhance the effectiveness of RNAi trigger molecule oligonucleotides and overcome these problems have taken many forms. These modifications include base ring modifications, sugar moiety modifications, and sugar-phosphate backbone modifications, many of which are exemplified herein and used in the present disclosure. Previous sugar-phosphate backbone modifications, particularly on the phosphorus atom, have affected various degrees of nuclease resistance. However, while the ability of RNAi trigger oligonucleotides to load into RISC and direct the location of relevant mRNA sequences is the basis of RNAi trigger methodology, many modifications act in cross-purposes with each other to optimize the behavior of RNAi triggers. . This balancing act must be considered relative to the development of advanced and efficient RNAi molecules.

Another key factor is the stereochemical effects produced in oligomers with P-chiral centers. Typically, chiral mixtures will be constructed in sequential non-stereospecific chain synthesis of oligomers with n nucleosides in length. It has been observed that Rp and Sp homochiral chains, the absolute configuration of the methanephosphonate phosphorus atom in all internucleotides is Rp or Sp, and non-stereoregular chains show different physicochemical properties and oligos with complementary sequences. Different abilities of nucleotides to form adducts. Additionally, phosphorothioate analogs of nucleotides have shown substantial stereoselective differences in resistance to nuclease activity between Oligo-Rp and Oligo-Sp oligonucleotides (Potter, Biochemistry, 22:1369, ( 1983); Bryant et al., Biochemistry, 18:2825, (1979)). Lesnikowski (Nucl. Acids Res., 18:2109, (1990)) observed diastereomerically pure octathymidine methylphosphonate in which six of the seven methylphosphonate linkages reacted with the matrix. (when the phosphorus atoms have a defined configuration) exhibit substantial differences in melting temperatures. Based on the current disclosure, chirally pure nucleotide analogs or portions thereof are expected to provide trigger structures with improved properties, allowing the development of more potent and durable RNAi triggers.

In some of the presently disclosed embodiments, it may be advantageous to enhance stability by including specific bases in the overhang, or by including a modified core in a single stranded overhang, e.g., in the 5' or 3' overhang, or in both. nucleotide or nucleotide substitutions. Likewise, it may be desirable to include purine nucleotides in the overhang since they are more resistant to nuclease activity. In some embodiments, all or some of the bases of the 3' or 5' overhang will be modified with modifications described herein. Modifications may include the use of modifications on the 2'OH group of ribose, deoxythymidine rather than ribonucleotides, and the use of modifications on the phosphate group, also known as phosphorothioate modifications. The overhang need not be homologous to the target sequence. Instructions Reduce target gene expression

In some embodiments, the present disclosure provides methods of contacting or delivering an effective amount of any of the lipid-conjugated RNAi oligonucleotides herein to a cell or population of cells to reduce expression of a target gene.

In some embodiments, expression of the target gene is reduced in one or more tissues or cells of the individual. In some embodiments, expression of the target gene is reduced in the central nervous system (CNS). In some embodiments, expression of the target gene is reduced in eye tissue. In some embodiments, expression of the target gene is reduced in the liver. In some embodiments, the expression of the target gene is reduced in adipose tissue. In some embodiments, expression of the target gene is reduced in adrenal tissue. In some embodiments, expression of the target gene is reduced in skeletal muscle tissue. In some embodiments, expression of the target gene is reduced in the heart. In some embodiments, expression of the target gene is reduced in the lungs.

In some embodiments, reduced target gene expression is determined by measuring a reduction in the amount or level of target mRNA, protein encoded by target mRNA, or target gene (mRNA or protein) activity in the cell. Such methods include those described herein and known to those of ordinary skill in the art.

The methods provided herein can be used with any appropriate cell type. In some embodiments, the cell line is any cell expressing the target mRNA. In some embodiments, the cell line is obtained from primary cells of an individual. In some embodiments, the primary cells have undergone a limited number of passages such that the cells substantially maintain native phenotypic properties. In some embodiments, the cell line to which the oligonucleotide is delivered is ex vivo or ex vivo (ie, delivery can be made to cells in culture or to an organism in which the cells reside).

In some embodiments, lipid-conjugated RNAi oligonucleotides disclosed herein are delivered to cells or populations of cells using nucleic acid delivery methods known in the art, including but not limited to injecting RNAi containing the lipid-conjugated RNAi. Solutions or pharmaceutical compositions of oligonucleotides, bombardment of particles covered with the lipid-conjugated RNAi oligonucleotide, exposure of cells or cell populations to lipid-conjugated RNAi oligonucleotides The cell membrane is electroporated in a solution or in the presence of the lipid-conjugated RNAi oligonucleotide. Other methods known in the art for delivering oligonucleotides to cells may be used, such as lipid-mediated carrier delivery, chemical-mediated delivery, and cationic liposome transfection (such as calcium phosphate), and others.

In some embodiments, target gene expression is reduced by an assay or technique that assesses one or more molecules, properties, or characteristics of a cell or cell population associated with target gene expression, or by an assessment that directly indicates that the cell or Assays or techniques for measuring molecules (e.g., target mRNA or protein) expressed by a target gene in a population of cells. In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein reduces target gene expression in a cell to an extent by contacting the cell or cells with the lipid-conjugated RNAi oligonucleotide. The target gene expression of the population is compared to that of a control cell or population of cells (e.g., cells or a population of cells that are not contacted with the lipid-conjugated RNAi oligonucleotide or that are contacted with the control lipid-conjugated RNAi oligonucleotide) evaluate. In some embodiments, control amounts or levels of target gene expression in control cells or cell populations are predetermined such that control amounts or levels do not need to be measured each time an assay or technique is performed. The predetermined level or value can take a variety of forms. In some embodiments, the predetermined level or value may be a single cutoff value, such as the median or mean.

In some embodiments, contacting or delivering a lipid-conjugated RNAi oligonucleotide described herein to a cell or population of cells results in reduced expression of the target gene. In some embodiments, the target gene expression is reduced relative to the target gene in cells or cell populations that are not exposed to the lipid-conjugated RNAi oligonucleotide or are exposed to a control lipid-conjugated RNAi oligonucleotide. Performance relative to quantity or level. In some embodiments, the reduction in target gene expression is about 1% or less, about 5% or less, about 10% or less, about 15% relative to a control amount or level of target gene expression. % or less, about 20% or less, about 25% or less, about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or lower, about 55% or lower, about 60% or lower, about 70% or lower, about 80% or lower or about 90% or lower. In some embodiments, the control amount or level of target gene expression is the amount or level of target mRNA and/or protein in cells or cell populations that have not been contacted with the lipid-conjugated RNAi oligonucleotides herein. In some embodiments, the effect of delivering a lipid-conjugated RNAi oligonucleotide to a cell or cell population according to the methods herein is over any limited period or amount of time (e.g., minutes, hours, days, Weeks, months) later. For example, in some embodiments, the lipid-conjugated RNAi oligonucleotide is contacted or delivered to the cell or cell population for at least about 4 hours, about 8 hours, about 12 hours, about 18 hours, about 24 hours; or At least about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, about 56 days, about 63 days, about 70 days, about 77 days, or about 84 days or more Then, the target gene expression of the cell or cell population is determined. In some embodiments, the lipid-conjugated RNAi oligonucleotide is contacted or delivered to the cell or cell population for at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months. Months or approximately 6 months or more later, the target gene expression of the cell or cell population is determined. Reduce CNS target gene expression

In some embodiments, expression of the target gene (neuronal target gene) is reduced in regions of the CNS. In some embodiments, expression of the target gene is reduced in at least one region of the CNS. In some embodiments, a region of the CNS is one or more tissues of the CNS. In some embodiments, regions of the CNS include, but are not limited to, the brain, prefrontal cortex, frontal cortex, motor cortex, temporal cortex, parietal cortex, occipital cortex, sensory cortex, hippocampus, caudate Caudate, striatum, globus pallidus, optic thalamus, midbrain, tegmentum, substantia nigra, pons, brainstem, cerebellar white matter, cerebellum, Dentate nucleus, medulla oblongata, cervical spinal cord, thoracic spinal cord, lumbar spinal cord, cervical dorsal root ganglion, thoracic dorsal root ganglion, lumbar dorsal root ganglion, sacral dorsal root nerve Sacral dorsal root ganglion, nodose ganglia, femoral nerve, sciatic nerve, sural nerve, amygdala, hypothalamus, putamen, callosum, and cranial nerves. In some embodiments, the CNS region is selected from the group consisting of spinal cord, lumbar root ganglia, medulla oblongata, hippocampus, frontal cortex, brainstem, cerebellum, and combinations thereof. In one embodiment, expression of the target gene is reduced in at least one region of the CNS selected from the group consisting of frontal cortex, medulla oblongata, hippocampus, hypothalamus, cerebellum, lumbar spinal cord, lumbar dorsal root ganglia, and any combination thereof .

In some embodiments, expression of a neuronal target gene is reduced in at least one tissue of the CNS. In some embodiments, expression of a stellate cell target gene is reduced in at least one tissue of the CNS. In some embodiments, expression of the oligodendritic cell target gene is reduced in at least one tissue of the CNS. In some embodiments, expression of neuronal target mRNA is reduced in at least one tissue of the CNS. In some embodiments, expression of stellate cell target mRNA is reduced in at least one tissue of the CNS. In some embodiments, expression of target mRNA by oligodendritic cells is reduced in at least one tissue of the CNS.

In one embodiment, the neuronal target mRNA is expressed in at least one of the CNS selected from the group consisting of frontal cortex, medulla oblongata, hippocampus, hypothalamus, cerebellum, lumbar spinal cord, lumbar dorsal root ganglia, and any combination thereof. A tissue reduction. In one embodiment, the stellate cell target mRNA is expressed in the CNS selected from the group consisting of frontal cortex, medulla oblongata, hippocampus, hypothalamus, cerebellum, lumbar spinal cord, lumbar dorsal root ganglia, and any combination thereof. Reduction in at least one tissue. In one embodiment, the target mRNA of the oligodendritic cells is expressed in the CNS selected from the group consisting of frontal cortex, medulla oblongata, hippocampus, hypothalamus, cerebellum, lumbar spinal cord, lumbar dorsal root ganglia, and any combination thereof. at least one tissue is reduced.

In some embodiments, the subject's expression of the target gene in the CNS is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50% when compared to the expression of the target gene in control tissue. , about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

In some embodiments, the subject's expression of a target gene in stellate cells is reduced by at least about 30%, about 35%, about 40%, about 45% when compared to expression of a target gene in non-target cells. , about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

In some embodiments, the subject's target gene expression in oligodendritic cells is reduced by at least about 30%, about 35%, about 40%, about 45% when compared to target gene expression in non-target cells. %, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

In some embodiments, the individual's expression of a target gene in neurons is reduced by at least about 30%, about 35%, about 40%, about 45%, when compared to expression of a target gene in non-target cells. About 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99%.

In some embodiments, contacting or delivering a lipid-conjugated RNAi oligonucleotide described herein to a cell or population of cells results in reduced expression of a target gene in neurons. In some embodiments, the reduction in target gene expression in neurons is relative to the amount or level of target gene expression in stellate cells contacted with the lipid-conjugated RNAi oligonucleotide. In some embodiments, the reduction in expression of a target gene in a neuron relative to the amount or level of expression of a target gene in a stellate cell is about 1% or less, about 5% or less, about 10 % or less, about 15% or less, about 20% or less, about 25% or less, about 30% or less, about 35% or less, about 40% or less, about 45% or lower, about 50% or lower, about 55% or lower, about 60% or lower, about 70% or lower, about 80% or lower or about 90% or lower. In some embodiments, the reduction in target gene expression in neurons is relative to the amount or level of target gene expression in oligodendritic cells contacted with the lipid-conjugated RNAi oligonucleotide. In some embodiments, the reduction in expression of a target gene in a neuron relative to the amount or level of expression of a target gene in an oligodendritic cell is about 1% or less, about 5% or less, about 10% or less, about 15% or less, about 20% or less, about 25% or less, about 30% or less, about 35% or less, about 40% or less, about 45 % or less, about 50% or less, about 55% or less, about 60% or less, about 70% or less, about 80% or less, or about 90% or less.

In some embodiments, the reduction in target gene expression in stellate cells is increased by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, relative to the reduction in target gene expression in neurons. At least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, or at least 90%. In some embodiments, the reduction in target gene expression in oligodendritic cells is increased by at least 1%, at least 5%, at least 10%, at least 15%, at least 20% relative to the reduction in target gene expression in neurons. , at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80% or at least 90%.

In some embodiments, contacting or delivering a lipid-conjugated RNAi oligonucleotide described herein to a cell or population of cells results in reduced expression of target genes by stellate cells. In some embodiments, the reduction in target gene expression in stellate cells is relative to the amount or level of target gene expression in neurons contacted with the lipid-conjugated RNAi oligonucleotide. In some embodiments, the reduction in expression of a target gene in a stellate cell relative to the amount or level of expression of a target gene in a neuron is about 1% or less, about 5% or less, about 10 % or less, about 15% or less, about 20% or less, about 25% or less, about 30% or less, about 35% or less, about 40% or less, about 45% or lower, about 50% or lower, about 55% or lower, about 60% or lower, about 70% or lower, about 80% or lower or about 90% or lower. In some embodiments, the reduction in target gene expression in stellate cells is relative to the amount or level of target gene expression in oligodendritic cells contacted with the lipid-conjugated RNAi oligonucleotide. In some embodiments, the reduction in expression of a target gene in stellate cells is about 1% or less, about 5% or less, relative to the amount or level of expression of the target gene in oligodendritic cells. About 10% or less, about 15% or less, about 20% or less, about 25% or less, about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or less, about 60% or less, about 70% or less, about 80% or less, or about 90% or less.

In some embodiments, the reduction in target gene expression in neurons is increased by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, relative to the reduction in target gene expression in stellate cells. At least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, or at least 90%. In some embodiments, the reduction in target gene expression in oligodendritic cells is increased by at least 1%, at least 5%, at least 10%, at least 15%, at least 20% relative to the reduction in target gene expression in stellate cells. %, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80% or at least 90%.

In some embodiments, contacting or delivering a lipid-conjugated RNAi oligonucleotide described herein to a cell or population of cells results in reduced expression of a target gene by oligodendritic cells. In some embodiments, the reduction in oligodendritic cell target gene expression is relative to the amount or level of target gene expression in neurons contacted with the lipid-conjugated RNAi oligonucleotide. In some embodiments, the reduction in target gene in oligodendritic cells is about 1% or less, about 5% or less, about 10% relative to the amount or level of target gene expression in neurons. or lower, about 15% or lower, about 20% or lower, about 25% or lower, about 30% or lower, about 35% or lower, about 40% or lower, about 45% or Lower, about 50% or lower, about 55% or lower, about 60% or lower, about 70% or lower, about 80% or lower, or about 90% or lower. In some embodiments, the reduction in target gene expression in oligodendritic cells is relative to the amount or level of target gene expression in stellate cells contacted with the lipid-conjugated RNAi oligonucleotide. In some embodiments, the reduction in expression of a target gene in oligodendritic cells relative to the amount or level of expression of a target gene in stellate cells is about 1% or less, about 5% or less, About 10% or less, about 15% or less, about 20% or less, about 25% or less, about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or less, about 60% or less, about 70% or less, about 80% or less, or about 90% or less.

In some embodiments, the reduction in target gene expression in neurons is increased by at least 1%, at least 5%, at least 10%, at least 15%, at least 20% relative to the reduction in target gene expression in oligodendritic cells. , at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80% or at least 90%. In some embodiments, the reduction in target gene expression in stellate cells is increased by at least 1%, at least 5%, at least 10%, at least 15%, at least 20% relative to the reduction in target gene expression in oligodendritic cells. %, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80% or at least 90%.

In some embodiments, contacting or delivering an oligonucleotide described herein to a cell or population of cells results in reduced expression of a target gene in the CNS.

In one embodiment, differences in target mRNA expression between cell types or tissue types are measured using methods known in the art. In one embodiment, the difference in target mRNA expression between cell types or tissue types is measured as a decrease in target mRNA in a first cell/tissue type compared to a decrease in target mRNA in a second cell/tissue type.

For example, differences in target mRNA expression between cell types or tissue types are measured using polymerase chain reaction methods (eg, RT-PCR) to compare the relative performance between different tissues or cell types. In one embodiment, differences in target mRNA expression between cell types or tissue types are measured using Northern blot analysis, in situ hybridization, RT-PCR, RNA sequencing, or other methods known in the art. In some embodiments, the relative amounts of target mRNA expression are compared between cell or tissue types. In some embodiments, the absolute amount of target mRNA expression is compared between cell or tissue types. Reduce expression of target genes in the eye

In some embodiments, expression of the target gene is reduced in the eye region. In some embodiments, areas of the eye include, but are not limited to, the optic nerve and retina.

In some embodiments, the individual's target gene expression in eye tissue is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50% when compared to target gene expression in other control tissues. %, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%. Reduce liver target gene expression

In some embodiments, expression of the target gene is reduced in liver cells. In some embodiments, the liver cells are macrophages located in the liver. In some embodiments, expression of the target gene is reduced in regions of the liver.

In some embodiments, the subject's target gene expression in the liver is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, when compared to target gene expression in other tissues. About 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%. Reduce target gene expression in muscle tissue

In some embodiments, expression of the target gene is reduced in cells of muscle tissue (eg, skeletal muscle). In some embodiments, the subject's target gene expression in muscle tissue (e.g., skeletal muscle) is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more than 99% . Reduce target gene expression in adipose tissue

In some embodiments, the expression of the target gene is reduced cells in adipose tissue. In some embodiments, the subject's target gene expression in adipose tissue is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50% when compared to target gene expression in other tissues. , about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%. Reduce target gene expression in cardiac tissue

In some embodiments, expression of the target gene is reduced in cells in cardiac tissue. In some embodiments, expression of the target gene is reduced in regions of the heart. In some embodiments, the subject's target gene expression in cardiac tissue is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50% when compared to target gene expression in other tissues. , about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%. Reduce target gene expression in lung tissue

In some embodiments, the expression of the target gene is a decrease in cells in the lung tissue. In some embodiments, expression of the target gene is reduced in regions of the lung. In some embodiments, the subject's target gene expression in cardiac tissue is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50% when compared to target gene expression in other tissues. , about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%. Treatment

In some embodiments, the present disclosure provides methods for treating a disease, disorder, or condition associated with expression of a target gene in one or more tissues or cells. In some embodiments, the present disclosure provides methods for treating diseases, disorders, or conditions associated with expression of target genes of the CNS (eg, neuronal genes). In some embodiments, the present disclosure provides methods for treating diseases, disorders, or conditions associated with expression of target genes in ocular tissue. In some embodiments, the present disclosure provides methods for treating a disease, disorder, or condition associated with expression of a liver target gene (eg, a macrophage target gene). In some embodiments, the present disclosure provides methods for treating diseases, disorders, or conditions associated with expression of target genes in adipose tissue. In some embodiments, the present disclosure provides methods for treating diseases, disorders, or conditions associated with expression of target genes in adrenal tissue. In some embodiments, the present disclosure provides methods for treating diseases, disorders, or conditions associated with expression of target genes in skeletal muscle tissue. In some embodiments, the present disclosure provides methods for treating diseases, disorders, or conditions associated with expression of target genes in lung tissue. In some embodiments, the present disclosure provides methods for treating diseases, disorders, or conditions associated with expression of target genes in cardiac tissue.

The methods described herein typically involve administering to an individual a therapeutically effective amount of a lipid-conjugated RNAi oligonucleotide herein, that is, an amount capable of producing the desired therapeutic outcome. A therapeutically acceptable amount may be an amount that therapeutically treats a disease or condition. The appropriate dosage for any individual will depend on factors including the individual's size, body surface area, age, the composition to be administered, the active ingredients in the composition, time and route of administration, and general health. , and other drugs to be administered at the same time.

In some embodiments, the subject is administered enterally (e.g., orally, via a gastric feeding tube, via a duodenal feeding tube, via a gastrostomy, or transrectally), parenterally (e.g., subcutaneous injection, intravenous injection or infusion, intraarterial injection or infusion, intraosseous infusion, intramuscular injection, intracranial injection, intracerebroventricular injection, intrathecal), topical (e.g., epidermal, inhalation, via eye drops) , or through mucosal membranes), or by direct injection into a target organ (e.g., the brain of an individual).

In some embodiments, the lipid-conjugated RNAi oligonucleotides herein, or compositions thereof, are administered annually, every 6 months, every 4 months, quarterly (every three months), Give every two months (every two months), monthly or weekly. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein, or compositions thereof, are administered weekly or at intervals of two or three weeks. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein, or compositions thereof, are administered daily. In some embodiments, a subject is administered one or more loading doses of a lipid-conjugated RNAi oligonucleotide herein, or a composition thereof, followed by one or more maintenance doses ( maintenance dose) of the lipid-conjugated RNAi oligonucleotide, or a composition thereof.

In some embodiments, the subject to be treated is a human, or non-human primate, or other mammalian subject. Other exemplary individuals include domestic animals, such as dogs and cats; domestic animals, such as horses, cattle, pigs, sheep, goats, and chickens; and animals, such as mice, rats, guinea pigs, and hamsters. Treatment methods in the CNS

The present disclosure provides oligonucleotides for use as medicaments, and particularly for use in methods of treating diseases, disorders and conditions associated with the CNS. The present disclosure also provides lipid-conjugated RNAi oligonucleotides for use in, or suitable for use in, the treatment of individuals suffering from a disease, disorder or condition associated with the expression of a target gene (e.g., a neuronal gene) (e.g. , human), the disease, disorder or condition would benefit from reduced expression of the target gene. In some embodiments, the present disclosure provides lipid-conjugated RNAi oligonucleotides for use, or may be adapted to treat individuals suffering from a disease, disorder, or condition associated with expression of a target gene in the CNS. The present disclosure also provides lipid-conjugated RNAi oligonucleotides for use in, or may be adapted for use in the manufacture of medicaments or pharmaceutical compositions for treating diseases, disorders, or conditions associated with expression of target genes in the CNS. In some embodiments, the lipid-conjugated RNAi oligonucleotides are, or are suitable for, targeting mRNA and reducing expression of target genes in the CNS (eg, via the RNAi pathway). In some embodiments, the lipid-conjugated RNAi oligonucleotides are used, or may be adapted, to target mRNA and reduce the amount or level of target mRNA, protein, and/or activity.

Furthermore, in some embodiments of the methods herein, individuals who have or are susceptible to a disease, disorder, or condition associated with expression of a target gene in the CNS are selected for use with the lipids herein Conjugated RNAi oligonucleotide therapy. In some embodiments, methods include selecting individuals who have markers (eg, biomarkers) of, or are susceptible to, a disease, disorder, or condition associated with expression of a target gene in the CNS, such markers such as But it is not limited to target mRNA, protein, or combinations thereof. Likewise, and as described in detail below, some embodiments of the methods provided by the present disclosure include steps such as measuring or obtaining a baseline value for a marker of expression of a target gene in the CNS and then comparing the value so obtained with that found in that individual. One or more other baseline values are obtained following administration of the lipid-conjugated RNAi oligonucleotide to assess the effectiveness of the treatment.

The present disclosure also provides for the use of lipid-conjugated RNAi oligonucleotides provided herein for the treatment of patients with, suspected of having, or developing diseases, disorders, or conditions associated with expression of target genes in the CNS. Individuals at risk for a disease, disorder, or condition associated with expression of the target gene. In some embodiments, the present disclosure provides methods of treating or attenuating the onset or progression of a disease, disorder, or condition associated with expression of a target gene in the CNS using lipid-conjugated RNAi oligonucleotides provided herein . In some embodiments, the present disclosure provides for the use of lipid-conjugated RNAi oligonucleotides provided herein to achieve a goal in an individual suffering from a disease, disorder, or condition associated with expression of a target gene in the CNS. or a variety of therapeutic benefits.

In some embodiments of the methods herein, a subject is treated by administering a therapeutically effective amount of any one or more of the lipid-conjugated RNAi oligonucleotides provided herein. In some embodiments, treatment includes reducing expression of CNS target genes (eg, neuronal target genes). In some implementations, the individual is treated therapeutically. In some implementations, the individual is treated prophylactically.

In some embodiments of the methods herein, a lipid provided herein is administered to an individual suffering from a disease, disorder, or condition associated with expression of a target gene of the CNS (eg, a neuronal target gene). The conjugated RNAi oligonucleotide, or a pharmaceutical composition containing the lipid-conjugated RNAi oligonucleotide, reduces the expression of the target gene in the individual, thereby treating the individual. In some embodiments, the amount or level of target mRNA is reduced in the individual. In some embodiments, the amount or level of protein encoded by the target mRNA is reduced in the individual.

In some embodiments of the methods herein, a lipid provided herein is administered to an individual suffering from a disease, disorder, or condition associated with expression of a target gene of the CNS (eg, a neuronal target gene). Conjugated RNAi oligonucleotides, or pharmaceutical compositions containing the lipid-conjugated RNAi oligonucleotides, such that the target gene expression of the individual is equivalent to administration of the lipid-conjugated RNAi oligonucleotides or pharmaceuticals The target gene expression before the composition is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99%. In some embodiments, the subject's CNS target genes (e.g., neuronal target genes) are expressed when the individual does not receive the lipid conjugated RNAi oligonucleotide or pharmaceutical composition or receives the control lipid conjugate. The RNAi oligonucleotide, pharmaceutical composition, or target gene expression of the treated individual (e.g., a reference or control individual) is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

In some embodiments of the methods herein, a lipid conjugate herein is administered to an individual suffering from a disease, disorder, or condition associated with expression of a target gene of the CNS (eg, a neuronal target gene). RNAi oligonucleotides, or pharmaceutical compositions containing the lipid-conjugated RNAi oligonucleotides, such that the amount or level of target mRNA in the individual is equivalent to administration of the lipid-conjugated RNAi oligonucleotide or The amount or level of target mRNA before the pharmaceutical composition is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%. In some embodiments, the amount or level of target mRNA in the individual is equivalent to that of the individual who did not receive the lipid-conjugated RNAi oligonucleotide or pharmaceutical composition or who received a control lipid-conjugated RNAi oligonucleotide or pharmaceutical composition. The amount or level of target mRNA in a substance or treated individual (e.g., a reference or control individual) is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55% compared to , about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

In some embodiments of the methods herein, a lipid conjugate herein is administered to an individual suffering from a disease, disorder, or condition associated with expression of a target gene of the CNS (eg, a neuronal target gene). RNAi oligonucleotides, or pharmaceutical compositions comprising the lipid-conjugated RNAi oligonucleotides, such that the amount or level of the protein encoded by the target gene in the individual is equivalent to that administered to the lipid-conjugated The amount or level of the protein encoded by the target gene before the RNAi oligonucleotide or pharmaceutical composition is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%. In some embodiments, the amount or level of protein encoded by a CNS target gene (e.g., a neuronal target gene) in the subject is equal to that of the subject who does not receive the lipid-conjugated RNAi oligonucleotide or pharmaceutical composition. The amount or level of the protein encoded by the target gene is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% , about 95%, about 99% or greater than 99%.

In some embodiments of the methods herein, a lipid conjugate herein is administered to an individual suffering from a disease, disorder, or condition associated with expression of a target gene of the CNS (eg, a neuronal target gene). RNAi oligonucleotides, or pharmaceutical compositions containing the lipid-conjugated RNAi oligonucleotides, such that the amount or level of target gene activity in the individual is equivalent to administration of the lipid-conjugated RNAi oligonucleotides Or the amount or level of target gene activity before the pharmaceutical composition is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%. , about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%. In some embodiments, the amount or level of target gene activity in the individual is equivalent to that of the individual who did not receive the lipid-conjugated RNAi oligonucleotide or pharmaceutical composition or who received a control lipid-conjugated RNAi oligonucleotide, pharmaceutical composition. The amount or level of target gene activity of the composition or treated individual (e.g., a reference or control individual) is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

Suitable methods for determining the expression of a target gene, the amount or level of target mRNA, the amount or level of protein encoded by the target gene, and/or the amount or level of target gene activity in an individual or a sample from an individual are the present invention. known in the technical field. Furthermore, the examples shown herein illustrate exemplary methods of determining target gene expression.

In some embodiments, target gene expression, the amount or level of target gene mRNA, the amount or level of protein encoded by the target gene, the amount or level of target gene activity, or any combination thereof, is present in the cell. (e.g., neurons), a population or group of cells (e.g., organoids), an organ (e.g., CNS), blood or a portion thereof (e.g., plasma), a tissue (e.g., brain tissue), a sample (e.g., CSF sample or brain biopsy sample), or any other biological material obtained or isolated from the individual. In some embodiments, the expression of a CNS target gene (e.g., a neuronal target gene), the amount or level of target gene mRNA, the amount or level of protein encoded by the target gene, the target gene The amount or level of activity, or any combination thereof, in more than one type of cell (e.g., neurons), in more than one group of cells, in more than one organ (e.g., the brain and one or more other organs), in more than one type of blood Parts (e.g., plasma and one or more other blood fractions), more than one type of tissue (e.g., brain tissue and one or more other types of tissue), more than one type of sample obtained or isolated from the individual (e.g., Brain biopsy samples and one or more other types of biopsies) are reduced.

In some embodiments, expression of the target mRNA is reduced in one or more of the individual's stellate cells, neurons, or oligodendritic cells. In some embodiments, expression of the target mRNA is reduced in stellate cells. In some embodiments, the expression of the target mRNA is reduced in neurons. In some embodiments, expression of the target mRNA is reduced in oligodendritic cells. In some embodiments, expression of the target mRNA is reduced in stellate cells and neurons. In some embodiments, expression of the target mRNA is reduced in stellate cells and oligodendritic cells. In some embodiments, expression of the target mRNA is reduced in neurons and oligodendritic cells. In some embodiments, expression of the target mRNA is reduced in stellate cells, oligodendritic cells, and neurons.

In some embodiments, CNS target genes (e.g., neuronal target genes) are expressed in the spinal cord, dorsal root ganglia (DRG), medulla oblongata, hippocampus, frontal cortex, brainstem, or cerebellum One or more are reduced. In some embodiments, CNS target genes (e.g., neuronal target genes) are expressed in the spinal cord, lumbar spinal cord, thoracic spinal cord, cervical spinal cord, lumbar root ganglia (DRG), medulla oblongata, hippocampus, Reduction in one or more of the frontal cortex, brainstem, hypothalamus, or cerebellum. In some embodiments, expression of CNS target genes (eg, neuronal target genes) is reduced in the spinal cord. In some embodiments, expression of CNS target genes (eg, neuronal target genes) is reduced in the lumbar spinal cord. In some embodiments, expression of CNS target genes (eg, neuronal target genes) is reduced in the thoracic spinal cord. In some embodiments, expression of CNS target genes (eg, neuronal target genes) is reduced in the cervical spinal cord. In some embodiments, expression of CNS target genes (eg, neuronal target genes) is reduced in lumbar dorsal root ganglia. In some embodiments, expression of CNS target genes (eg, neuronal target genes) is reduced in the medulla oblongata. In some embodiments, expression of CNS target genes (eg, neuronal target genes) is reduced in the hippocampus. In some embodiments, expression of CNS target genes (eg, neuronal target genes) is reduced in the frontal cortex. In some embodiments, expression of CNS target genes (eg, neuronal target genes) is reduced in the brainstem. In some embodiments, expression of CNS target genes (eg, neuronal target genes) is reduced in the hypothalamus. In some embodiments, expression of CNS target genes (eg, neuronal target genes) is reduced in the cerebellum.

In some embodiments, the present disclosure provides a method of reducing the expression of target mRNA in stellate cells, which includes administering a blunt-ended double-stranded oligonucleotide, wherein the oligonucleotide includes: (i) a sense strand, wherein the sense strand is 20 nucleotides in length, wherein the sense strand comprises at least one lipid moiety conjugated to the 5' end, 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 includes a region complementary to the target mRNA of the stellate cell, and wherein the sense strand and the antisense strand are separate strands forming an asymmetric duplex region having a 2 nucleotide overhang at the 3' end of the antisense strand, wherein the duplex region is 20 nucleotides, and Wherein the oligonucleotide does not reduce the target mRNA in neurons to the same amount as the reduction in stellate cells.

In some embodiments, the present disclosure provides a method of reducing the expression of target mRNA in stellate cells, which includes administering a double-stranded oligonucleotide, wherein the oligonucleotide includes: (i) a sense strand, wherein the sense strand is 14 nucleotides in length, wherein the sense strand comprises at least one lipid moiety conjugated to the 5' end, 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 includes a region complementary to the target mRNA of the stellate cell, and wherein the sense strand and the antisense strand are separate strands forming an asymmetric duplex region having an overhang of 8 nucleotides at the 3' end of the antisense strand, wherein the duplex region is 14 nucleotides, Wherein the oligonucleotide does not reduce the target mRNA in oligodendritic cells to the same or a similar amount as the reduction in stellate cells.

In some embodiments, the present disclosure provides a method of reducing target mRNA of stellate cells and oligodendritic cells to the same or similar amounts, comprising administering a double-stranded oligonucleotide, wherein the oligonucleotide comprises : (i) a sense strand, wherein the sense strand is 20 nucleotides in length, wherein the sense strand comprises at least one lipid moiety conjugated to the 5' end, 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 includes a region complementary to the target mRNA of the stellate cells and oligodendritic cells, and wherein the sense strand and the antisense strand are separate strands forming an asymmetric duplex region having a 2 nucleotide overhang at the 3' end of the antisense strand, wherein the duplex region is 20 nucleotides, thereby reducing the expression of the target mRNA in the stellate cells and oligodendritic cells to the same or similar amount.

In some embodiments, the present disclosure provides a method of reducing the expression of target mRNA in stellate cells and neurons, comprising administering a double-stranded oligonucleotide, wherein the oligonucleotide comprises: (i) a sense strand, wherein the sense strand is 14 nucleotides in length, wherein the sense strand comprises at least one lipid moiety conjugated to the 5' end, 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 includes a region complementary to the target mRNA of the stellate cells and neurons, and wherein the sense strand and the antisense strand are separate strands forming an asymmetric duplex region having an overhang of 8 nucleotides at the 3' end of the antisense strand, wherein the duplex region is 14 nucleotides, Wherein the oligonucleotide does not reduce the target mRNA in oligodendritic cells to the same amount as in the stellate cells and neurons.

In some embodiments, the present disclosure provides a method of reducing the expression of target mRNA in neurons, which includes administering a double-stranded oligonucleotide, wherein the oligonucleotide includes: (i) A sense strand, wherein the sense strand is 14 nucleotides in length, wherein the sense strand contains at least one lipid moiety conjugated to the 5' end, and wherein the sense strand contains at least one locked nucleic acid and no more than 5 locked nucleic acids, and (ii) an antisense strand, wherein the antisense strand is 22 nucleotides in length, and wherein the antisense strand includes a region complementary to the target mRNA of the neuron, and wherein the sense strand and antisense strand are separate strands forming an asymmetric duplex region having an overhang of approximately 8 nucleotides at the 3' end of the antisense strand, wherein the duplex region is approximately 14 nucleotides.

In some embodiments, the present disclosure provides a method of reducing the expression of target mRNA in neurons, which includes administering a double-stranded oligonucleotide, wherein the oligonucleotide includes: (i) a sense strand, wherein the sense strand is 14 nucleotides in length, wherein the sense strand comprises at least one lipid moiety conjugated to the 5' end, 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 includes a region complementary to the target mRNA of the neuron, and wherein the sense strand and antisense strand are separate strands forming an asymmetric duplex region having an overhang of approximately 8 nucleotides at the 3' end of the antisense strand, wherein the duplex region is approximately 14 nucleotides, thereby reducing the expression of the target mRNA in the neuron, wherein the oligonucleotide does not reduce the target mRNA in the oligodendritic cells to the same amount as in the neuron.

In some embodiments, the present disclosure provides a method of reducing the expression of target mRNA in neurons of the lumbar root ganglion (DRG), comprising administering a double-stranded oligonucleotide, wherein the oligonucleotide comprises : (i) a sense strand, wherein the sense strand is 26 nucleotides in length, wherein the sense strand includes a backbone loop that includes at least one lipid moiety conjugated thereto, and wherein the sense strand is at 5' contains locked nucleic acid, and (ii) an antisense strand, wherein the length of the antisense strand is 22 nucleotides, wherein the antisense strand is at positions 1 and 2, positions 2 and 3, positions 3 and 4, positions 12 and 13, positions 13 and 14. Contains phosphorothioate linkages between positions 14 and 15, positions 15 and 16, positions 16 and 17, positions 17 and 18, positions 18 and 19, positions 19 and 20, positions 20 and 21, and positions 21 and 22 , and wherein the antisense strand includes a region complementary to the target mRNA of the neuron, and wherein the sense strand and antisense strand are separate strands forming an asymmetric duplex region having an overhang of approximately 8 nucleotides at the 3' end of the antisense strand, wherein the duplex region is approximately 14 nucleotides.

Examples of diseases, disorders, or conditions associated with expression of CNS target genes (eg, neuronal target genes) include, but are not limited to, Pelizaeus-Merzbacher Disease, spinal cord injury, and stroke, Krabbe Disease, Metachromatic Leukodystrophy, Adult-Onset Leukodystrophy, Multiple Sclerosis, X-linked Adrenal Leukodystrophy, Progressive Supranuclear palsy (PSP), corticobasal degeneration (CBD), argyrophilic granular disease (AGD), global glial tauopathy (GGT), aging-related tau astrocytes ARTAG, FTD-17, neurodegenerative disease with respiratory failure, Dementia with Seizures, Pick's disease , myotonic dystrophy type 1 or 2 (MD1 or MD2), Down syndrome, spastic paraplegia (SP), Niemann-Pick disease type C, dementia with Lewy bodies (DLB), Lewy bodies Dysphagia, Lewy body disease, olivopontocerebellar atrophy, striatonigral degeneration, Schaye-Dreiger syndrome, spinal muscular atrophy V (SMAV), Huntington's disease (HD), Alzheimer's disease, SCA1, SCA2, SCA3, SCA7, SCA10 (spinocerebellar dyskinesia type 1, 2, 3, 7 or 10), multiple system atrophy (MSA), spinobulbar muscle disease SBMA (Kennedy disease), Friedrich Ataxia, Fragile X-associated tremor/ataxia syndrome (FXTAS), Fragile Fragile DRPLA, recessive CNS disorders, ALS (amyotrophic lateral sclerosis), M2DS (MECP2 duplication syndrome), FTD (frontotemporal dementia), Prion disease, Adult-onset leukodystrophy, Alexander's disease, Krabbe's disease, chronic traumatic encephalopathy, familial middle lobar sclerosis (PMD), Lafora disease, stroke, amyloid cerebrovascular disease ( CAA), and metachromatic leukodystrophy (MLD).

In some embodiments, a CNS target gene (eg, a neuronal target gene) can be a target gene from any mammal, such as a human. Any CNS target gene (eg, neuronal target gene) can be silenced according to the methods described herein.

In some embodiments, the lipid-conjugated RNAi oligonucleotides herein, or compositions thereof, are administered intrathecally into the cerebrospinal fluid (CSF) (e.g., injected or infused into the subarachnoid space in body fluids). In some embodiments, intrathecal administration of the lipid-conjugated RNAi oligonucleotides herein, or compositions thereof, is performed as a single bolus injection into the subarachnoid space. In some embodiments, intrathecal administration of the lipid-conjugated RNAi oligonucleotides herein, or compositions thereof, is performed by infusion into the subarachnoid space. In some embodiments, intrathecal administration of the lipid-conjugated RNAi oligonucleotides herein, or compositions thereof, is performed via a catheter into the subarachnoid space. In some embodiments, intrathecal administration of the lipid-conjugated RNAi oligonucleotides herein, or compositions thereof, is via a pump. In some embodiments, intrathecal administration of the lipid-conjugated RNAi oligonucleotides herein, or compositions thereof, is via an implantable pump. In some embodiments, administration is via an implantable device that operates or functions as a reservoir.

In some embodiments, the lipid-conjugated RNAi oligonucleotides herein, or compositions thereof, are administered intrathecally into the cisterna magna (also known as the cisterna magna). Intrathecal administration into the cisterna magna is called "intracisternal administration" or "intracisternal magna (i.c.m.) administration." In some embodiments, the lipid-conjugated RNAi oligonucleotides herein, or compositions thereof, are administered intrathecally into the subarachnoid space of the lumbar spinal cord. Intrathecal administration into the subarachnoid space of the lumbar spinal cord is called "lumbar intrathecal (i.t.) administration." In some embodiments, the lipid-conjugated RNAi oligonucleotides herein, or compositions thereof, are administered intrathecally into the subarachnoid space of the cervical spinal cord. Intrathecal administration into the subarachnoid space of the cervical spinal cord is called "cervical intrathecal (i.t.) administration." In some embodiments, the lipid-conjugated RNAi oligonucleotides herein, or compositions thereof, are administered intrathecally into the subarachnoid space of the thoracic spinal cord. Intrathecal administration into the subarachnoid space of the thoracic spinal cord is referred to as "thoracic intrathecal (i.t.) administration." In some embodiments, the lipid-conjugated RNAi oligonucleotides herein, or compositions thereof, are administered by intracerebroventricular injection or infusion into the cerebral ventricles. Intraventricular administration into the ventricular space is called "intracerebroventricular (i.c.v.) administration." In some embodiments, the Ommaya reservoir is used to administer lipid-conjugated RNAi oligonucleotides, or compositions thereof, by intracerebroventricular injection or infusion.

In some embodiments, the lipid-conjugated RNAi oligonucleotides herein, or compositions thereof, are administered ophthalmic, intraocular, subconjunctival, intravitreal, retrobulbar, or intracameral.

In some embodiments, the lipid-conjugated RNAi oligonucleotides herein, or compositions thereof, are administered epidurally. Treatment methods in ocular tissue

The present disclosure provides oligonucleotides for use as medicaments, and particularly for use in methods of treating diseases, disorders and conditions associated with ocular tissue. The present disclosure also provides oligonucleotides for use in, or that may be suitable for, the treatment of an individual (eg, a human) suffering from a disease, disorder, or condition associated with expression of an ocular target gene. would benefit from reduced expression of eye target genes. In some embodiments, the present disclosure provides oligonucleotides for use in, or applicable for, the treatment of individuals suffering from diseases, disorders, or conditions associated with expression of ocular target genes. The present disclosure also provides oligonucleotides for use in, or that may be adapted for use in the manufacture of medicaments or pharmaceutical compositions for the treatment of diseases, disorders or conditions associated with expression of ocular target genes. In some embodiments, the oligonucleotides are, or are suitable for, targeting mRNA and reducing expression of target genes (eg, via the RNAi pathway). In some embodiments, the oligonucleotides are, or are suitable for, targeting mRNA and reducing the amount or level of target mRNA, protein, and/or activity.

Furthermore, in some embodiments of the methods herein, individuals who have, or are susceptible to, a disease, disorder, or condition associated with expression of an ocular target gene are selected for use with the oligonucleotides herein. Glycoside treatment. In some embodiments, methods include selecting individuals who have markers (e.g., biomarkers) of, or are susceptible to, a disease, disorder, or condition associated with expression of an ocular target gene, such as but Not limited to target mRNA, protein, or combinations thereof. Likewise, and as described in detail below, some embodiments of the methods provided by the present disclosure include steps such as measuring or obtaining a baseline value for a marker of expression of an ocular target gene and then comparing the value so obtained to that experienced by the individual. One or more other baseline values are obtained after administration of the oligonucleotide to assess the effectiveness of the treatment.

The present disclosure also provides for the use of oligonucleotides provided herein to treat patients with, suspected of having, or developing a disease, disease, or condition associated with expression of an ocular target gene. An individual who is at risk of a disease, illness or condition. In some embodiments, the present disclosure provides methods of using oligonucleotides provided herein to treat or attenuate the onset or progression of a disease, disorder, or condition associated with expression of an ocular target gene. In some embodiments, the present disclosure provides methods of using oligonucleotides provided herein to achieve one or more therapeutic benefits in individuals suffering from a disease, disorder, or condition associated with expression of an ocular target gene. .

In some embodiments of the methods herein, an individual is treated by administering a therapeutically effective amount of any one or more of the oligonucleotides provided herein. In some embodiments, treatment includes reducing expression of an ocular target gene. In some implementations, the individual is treated therapeutically. In some implementations, the individual is treated prophylactically.

In some embodiments of the methods herein, an oligonucleotide provided herein, or comprising the oligonucleotide, is administered to an individual suffering from a disease, disorder, or condition associated with expression of an ocular target gene. The pharmaceutical composition of the acid reduces the expression of the target gene in the individual, thereby treating the individual. In some embodiments, the amount or level of target mRNA is reduced in the individual. In some embodiments, the amount or level of protein encoded by the target mRNA is reduced in the individual.

In some embodiments of the methods herein, an oligonucleotide provided herein, or comprising the oligonucleotide, is administered to an individual suffering from a disease, disorder, or condition associated with expression of an ocular target gene. A pharmaceutical composition of an acid that causes the individual's target gene expression to be reduced by at least about 30%, about 35%, about 40%, or about 40% compared to the target gene expression before administration of the oligonucleotide or pharmaceutical composition. About 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99 %. In some embodiments, the expression of the ocular target gene in the individual is equivalent to that of an individual who did not receive the oligonucleotide or pharmaceutical composition or who received a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or control The target gene expression of an individual) is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99%.

In some embodiments of the methods herein, an oligonucleotide herein, or an oligonucleotide comprising the oligonucleotide, is administered to an individual suffering from a disease, disorder, or condition associated with expression of an ocular target gene. A pharmaceutical composition that reduces the amount or level of target mRNA in the individual by at least about 30%, about 35%, or about 35% compared to the amount or level of target mRNA before administration of the oligonucleotide or pharmaceutical composition. About 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99 % or greater than 99%. In some embodiments, the amount or level of target mRNA in the individual is equivalent to that in an individual who did not receive the oligonucleotide or pharmaceutical composition or who received a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or The amount or level of target mRNA is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

In some embodiments of the methods herein, an oligonucleotide herein, or an oligonucleotide comprising the oligonucleotide, is administered to an individual suffering from a disease, disorder, or condition associated with expression of an ocular target gene. A pharmaceutical composition such that the amount or level of the protein encoded by the eye target gene in the individual is equivalent to the amount or level of the protein encoded by the target gene before administration of the oligonucleotide or pharmaceutical composition. Compared with the system, it is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99%. In some embodiments, the amount or level of the protein encoded by the eye target gene is equivalent to that of a subject who did not receive the oligonucleotide or pharmaceutical composition or who received a control oligonucleotide, pharmaceutical composition, or treatment. The amount or level of the protein encoded by the target gene in an individual (e.g., a reference or control individual) is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

In some embodiments of the methods herein, an oligonucleotide herein, or an oligonucleotide comprising the oligonucleotide, is administered to an individual suffering from a disease, disorder, or condition associated with expression of an ocular target gene. A pharmaceutical composition that reduces the amount or level of target gene activity in the individual by at least about 30% or about 35% compared to the amount or level of target gene activity before administration of the oligonucleotide or pharmaceutical composition. %, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, About 99% or greater than 99%. In some embodiments, the amount or level of target gene activity in the individual is equivalent to that of an individual who did not receive the oligonucleotide or pharmaceutical composition or who received a control oligonucleotide, pharmaceutical composition, or treatment (e.g., reference or control individual), the amount or level of target gene activity is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65% compared to , about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

Suitable methods for determining the expression of a target gene, the amount or level of target mRNA, the amount or level of protein encoded by the target gene, and/or the amount or level of target gene activity in an individual or a sample from an individual are the present invention. known in the technical field. Furthermore, the examples shown herein illustrate exemplary methods of determining target gene expression.

In some embodiments, target gene expression, the amount or level of target gene mRNA, the amount or level of protein encoded by the target gene, the amount or level of target gene activity, or any combination thereof, is present in the cell. (e.g., retinal cells), cell population or group of cells (e.g., organoids), organ (e.g., eye), blood or portion thereof (e.g., plasma), tissue (e.g., eye tissue), sample (e.g., eye biological test specimen), or any other biological material obtained or isolated from the individual. In some embodiments, the expression of a target gene, the amount or level of target gene mRNA, the amount or level of protein encoded by the target gene, the amount or level of target gene activity, or any combination thereof Tethered to more than one type of cell (e.g., retinal cells), more than one group of cells, more than one organ (e.g., eye and one or more other organs), more than one part of blood (e.g., plasma and one or more other blood portion), more than one type of tissue (e.g., ocular tissue and one or more other types of tissue), more than one type of sample obtained or isolated from the individual (e.g., ocular biopsy sample and one or more other types of biological examination samples) decreased.

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

In some embodiments, the oligonucleotide conjugates herein, or compositions thereof, are administered ophthalmic, intraocular, subconjunctival, intravitreal, retrobulbar, or intracameral. Treatment methods in the liver

The present disclosure provides oligonucleotides for use as medicaments, and particularly for use in methods of treating diseases, disorders and conditions associated with the liver. The present disclosure also provides oligonucleotides for use in, or suitable for use in, treating individuals suffering from a disease, disorder or condition associated with expression of a liver target gene (e.g., a liver macrophage target gene) (e.g., a liver macrophage target gene). , human), the disease, disorder or condition would benefit from reduced expression of liver target genes. In some embodiments, the present disclosure provides oligonucleotides for use in, or suitable for treating, patients with diseases associated with expression of target genes of liver target genes (e.g., liver macrophage target genes), An individual with a disease or condition. The present disclosure also provides oligonucleotides for use in, or that may be adapted for use in the manufacture of medicaments for the treatment of diseases, disorders or conditions associated with expression of liver target genes (e.g., liver macrophage target genes), or Pharmaceutical compositions. In some embodiments, the oligonucleotides are, or are suitable for, targeting mRNA and reducing expression of liver target genes (eg, via the RNAi pathway). In some embodiments, the oligonucleotides are, or are suitable for, targeting mRNA and reducing the amount or level of liver target mRNA, protein, and/or activity.

Furthermore, in some embodiments of the methods herein, the selection is for having or being susceptible to a disease, disorder, or condition associated with expression of a liver target gene (e.g., a liver macrophage target gene). Subjects with the condition are subject to treatment with the oligonucleotides herein. In some embodiments, methods include selecting for markers (e.g., biomarkers) of a disease, disorder, or condition associated with expression of a liver target gene (e.g., a liver macrophage target gene) or susceptibility to the disease, Individuals with a disease or condition, such markers such as, but not limited to, target mRNA, protein, or combinations thereof. Likewise, and as described in detail below, some embodiments of the methods provided by the present disclosure include steps such as measuring or obtaining a baseline value for a marker of expression of a liver target gene, and then comparing the value so obtained to that experienced by the individual. One or more other baseline values are obtained after administration of the oligonucleotide to assess the effectiveness of the treatment.

The present disclosure also provides for the use of oligonucleotides provided herein to treat patients with, suspected of having, or developing a disease, disorder, or condition associated with expression of a liver target gene. An individual who is at risk of a disease, illness or condition. In some embodiments, the present disclosure provides for the use of oligonucleotides provided herein to treat or attenuate diseases, disorders, or conditions associated with expression of liver target genes (e.g., liver macrophage target genes). Method of onset or progression. In some embodiments, the present disclosure provides for the use of oligonucleotides provided herein to treat patients with a disease, disorder, or condition associated with expression of a liver target gene (e.g., a liver macrophage target gene). A method of achieving one or more therapeutic benefits in an individual.

In some embodiments of the methods herein, an individual is treated by administering a therapeutically effective amount of any one or more of the oligonucleotides provided herein. In some embodiments, treatment includes reducing expression of a liver target gene (eg, a liver macrophage target gene). In some implementations, the individual is treated therapeutically. In some implementations, the individual is treated prophylactically.

In some embodiments of the methods herein, an individual suffering from a disease, disorder, or condition associated with expression of a liver target gene (eg, a liver macrophage target gene) is administered as provided herein. The oligonucleotide, or a pharmaceutical composition containing the oligonucleotide, reduces the expression of the target gene in the individual, thereby treating the individual. In some embodiments, the amount or level of target mRNA is reduced in the individual. In some embodiments, the amount or level of protein encoded by the target mRNA is reduced in the individual.

In some embodiments of the methods herein, an individual suffering from a disease, disorder, or condition associated with expression of a liver target gene (eg, a liver macrophage target gene) is administered as provided herein. An oligonucleotide, or a pharmaceutical composition containing the oligonucleotide, such that the target gene expression of the individual is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% , about 95%, about 99% or greater than 99%. In some embodiments, the expression of liver target genes (e.g., liver macrophage target genes) of the individual is equivalent to not receiving the oligonucleotide or pharmaceutical composition or receiving a control oligonucleotide, pharmaceutical composition. Target gene expression in a drug or treated individual (e.g., a reference or control individual) is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60% compared to the previous time. %, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

In some embodiments of the methods herein, an oligonucleotide herein is administered to an individual suffering from a disease, disorder, or condition associated with expression of a liver target gene (eg, a liver macrophage target gene). oligonucleotide, or a pharmaceutical composition containing the oligonucleotide, such that the amount or level of target mRNA in the individual is compared to the amount or level of target mRNA before administration of the oligonucleotide or pharmaceutical composition. system is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85% , about 90%, about 95%, about 99% or greater than 99%. In some embodiments, the amount or level of target mRNA in the individual is equivalent to that in an individual who did not receive the oligonucleotide or pharmaceutical composition or who received a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or The amount or level of target mRNA is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

In some embodiments of the methods herein, an oligonucleotide herein is administered to an individual suffering from a disease, disorder, or condition associated with expression of a liver target gene (eg, a liver macrophage target gene). oligonucleotide, or a pharmaceutical composition containing the oligonucleotide, such that the amount or level of the protein encoded by the liver target gene in the individual is equivalent to the amount or level of the protein encoded by the liver target gene before administration of the oligonucleotide or pharmaceutical composition. The amount or level of the protein encoded by the target gene is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70 %, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%. In some embodiments, the amount or level of protein encoded by a liver target gene (e.g., a liver macrophage target gene) in the subject is equivalent to that of not receiving the oligonucleotide or pharmaceutical composition or receiving a control. The amount or level of the protein encoded by the target gene in the oligonucleotide, pharmaceutical composition, or treated individual (e.g., a reference or control individual) is reduced by at least about 30%, about 35%, about 40% compared to %, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or Greater than 99%.

In some embodiments of the methods herein, an oligonucleotide herein is administered to an individual suffering from a disease, disorder, or condition associated with expression of a liver target gene (eg, a liver macrophage target gene). oligonucleotide, or a pharmaceutical composition containing the oligonucleotide, such that the amount or level of target gene activity in the individual is equivalent to the amount or level of target gene activity before administration of the oligonucleotide or pharmaceutical composition. Compared with the system, it is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99%. In some embodiments, the amount or level of target gene activity in the individual is equivalent to that of an individual who did not receive the oligonucleotide or pharmaceutical composition or who received a control oligonucleotide, pharmaceutical composition, or treatment (e.g., reference or control individual), the amount or level of target gene activity is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65% compared to , about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

Suitable methods for determining the expression of a target gene, the amount or level of target mRNA, the amount or level of protein encoded by the target gene, and/or the amount or level of target gene activity in an individual or a sample from an individual are the present invention. known in the technical field. Furthermore, the examples shown herein illustrate exemplary methods of determining target gene expression.

In some embodiments, target gene expression, the amount or level of target gene mRNA, the amount or level of protein encoded by the target gene, the amount or level of target gene activity, or any combination thereof, is present in the cell. (e.g., liver macrophages), a population or population of cells (e.g., organoids), an organ (e.g., liver), blood or a portion thereof (e.g., plasma), a tissue (e.g., liver tissue), a sample (e.g., liver tissue) , liver biopsy sample), or any other biological material obtained or isolated from the individual. In some embodiments, the expression of a liver target gene, the amount or level of target gene mRNA, the amount or level of protein encoded by the target gene, the amount or level of target gene activity, or any combination thereof Tethered to more than one type of cell (e.g., liver macrophages), more than one group of cells, more than one organ (e.g., liver and one or more other organs), more than one part of blood (e.g., plasma and one or more multiple other blood fractions), more than one type of tissue (e.g., liver tissue and one or more other types of tissue), more than one type of sample obtained or isolated from the individual (e.g., liver biopsy sample and one or more Other types of biological examination samples) decreased.

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

In some embodiments, the oligonucleotide conjugates herein, or compositions thereof, are administered via subcutaneous or intravenous administration. Treatment methods in adipose tissue

The present disclosure provides oligonucleotides for use as medicaments, and particularly for use in methods of treating diseases, disorders and conditions associated with adipose tissue. The present disclosure also provides oligonucleotides for use in, or that may be applicable to, the treatment of an individual (eg, a human) suffering from a disease, disorder, or condition associated with expression of an adipose tissue target gene. The condition would benefit from reducing the expression of adipose tissue target genes. In some embodiments, the present disclosure provides oligonucleotides for use in, or applicable for, the treatment of individuals suffering from diseases, disorders, or conditions associated with expression of adipose tissue target genes. The present disclosure also provides oligonucleotides for use in, or that may be adapted for use in the manufacture of medicaments or pharmaceutical compositions for treating diseases, disorders, or conditions associated with expression of adipose tissue target genes. In some embodiments, the oligonucleotides are, or are suitable for, targeting mRNA and reducing expression of adipose tissue target genes (eg, via the RNAi pathway). In some embodiments, the oligonucleotide is, or is suitable for, targeting mRNA and reducing the amount or level of adipose tissue target mRNA, protein, and/or activity.

Furthermore, in some embodiments of the methods herein, individuals who have or are susceptible to a disease, disorder, or condition associated with expression of an adipose tissue target gene are selected for use with a glycoside herein. Acid treatment. In some embodiments, methods include selecting individuals who have markers (eg, biomarkers) of, or are susceptible to, a disease, disorder, or condition associated with expression of an adipose tissue target gene, such markers such as But it is not limited to target mRNA, protein, or combinations thereof. Likewise, and as described in detail below, some embodiments of the methods provided by the present disclosure include steps such as measuring or obtaining a baseline value for a marker of expression of an adipose tissue target gene and then comparing the value so obtained to that found in that individual. One or more other baseline values are obtained after administration of the oligonucleotide to assess the effectiveness of the treatment.

The present disclosure also provides for the use of oligonucleotides provided herein to treat patients with, suspected of having, or developing disease, disease, or condition associated with expression of adipose tissue target genes. Individuals at risk of related diseases, illnesses or conditions. In some embodiments, the present disclosure provides methods of using oligonucleotides provided herein to treat or attenuate the onset or progression of a disease, disorder, or condition associated with expression of adipose tissue target genes. In some embodiments, the present disclosure provides for the use of oligonucleotides provided herein to achieve one or more therapeutic benefits in individuals suffering from a disease, disorder, or condition associated with expression of an adipose tissue target gene. method.

In some embodiments of the methods herein, an individual is treated by administering a therapeutically effective amount of any one or more of the oligonucleotides provided herein. In some embodiments, treatment includes reducing expression of adipose tissue target genes. In some implementations, the individual is treated therapeutically. In some implementations, the individual is treated prophylactically.

In some embodiments of the methods herein, an oligonucleotide provided herein, or comprising the oligonucleotide, is administered to an individual suffering from a disease, disorder, or condition associated with expression of an adipose tissue target gene. The pharmaceutical composition of glycosides reduces the expression of the target gene in the individual, thereby treating the individual. In some embodiments, the amount or level of target mRNA is reduced in the individual. In some embodiments, the amount or level of protein encoded by the target mRNA is reduced in the individual.

In some embodiments of the methods herein, an oligonucleotide provided herein, or comprising the oligonucleotide, is administered to an individual suffering from a disease, disorder, or condition associated with expression of an adipose tissue target gene. Pharmaceutical compositions of oligonucleotides that reduce the target gene expression of the individual by at least about 30%, about 35%, or about 40% compared to the target gene expression before administration of the oligonucleotide or pharmaceutical composition. , about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater 99%. In some embodiments, the expression of the adipose tissue target gene in the individual is equivalent to that of an individual who did not receive the oligonucleotide or pharmaceutical composition or who received a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or The target gene expression of the control individual) is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, About 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

In some embodiments of the methods herein, an oligonucleotide herein, or comprising the same, is administered to an individual suffering from a disease, disorder, or condition associated with expression of an adipose tissue target gene. A pharmaceutical composition that reduces the amount or level of target mRNA in the individual by at least about 30% or about 35% compared to the amount or level of target mRNA before administration of the oligonucleotide or pharmaceutical composition. , about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%. In some embodiments, the amount or level of target mRNA in the individual is equivalent to that in an individual who did not receive the oligonucleotide or pharmaceutical composition or who received a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or The amount or level of target mRNA is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

In some embodiments of the methods herein, an oligonucleotide herein, or comprising the same, is administered to an individual suffering from a disease, disorder, or condition associated with expression of an adipose tissue target gene. A pharmaceutical composition such that the amount or level of the protein encoded by the adipose tissue target gene in the individual is equivalent to the amount of protein encoded by the target gene before administration of the oligonucleotide or pharmaceutical composition Or the level is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80% , about 85%, about 90%, about 95%, about 99% or greater than 99%. In some embodiments, the amount or level of the protein encoded by the adipose tissue target gene is equivalent to that of the subject who did not receive the oligonucleotide or pharmaceutical composition or who received a control oligonucleotide, pharmaceutical composition, or treatment. The amount or level of the protein encoded by the target gene in an individual (e.g., a reference or control individual) is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, About 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

In some embodiments of the methods herein, an oligonucleotide herein, or comprising the same, is administered to an individual suffering from a disease, disorder, or condition associated with expression of an adipose tissue target gene. A pharmaceutical composition that reduces the amount or level of target gene activity in the individual by at least about 30% or about 30% compared to the amount or level of target gene activity before administration of the oligonucleotide or pharmaceutical composition. 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% , about 99% or greater than 99%. In some embodiments, the amount or level of target gene activity in the individual is equivalent to that of an individual who did not receive the oligonucleotide or pharmaceutical composition or who received a control oligonucleotide, pharmaceutical composition, or treatment (e.g., reference or control individual), the amount or level of target gene activity is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65% compared to , about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

Suitable methods for determining the expression of a target gene, the amount or level of target mRNA, the amount or level of protein encoded by the target gene, and/or the amount or level of target gene activity in an individual or a sample from an individual are the present invention. known in the technical field. Furthermore, the examples shown herein illustrate exemplary methods of determining target gene expression.

In some embodiments, target gene expression, the amount or level of target gene mRNA, the amount or level of protein encoded by the target gene, the amount or level of target gene activity, or any combination thereof, is present in the cell. , cell population or group of cells (e.g., organoids), organ, blood or portion thereof (e.g., plasma), tissue (e.g., adipose tissue), sample (e.g., adipose biopsy sample), or obtained from the individual or Isolation of any other biological material is reduced. In some embodiments, the expression of a target gene in adipose tissue, the amount or level of target gene mRNA, the amount or level of protein encoded by the target gene, the amount or level of target gene activity, or any other The combination is in more than one type of cell, more than one group of cells, more than one organ (e.g., fat and one or more other organs), more than one part of blood (e.g., plasma and one or more other blood parts), more than one One type of tissue (for example, adipose tissue and one or more other types of tissue), more than one type of sample obtained or isolated from the individual (for example, a fat biopsy sample and one or more other types of biopsy samples) Reduce.

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

In some embodiments, the oligonucleotide conjugates herein, or compositions thereof, are administered via subcutaneous or intravenous administration. Treatment methods in adrenal tissue

The present disclosure provides oligonucleotides for use as medicaments, and particularly for use in methods of treating diseases, disorders and conditions associated with adrenal tissue. The present disclosure also provides oligonucleotides for use in, or that may be applicable to, the treatment of an individual (e.g., a human) suffering from a disease, disorder, or condition associated with expression of an adrenal tissue target gene. conditions would benefit from reduced expression of target genes in adrenal tissue. In some embodiments, the present disclosure provides oligonucleotides for use in, or suitable for use in, the treatment of individuals suffering from diseases, disorders, or conditions associated with expression of target genes in adrenal tissue. The present disclosure also provides oligonucleotides for use in, or that may be adapted for use in, the manufacture of medicaments or pharmaceutical compositions for the treatment of diseases, disorders, or conditions associated with expression of adrenal tissue target genes. In some embodiments, the oligonucleotides are, or may be, used to target mRNA and reduce expression of target genes in adrenal tissue (e.g., via the RNAi pathway). In some embodiments, the oligonucleotide is, or is suitable for, targeting mRNA and reducing the amount or level of target mRNA, protein, and/or activity in adrenal tissue.

Furthermore, in some embodiments of the methods herein, individuals who have, or are susceptible to, a disease, disorder, or condition associated with expression of the adrenal tissue target gene are selected for use with the oligosactics herein. Nucleotide therapy. In some embodiments, methods include selecting individuals who have markers (eg, biomarkers) of, or are susceptible to, a disease, disorder, or condition associated with expression of an adrenal tissue target gene, such markers such as But it is not limited to target mRNA, protein, or combinations thereof. Likewise, and as described in detail below, some embodiments of the methods provided by the present disclosure include steps such as measuring or obtaining a baseline value for a marker of expression of a target gene in adrenal tissue and then comparing the value so obtained with the expression of a target gene in the individual. One or more other baseline values are obtained after administration of the oligonucleotide to assess the effectiveness of the treatment.

The present disclosure also provides for the use of oligonucleotides provided herein to treat patients with, suspected of having, or developing disease, disease, or condition associated with expression of adrenal tissue target genes. Individuals at risk of related diseases, illnesses or conditions. In some embodiments, the present disclosure provides methods of using oligonucleotides provided herein to treat or attenuate the onset or progression of a disease, disorder, or condition associated with expression of adrenal tissue target genes. In some embodiments, the present disclosure provides for the use of oligonucleotides provided herein to achieve one or more therapeutic benefits in individuals suffering from a disease, disorder, or condition associated with expression of an adrenal tissue target gene. method.

In some embodiments of the methods herein, an individual is treated by administering a therapeutically effective amount of any one or more of the oligonucleotides provided herein. In some embodiments, treatment includes reducing expression of a target gene in adrenal tissue. In some implementations, the individual is treated therapeutically. In some implementations, the individual is treated prophylactically.

In some embodiments of the methods herein, an oligonucleotide provided herein, or comprising the oligonucleotide, is administered to an individual suffering from a disease, disorder, or condition associated with expression of an adrenal tissue target gene. The pharmaceutical composition of glycosides reduces the expression of the target gene in the individual, thereby treating the individual. In some embodiments, the amount or level of target mRNA is reduced in the individual. In some embodiments, the amount or level of protein encoded by the target mRNA is reduced in the individual.

In some embodiments of the methods herein, an oligonucleotide provided herein, or comprising the oligonucleotide, is administered to an individual suffering from a disease, disorder, or condition associated with expression of an adrenal tissue target gene. Pharmaceutical compositions of oligonucleotides that reduce the target gene expression of the individual by at least about 30%, about 35%, or about 40% compared to the target gene expression before administration of the oligonucleotide or pharmaceutical composition. , about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater 99%. In some embodiments, the expression of the target gene in the adrenal tissue of the individual is equivalent to that of an individual who did not receive the oligonucleotide or pharmaceutical composition or who received a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or The target gene expression of the control individual) is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, About 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

In some embodiments of the methods herein, an oligonucleotide herein, or comprising the same, is administered to an individual suffering from a disease, disorder, or condition associated with expression of an adrenal tissue target gene. A pharmaceutical composition that reduces the amount or level of target mRNA in the individual by at least about 30% or about 35% compared to the amount or level of target mRNA before administration of the oligonucleotide or pharmaceutical composition. , about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%. In some embodiments, the amount or level of target mRNA in the individual is equivalent to that in an individual who did not receive the oligonucleotide or pharmaceutical composition or who received a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or The amount or level of target mRNA is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

In some embodiments of the methods herein, an oligonucleotide herein, or comprising the same, is administered to an individual suffering from a disease, disorder, or condition associated with expression of an adrenal tissue target gene. A pharmaceutical composition such that the amount or level of the protein encoded by the adrenal target gene in the individual is equivalent to the amount or level of the protein encoded by the target gene before administration of the oligonucleotide or pharmaceutical composition. The level is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, About 85%, about 90%, about 95%, about 99%, or greater than 99%. In some embodiments, the amount or level of the protein encoded by the adrenal tissue target gene is equivalent to that of the subject who did not receive the oligonucleotide or pharmaceutical composition or who received a control oligonucleotide, pharmaceutical composition, or treatment. The amount or level of the protein encoded by the target gene in an individual (e.g., a reference or control individual) is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, About 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

In some embodiments of the methods herein, an oligonucleotide herein, or comprising the same, is administered to an individual suffering from a disease, disorder, or condition associated with expression of an adrenal tissue target gene. A pharmaceutical composition that reduces the amount or level of target gene activity in the individual by at least about 30% or about 30% compared to the amount or level of target gene activity before administration of the oligonucleotide or pharmaceutical composition. 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% , about 99% or greater than 99%. In some embodiments, the amount or level of target gene activity in the individual is equivalent to that of an individual who did not receive the oligonucleotide or pharmaceutical composition or who received a control oligonucleotide, pharmaceutical composition, or treatment (e.g., reference or control individual), the amount or level of target gene activity is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65% compared to , about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

Suitable methods for determining the expression of a target gene, the amount or level of target mRNA, the amount or level of protein encoded by the target gene, and/or the amount or level of target gene activity in an individual or a sample from an individual are the present invention. known in the technical field. Furthermore, the examples shown herein illustrate exemplary methods of determining target gene expression.

In some embodiments, target gene expression, the amount or level of target gene mRNA, the amount or level of protein encoded by the target gene, the amount or level of target gene activity, or any combination thereof, is present in the cell. , cell population or group of cells (e.g., organoids), organ, blood or portion thereof (e.g., plasma), tissue (e.g., adrenal tissue), sample (e.g., adrenal biopsy sample), or obtained from the individual or Isolation of any other biological material is reduced. In some embodiments, the expression of a target gene in adrenal tissue, the amount or level of target gene mRNA, the amount or level of protein encoded by the target gene, the amount or level of target gene activity, or any other The combination is in more than one type of cell, more than one group of cells, more than one organ (e.g., adrenal glands and one or more other organs), more than one part of blood (e.g., plasma and one or more other parts of blood), more than one One type of tissue (e.g., adrenal tissue and one or more other types of tissue), more than one type of sample obtained or isolated from the individual (e.g., an adrenal biopsy sample and one or more other types of biopsy samples) Reduce.

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

In some embodiments, the oligonucleotide conjugates herein, or compositions thereof, are administered via subcutaneous or intravenous administration. Treatment methods in muscle tissue

The present disclosure provides oligonucleotides for use as medicaments, and particularly for use in methods of treating diseases, disorders and conditions associated with muscle tissue. The present disclosure also provides oligonucleotides for use in, or suitable for use in, treating an individual (e.g., a human) suffering from a disease, disorder, or condition associated with expression of a muscle target gene. would benefit from reduced expression of muscle target genes. In some embodiments, the present disclosure provides oligonucleotides for use in, or applicable for, the treatment of individuals suffering from diseases, disorders, or conditions associated with expression of muscle target genes. The present disclosure also provides oligonucleotides for use in, or that may be adapted for use in the manufacture of medicaments or pharmaceutical compositions for treating diseases, disorders, or conditions associated with expression of muscle target genes. In some embodiments, the oligonucleotides are, or are suitable for, targeting mRNA and reducing expression of muscle target genes (eg, via the RNAi pathway). In some embodiments, the oligonucleotides are, or are suitable for, targeting mRNA and reducing the amount or level of muscle target mRNA, protein, and/or activity.

Furthermore, in some embodiments of the methods herein, individuals who have, or are susceptible to, a disease, disorder, or condition associated with expression of a muscle target gene are selected for use with the oligonucleotides herein. Glycoside treatment. In some embodiments, methods include selecting individuals who have markers (e.g., biomarkers) of, or are susceptible to, a disease, disorder, or condition associated with expression of a muscle target gene, such as but Not limited to target mRNA, protein, or combinations thereof. Likewise, and as described in detail below, some embodiments of the methods provided by the present disclosure include steps such as measuring or obtaining a baseline value for a marker of expression of a muscle target gene, and then comparing the value so obtained to that experienced by the individual. One or more other baseline values are obtained after administration of the oligonucleotide to assess the effectiveness of the treatment.

The present disclosure also provides for the use of oligonucleotides provided herein to treat patients with, suspected of having, or developing a disease, disorder, or condition associated with expression of a muscle target gene. An individual who is at risk of a disease, illness or condition. In some embodiments, the present disclosure provides methods of using oligonucleotides provided herein to treat or attenuate the onset or progression of a disease, disorder, or condition associated with expression of a muscle target gene. In some embodiments, the present disclosure provides methods of using oligonucleotides provided herein to achieve one or more therapeutic benefits in an individual suffering from a disease, disorder, or condition associated with expression of a muscle target gene. .

In some embodiments of the methods herein, an individual is treated by administering a therapeutically effective amount of any one or more of the oligonucleotides provided herein. In some embodiments, treatment includes reducing expression of muscle target genes. In some implementations, the individual is treated therapeutically. In some implementations, the individual is treated prophylactically.

In some embodiments of the methods herein, an oligonucleotide provided herein, or comprising the oligonucleotide, is administered to an individual suffering from a disease, disorder, or condition associated with expression of a muscle target gene. The pharmaceutical composition of the acid reduces the expression of the target gene in the individual, thereby treating the individual. In some embodiments, the amount or level of target mRNA is reduced in the subject. In some embodiments, the amount or level of protein encoded by the target mRNA is reduced in the individual.

In some embodiments of the methods herein, an oligonucleotide provided herein, or comprising the oligonucleotide, is administered to an individual suffering from a disease, disorder, or condition associated with expression of a muscle target gene. A pharmaceutical composition of an acid that causes the individual's target gene expression to be reduced by at least about 30%, about 35%, about 40%, or about 40% compared to the target gene expression before administration of the oligonucleotide or pharmaceutical composition. About 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99 %. In some embodiments, the expression of the muscle target gene in the individual is equivalent to that of an individual who did not receive the oligonucleotide or pharmaceutical composition or who received a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or control The target gene expression of an individual) is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99%.

In some embodiments of the methods herein, an oligonucleotide herein, or an oligonucleotide comprising the oligonucleotide, is administered to an individual suffering from a disease, disorder, or condition associated with expression of a muscle target gene. A pharmaceutical composition that reduces the amount or level of target mRNA in the individual by at least about 30%, about 35%, or about 35% compared to the amount or level of target mRNA before administration of the oligonucleotide or pharmaceutical composition. About 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99 % or greater than 99%. In some embodiments, the amount or level of target mRNA in the individual is equivalent to that in an individual who did not receive the oligonucleotide or pharmaceutical composition or who received a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or The amount or level of target mRNA is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

In some embodiments of the methods herein, an oligonucleotide herein, or an oligonucleotide comprising the oligonucleotide, is administered to an individual suffering from a disease, disorder, or condition associated with expression of a muscle target gene. A pharmaceutical composition such that the amount or level of the protein encoded by the muscle target gene in the individual is equivalent to the amount or level of the protein encoded by the target gene before administration of the oligonucleotide or pharmaceutical composition. Compared with the system, it is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99%. In some embodiments, the amount or level of the protein encoded by the muscle target gene in the subject is equivalent to that of a subject who did not receive the oligonucleotide or pharmaceutical composition or who received a control oligonucleotide, pharmaceutical composition, or treatment. The amount or level of the protein encoded by the target gene in an individual (e.g., a reference or control individual) is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

In some embodiments of the methods herein, an oligonucleotide herein, or an oligonucleotide comprising the oligonucleotide, is administered to an individual suffering from a disease, disorder, or condition associated with expression of a muscle target gene. A pharmaceutical composition that reduces the amount or level of target gene activity in the individual by at least about 30% or about 35% compared to the amount or level of target gene activity before administration of the oligonucleotide or pharmaceutical composition. %, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, About 99% or greater than 99%. In some embodiments, the amount or level of target gene activity in the individual is equivalent to that of an individual who did not receive the oligonucleotide or pharmaceutical composition or who received a control oligonucleotide, pharmaceutical composition, or treatment (e.g., reference or control individual), the amount or level of target gene activity is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65% compared to , about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

Suitable methods for determining the expression of a target gene, the amount or level of target mRNA, the amount or level of protein encoded by the target gene, and/or the amount or level of target gene activity in an individual or a sample from an individual are the present invention. known in the technical field. Furthermore, the examples shown herein illustrate exemplary methods of determining target gene expression.

In some embodiments, target gene expression, the amount or level of target gene mRNA, the amount or level of protein encoded by the target gene, the amount or level of target gene activity, or any combination thereof, is present in the cell. , cell population or group of cells (e.g., organoids), organ, blood or portion thereof (e.g., plasma), tissue (e.g., muscle tissue), sample (e.g., muscle biopsy sample), or obtained from the individual or Isolation of any other biological material is reduced. In some embodiments, the expression of a muscle target gene, the amount or level of target gene mRNA, the amount or level of protein encoded by the target gene, the amount or level of target gene activity, or any combination thereof Tethered to more than one type of cell, more than one group of cells, more than one organ (e.g., muscle and one or more other organs), more than one part of blood (e.g., plasma and one or more other blood parts), more than one A decrease in the number of types of tissue (e.g., muscle tissue and one or more other types of tissue), or in more than one type of sample obtained or isolated from the individual (e.g., a muscle biopsy sample and one or more other types of biopsies) .

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

In some embodiments, the oligonucleotide conjugates herein, or compositions thereof, are administered via subcutaneous or intravenous administration. Treatment methods in cardiac tissue

The present disclosure provides oligonucleotides for use as medicaments, and particularly for use in methods of treating diseases, disorders and conditions associated with cardiac tissue. The present disclosure also provides oligonucleotides for use in, or suitable for use in, the treatment of an individual (e.g., a human) suffering from a disease, disorder, or condition associated with expression of a cardiac target gene. would benefit from reduced expression of cardiac target genes. In some embodiments, the present disclosure provides oligonucleotides for use in, or applicable to, methods of treating patients with diseases, disorders, or conditions associated with expression of cardiac target genes. The present disclosure also provides oligonucleotides for use in, or suitable for use in, the manufacture of medicaments or pharmaceutical compositions for the treatment of diseases, disorders, or conditions associated with expression of cardiac target genes. In some embodiments, the oligonucleotides are, or are suitable for, targeting mRNA and reducing expression of cardiac target genes (eg, via the RNAi pathway). In some embodiments, the oligonucleotides are, or are suitable for, targeting mRNA and reducing the amount or level of cardiac target mRNA, protein, and/or activity.

Furthermore, in some embodiments of the methods herein, individuals who have or are susceptible to a disease, disorder, or condition associated with expression of a cardiac target gene are selected for use with the oligonucleotides herein. Glycoside treatment. In some embodiments, methods include selecting individuals who have markers (e.g., biomarkers) of, or are susceptible to, a disease, disorder, or condition associated with expression of a cardiac target gene, such as but Not limited to target mRNA, protein, or combinations thereof. Likewise, and as described in detail below, some embodiments of the methods provided by the present disclosure include steps such as measuring or obtaining a baseline value for a marker of expression of a cardiac target gene, and then comparing the value so obtained to that experienced by the individual. One or more other baseline values are obtained after administration of the oligonucleotide to assess the effectiveness of the treatment.

The present disclosure also provides for the use of oligonucleotides provided herein to treat patients with, suspected of having, or developing a disease, disorder, or condition associated with expression of a cardiac target gene. An individual who is at risk of a disease, illness or condition. In some embodiments, the present disclosure provides methods of using oligonucleotides provided herein to treat or attenuate the onset or progression of a disease, disorder, or condition associated with expression of a cardiac target gene. In some embodiments, the present disclosure provides methods of using oligonucleotides provided herein to achieve one or more therapeutic benefits in an individual suffering from a disease, disorder, or condition associated with expression of a cardiac target gene. .

In some embodiments of the methods herein, an individual is treated by administering a therapeutically effective amount of any one or more of the oligonucleotides provided herein. In some embodiments, treatment includes reducing expression of cardiac target genes. In some implementations, the individual is treated therapeutically. In some implementations, the individual is treated prophylactically.

In some embodiments of the methods herein, an oligonucleotide provided herein, or comprising the oligonucleotide, is administered to an individual suffering from a disease, disorder, or condition associated with expression of a cardiac target gene. The pharmaceutical composition of the acid reduces the expression of the target gene in the individual, thereby treating the individual. In some embodiments, the amount or level of target mRNA is reduced in the individual. In some embodiments, the amount or level of protein encoded by the target mRNA is reduced in the individual.

In some embodiments of the methods herein, an oligonucleotide provided herein, or comprising the oligonucleotide, is administered to an individual suffering from a disease, disorder, or condition associated with expression of a cardiac target gene. A pharmaceutical composition of an acid that causes the individual's target gene expression to be reduced by at least about 30%, about 35%, about 40%, or about 40% compared to the target gene expression before administration of the oligonucleotide or pharmaceutical composition. About 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99 %. In some embodiments, the expression of the cardiac target gene in the individual is equivalent to that of an individual who did not receive the oligonucleotide or pharmaceutical composition or who received a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or control The target gene expression of an individual) is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

In some embodiments of the methods herein, an oligonucleotide herein, or an oligonucleotide comprising the oligonucleotide, is administered to an individual suffering from a disease, disorder, or condition associated with expression of a cardiac target gene. A pharmaceutical composition that reduces the amount or level of target mRNA in the individual by at least about 30%, about 35%, or about 35% compared to the amount or level of target mRNA before administration of the oligonucleotide or pharmaceutical composition. About 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99 % or greater than 99%. In some embodiments, the amount or level of target mRNA in the individual is equivalent to that in an individual who did not receive the oligonucleotide or pharmaceutical composition or who received a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or The amount or level of target mRNA is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

In some embodiments of the methods herein, an oligonucleotide herein, or an oligonucleotide comprising the oligonucleotide, is administered to an individual suffering from a disease, disorder, or condition associated with expression of a cardiac target gene. A pharmaceutical composition such that the amount or level of the protein encoded by the cardiac target gene in the individual is equivalent to the amount or level of the protein encoded by the target gene before administration of the oligonucleotide or pharmaceutical composition. Compared with the system, it is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99%. In some embodiments, the amount or level of the protein encoded by the cardiac target gene is equivalent to that of a subject who did not receive the oligonucleotide or pharmaceutical composition or who received a control oligonucleotide, pharmaceutical composition, or treatment. The amount or level of the protein encoded by the target gene in an individual (e.g., a reference or control individual) is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

In some embodiments of the methods herein, an oligonucleotide herein, or an oligonucleotide comprising the oligonucleotide, is administered to an individual suffering from a disease, disorder, or condition associated with expression of a cardiac target gene. A pharmaceutical composition that reduces the amount or level of target gene activity in the individual by at least about 30% or about 35% compared to the amount or level of target gene activity before administration of the oligonucleotide or pharmaceutical composition. %, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, About 99% or greater than 99%. In some embodiments, the amount or level of target gene activity in the individual is equivalent to that of an individual who did not receive the oligonucleotide or pharmaceutical composition or who received a control oligonucleotide, pharmaceutical composition, or treatment (e.g., reference or control individual), the amount or level of target gene activity is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65% compared to , about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99%.

Suitable methods for determining the expression of a target gene, the amount or level of target mRNA, the amount or level of protein encoded by the target gene, and/or the amount or level of target gene activity in an individual or a sample from an individual are the present invention. known in the technical field. Furthermore, the examples shown herein illustrate exemplary methods of determining target gene expression.

In some embodiments, target gene expression, the amount or level of target gene mRNA, the amount or level of protein encoded by the target gene, the amount or level of target gene activity, or any combination thereof, is present in the cell. , cell population or group of cells (e.g., organoids), organ, blood or portion thereof (e.g., plasma), tissue (e.g., heart tissue), sample (e.g., cardiac biopsy sample), or obtained from the individual or Isolation of any other biological material is reduced. In some embodiments, the expression of a cardiac target gene, the amount or level of target gene mRNA, the amount or level of protein encoded by the target gene, the amount or level of target gene activity, or any combination thereof Tethered to more than one type of cell, more than one group of cells, more than one organ (e.g., heart and one or more other organs), more than one part of blood (e.g., plasma and one or more other parts of blood), more than one A decrease in the number of types of tissue (e.g., cardiac tissue and one or more other types of tissue), or more than one type of sample obtained or isolated from the individual (e.g., a cardiac biopsy sample and one or more other types of biopsy samples) .

In some embodiments, the cardiac target gene can be a target gene from any mammal, such as a human. Any cardiac gene can be silenced according to the methods described herein. In some embodiments, the oligonucleotide conjugates herein, or compositions thereof, are administered via subcutaneous or intravenous administration. Test kit

In some embodiments, the present disclosure provides a kit comprising a lipid-conjugated RNAi oligonucleotide or a composition thereof herein, and instructions for use. In some embodiments, the kit includes a lipid-conjugated RNAi oligonucleotide or a composition thereof herein, and a package insert containing instructions for use of the kit and/or any of its components. In some embodiments, the kit includes a lipid-conjugated RNAi oligonucleotide or composition thereof herein, one or more controls, and various buffers, reagents well known in the art, in a suitable container. Enzymes and other standard components. In some embodiments, the container includes at least one vial, well, test tube, flask, bottle, syringe or other container device in which a lipid-conjugated RNAi oligonucleotide or composition thereof herein is placed, and in some case, be appropriately aliquoted. In some embodiments in which additional components are provided, the kit contains additional containers in which such components are placed. The kit may also include a device containing hermetically contained lipid-conjugated RNAi oligonucleotides or compositions thereof herein and any other reagents for commercial sale. Such containers may include injection molded or blow molded plastic containers in which the desired vials are received. The container and/or kit may include labels with instructions for use and/or warnings.

In some embodiments, a kit includes a lipid-conjugated RNAi oligonucleotide or a composition thereof herein, and a pharmaceutically acceptable carrier, or a pharmaceutical containing the lipid-conjugated RNAi oligonucleotide. Compositions and instructions for treating or delaying the progression of a disease, disorder or condition associated with the expression of a target gene expressed in one or more tissues or cells in an individual in need thereof.

In some embodiments, a kit includes a lipid-conjugated RNAi oligonucleotide or a composition thereof herein, and a pharmaceutically acceptable carrier, or a pharmaceutical containing the lipid-conjugated RNAi oligonucleotide. Compositions and instructions for treating or delaying the progression of a disease, disorder, or condition associated with the expression of a target gene (eg, a neuronal target gene) expressed in the CNS in an individual in need thereof.

In some embodiments, a kit includes a lipid-conjugated RNAi oligonucleotide or a composition thereof herein, and a pharmaceutically acceptable carrier, or a pharmaceutical containing the lipid-conjugated RNAi oligonucleotide. Compositions and instructions for treating or delaying the progression of a disease, disorder or condition associated with the expression of a target gene expressed in ocular tissue in an individual in need thereof.

In some embodiments, a kit includes a lipid-conjugated RNAi oligonucleotide or a composition thereof herein, and a pharmaceutically acceptable carrier, or a pharmaceutical containing the lipid-conjugated RNAi oligonucleotide. Compositions and instructions for treating or delaying the progression of a disease, disorder or condition associated with the expression of a target gene expressed in macrophages in an individual in need thereof.

In some embodiments, a kit includes a lipid-conjugated RNAi oligonucleotide or a composition thereof herein, and a pharmaceutically acceptable carrier, or a pharmaceutical containing the lipid-conjugated RNAi oligonucleotide. Compositions and instructions for treating or delaying the progression of a disease, disorder or condition associated with expression of target genes expressed in macrophages in the liver in an individual in need thereof.

In some embodiments, a kit includes a lipid-conjugated RNAi oligonucleotide or a composition thereof herein, and a pharmaceutically acceptable carrier, or a pharmaceutical containing the lipid-conjugated RNAi oligonucleotide. Compositions and instructions for treating or delaying the progression of a disease, disorder or condition associated with the expression of a target gene expressed in adipose tissue in an individual in need thereof.

In some embodiments, a kit includes a lipid-conjugated RNAi oligonucleotide or a composition thereof herein, and a pharmaceutically acceptable carrier, or a pharmaceutical containing the lipid-conjugated RNAi oligonucleotide. Compositions and instructions for treating or delaying the progression of a disease, disorder or condition associated with the expression of a target gene expressed in cardiac tissue in an individual in need thereof.

In some embodiments, a kit includes a lipid-conjugated RNAi oligonucleotide or a composition thereof herein, and a pharmaceutically acceptable carrier, or a pharmaceutical containing the lipid-conjugated RNAi oligonucleotide. Compositions and instructions for treating or delaying the progression of a disease, disorder, or condition associated with the expression of a target gene expressed in muscle tissue (eg, skeletal muscle) in an individual in need thereof.

In some embodiments, a kit includes a lipid-conjugated RNAi oligonucleotide or a composition thereof herein, and a pharmaceutically acceptable carrier, or a pharmaceutical containing the lipid-conjugated RNAi oligonucleotide. Compositions and instructions for treating or delaying the progression of a disease, disorder or condition associated with the expression of a target gene expressed in adrenal tissue in an individual in need thereof. definition

As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. In addition, the singular form and the articles "a/an/" and "the" are intended to include the plural form as well, unless expressly stated otherwise. It should further be understood that the terms include, comprise, including and/or comprising, when used in this specification, indicate the presence of the indicated features, integers, steps, operations, elements, and /or components, but does not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. Furthermore, it will be understood that when an element (including components or subsystems) is referred to and/or shown as being connected or coupled to another element, it can be directly connected or coupled to the other element or intervening elements may be present.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to the subject of this disclosure. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods or compositions, the methods and materials described herein are exemplary.

General textbooks describing molecular biology techniques useful in this article, including the use of vectors, promoters, and many other related topics, include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, volume 152, (Academic Press, Inc. , San Diego, Calif.)("Berger"); Sambrook et al., Molecular Cloning--A Laboratory Manual, 2d ed., Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, 1989("Sambrook" ) and Current Protocols in Molecular Biology, FMAusubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley and Sons, Inc., (1999 Supplement) ("Ausubel"). It is sufficient to guide those skilled in the art through in vitro amplification methods (including polymerase chain reaction (PCR), ligase chain reaction (LCR), Q.beta replicase amplification and other RNA polymerase Examples of mediating techniques (e.g., NASBA) such as procedures for producing homologous nucleic acids of the present disclosure are found in Berger, Sambrook, and Ausubel, and in Mullis et al., (1987) U.S. Patent No. 4,683,202 No.; Innis et al., eds. (1990); PCR Protocols: A Guide to Methods and Applications (Academic Press Inc. San Diego, Calif.) ("Innis"); Arnheim and Levinson (Oct. 1, 1990) CandEN 36-47; J.NIH Res.(1991)3: 81-94;Kwoh et al.,(1989) Proc. Natl.Acad.Sci.USA 86: 1173; Guatelliet al(1990) Proc. Nat'l. Acad.Sci.USA 87: 1874; Lomell et al., (1989) J. Clin. Chem 35: 1826; Landegren et al., (1988) Science 241: 1077-80; Van Brunt (1990) Biotechnology 8: 291 -94; Wu and Wallace (1989) Gene 4: 560; Barringer et al., (1990) Gene 89: 117; and Sooknanan and Malek (1995) Biotechnology 13: 563-564. Methods for improving the in vitro amplification of nucleic acids are described in Wallace et al., U.S. Patent No. 5,426,039. Improved methods for amplifying large nucleic acids by PCR are summarized in Cheng et al., (1994) Nature 369: 684-85 and references cited therein, in which PCR amplicons of up to 40 kb are generated.

As used in this specification and the accompanying claims, the singular forms "a/an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pharmaceutical carrier" includes a mixture of two or more such carriers and the like.

Ranges may be expressed herein as from "about" one value, and/or to "about" another value. When expressed as such a range, another embodiment includes from one value and/or to the other value. Similarly, when an approximation of a numerical value is expressed by use of the preceding "approximately", it should be understood that the value forms another implementation aspect. It is further understood that the endpoints of each range are meaningful both with respect to and independently of the other endpoint. It is also understood that every value disclosed herein and in addition to the value itself, each value disclosed herein is also "about" that value. For example, if "10" is revealed, "approximately 10" is also revealed. It is also to be understood that when a value is disclosed, "less than or equal to" the value, "greater than or equal to" the value, and possible ranges between values are also disclosed, as would be understood by one of ordinary skill in the art. For example, if the value "10" is revealed, then "less than or equal to 10" and "greater than or equal to 10" are also revealed. Also understand that throughout the application, data is provided in several different formats, and that this data represents endpoints and starting points, as well as ranges for any combination of those data points. For example, if a particular data point of "10" is disclosed and a particular data point of "15" is disclosed, it will be understood that the disclosure is considered to be greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15, and between 10 and 15. Also understand that units between two specific units are also revealed. For example, if 10 and 15 are disclosed, 11, 12, 13, and 14 are also disclosed.

As used herein, the term "amount" means an absolute amount (e.g., the absolute amount of an mRNA or protein), a relative amount (e.g., the relative amount of a target mRNA or protein when measured by a PCR assay), or Concentrations (e.g., the concentration of lipid-conjugated RNA in a composition), whether the amounts referred to in a given instance refer to absolute amounts, concentrations, or both, are generally relevant in the art based on the context provided herein. It will be clear to those who know.

As used herein, "bicyclic nucleotide" refers to a nucleotide that contains a bicyclic sugar moiety.

As used herein, "bicyclic sugar moiety" refers to a modified sugar moiety (including but not limited to furanosyl) containing a 4- to 7-membered ring that includes a bridge that includes a bridge connecting the 4- to 7-membered rings. Two atoms of the 7-membered ring form a second ring, resulting in a bicyclic structure. Typically, the 4 to 7 membered ring is a sugar. In some embodiments, the 4- to 7-membered ring is furanosyl. In certain embodiments, the bridge connects the 2'-carbon and 4'-carbon of the furanosyl group.

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

As used herein, "deoxyribonucleotide" refers to a nucleotide that has a hydrogen in place of a hydroxyl group at the 2' position of its pentose sugar when compared to ribonucleotides. Modified deoxyribonucleotides are deoxyribonucleotides having modifications or substitutions of one or more atoms other than at the 2' position, including modifications within or on sugars, phosphate groups or bases or replace.

As used herein, "double-stranded RNA" or "dsRNA" refers to an RNA oligonucleotide that is substantially in the form of a duplex. In some embodiments, one of the dsRNA oligonucleotides Complementary base pairing of duplex regions or regions is formed between antiparallel sequences of nucleotides in covalently separated nucleic acid strands. In some embodiments, one or more of the dsRNA Complementary base pairing of duplex regions is formed between antiparallel sequences of nucleotides of covalently linked nucleic acid strands. In some embodiments, complementary base pairing of one or more duplex regions of dsRNA Base pairing is formed from a single nucleic acid strand that is folded (e.g., via a hairpin) to provide complementary antiparallel sequences of nucleotides that are base paired together. In some embodiments, the dsRNA includes two strands of each other. Fully duplexed covalently isolated nucleic acid strands. However, in some embodiments, the dsRNA includes two partially duplexed (e.g., having an overhang at one or both ends) covalently isolated nucleic acid strands. In some embodiments, the dsRNA includes antiparallel sequences of partially complementary nucleotides and, therefore, may have one or more mismatches, which may include internal mismatches or terminal mismatches.

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

As used herein, "excipient" refers to a non-therapeutic agent that may be included in a composition, for example, to provide or contribute to a desired consistency or stabilizing effect. As used herein, a "loop" refers to an unpaired region of a nucleic acid (e.g., an oligonucleotide) that is flanked by antiparallel regions of two nucleic acids that are antiparallel to each other. The parallel regions are sufficiently complementary to each other that under appropriate hybridization conditions (e.g., in phosphate buffer solution, in cells), two antiparallel regions flanking the unpaired region hybridize to form a duplex (called a duplex). "trunk").

As used herein, "melting temperature" or " _Tm " means the temperature at which the two strands of a duplex nucleic acid separate. _Tm is often used as a parameter for duplex stability or for complementing the two strands of a nucleic acid or portion thereof. A measure of bond affinity. _{T m} can be measured by using UV spectroscopy to determine the formation and disintegration (melting) of hybridization. The base stacking that occurs during hybridization is accompanied by a decrease in UV absorption (light coloration). Therefore, UV A decrease in absorption indicates a higher _Tm .

As used herein, "modified internucleotide linkage" refers to one or more chemical modifications when compared to a reference internucleotide linkage that includes a phosphodiester bond linkages between nucleotides. In some embodiments, the modified nucleotides have non-naturally occurring linkages. Typically, a modified internucleotide linkage confers one or more desirable properties to the nucleic acid in which the modified internucleotide linkage is present. For example, modified nucleotides can improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, biological activity, reduced immunogenicity, etc.

As used herein, "modified nucleotide" refers to a nucleotide that has one or more chemical modifications when compared to a corresponding reference nucleotide selected from: adenine ribose Nucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, adenine deoxyribonucleotides, guanine deoxyribonucleotides, cytosine deoxyribonucleotides acid, and thymidine deoxyribonucleotides. In some embodiments, the modified nucleotides are non-naturally occurring nucleotides. In some embodiments, modified nucleotides have one or more chemical modifications in their sugar, nucleobase, and/or phosphate groups. In some embodiments, a modified nucleotide has one or more chemical moieties that are conjugated to the corresponding reference nucleotide. Typically, modified nucleotides confer one or more desired properties to the nucleic acid in which the modified nucleotide is present. For example, modified nucleotides can improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, biological activity, reduced immunogenicity, etc.

As used herein, "neuronal mRNA" and "neuronal gene" refer to any gene, mRNA and/or protein encoded/expressed by genes of neurons in the central nervous system. In some embodiments, the neuronal mRNA or neuronal gene is predominantly expressed in neurons relative to other cell types, such as other cell types of the CNS.

As used herein, "ocular mRNA" and "ocular gene" refer to any gene, mRNA and/or protein encoded/expressed by genes of cells of eye tissue. In some embodiments, the ocular mRNA or ocular gene is predominantly expressed in cells of the ocular tissue relative to other cell types, such as cell types of other organs.

As used herein, "macrophage mRNA" and "macrophage gene" refer to any gene encoded/expressed by the genes of macrophages in a tissue (e.g., liver), mRNA and/or protein. In some embodiments, the macrophage mRNA or macrophage gene is predominantly expressed in macrophages relative to other cell types, such as non-macrophage immune cells.

As used herein, "nicked tetraloop structure" refers to the structure of an RNAi oligonucleotide characterized by the separation of the sense (passenger) strand and the antisense (guide) strand, where the sense strand Having regions that are complementary to the antisense strands, and wherein at least one of the strands (generally speaking, the sense strand) has a four-member ring configured to be stable within the at least one strand adjacent to the main area.

As used herein, "oligonucleotide" refers to a short nucleic acid (eg, less than about 100 nucleotides long). Oligonucleotides can be single-stranded (ss) or ds. Oligonucleotides may or may not have double-stranded regions. As a non-limiting set of examples, oligonucleotides may be, but are not limited to, small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), endonuclease matrix interfering RNA (dsiRNA), reverse sense oligonucleotide, short siRNA or ss siRNA. In some embodiments, the double-stranded (dsRNA) is an RNAi oligonucleotide.

The terms "lipid-conjugated RNAi oligonucleotide" and "oligonucleotide-ligand conjugate" are used interchangeably and refer to a compound containing one or more Oligonucleotides are nucleotides conjugated to one or more targeting ligands (eg, lipids).

As used herein, "overhang" refers to end-to-end non-base pairing resulting from a strand or region that extends beyond the end of the complementary strand with which it forms a duplex. In some embodiments, the overhang includes one or more unpaired nucleotides extending from the duplex region at the 5' end or 3' end of the dsRNA. In some embodiments, the overhang is a 3' or 5' overhang on the antisense or sense strand of the dsRNA.

As used herein, "phosphate analog" refers to a chemical moiety that mimics the electrostatic and/or steric properties of a phosphate group. In some embodiments, the phosphate analog is positioned at the 5' terminal nucleotide of the oligonucleotide, replacing the 5'-phosphate that is normally susceptible to enzymatic removal. In some embodiments, the 5' phosphate analog contains a phosphatase-resistant linkage. Examples of phosphate analogs include, but are not limited to, 5'phosphonate esters such as 5'methylenephosphonate (5'-MP) and 5'-(E)-vinylphosphonate (5'-VP ). In some embodiments, the oligonucleotide has a phosphate analog at the 4'-carbon position of the sugar at the 5' terminal nucleotide (termed a "4'-phosphate analog"). An example of a 4'-phosphate analog is an oxymethylphosphonate in which the oxygen atom of the oxymethyl group is bonded to a sugar moiety (eg, at its 4' carbon) or analog thereof. See, for example, U.S. Provisional Patent Application Nos. 62/383,207 (filed on September 2, 2016) and 62/393,401 (filed on September 12, 2016). Other modifications targeting the 5' end of oligonucleotides have been developed (see, e.g., International Patent Application No. WO 2011/133871; U.S. Patent No. 8,927,513; and Prakash et al., (2015) Nucleic Acids Res. 43 : 2993-3011).

As used herein, "reduced expression" of a target gene refers to a cell, cell population, sample, or individual when compared to an appropriate reference (e.g., a reference cell, cell population, sample, or individual). Or the amount or level of RNA transcript (eg, target mRNA) or protein encoded by the target gene is reduced and/or the amount or level of gene activity is reduced in the individual. For example, when compared to cells not treated with a double-stranded oligonucleotide, an oligonucleotide or conjugate herein (e.g., a lipid-conjugated RNAi oligonucleotide that contains a Contact of an antisense strand with a nucleotide sequence complementary to the nucleotide sequence of the target mRNA) to the cell can result in the activity of the target mRNA, the protein encoded by the target gene, and/or the target gene (e.g., via the RNAi pathway Inactivating and/or degrading target mRNA) is reduced in amount or level. Similarly, and as used herein, "reducing expression" refers to actions that result in reduced expression of a target gene.

As used herein, a "region of complementarity" refers to a nucleotide sequence of a nucleic acid (e.g., dsRNA) that is sufficiently complementary to an antiparallel sequence of nucleotides to permit hybridization under appropriate hybridization conditions ( For example, hybridization between two nucleotide sequences in phosphate buffer, in cells, etc.). In some embodiments, the oligonucleotides herein comprise targeting sequences having regions that are complementary to the mRNA target sequence.

As used herein, "ribonucleotide" refers to a nucleotide having a ribose sugar in the form of a pentose sugar containing a hydroxyl group at its 2' position. Modified ribonucleotides are ribonucleotides that have modifications or substitutions of one or more atoms other than at the 2' position, including modifications or substitutions within or themselves of the ribose, phosphate group, or base. As used herein, "RNAi oligonucleotide" refers to (a) a dsRNA having a sense strand (passenger) and an antisense strand (guide), where the antisense strand or a portion of the antisense strand is composed of Al Ancient 2 (Ago2) endonuclease is used in the cleavage of target mRNA, or (b) ss oligonucleotide with a single antisense strand, wherein the antisense strand (or a portion of the antisense strand) is composed of Ago2 nucleic acid Endonucleases are used to cleave target mRNA. As used herein, "strand" refers to a single contiguous sequence of nucleotides linked together by internucleotide linkages (eg, phosphodiester linkages or phosphorothioate linkages). In some embodiments, a strand has two free ends (eg, a 5' end and a 3' end).

As used herein, "subject" means any mammal, including mice, rabbits, and humans. In one implementation, the individual system person or NHP. In addition, "individual" or "patient" may be used interchangeably with "subject".

As used herein, "synthetic" means artificially synthesized (e.g., using a machine (e.g., a solid-state nucleic acid synthesizer)) or otherwise not derived from a natural source (e.g., a cell or organism) from which the molecule is typically produced. ) of nucleic acids or other molecules.

As used herein, "targeting ligand" refers to a molecule that selectively binds to a homologous molecule (e.g., a receptor) of a tissue or cell of interest and/or can be conjugated to another substance to A molecule or "moiety" (e.g., carbohydrate, amino sugar, cholesterol, peptide, or lipid) that serves the purpose of targeting another substance to a tissue or cell of interest. For example, in some embodiments, a targeting ligand can be conjugated to an oligonucleotide for the purpose of targeting the oligonucleotide to a specific tissue or cell of interest. In some embodiments, targeting ligands selectively engage cell surface receptors. Thus, in some embodiments, a targeting ligand, when conjugated to an oligonucleotide, selectively binds to a receptor expressed on the cell surface and is composed of an oligonucleotide, a targeting ligand, and a receptor. The cells of the somatic complex undergo endosomal internalization to facilitate delivery of oligonucleotides to specific cells. In some embodiments, the targeting ligand is conjugated to the oligonucleotide via a linker that is cleaved after or during cellular internalization, allowing the oligonucleotide to release the targeting ligand in the cell.

As used herein, "tetraloop" refers to a loop that increases the stability of adjacent duplexes formed by hybridization of flanking nucleotide sequences. When the melting temperature (T _m ) of adjacent backbone duplexes increases, that is, the melting temperature is higher than that expected on average from a set of loops of comparable length consisting of a randomly selected set of nucleotide sequences. The increase in stability is detectable at the _Tm of adjacent backbone duplexes. For example, _a four-member loop can impart at least about 50°C, at least about 55°C, at least about 56°C, at least A _Tm of about 58°C, at least about 60°C, at least about 65°C, or at least about 75°C. In some embodiments, four-member loops can stabilize bp in adjacent backbone duplexes through stacking interactions. In addition, the interactions between nucleotides in the four-member loop include but are not limited to non-Watson-Crick base pairing, stacking interactions, hydrogen bonding and contact interactions (Cheong et al. , (1990) Nature 346: 680-82; Heus and Pardi (1991) Science 253: 191-94). In some embodiments, the four-member loop contains or consists of 3 to 6 nucleotides, and typically 4 to 5 nucleotides. In some embodiments, a four-member loop includes or consists of 3, 4, 5, or 6 nucleotides, which may or may not be modified (e.g., they may or may not be associated with Targeting moiety conjugation). In one embodiment, the four-member loop consists of 4 nucleotides. Any nucleotide can be used in the four-member loop, and standard IUPAC-IUB notation for such nucleotides can be used as described in Cornish-Bowden ((1985) Nucleic Acids Res. 13: 3021-3030) By. For example, the letter "N" can be used to mean that any base can be at that position, the letter "R" can be used to show that A (adenine) or G (guanine) can be at that position, and "B" can be used to show that C (cytosine), G (guanine), or T (thymine) can be at this position. Examples of four-member hoops include the UNCG family of four-member hoops (e.g., UUCG), the GNRA family of four-member hoops (e.g., GAAA), and the CUUG four-member hoop (Woese et al., (1990) Proc. . Natl. Acad. Sci. USA 87: 8467-71; Antao et al., (1991) Nucleic Acids Res. 19: 5901-05). Examples of DNA four-member loops include the d(GNNA) family of four-member loops (eg, d(GTTA), the d(GNRA) family of four-member loops, the d(GNAB) family of four-member loops, The d(CNNG) family of four-member circles, and the d(TNCG) family of four-member circles (for example, d(TTCG)). (See, e.g., Nakano et al., (2002) Biochem. 41: 4281-92; Shinji et al., (2000) Nippon Kagakkai Koen Yokoshu 78: 731). In some implementations, the four-member loop system includes a four-member loop structure with a cutout.

As used herein, " _{Tm -} increasing nucleotide" refers to an increase in the oligonucleotide when compared to an oligonucleotide duplex without the _Tm -increasing nucleotide. Nucleotides that increase the melting temperature ( _Tm ) of the nucleotide duplex. Nucleotides that increase _Tm include, but are not limited to, bicyclic nucleotides, tricyclic nucleotides, G-clamps and their analogs, and hexitol cores oligonucleotides. Certain modified nucleotides with modified sugar moieties or modified nucleobases can also be used to increase the T _m of the oligonucleotide duplex. As used herein, "increasing T _{The m} -increasing nucleotide specifically excludes nucleotides modified with 2'-OMe or 2'-F at the 2'-position of the sugar moiety.

As used herein, "treating" means treating an existing condition (e.g., disease, illness) for the purpose of improving an individual's health and/or well-being or preventing or reducing the likelihood of the condition occurring. The act of providing care to an individual in need thereof, such as by administering a therapeutic agent (eg, an oligonucleotide herein) to the individual. In some embodiments, treatment involves reducing the frequency or severity of at least one sign, symptom, or contributing factor of a condition (eg, disease, disorder) experienced by an individual. EXAMPLES Example 1: Preparation of RNAi oligonucleotide Oligonucleotide synthesis and purification

The oligonucleotides (RNAi oligonucleotides) described in the Examples were chemically synthesized using methods described herein. In general, RNAi oligonucleotides are synthesized using known phosphoramidite synthesis methods (see, e.g., Hughes and Ellington (2017) Cold Spring Harb Perspect Biol . 9(1):a023812; Beaucage SL, Caruthers MH STUDIES ON NUCLEOTIDE CHEMISTRY V : Deoxynucleoside Phosphoramidites-A New Class of Key Intermediates for Deoxypolynucleotide Synthesis , Tetrahedron Lett. 1981; 22:1859-62. doi: 10.1016/S0040-4039(01)90461-7); PCT application No. PCT/US2021/ 42469 (each of which is incorporated herein by reference)), solid-phase oligonucleotide synthesis methods as described for 19 to 23mer RNAi oligonucleotides were also used (see, e.g., Scaringe et al . al. (1990) Nucleic Acids Res . 18: 5433-41 and Usman et al. (1987) J. Am. Chem. Soc . 109:7845-45, see also, U.S. Patent Nos. 5,804,683; 5,831,071; No. 5,998,203; No. 6,008,400; No. 6,111,086; No. 6,117,657; No. 6,353,098; No. 6,362,323; No. 6,437,117 and No. 6,469,158).

Individual RNA strands were synthesized and purified by HPLC according to standard methods (Integrated DNA Technologies; Coralville, IA). For example, RNA oligonucleotides were synthesized using solid phase phosphoramidite chemistry, deprotected, and deprotected on a NAP-5 column (Amersham Pharmacia Biotech; Piscataway, NJ) using standard techniques. salt (Damha & Olgivie (1993) Methods Mol. Biol. 20: 81-114; Wincott et al. (1995) Nucleic Acids Res. 23: 2677-84), while the phosphoramidite synthesis is as follows: Formic acid 2- (2-((((6aR,8R,9R,9aR)-8-(6- phenylamino -9H- purin -9- yl )-2,2,4,4 - tetraisopropyltetrahydro- 6H- Furo [3,2-f][1,3,5,2,4] trioxadisiloctane (trioxadisilocin-9- yl ) oxy ) methoxy ) ethoxy ) Synthesis of ethy -1- ammonium (1-6)

A solution of compound 1-1 (25.00 g, 67.38 mmol) in 20 mL of DMF was treated with pyridine (11 mL, 134.67 mmol) and tetraisopropyldisiloxane dichloride (tetraisopropyldisiloxane dichloride) at 10°C. 22.63 mL, 70.75 mmol). The resulting mixture was stirred at 25 °C for 3 h and quenched with 20% citric acid (50 mL). The aqueous layer was extracted with EtOAc (3X50 mL) and the combined organic layers were concentrated in vacuo. The crude residue was recrystallized from a mixture of MTBE and n-heptane (1:15, 320 mL) to give compound 1-2 as a white oily solid (37.20 g, 90%).

A solution of compound 1-2 (37.00 g, 60.33 mmol) in 20 mL of DMSO was treated with AcOH (20 mL, 317.20 mmol) and _Ac2O (15 mL, 156.68 mmol). The mixture was stirred at 25 °C for 15 h. The reaction was diluted with EtOAc (100 mL) and quenched with sat _{. K2CO3} (50 mL). The aqueous layer was extracted with EtOAc (3X50 mL). The combined organic layers were concentrated and recrystallized from ACN (30 mL) to give compound 1-3 as a white solid (15.65 g, 38.4%).

A solution of compound 1-3 (20.00 g, 29.72 mmol) in 120 mL of DCM was treated with Fmoc-amino-ethoxyethanol (11.67 g, 35.66 mmol) at 25°C. The mixture was stirred to obtain a clear solution and then treated with 4Å molecular sieves (20.0 g), N -iodosuccinimide (8.02 g, 35.66 mmol), and TfOH (5.25 mL, 59.44 mmol). The mixture was stirred at 30°C until HPLC analysis indicated >95% consumption of compound 1-3. The reaction was quenched with TEA (6 mL) and filtered. The filtrate was diluted with EtOAc, washed with saturated NaHCO ₃ (2×100 mL), saturated Na ₂ SO ₃ (2×100 mL), and water (2×100 mL) and concentrated in vacuo to give crude compound 1-4 ( 26.34 g, 93.9%), which was used directly in the next step without further purification.

A solution of compound 1-4 (26.34 g, 27.62 mmol) in a mixture of DCM/water (10:7, 170 mL) was treated with DBU (7.00 mL, 45.08 mmol) at 5°C. The mixture was stirred at 5 to 25 °C for 1 h. The organic layer was then separated, washed with water (100 mL), and diluted with DCM (130 mL). The solution was treated with fumaric acid (7.05 g, 60.76 mmol) and 4Å molecular sieve (26.34 g) in four portions. The mixture was stirred for 1 h, concentrated, and recrystallized from a mixture of MTBE and DCM (5:1) to obtain compound 1-6 (14.74 g, 62.9%) as a white solid: ¹ H NMR (400 MHz, d ₆ -DMSO) 8.73 (s, 1H), 8.58 (s, 1H), 8.15-8.02 (m, 2H), 7.65-7.60 (m, 1H), 7.59-7.51 (m, 2H), 6.52 (s, 2H) , 6.15(s, 1H), 5.08-4.90 (m, 3H), 4.83-4.78 (m, 1H), 4.15-3.90 (m, 3H), 3.79-3.65 (m, 2H), 2.98-2.85 (m, 6H), 1.20-0.95 (m, 28H). (2R,3R,4R,5R)-5-(6- phenylamino -9H- purin -9- yl )-2-(( bis (4- methoxyphenyl )( phenyl ) methoxy ) Methyl )-4-((2-(2-[ lipid ] -acylaminoethoxy ) ethoxy ) methoxy ) tetrahydrofuran -3- yl (2- cyanoethyl ) diisopropyl Synthesis of phosphatamides (2-4a to 2-4e)

A solution of compound 1-6 (50.00 g, 59.01 mmol) in 150 mL of 2-methyltetrahydrofuran was washed with ice-cold _{aqueous K2HPO4} (6%, 100 mL) and brine (20%, 2X100 mL). The organic layer was separated and treated with caproic acid (10.33 mL, 82.61 mmol), HATU (33.66 g, 88.52 mmol), and DMAP (10.81 g, 147.52 mmol) at 0°C. The resulting mixture was allowed to warm to 25°C and stirred for 1 h. The solution was washed with water (2X100 mL), brine (100 mL), and concentrated in vacuo to give a crude residue. Flash chromatography on silica gel (1:1 hexane/acetone) gave compound 2-1a (34.95 g, 71.5%) as a white solid.

A mixture of compound 2-1a (34.95 g, 42.19 mmol) and TEA (9.28 mL, 126.58 mmol) in 80 mL of THF was added dropwise with triethylamine trihydrofuride (20.61 mL, 126.58 mmol) at 10°C. Add processing. The mixture was allowed to warm to 25°C and stirred for 2 h. The reaction was concentrated, dissolved in DCM (100 mL) and washed with saturated _NaHCO3 (5X20 mL) and brine (50 mL). The organic layer was concentrated in vacuo to give crude compound 2-2a (24.72 g, 99%), which was used directly in the next step without further purification.

A solution of compound 2-2a (24.72 g, 42.18 mmol) in 50 mL of DCM was treated with N -methylmalin (18.54 mL, 168.67 mmol) and DMTr-Cl (15.69 g, 46.38 mmol). The mixture was stirred at 25°C for 2 h and quenched with saturated _NaHCO3 (50 mL). The organic layer was separated, washed with water, and concentrated to obtain a crude syrup. Flash chromatography on silica gel (1:1 hexane/acetone) gave compound 2-3a (30.05 g, 33.8 mmol, 79.9%) as a white solid.

A solution of compound 2-3a (25.00 g, 28.17 mmol) in 50 mL of DCM was treated with N -methylmalin (3.10 mL, 28.17 mmol) and tetrazole (0.67 mL, 14.09 mmol) under nitrogen atmosphere. Bis(diisopropylamino)phosphine chloride (9.02 g, 33.80 mmol) was added dropwise to the solution and the resulting mixture was stirred at 25 °C for 4 h. The reaction was quenched with water (15 mL) and the aqueous layer was extracted with DCM (3X50 mL). The combined organic layers were washed with saturated NaHCO ₃ (50 mL) and concentrated to obtain a crude solid, which was recrystallized from a mixture of DCM/MTBE/n-hexane (1:4:40) to obtain a white color. Solid compound 2-4a (25.52 g, 83.4%): ¹ H NMR (400 MHz, d ₆ -DMSO) 11.25 (s, 1H), 8.65-8.60 (m, 2 H), 8.09-8.02 (m, 2H ), 7.71 (s, 1H), 7.67-7.60 (m, 1H), 7.59-7.51 (m, 2H), 7.38-7.34 (m, 2H), 7.30-7.25 (m, 7H), 6.85-6.79 (m , 4H), 6.23-6.20 (m, 1H), 5.23-5.14 (m, 1H), 4. 80-4.69 (m, 3H), 4.33-4.23 (m, 2H), 3.90-3.78 (m, 1H) , 3.75 (s, 6H), 3.74-3.52 (m, 3H), 3.50-3.20 (m, 6H), 3.14-3.09 (m, 2H), 3.09 (s, 1H), 2.82-2.80 (m, 1H) , 2.65-2.60 (m, 1H), 2.05-1.96 (m, 2H), 1.50-1.39 (m, 2H), 1.31-1.10 (m, 14H), 1.08-1.05 (m, 2 H), 0.85-0.79 (m, 3H); ³¹ P NMR (162 MHz, d ₆ -DMSO) 149.43, 149.18.

Compounds 2-4b, 2-4c, 2-4d, and 2-4e were prepared using similar procedures described above for compound 2-4a. Compound 2-4b was obtained as a white solid (25.50 g, 85.4%): ¹ H NMR (400 MHz, d ₆ -DMSO) 11.23 (s, 1H), 8.65-8.60 (m, 2 H), 8.05-8.02 ( m, 2H), 7.73-7.70 (m, 1H), 7.67-7.60 (m, 1H), 7.59-7.51 (m, 2H), 7.38-7.34 (m, 2H), 7.30-7.25 (m, 7H), 6.89-6.80 (m, 4H), 6.21-6.15 (m, 1H), 5.23-5.17 (m, 1H), 4. 80-4.69 (m, 3H), 4.40-4.21 (m, 2H), 3.91-3.80 (m, 1H), 3.74 (s, 6H), 3.74-3.52 (m, 3H), 3.50-3.20 (m, 6H), 3.14-3.09 (m, 2H), 3.09 (s, 1H), 2.83-2.79 (m, 1H), 2.68-2.62 (m, 1H), 2.05-1.97 (m, 2H), 1.50-1.38 (m, 2H), 1.31-1.10 (m, 18H), 1.08-1.05 (m, 2H) , 0.85-0.78 (m, 3H); ³¹ P NMR (162 MHz, d ₆ -DMSO) 149.43, 149.19.

Compound 2-4c was obtained as an off-white solid (36.60 g, 66.3%): ¹ H NMR (400 MHz, d ₆ -DMSO) 11.22 (s, 1H), 8.64-8.59 (m, 2H), 8.05-8.00 (m , 2H), 7.73-7.70 (m, 1H), 7.67-7.60 (m, 1H), 7.59-7.51 (m, 2H), 7.38-7.34 (m, 2H), 7.30-7.25 (m, 7H), 6.89 -6.80 (m, 4H), 6.21-6.15 (m, 1H), 5.25-5.17 (m, 1H), 4.80-4.69 (m, 3H), 4.40-4.21 (m, 2H), 3.91-3.80 (m, 1H), 3.74 (s, 6H), 3.74-3.50 (m, 3H), 3.50-3.20 (m, 6H), 3.14-3.09 (m, 2H), 3.09 (s, 1H), 2.83-2.79 (m, 1H), 2.68-2.62 (m, 1H), 2.05-1.99 (m, 2H), 1.50-1.38 (m, 2H), 1.33-1.12 (m, 38H), 1.08-1.05 (m, 2 H), 0.86 -0.80 (m, 3H); ³¹ P NMR (162 MHz, d ₆ -DMSO) 149.42, 149.17.

Compound 2-4d was obtained as an off-white solid (26.60 g, 72.9%): ¹ H NMR (400 MHz, d ₆ -DMSO) 11.22 (s, 1H), 8.64-8.59 (m, 2H), 8.05-8.00 (m , 2H), 7.73-7.70 (m, 1H), 7.67-7.60 (m, 1H), 7.59-7.51 (m, 2H), 7.38-7.33 (m, 2H), 7.30-7.25 (m, 7H), 6.89 -6.80 (m, 4H), 6.21-6.15 (m, 1H), 5.22-5.17 (m, 1H), 4.80-4.69 (m, 3H), 4.40-4.21 (m, 2H), 3.91-3.80 (m, 1H), 3.74 (s, 6H), 3.74-3.52 (m, 3H), 3.50-3.20 (m, 6H), 3.14-3.09 (m, 2H), 3.09 (s, 1H), 2.83-2.79 (m, 1H), 2.68-2.62 (m, 1H), 2.05-1.99 (m, 2H), 1.50-1.38 (m, 2H), 1.35-1.08 (m, 38H), 1.08-1.05 (m, 2 H), 0.85 -0.79 (m, 3H); ³¹ P NMR (162 MHz, d ₆ -DMSO) 149.47, 149.22.

Compound 2-4e was obtained as a white solid (38.10 g, 54.0%): ¹ H NMR (400 MHz, d ₆ -DMSO) 11.21 (s, 1H), 8.64-8.59 (m, 2H), 8.05-8.00 (m , 2H), 7.73-7.70 (m, 1H), 7.67-7.60 (m, 1H), 7.59-7.51 (m, 2H), 7.38-7.34 (m, 2H), 7.30-7.25 (m, 7H), 6.89 -6.80 (m, 4H), 6.21-6.15 (m, 1H), 5.23-5.17 (m, 1H), 4.80-4.69 (m, 3H), 4.40-4.21 (m, 2H), 3.91-3.80 (m, 1H), 3.73 (s, 6H), 3.74-3.52 (m, 3H), 3.47-3.22 (m, 6H), 3.14-3.09 (m, 2H), 3.09 (s, 1H), 2.83-2.79 (m, 1H), 2.68-2.62 (m, 1H), 2.05-1.99 (m, 2H), 1.50-1.38 (m, 2H), 1.35-1.06 (m, 46H), 1.08-1.06 (m, 2 H), 0.85 -0.77 (m, 3H); ³¹ P NMR (162 MHz, d ₆ -DMSO) 149.41, 149.15.

The oligomers were purified using ion exchange high-performance liquid chromatography (IE-HPLC) on an Amersham Source 15Q column (1.0 cm × 25 cm; Amersham Pharmacia Biotech) using a 15-minute step linear gradient. The gradient varied from 90:10 buffer A:B to 52:48 buffer A:B, where buffer A was 100 mM Tris pH 8.5 and buffer B was 100 mM Tris pH 8.5, 1 M NaCl. Samples were monitored at 260 nm and peaks corresponding to full-length oligonucleotide species were collected, poured together, desalted on a NAP-5 column, and freeze-dried.

The purity of each oligomer was determined by capillary electrophoresis (CE) on a Beckman PACE 5000 (Beckman Coulter, Inc.; Fullerton, CA). CE capillaries have an inner diameter of 100 μm and contain ssDNA 100R gel (Beckman-Coulter). Typically, approximately 0.6 nmole of oligonucleotide is injected into a capillary, run in an electric field of 444 V/cm, and detected by UV absorbance at 260 nm. Denatured Tris-borate-7 M-urea running buffer was purchased from Beckman-Coulter. The oligonucleotides obtained were at least 90% pure as assessed by CE and were used in the experiments described below. Compound identity was verified by a matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer on the Voyager DE™ Biospectometry Work Station (Applied Biosystems; Foster City, CA) following the manufacturer's recommended procedures. The relative molecular weights of all oligomers were obtained and were usually within 0.2% of the expected molecular weight. Preparation of duplex

Resuspend the single-stranded RNA oligos (eg, at 100 μM concentration) in duplex buffer consisting of 100 mM potassium acetate, 30 mM HEPES (pH 7.5). The complementary sense and antisense strands are mixed in equimolar amounts to yield, for example, a 50 μM final solution of the duplex. Samples were heated to 100°C in RNA buffer (IDT) for 5 minutes and allowed to cool to room temperature before use. Store RNAi oligonucleotides at -20°C. Store single-stranded RNA oligos lyophilized or in nuclease-free water at -80°C.

The synthetic methods described herein were used to generate the lipid-conjugated oligonucleotides described in the examples below. Example 2: RNAi oligonucleotide-lipid conjugates with a backbone-four-member loop at the 5' end of the sense strand provide reduced expression of neuronal target genes in the CNS

To identify RNAi oligonucleotide-lipid conjugates capable of reducing mRNA expression in CNS neurons, a series of C16-conjugated RNAi oligonucleotides were generated by the method described in Example 1. Each oligonucleotide tested contained an antisense strand with a region complementary to the mRNA encoding class III β3 tubulin (Tubb3). Tubb3 is a protein expressed primarily in neurons and was targeted herein to demonstrate the delivery of lipid-conjugated RNAi oligonucleotides to neurons in the CNS.

Comparison was made to the parent oligonucleotide-lipid conjugate (having the structure of compound 1 shown in Figure 1A) evaluated in a previous study. Compound 1 contains a backbone loop at the 3' end of the sense strand and a 2-nt overhang at the 3' end of the antisense strand. The backbone loop contains a four-member loop having the nucleotide sequence 5'-GAAA-3', a 6-nt backbone, and a second nucleotide conjugated to the four-member loop (i.e., 5 '-G A AA-3' is the C16 lipid of the underlined "A").

The RNAi oligonucleotide-lipid conjugates evaluated compared to Compound 1 had the structures of Compounds 2 to 14 as shown in Figures 1A to 1C. These structures contain the structural features described below: (i) Compounds 2 to 5 as shown in Figure 1A have a sense strand with a backbone loop at the 5' end and a blunt end at the 3' end. The backbone loop has (a) a four-member loop with the sequence 5'-GAAA-3'; and (b) a backbone of 6-nt in length (Compound 2) or 4-nt in length (Compounds 3 to 5) . Compounds 2 to 4 have a C16 lipid at the second nucleotide of the four-member loop (i.e., the underlined "A" in 5'-GAAA-3'), while compound 5 has a C16 lipid between the sense strand and C16 lipid at position 15. The 5' end of the sense strand of compound 4 has a series of phosphorothioate (PS) backbone linkages (compound 4). (ii) Compounds 6 to 9 as shown in Figure 1B contain a sense strand with a backbone loop at the 5' end, which backbone loop contains (a) a four-member loop with the sequence 5'-UACG-3'; ( b) a backbone of 4-nt length (compound 6) or a backbone of 2-nt length (compounds 7 to 9); and (c) the second nucleotide conjugated to the four-member loop (i.e., 5 '-U A CG-3' is the C16 lipid of the underlined "A" in the four-membered circle. Compounds 8 and 9 have locked nucleic acids (LNA) at the base of this backbone. The 3' end of the sense strand has a blunt end (compounds 6 to 8) or a 2-nucleotide truncation that results in a 2-nt overhang at the 5' end of the antisense strand (compound 9). (iii) Compound 10 as shown in Figure 1 contains a sense strand with a backbone loop at the 5' end and a blunt end at the 3' end. The backbone loop contains (a) a four-member loop with the sequence 5'-GAAA-3'; and (b) a backbone 6-nt in length. The 3' end of the sense strand has a C16 lipid conjugate. (iv) Compounds 11 to 13 as shown in Figure 1C have a sense strand with a backbone loop at the 5' end and a blunt end at the 3' end. The backbone loop has (a) a four-member loop with the sequence 5'-GAAA-3'; (b) a backbone with 1 to 3 LNAs and a length of 4-nt in the backbone (compounds 11 to 13, respectively) ; and (c) a C16 lipid conjugate located at the second nucleotide of the four-member loop (i.e., the underlined "A" in 5'-GA AA -3'). The 3' backbone of compound 13 contains an LNA with a thymine nucleobase instead of a uridine nucleobase. (v) Compound 14 as shown in Figure 1C has a positive strand with a backbone loop at the 5' end and a blunt end at the 3' end. The backbone loop has (a) a four-member loop with the sequence 5'-UACG-3'; (b) a backbone composed of LNA and 2-nt in length; and (c) located in the four-member loop. C16 lipid conjugate of the second nucleotide (i.e., the underlined "A" in 5'- UACG -3'). The 5' end of the antisense strand is pre-trimmed to expose the 5'OH.

To evaluate these RNAi oligonucleotide-lipid conjugates, 6- to 8-week-old C57BL/6 female mice were treated with 500 µg of oligonucleotide-lipid conjugates in artificial cerebrospinal fluid (aCSF). Rats were given a single intrathecal (it) injection. The RNAi oligonucleotide-lipid conjugate systems administered to each treatment group are summarized in Table 1. Group A were control mice receiving aCSF.

Target attenuation was assessed 7 days after injection. RNA was extracted from the lumbar spinal cord, lumbar dorsal root ganglia (DRG), medulla oblongata, hippocampus, and frontal cortex to determine murine Tubb3 mRNA levels (normalized to the endogenous housekeeping gene Rpl23) by qPCR. Perform qPCR using the PrimeTime™ qPCR Probe Assay. The percentage of residual murine Tubb3 mRNA in samples from treated mice was determined using the 2-ΔΔCt (“delta-delta Ct”) method (Livak and Schmittgen (2001) METHODS 25:402–408).

The expression percentages of TUBB3 mRNA in the frontal cortex and hippocampus are shown in Figures 2A to 2B respectively. Attenuation was observed in these tissues for most treatment groups.

The expression percentages of TUBB3 mRNA in the cerebellum, brainstem, lumbar DRG and lumbar spinal cord are shown in Figures 2C to 2F respectively. Attenuation was observed in the cerebellum and brainstem for most treatment groups (Figure 2C and Figure 2D). All treatment groups demonstrated silencing of TUBB3 mRNA expression in the lumbar DRG and lumbar spinal cord (Figure 2E and Figure 2F). The presence of a highly stable stem loop at the 5' end of the sense strand increases the attenuation of TUBB3 mRNA expression levels in several CNS tissues. Similarly, the presence of LNA in the backbone of the backbone loop structure increases silencing activity. Example 3: Reduced expression of CNS neuronal target genes produced by RNAi oligonucleotide-lipid conjugates with overhangs in the antisense strand

RNAi oligonucleotide-lipid conjugates with the structures of compounds 6 to 8, which resulted in efficient inhibition of TUBB3 expression in CNS tissues as described in Example 2, were co-conjugated with TUBB3-targeting oligonucleotide-lipid conjugates with additional modifications. Compared with the yoke structure.

RNAi oligonucleotide-lipid conjugates according to compounds 7 to 9 were compared to those having the structure of compounds 15 to 17 as shown in Figure 3 and further described below. Each compound has an antisense strand with a region complementary to TUBB3 mRNA.

(i) Compound 15 has a positive strand with a backbone loop at the 5' end and a blunt end at the 3' end. The backbone loop contains (a) a four-member loop with the sequence 5'-UACG-3'; (b) a 2-nt-length backbone with 1 LNA; and (iii) the fourth member of the four-member loop. C16 lipid conjugate of two nucleotides (i.e., the underlined "A" in 5'- UACG -3'). The 5' end of the antisense strand is pre-trimmed to expose the 5'OH.

(ii) Compound 16 has a sense strand with a 2-nt truncation at the 5' end and a 2-nt truncation at the 3' end (i.e., resulting in a 2-nt truncation at the 5' and 3' ends of the antisense strand) 2-nt overhang). The C16 lipid conjugation system is located at the 5' end of the sense strand.

(iii) Compound 17 has a 2-nt truncation at the 5' end (i.e., resulting in an overhanging antisense strand at the 3' end), a blunt end at the 3' end, and a C16 lipid at the 5' end The justice strand of the conjugate body. The 5' end of the antisense strand was truncated to 20-nt in length and pre-trimmed to expose the 5'OH.

(iv) Compound 18 has a 2-nt truncation at the 5' end (ie, resulting in an antisense strand with an overhang at the 3' end) and a blunt sense strand at the 3' end. The C16 lipid conjugation system is located at the 5' end of the sense strand.

To evaluate these RNAi oligonucleotide-lipid conjugates, 6- to 8-week-old C57BL/6 female mice were given a single intravenous dose of 500 µg of the oligonucleotide-lipid conjugate in aCSF. injection. The RNAi oligonucleotide-lipid conjugate systems administered to each animal group are shown in Table 2. Group A were control mice receiving aCSF.

Target attenuation was assessed 7 days after injection. RNA was extracted from various CNS tissues to determine murine TUBB3 mRNA levels (normalized to the endogenous housekeeping gene Rpl23) by qPCR as described in Example 2. The expression percentages of TUBB3 mRNA in the frontal cortex, hippocampus, medulla oblongata, cerebellum, lumbar DRG and lumbar spinal cord are shown in Figures 4A to 4F respectively. Decreased expression of TUBB3 mRNA was observed in various tissues for all or most treatment groups. Example 4: RNAi oligonucleotide-lipid conjugates with truncations at the 3' end of the sense strand provide enhanced activity for reducing expression of neuronal target genes in the CNS

Preparation of RNAi oligonucleotide-lipid conjugates designed to target TUBB3 mRNA and having a 2-nt overhang at the 3' end of the antisense strand to assess the impact of truncation at the 3' end of the sense strand on TUBB3 mRNA in the CNS performance effect.

Comparison was made to the parent RNAi oligonucleotide-lipid conjugate having the structure of compound 18 as shown in Figure 5. Compound 18 has an antisense strand with a 2-nt overhang at the 3' end and a sense strand with a blunt end at the 3' end and a C16 lipid conjugate at the 5' end.

RNAi oligonucleotide-lipid conjugates compared to compound 18 had structures according to compounds 19 to 28 as shown in Figure 5.

Each of compounds 19 to 26 has an antisense strand with a 2-nt overhang at the 3' end and a sense strand with a C16 lipid conjugate at the 5' end. Compounds 19 to 26 further contained overhangs at the 5' end of the antisense strand, with these overhangs ranging from 1-nt to 8-nt respectively. Compounds 27 and 28 contain a 4-nt or 5-nt overhang, respectively, at the 5' end of the antisense strand, and the nucleotides at positions 2 to 5 of the exposed overhang have PS-backbone linkages.

To evaluate these RNAi oligonucleotide-lipid conjugates, C57BL/6 female mice were given a single waist injection with 500 µg of oligonucleotide-lipid conjugate formulated in aCSF. The RNAi oligonucleotide-lipid conjugate system administered to each animal group is shown in Table 3. Group A were control mice receiving aCSF.

Target attenuation was assessed 7 days after injection. RNA was extracted from various CNS tissues to determine murine TUBB3 mRNA levels (normalized to the endogenous housekeeping gene Rpl23) by qPCR as described in Example 2. The percentages of TUBB3 mRNA expression in cerebellar and lumbar DRGs are shown in Figures 6A to 6B, respectively.

It was observed that, compared to compound 18, up to 3-nt truncation at the 3' end of the sense strand was tolerated without reducing TUBB3 mRNA repression. See Figures 6A to 6B for 3'p-3; 3'p-2; and 3'p-1 compared to the parent. In contrast, 4-nt or more truncations at the 3' end of the sense strand generally reduce but do not eliminate reduced expression of the target mRNA. See Groups F to J compared to Group B of Figures 6A to 6B.

Evaluate the effect of a truncated combination at the 3' end of the justice strand with an LNA positioned within the justice strand. The RNAi oligonucleotide-lipid conjugates evaluated had structures according to compounds 29 to 38 as shown in Figure 7.

Compound 29 has a structure identical to compound 18 but includes LNA at positions 2, 15, 16, 18 and 19 of the sense strand. Compounds 30 to 37 further include truncations at the 3' end of the sense strand, the lengths of these truncations being 1-nt to 8-nt respectively. Compound 38 contains a 4-nt truncation at the 3' end of the sense strand and contains PS-backbone linkages to nucleotides at positions 2 to 5 of the exposed overhang of the antisense strand.

To evaluate these RNAi oligonucleotide-lipid conjugates, C57BL/6 female mice were given a single waist injection with 500 µg of oligonucleotide-lipid conjugate formulated in aCSF. The RNAi oligonucleotide-lipid conjugate systems administered to each animal group are shown in Table 4. Group A were control mice receiving aCSF.

Target attenuation was assessed 7 days after injection. RNA was extracted from various CNS tissues to determine murine TUBB3 mRNA levels (normalized to the endogenous housekeeping gene Rpl23) by qPCR as described in Example 2. The percentages of TUBB3 mRNA expression in cerebellar and lumbar DRGs are shown in Figures 8A to 8B, respectively. A decrease in TUBB3 mRNA expression was observed in both tissues for all or most treatment groups. Example 5: RNAi oligonucleotide-lipid conjugates evaluated for reduction of target gene expression in ocular tissue

To identify RNAi oligonucleotide-lipid conjugates capable of reducing mRNA expression in eye tissue, a series of lipid-conjugated RNAi oligonucleotides were generated by the method described in Example 1. Each oligonucleotide tested had an antisense strand with a region complementary to the mRNA encoding rat reticulin 4 (RTN4), a regulator of developmental neuronal outgrowth expressed in the eye.

Comparison with the parent RNAi oligonucleotide-lipid conjugate having a structure according to compound 39 shown in Figure 9. Compound 39 has an antisense strand with a 2-nt overhang at the 3' end and a blunt end at the 5' end. Compound 39 further contains a C16 lipid conjugate at the 5' end of the sense strand.

The RNAi oligonucleotide-lipid conjugates evaluated had variations in the length of the aliphatic lipid, incorporation of LNA, and truncation at the 5' and/or 3' end of the sense strand. The structure of the oligonucleotide-lipid conjugate is shown in Figure 9 and further described below: (i) Compound 40 contains a 5-nt truncation at the 5' end, a blunt end at the 3' end, and a sense strand in has the sense strand of the C16 lipid conjugate at position 1; (ii) Compound 41 contains a 2-nt truncation at the 5' end, a 2-nt truncation at the 3' end and a sense strand at position 1 of the sense strand. The sense strand of the C16 lipid conjugate; (iii) Compound 42 contains a 5-nt truncation at the 5' end, a 2-nt truncation at the 3' end and a C16 lipid conjugate at position 1 of the sense strand (iv) Compound 43 contains a sense strand with a 2-nt truncation at the 5' end, a 2-nt truncation at the 3' end, and a C18 lipid conjugate at position 1 of the sense strand ; (v) Compound 44 contains a sense strand with a 2-nt truncation at the 5' end, a 2-nt truncation at the 3' end, and a C22 lipid conjugate at position 1 of the sense strand; (vi) Compound 45 contains a sense strand with a 5-nt truncation at the 5' end, a blunt end at the 3' end, and a C16 lipid conjugate at position 1 of the sense strand. The exposed overhang of the antisense strand contains PS linkages between adjacent residues; (vii) Compounds 46 to 47 each contain a backbone loop at the 5' end and a 2-nt truncation at the 3' end. Justice shares. The backbone loop has (i) a four-member loop with the sequence 5'-UACG-3'; and (ii) a backbone composed of LNA and 2-nt in length. Compound 46 contains a C16 lipid conjugate at position 4 of the sense strand (i.e., the underlined "A" in the 5'-U A CG-3' four-membered ring), whereas compound 47 does not contain a lipid conjugate. . (viii) Compound 48 contains a sense strand with a 2-nt truncation at the 5' end, a blunt end at the 3' end, a C16 lipid conjugate at position 1, and LNA at positions 2, 15, and 16; and (ix) Compound 49 contains a sense strand with a 5-nt truncation at the 5' end, a blunt end at the 3' end, a C16 lipid conjugate at position 1 and LNA at positions 2, 10 and 11.

To evaluate these RNAi oligonucleotide-lipid conjugates, Sprague-Dawley rats were given intravitreal injections of 250 µg of oligonucleotide-lipid conjugate in each eye. . The RNAi oligonucleotide-lipid conjugate systems administered to each animal group are summarized in Table 5. Group A is control rats receiving PBS.

Target attenuation was assessed 14 days after injection. RNA was extracted from the retina or optic nerve of one eye and rat RTN4 mRNA levels (normalized to the endogenous housekeeping gene PPIB) were determined by qPCR. qPCR was performed using the PrimeTime™ qPCR Probe Assay, which consists of a pair of primers and a fluorescently labeled 5' nuclease probe specific for rat RTN4 mRNA. In samples from treated rats, the percentage of residual rat RTN4 mRNA was determined using the 2-ΔΔCt (“delta-delta Ct”) method (Livak and Schmittgen (2001) METHODS 25:402–408). The expression percentages of RTN4 mRNA in the retina and optic nerve are shown in Figures 10A to 10B respectively. Inhibition of RTN4 mRNA expression was observed in both retinal and optic nerve tissue for each treatment group. Example 6: RNAi oligonucleotide-GalNAc conjugates with sense strand truncations and LNA assessed for target attenuation in liver

RNAi oligonucleotide-GalNAc conjugates were prepared using silencing of target genes in the liver with a backbone-four-member loop at the 3' end and a truncated 5' end incorporating the sense strand of LNA. Each oligonucleotide-lipid conjugate tested contained an antisense strand with a region complementary to the mRNA encoding Aldh2.

Compare to the parent oligonucleotide-GalNAc conjugate having (a) no truncation at the 5' end, thereby providing a 2-nt overhang at the 3' end of the antisense strand; and (b) At the 3' end there is a righteous strand with a notched trunk ring. The backbone is 3 bp in length and is composed of LNA. The backbone contains the sequence 5'-GAA-3', with a 2'-aminodiamine group at positions 2 and 3 from the 5' end (i.e., the underlined nucleotides in the 5'- GAA -3' loop). Ethoxymethanol-GalNAc.

Oligonucleotide-GalNAc conjugated systems evaluated included those having the structures of compounds 57 and 98 to 108 as shown in Figure 10C and further described below: (i) Compounds 57 and 98 to 100 contain a sense strand with a 6-nt truncation at (a) the 5' end to provide an 8-nt overhang at the 3' end of the antisense strand; and (b) There is the same nicked backbone loop at the 3' end as that found in the parent oligonucleotide-GalNAc conjugate. Compound 98 contains one additional LNA at the 5' end of the sense strand; compound 99 contains two additional LNAs, one at the 5' end and the other 9-nt from the 5' end of the sense strand; compound 100 contains three Additional LNA, one at the 5' end and the other two located at 9-nt and 10-nt from the 5' end of the sense strand; Compound 101 contains five additional LNA, one at the 5' end and the other four They are respectively located at 9-nt, 10-nt, 12-nt and 13-nt from the 5' end of the sense strand. (ii) Compounds 101 to 104 contain a sense strand with a 7-nt truncation at (a) the 5' end to provide a 9-nt overhang at the 3' end of the antisense strand; and (b) at the 3' end of the antisense strand The 'end has the same nicked backbone loop as that found in the parent oligonucleotide-GalNAc conjugate. Compound 102 contains two additional LNAs, one at the 5' end and the other 8-nt from the 5' end of the sense strand; compound 103 contains three additional LNAs, one at the 5' end and the other two are located 8-nt from the 5' end of the sense strand. at 8-nt and 9-nt from the 5' end of the sense strand; and compound 104 contains five additional LNAs, one at the 5' end and the other four at the 8-nt, 5' end from the sense strand. at 9-nt, 11-nt and 12-nt. (iii) Compounds 105 to 108 contain a sense strand with an 8-nt truncation at the 5' end of (a) the antisense strand to provide a 10-nt overhang at the 3' end of the antisense strand; and (b) at the 3' end of the antisense strand The 'end has the same nicked backbone loop as that found in the parent oligonucleotide-GalNAc conjugate. Compound 106 contains two additional LNAs, one at the 5' end and the other 7-nt from the 5' end of the sense strand; compound 107 contains three additional LNAs, one at the 5' end and the other two are located 7-nt from the 5' end of the sense strand. At 7-nt and 8-nt from the 5' end of the sense strand; and compound 108 contains five additional LNAs, one at the 5' end and the other four at the 7-nt, 5' end from the sense strand. at 8-nt, 10-nt and 11-nt.

To evaluate these RNAi oligonucleotide-GalNAc conjugates, mice were given a single intravenous (iv) injection of 0.5 mg/kg oligonucleotide-GalNAc conjugate in PBS. The RNAi oligonucleotide-GalNAc conjugate systems administered to each of the treatment groups are summarized in Table 6. Group A were control mice that received PBS only.

Target attenuation was assessed 4 days after injection. RNA was extracted from liver tissue, and murine ALDH2 mRNA levels were measured by qPCR. Target mRNA reduction was measured by qPCR using the CFX384 TOUCH™ Real-Time PCR Detection System (BioRad Laboratories, Inc., Hercules, CA). All samples were normalized to PBS-treated control animals and plotted using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA). The normalized ALDH2 mRNA expression in the liver measured for each treatment group relative to the control is shown in Figure 10D. Suppression of ALDH2 mRNA expression was observed for most treatment groups. Example 7: RNAi oligonucleotide-lipid conjugates for reducing expression of markers highly expressed by non-hepatocytes of the liver

The liver has a cellular composition of approximately 60% hepatocytes and 35% non-organic cells (NPCs). NPC is composed of macrophages (also known as Kupffer cells), liver sinusoidal endothelial cells (LSEC), liver satellite cells and other white blood cells. Macrophages in the liver have the function of maintaining tissue homeostasis and responding to tissue damage (see Braet et al., Seminars in Cell Developmental Biology, 2017). We assessed whether systemic delivery of RNAi oligonucleotide-lipid conjugates directed to target genes highly expressed by hepatic NPCs would induce silencing of hepatic target genes. Target genes selected for evaluation included PECAM1 and CD68, which are highly represented by NPCs of the liver (specifically, LSECs and macrophages, respectively) (see Ouyang et al (2014) Oncology Lett; Klein, et al (2021) Mol.Ther).

The RNAi oligonucleotide lipid conjugates evaluated had the structures of compounds 120 to 122 as shown in Figure 10E. Each contains a nicked backbone-four-member loop at the 3' end of the sense strand and a 2-nucleotide overhang at the 3' end of the antisense strand. The backbone-four-member loop contains a four-member loop with the nucleotide sequence 5'-GAAA-3' and a backbone of 6 nt in length. The 5' end of the sense strand contains a C22 lipid conjugate. Compound 120 has an antisense strand, which has a region complementary to the first target sequence (PECAM1-2392) in the mRNA encoding PECAM1; compound 121 has an antisense strand, which has a region complementary to the first target sequence (PECAM1-2392) in the encoding mRNA. The two target sequences (PECAM1-3222) are complementary to each other; while compound 122 has an antisense strand that is complementary to the target sequence in the mRNA encoding CD68 (CD68-0815).

To evaluate RNAi oligonucleotide-lipid conjugates targeting PECAM1 and CD68, female CD-1 mice were given a single subcutaneous injection of 30 mg/kg oligonucleotide-lipid conjugates in PBS. . The RNAi oligonucleotide-lipid conjugate systems administered to each of the treatment groups are summarized in Table 7. Group A were control mice that received PBS only.

Target attenuation was assessed 7 days after injection. RNA was extracted from liver and extrahepatic tissues, and murine PECAM and CD68 mRNA levels (normalized to the endogenous housekeeping gene Ppib) were measured by qPCR. In samples from treated mice, the percentage of residual murine mRNA was determined using the 2-ΔΔCt ("delta-delta Ct") method (Livak and Schmittgen (2001) METHODS 25:402-408).

The measured PECAM1 and CD68 mRNA expression percentages for each treatment group are shown in Figures 10F to 10G, respectively. For mice receiving Compound 120, the expression of the mRNA encoding PECAM1 was reduced by approximately 50%. Expression of the mRNA encoding CD68 was reduced by approximately 75% in mice receiving Compound 122. Example 8: Effect of dosage on silencing mediated by RNAi oligonucleotide-lipid conjugates targeting markers abundantly expressed by liver macrophages

Doses of the CD68-targeting RNAi oligonucleotide-lipid conjugates described in Example 7 were titrated to determine the effect on silencing of CD68 expression by liver macrophages. Specifically, the RNAi oligonucleotide-lipid conjugate evaluated had the structure of compound 122 as shown in Figure 10E.

Female CD-1 mice were administered a single subcutaneous injection of the oligonucleotide-lipid conjugate at 3 to 90 mg/kg in PBS, as shown in Table As outlined in 8. Group A were control mice that received PBS only.

Target attenuation was assessed 7 days after injection. RNA was extracted from liver and extrahepatic tissue, and murine CD68 mRNA levels were determined by qPCR as described in Example 7. Livers were also collected for immunohistochemistry (IHC) analysis of CD68 protein levels. Livers were fixed in formalin and stained using a CD68-specific primary antibody. (See, e.g., Li, et al (1996) Mod Pathol 9:982; Kunisch et al (2003) Annu Rheum Disc 63:774; Caffo et al (2005) Neurosurgery 57:551). CD68 expression was quantified in three randomly selected tissue regions and presented as mean values.

As shown in Figure 10H, a dose-dependent decrease in the expression of CD68 mRNA was observed in the liver tissue of mice administered Compound 122. An _ED50 of approximately 10 mg/kg was measured, and approximately 85% reduction in CD68 mRNA was observed at the maximum dose (90 mg/kg).

As shown in Figure 10I, a dose-dependent decrease in CD68 protein expression measured by IHC was also observed in the liver tissue of mice administered Compound 122. Representative IHC images are shown in Figure 10J, demonstrating that CD68 protein is abundant in liver macrophages. As shown in Figure 10K, as the dose of compound 122 increased, CD68 protein expression decreased in the liver. An _ED50 of approximately 3 mg/kg was measured, and approximately 93% reduction in CD68 protein was observed at the maximum dose (90 mg/kg). Example 9: RNAi oligonucleotide-lipid conjugates for reducing markers highly expressed by liver macrophages

RNAi oligonucleotide-lipid conjugates were evaluated to determine the effect of truncation at the 5' and/or 3' end of the sense strand on activity for silencing target genes expressed by liver macrophages. Each oligonucleotide-lipid conjugate tested contained an antisense strand with a region complementary to the mRNA encoding CD68, a protein highly expressed in liver macrophages.

Comparison was made with an oligonucleotide-lipid conjugate evaluated in a previous study with the structure of compound 58 shown in Figure 11A. Compound 58 contains a backbone loop at the 3' end of the sense strand and a 2-nucleotide overhang at the 3' end of the antisense strand. The backbone loop contains a four-member loop having the nucleotide sequence 5'-GAAA-3', a backbone of 6 nt in length, and a second nucleotide associated with the four-member loop (i.e., 5'-G A AA - The underlined "A" in the 3' four-membered ring) conjugated C22 lipid.

Oligonucleotide-lipid conjugate systems evaluated included those having the structures of compounds 59 to 97 as shown in Figures 11A to 11B and further described below: (i) Compounds 59 to 64 as shown in Figure 11A contain truncated sense strands with a blunt end at the 3' end and 0 to 5 nucleotides at the 5' end, respectively, yielding 2-nt to 7-nt overhanging antisense strand. Each contains a C22 lipid conjugate located 14-nt 3' from the sense strand. (ii) Compounds 65 to 69 as shown in Figure 11A contain a sense strand with a 2-nt truncation at the 3' end and a truncated sense strand of 1 to 5 nucleotides at the 5' end, respectively, yielding 5 The antisense strand has a 2-nt overhang at the 'end and a 3-nt to 7-nt overhang at the 3' end, respectively. Each contains a C22 lipid conjugate located 12-nt 3' from the sense strand. (iii) Compounds 70 to 74 as shown in Figure 11A contain a sense strand with a 2-nt truncation at the 3' end and a truncated sense strand of 1 to 5 nucleotides at the 5' end, respectively, yielding 5 The antisense strand has a 2-nt overhang at the 'end and a 3-nt to 7-nt overhang at the 3' end, respectively. Each contains a sense strand with a C22 lipid conjugate located 12-nt from the 3' end and a single LNA at the 5' end. (iv) Compounds 75 to 79 as shown in Figure 11A contain a sense strand with a 2-nt truncation at the 3' end and a truncated sense strand of 1 to 5 nucleotides at the 5' end, respectively, yielding 5 The antisense strand has a 2-nt overhang at the 'end and a 3-nt to 7-nt overhang at the 3' end, respectively. Each contains a sense strand with a C22 lipid conjugate located 12-nt from the 3' end and two LNAs (one located at the 5' end and the other 3-nt from the 3' end). (v) Compounds 80 to 84 as shown in Figure 11B contain a sense strand with a 2-nt truncation at the 3' end and a truncated sense strand of 1 to 5 nucleotides at the 5' end, respectively, yielding 5 The antisense strand has a 2-nt overhang at the 'end and a 3-nt to 7-nt overhang at the 3' end, respectively. Each contains a C22 lipid conjugate located 12-nt from the 3' end and three LNAs (one at the 5' end, another 3-nt from the 3' end, and one at the 3' end). The 4-nt position of the 3' end) of the positive strand. (vi) Compounds 85 to 89 as shown in Figure 11B contain a sense strand with a 3-nt truncation at the 3' end and a truncated sense strand of 1 to 5 nucleotides at the 5' end, respectively, yielding 5 The antisense strand has a 3-nt overhang at the 'end and a 3-nt to 7-nt overhang at the 3' end, respectively. Each contains a C22 lipid conjugate located 11-nt from the 3' end and three LNAs (one at the 5' end, another 2-nt from the 3' end, and one located 2-nt from the 3' end). 3' end of the 3-nt) justice strand. An additional PS main link was installed so that two PS links appeared at the 3' end of the justice strand. (vii) Compounds 90 to 94 as shown in Figure 11B contain a sense strand with a 4-nt truncation at the 3' end and a truncated sense strand of 1 to 5 nucleotides at the 5' end, respectively, yielding at 5 The antisense strand has a 4-nt overhang at the 'end and a 3-nt to 7-nt overhang at the 3' end. Each contains a C22 lipid conjugate located 10-nt from the 3' end and three LNAs (one at the 5' end, another at the 3' end, and one at the 2-nt from the 3' end). nt) of the justice stock. An additional PS main link was installed so that two PS links appeared at the 3' end of the justice strand. (viii) Compounds 95 to 97 as shown in Figure 11B contain sense strands with a 4-nt truncation at the 3' end and a 6-nt to 8-nt truncation at the 5' end, respectively, yielding at The antisense strand has a 4-nt overhang at the 5' end and an 8-nt to 10-nt overhang at the 3' end. Each contains a C22 lipid conjugate at 7-nt from the 3' end and three LNAs (one at the 5' end, another at the 3' end, and one at the 2-nt from the 3' end). nt) of the justice stock. An additional PS main link was installed so that two PS links appeared at the 3' end of the justice strand.

To evaluate these RNAi oligonucleotide-lipid conjugates, CD-1 female mice (with a weight of 18 to 20 g) were given 15 mg/kg of oligonucleotide-lipid conjugates in PBS. A single subcutaneous (sc) injection. The RNAi oligonucleotide-lipid conjugate systems administered to each of the treatment groups are summarized in Table 9. Group A were control mice that received PBS only.

Target attenuation was assessed 7 days after injection. RNA was extracted from liver tissue and murine CD68 mRNA levels (normalized to the endogenous housekeeping gene Ppib) were measured by qPCR. Perform qPCR using the PrimeTime™ qPCR Probe Assay. The percentage of residual murine CD68 mRNA in samples from treated mice was determined using the 2-ΔΔCt (“delta-delta Ct”) method (Livak and Schmittgen (2001) METHODS 25:402–408). The percentage of liver CD68 mRNA expression measured for each treatment group is shown in Figure 12. Silencing of CD68 mRNA expression was observed for most treatment groups. Overall, truncation at the 5' end of the justice strand is well tolerated. All molecules in this example containing a 2-nt or 3-nt overhang at the 5' end of the antisense strand reduced CD68 mRNA expression. Compounds 90 to 94 containing a 4-nt overhang at the 5' end of the antisense strand reduced the performance of CD68 mRNA expression. Example 10: Liver-targeting RNAi oligonucleotide-lipid conjugates

RNAi oligonucleotide-lipid conjugates were evaluated to determine the effect of incorporating LNA and localizing the lipid conjugate on activity specifically targeting liver tissue. Each oligonucleotide-lipid conjugate tested contained an antisense strand with a region complementary to the mRNA encoding Aldh2, a protein expressed in the liver and other tissues.

Oligonucleotide-lipid conjugate systems evaluated included those with structures as shown in Figure 13 for compounds 109 to 117. The compounds each contain a sense strand with a blunt end at the 3' end, a truncation at the 5' end that results in an antisense strand with a 6-nt overhang at the 3' end, and a C16 lipid conjugate and at least one LNA share. The positioning of the lipid C16 conjugate and the amount of LNA vary with compound, as further described below:

(i) Compounds 109 to 111 contain a C16 lipid conjugate at the 3' end of the sense strand and an LNA at the 5' end of the sense strand. Compound 110 contains LNA at position 11 of the sense strand. Compound 111 contains LNA at positions 11 and 12 of the sense strand.

(ii) Compounds 112 to 114 contain a C16 lipid conjugate at the 5' end of the sense strand and an LNA at position 2 of the sense strand. Compound 113 contains LNA at position 11 of the sense strand. Compound 114 contains LNA at positions 11 and 12 of the sense strand.

(iii) Compounds 115 to 117 contain a C16 lipid conjugate at position 5 of the sense strand and an LNA at the 5' end of the sense strand. Compound 116 contains LNA at position 11 of the sense strand. Compound 117 contains LNA at positions 11 and 12 of the sense strand.

To evaluate these RNAi oligonucleotide-lipid conjugates, mice were given a single subcutaneous (sc) injection of 10 mg/kg oligonucleotide-lipid conjugates in PBS. The RNAi oligonucleotide-lipid conjugate systems administered to each animal group are summarized in Table 10. Group A were control mice that received PBS only.

Target attenuation was assessed 7 days after injection. RNA was extracted from various tissues and murine ALDH2 mRNA levels (normalized to the endogenous housekeeping gene Ppib) were measured by qPCR. qPCR was performed using the PrimeTime™ qPCR Probe Assay, which consists of a pair of primers and a fluorescently labeled 5' nuclease probe specific for murine ALDH2 mRNA. The percentage of residual murine ALDH2 mRNA in samples from treated mice was determined using the 2-ΔΔCt ("delta-delta Ct") method (Livak and Schmittgen (2001) METHODS 25:402-408).

Percent ALDH2 mRNA expression measured in liver, skeletal muscle, fat, and adrenal gland for each treatment group is shown in Figures 14A to 14D, respectively. Silencing of ALDH2 mRNA expression was observed in all tissues evaluated for most treatment groups. Example 11: RNAi oligonucleotide-lipid conjugates with double overhangs generate silencing of mRNA encoding liver targets

It was evaluated whether RNAi oligonucleotide-lipid conjugates with overhangs at both the 5' and 3' ends of the antisense strand would provide similar silencing activity against liver targets as conjugates with blunt ends. Silencing of the mRNA expression of four liver targets was assessed, including STAT3, SLC25A1, HMGB1, and ALDH2.

The RNAi oligonucleotide-lipid conjugates evaluated had the structures shown in Figure 15 for compounds 123 to 126. Compounds 123 to 126 had antisense strands with regions complementary to the mRNA encoding STAT3, SLC25A1, HMGB1 and ALDH2, respectively, with a 2-nt overhang at the 3' end and a blunt end at the 5' end. Compounds 127 to 130 have antisense strands with regions complementary to the mRNA encoding STAT3, SLC25A1, HMGB1 and ALDH2, respectively, and with 2-nt overhangs at both the 5' and 3' ends.

To evaluate these RNAi oligonucleotide-lipid conjugates, mice were given a single subcutaneous (sc) injection of 15 mg/kg of oligonucleotide-lipid conjugates in PBS. The RNAi oligonucleotide-lipid conjugate systems administered to each of the animal groups are summarized in Table 11. Group A were control mice that received PBS only.

Target attenuation was assessed 14 days after injection. RNA was extracted from liver tissue, and murine STAT3, SLC25A1, HMGB1, and ALDH2 mRNA levels (normalized to the endogenous housekeeping gene Ppib) were measured by qPCR. Perform qPCR using the PrimeTime™ qPCR Probe Assay. In samples from treated mice, the percentage of target mRNA remaining was determined using the 2-ΔΔCt ("delta-delta Ct") method (Livak and Schmittgen (2001) METHODS 25:402-408).

As shown in Figures 16A-16B, encoding STAT3 was observed for oligonucleotide-lipid conjugates with either a blunt end at the 5' end of the antisense strand or a double overhang at the 5' and 3' ends of the antisense strand. Similar levels of mRNA were reduced for (Fig. 16A), SLC25A1 (Fig. 16B), HMGB1 (Fig. 16C) and ALDH2 (Fig. 16D). Example 12: Four-member loop passenger truncations of neuronal target genes for CNS inhibition with and without phosphorothioate linkages on exposed guides

Evaluation of RNAi oligonucleotide-lipid conjugates to determine passenger strand truncation in oligonucleotides with four-member loops with and without phosphorothioate linkages on exposed guide strands in neurons Effect. Each oligonucleotide-lipid conjugate tested contained an antisense strand with a region complementary to the mRNA encoding TUBB3.

The oligonucleotide-lipid conjugate systems evaluated included those with the structures of compounds 131 to 136 as shown in Figure 17, which were similar to the parent oligonucleotide-lipid conjugate evaluated in a previous study (compound 1 , as mentioned above) compared to. Each of compounds 131 to 136 contains a sense strand with a four-member loop at the 3' end and a C16 lipid conjugated to the second nucleotide of the four-member loop. These compounds contain different passenger strand lengths and phosphorothioate linkages on each compound, as further described below:

(i) Compound 131 contains a 32-nucleotide sense strand with a 4-nt truncation (i.e., a 6-nt antisense strand overhang at the 3' end) between positions 1 and 2 of the sense strand and The antisense strand contains phosphorothioate linkages between positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22.

(ii) Compound 132 contains a 30-nucleotide sense strand with a 6-nt truncation (i.e., an 8-nt antisense strand overhanging the 3' end) between positions 1 and 2 of the sense strand and The antisense strand contains phosphorothioate linkages between positions 1 and 2, 2 and 3, 3 and 4, 13 and 14, 14 and 15, 20 and 21, and 21 and 22. Additionally, the justice strand contains the LNA at position 1.

(iii) Compound 133 contains a 28-nucleotide sense strand with an 8-nt truncation (i.e., a 10-nt antisense strand overhang at the 3' end) between positions 1 and 2 of the sense strand and The antisense strand contains phosphorothioate linkages between positions 1 and 2, 2 and 3, 3 and 4, 13 and 14, 14 and 15, 20 and 21, and 21 and 22. Additionally, the justice strand contains the LNA in position 1.

(iv) Compound 134 contains a 32-nucleotide sense strand with a 4-nt truncation (i.e., a 6-nt antisense strand overhang at the 3' end) between positions 1 and 2 of the sense strand and The antisense strand contains phosphorothioate linkages between positions 1 and 2, 2 and 3, 3 and 4, 16 and 17, 17 and 18, 18 and 19, 19 and 20, 20 and 21, and 21 and 22 .

(v) Compound 135 contains a 30-nucleotide sense strand with a 6-nt truncation (i.e., an 8-nt antisense strand overhang at the 3' end) between positions 1 and 2 of the sense strand and At positions 1 and 2, 2 and 3, 3 and 4, 13 and 14, 14 and 15, 15 and 16, 16 and 17, 17 and 18, 18 and 19, 19 and 20, 20 and 21 and Contains a phosphorothioate linkage between 21 and 22. Additionally, the justice strand contains the LNA in position 1.

(vi) Compound 136 contains a 28-nucleotide sense strand with an 8-nt truncation (i.e., a 10-nt antisense strand overhang at the 3' end) between positions 1 and 2 of the sense strand and At positions 1 and 2, 2 and 3, 3 and 4, 12 and 13, 13 and 14, 14 and 15, 15 and 16, 16 and 17, 17 and 18, 18 and 19, 19 and 20, Phosphorothioate linkages are included between 20 and 21 and 21 and 22. Additionally, the justice strand contains the LNA in position 1.

To evaluate these RNAi oligonucleotide-lipid conjugates, 6- to 10-week-old C57BL6 female mice were given 500 µg of oligonucleotide-lipid conjugates formulated in artificial cerebrospinal fluid (aCSF). Single intrathecal (it) injection. The RNAi oligonucleotide-lipid conjugate systems administered to each animal group are summarized in Table 12. Group A was control mice that received only artificial cerebrospinal fluid (aCSF).

Target attenuation was assessed 28 days after injection. RNA was extracted from various tissues and murine TUBB3 mRNA levels were determined as described in Example 2.

The percentage of TUBB3 mRNA expression measured in the frontal cortex, hippocampus, cerebellum, lumbar root ganglia, medulla oblongata, and lumbar spinal cord for each treatment group are shown in Figures 18A to 18F, respectively. Silencing of TUBB3 mRNA expression was observed in the cerebellum, lumbar DRG, medulla oblongata, and lumbar spinal cord. In particular, compound 136 (containing a P-8 truncation with a phosphorothioate linkage on the exposed antisense strand, i.e., a 10-nt antisense overhang) demonstrated increased inhibition of lumbar DRG, and in addition to There was limited inhibition outside the medulla, indicating a platform for attenuation of target genes for inhibition of the lumbar DRG. Example 13: 5' truncation of neuronal target genes for CNS inhibition and locked nucleic acid on the sense strand

Evaluation of RNAi oligonucleotide-lipid conjugates to determine the effects of blunt-ended passenger strand truncation versus locked nucleic acids on the guide strand in neurons. Each oligonucleotide-lipid conjugate tested contained an antisense strand with a region complementary to the mRNA encoding TUBB3.

The oligonucleotide-lipid conjugate systems evaluated included those with the structures of compounds 138 to 145 as shown in Figure 19, which were similar to the parent oligonucleotide-lipid conjugate evaluated in a previous study (compound 1 , as described above) compared to compound 137. Compound 137 contains a blunt end at the 3' end of the sense strand, a 2-nt overhang at the 3' end of the antisense strand, and a C16 lipid conjugated to the 5' end nucleotide of the sense strand. Each of compounds 138 to 145 contains a blunt end at the 3' end of the sense strand, a C16 lipid conjugated at the 5' end of the sense strand (position 1), and a phosphorothioate linkage at the sense strand strands 1 and 2, 18 and 19, 19 and 20; and antisense strands 1 and 2, 2 and 3, 3 and 4, 20 and 21, 21 and 22. Compounds 141 to 145 contain a truncation at the 5' end of the sense strand, which results in a 6-nt truncation (ie, an 8-nt antisense strand overhangs at the 3' end). Additionally, compounds 141 to 145 comprise two phosphorothioate linkages flanking the nucleotide at position 14 of the antisense strand (from 5' to 3') (i.e., positions 13 and 14 of the antisense strand) 14 and the phosphorothioate linkage between 14 and 15). These compounds contain locked nucleic acids at different positions, as further described below: (i) Compound 141 does not contain locked nucleic acid (LNA). (ii) Compounds 138 and 142 contain LNA at position 2 of the sense strand. (iii) Compound 139 contains LNA at positions 2 and 15. (iv) Compound 140 contains LNAs at positions 2, 15 and 16. (v) Compound 143 contains LNA at positions 2 and 9. (vi) Compound 144 contains LNAs at positions 2, 9 and 10. (vii) Compound 145 contains LNAs at positions 2, 9, 10, 12 and 13.

To evaluate these RNAi oligonucleotide-lipid conjugates, mice were given a single intrathecal (it) injection of 500 µg of oligonucleotide-lipid conjugates formulated in artificial cerebrospinal fluid (aCSF). . The RNAi oligonucleotide-lipid conjugate systems administered to each animal group are summarized in Table 13. Group A was control mice that received only artificial cerebrospinal fluid (aCSF).

Target attenuation was assessed 28 days after injection. RNA was extracted from various tissues and murine TUBB3 mRNA levels were determined as described in Example 2.

The percentage of TUBB3 mRNA expression measured in the frontal cortex, hippocampus, cerebellum, medulla oblongata, lumbar dorsal root ganglia, and lumbar spinal cord for each treatment group are shown in Figures 20A to 20F, respectively. Silencing of TUBB3 mRNA expression was observed in all tissues evaluated. Incorporation of at least one LNA with P-6 sense strand truncation resulted in attenuation of potentiation in most tissues evaluated (e.g., compound 142), while inclusion of 2 LNAs further enhanced attenuation. Example 14: 5' C16 lipid-conjugated blunt-ended oligonucleotide with sense-stranded locked nucleic acid for inhibition of neuronal target genes in the CNS

Evaluation of RNAi oligonucleotide-lipid conjugates to determine the neuronal effects of blunt-ended oligonucleotides and locked nucleic acids on the guide strand. Each oligonucleotide-lipid conjugate tested contained an antisense strand with a region complementary to the mRNA encoding TUBB3.

The oligonucleotide-lipid conjugate systems evaluated included those with the structures of compounds 138 to 140 and 146 to 148 as shown in Figure 21, with the parent oligonucleotide-lipid conjugates evaluated in previous studies. (Compounds 1 and 137, described above). Compounds 138 to 140 and 147 to 148 each contain a blunt end at the 3' end of the sense strand and a C16 lipid conjugated to a nucleotide at the 5' end of the sense strand. Compound 146 contains a blunt end at the 3' end of the sense strand and no lipid conjugate conjugated at the 5' end of the sense strand. Compounds 138 to 140 and 147 to 148 each comprise a phosphorothioate linkage between: sense strands 1 and 2, 18 and 19, and 19 and 20; and antisense strands 1 and 2, 2 and 3, 3 and 4, 20 and 21 and 21 and 22. These compounds contain locked nucleic acids at different positions, as further described below: (i) Compounds 137 and 146 do not contain locked nucleic acids. (ii) Compound 138 contains LNA at position 2 of the sense strand. (iii) Compound 139 contains LNA at positions 2 and 15. (iv) Compound 140 contains LNAs at positions 2, 15 and 16. (v) Compound 145 contains LNAs at positions 2, 15, 16 and 18. (vi) Compound 148 contains LNAs at positions 2, 15, 16, 18 and 19.

To evaluate these RNAi oligonucleotide-lipid conjugates, 6- to 10-week-old C57BL6 mice were given 500 µg of monomer of oligonucleotide-lipid conjugates formulated in artificial cerebrospinal fluid (aCSF). Intrathecal (it) injection. The RNAi oligonucleotide-lipid conjugate systems administered to each animal group are summarized in Table 14. Group A was control mice that received only artificial cerebrospinal fluid (aCSF).

Target attenuation was assessed 28 days after injection. RNA was extracted from various tissues and murine TUBB3 mRNA levels were determined as described in Example 2.

The percentage of TUBB3 mRNA expression measured in the frontal cortex, hippocampus, medulla oblongata, lumbar root ganglia, cerebellum, and lumbar spinal cord for each treatment group are shown in Figures 22A to 22F, respectively. For most treatment groups, silencing of TUBB3 mRNA expression was observed in all tissues evaluated. Conjugation of lipids (i.e., C16) on the 5' end of the sense strand enhances neuronal attenuation (see, eg, Compound 146 compared to Compounds E to I). Additionally, incorporation of at least 1 LNA increased oligonucleotide attenuation efficiency in deeper (e.g., frontal cortex and hippocampal gyrus) tissue compared to compounds without LNA, whereas inclusion of 2 to 4 LNA further enhanced attenuation. active. Example 15: 5' C16 lipid-conjugated P-4 truncated sense strand of locked nucleic acid for inhibition of neuronal target genes in the CNS

Evaluation of RNAi oligonucleotide-lipid conjugates to determine the effects of blunt-ended passenger strand truncation versus locked nucleic acids on the guide strand in neurons. Each oligonucleotide-lipid conjugate tested contained an antisense strand with a region complementary to the mRNA encoding TUBB3.

The oligonucleotide-lipid conjugate systems evaluated included those with the structures of compounds 149 to 154 as shown in Figure 23, which were similar to the parent oligonucleotide-lipid conjugate evaluated in a previous study (Compound 1 and 137, as mentioned above). Compounds 149 to 154 each contain a blunt end at the 3' end of the sense strand, and a truncation at the 5' end of the sense strand, which results in a 4-nt truncation (i.e., an antisense strand at the 3' end 6-nt overhang), and a C16 lipid conjugated at the 5' end of the sense strand. Each of compounds 149 to 154 contains a phosphorothioate linkage between sense strands 1 and 2, 14 and 15, and 15 and 16; and antisense strands 1 and 2, 2 and 3, and 3. and 4, 20 and 21 and 21 and 22. These compounds contain locked nucleic acids at different positions, as further described below: (i) Compounds 1, 137 and 149 do not contain locked nucleic acids. (ii) Compound 150 contains LNA at position 2 of the sense strand. (iii) Compound 151 contains LNA at positions 2 and 11 of the sense strand. (iv) Compound 152 contains LNA at positions 2, 11 and 12 of the sense strand. (v) Compound 153 contains LNA at positions 2, 11, 12 and 14 of the sense strand. (vi) Compound 154 contains LNA at positions 2, 11, 12, 14 and 15 of the sense strand.

To evaluate these RNAi oligonucleotide-lipid conjugates, 6- to 10-week-old C57BL6 female mice were given 500 µg of oligonucleotide-lipid conjugates formulated in artificial cerebrospinal fluid (aCSF). Single intrathecal (it) injection. The RNAi oligonucleotide-lipid conjugate systems administered to each animal group are summarized in Table 15. Group A was control mice that received only artificial cerebrospinal fluid (aCSF).

Target attenuation was assessed 28 days after injection. RNA was extracted from various tissues and murine TUBB3 mRNA levels were determined as described in Example 2.

The percentage of TUBB3 mRNA expression measured in the frontal cortex, hippocampus, medulla oblongata, lumbar root ganglia, cerebellum, and lumbar spinal cord for each treatment group are shown in Figures 24A to 24F, respectively. Compared to LNA-containing compounds, silencing of TUBB3 mRNA expression was observed in deeper (eg, frontal cortex and hippocampal gyrus) tissues. A decrease in the increase was observed with the addition of 2 to 3 LNAs. Example 16: 5' C16 lipid-conjugated P-8 truncated sense strand of locked nucleic acid for inhibition of neuronal target genes in the CNS

Evaluation of RNAi oligonucleotide-lipid conjugates to determine the effects of blunt-ended passenger strand truncation versus locked nucleic acids on the guide strand in neurons. Each oligonucleotide-lipid conjugate tested contained an antisense strand with a region complementary to the mRNA encoding TUBB3.

The oligonucleotide-lipid conjugate systems evaluated included those with the structures of compounds 155 to 160 as shown in Figure 25, which were similar to the parent oligonucleotide-lipid conjugate evaluated in a previous study (Compound 1 and 137, as mentioned above). Compounds 155 to 160 each contain a blunt end at the 3' end of the sense strand, and a truncation at the 5' end of the sense strand, which results in an 8-nt truncation (i.e., an antisense strand at the 3' end 10-nt overhang), and a C16 lipid conjugated at the 5' end of the sense strand. Additionally, compounds 155 to 160 comprise two phosphorothioate linkages flanking the nucleotide at position 14 of the antisense strand (from 5' to 3') (i.e., positions 13 and 14 of the antisense strand) 14 and the phosphorothioate linkage between 14 and 15). Each of compounds 155 to 160 contains a phosphorothioate linkage between: 1 and 2, 10 and 11, and 11 and 12 of the sense strand; and 1 and 2, 2 and 3, 3 of the antisense strand and 4, 13 and 14, 14 and 15, 20 and 21 and 21 and 22. These compounds contain locked nucleic acids at different positions, as further described below: (i) Compounds 1, 137 and 155 do not contain locked nucleic acids. (ii) Compound 156 contains LNA at position 2 of the sense strand. (iii) Compound 157 contains LNA at positions 2 and 7 of the sense strand. (iv) Compound 152 contains LNA at positions 2, 7 and 8 of the sense strand. (v) Compound 153 contains LNA at positions 2, 7, 8 and 10 of the sense strand. (vi) Compound 154 contains LNA at positions 2, 7, 8, 10 and 11 of the sense strand.

To evaluate these RNAi oligonucleotide-lipid conjugates, 6- to 10-week-old C57BL6 female mice were given 500 µg of oligonucleotide-lipid conjugates formulated in artificial cerebrospinal fluid (aCSF). Single intrathecal (it) injection. The RNAi oligonucleotide-lipid conjugate systems administered to each animal group are summarized in Table 16. Group A was control mice that received only artificial cerebrospinal fluid (aCSF).

Target attenuation was assessed 28 days after injection. RNA was extracted from various tissues and murine TUBB3 mRNA levels were determined as described in Example 2.

The percentage of TUBB3 mRNA expression measured in the frontal cortex, hippocampus, medulla oblongata, lumbar root ganglia, cerebellum, and lumbar spinal cord for each treatment group are shown in Figures 26A to 26F, respectively. Among molecules with P-8 truncations, the inclusion of at least one LNA lineage was beneficial for neuronal attenuation (Compound 155 compared to Compounds 156 to 160). In LNA-containing compounds, silencing of TUBB3 mRNA expression was observed in deeper (eg, frontal cortex and hippocampal gyrus) tissues. A decrease in the increase was observed with the addition of 2 to 3 LNAs. Example 17: 5' P-6 truncation of oligodendritic cell target genes for CNS inhibition and locked nucleic acid on the sense strand

Evaluation of RNAi oligonucleotide-lipid conjugates to determine the effect of blunt-ended passenger strand truncation versus locked nucleic acid on the exposed guide strand in oligodendritic cells. Each oligonucleotide-lipid conjugate tested contained an antisense strand with a region complementary to the mRNA encoding UGT8, a protein expressed on oligodendritic cells of the CNS.

Oligonucleotide-lipid conjugate systems evaluated included those having the structures of compounds 163 to 171 as shown in Figure 27, with the parent oligonucleotide-lipid conjugate compound 161 (having conjugation in four Compared to 162 (a blunt-end oligonucleotide with a C16 lipid conjugated to the 5' end nucleotide). Each of compounds 166 to 170 contains a blunt end at the 3' end of the sense strand and a truncation at the 5' end of the sense strand, which results in a 6-nt truncation (i.e., an 8-nt truncation at the 3' end). sense strand overhang), and C16 lipid conjugated at the 5' end of the sense strand. Each of compounds 162 to 165 contains a blunt end at the 3' end of the sense strand and a C16 lipid conjugated at the 5' end of the sense strand. Compound 171 contains a blunt end at the 3' end of the sense strand and does not contain lipid conjugated at the 5' end of the sense strand. Compounds 162 to 165 and 177 contain phosphorothioate linkages between sense strands 1 and 2, 18 and 19 and 19 and 20; and antisense strands 1 and 2, 2 and 3, 3 and 4, 20 and 21 and 21 and 22. Compounds 166 to 170 contain phosphorothioate linkages between sense strands 1 and 2, 18 and 19, and 19 and 20; and antisense strands 1 and 2, 2 and 3, 3 and 4, 13 and 14, 14 and 15, 20 and 21 and 21 and 22. These compounds contain locked nucleic acids at different positions, as further described below: (i) Compounds 161, 162, 166 and 171 do not contain locked nucleic acids. (ii) Compounds 163 and 167 contain LNA at position 2 of the sense strand. (iii) Compound 164 contains LNA at positions 2 and 15. (iv) Compound 165 contains LNAs at positions 2, 15 and 16. (v) Compound 168 contains LNA at positions 2 and 9. (vi) Compound 169 contains LNAs at positions 2, 9 and 10. (vii) Compound 170 contains LNAs at positions 2, 9, 10, 12 and 13.

To evaluate these RNAi oligonucleotide-lipid conjugates, 6- to 10-week-old C57BL6 female mice were given 500 µg of oligonucleotide-lipid conjugates formulated in artificial cerebrospinal fluid (aCSF). Single intrathecal (it) injection. The RNAi oligonucleotide-lipid conjugate systems administered to each animal group are summarized in Table 17. Group A was control mice that received only artificial cerebrospinal fluid (aCSF).

Target attenuation was assessed 28 days after injection. RNA was extracted from CNS tissue and murine UGT8 mRNA levels (normalized to the endogenous housekeeping gene Rp123) were measured by qPCR. In samples from treated mice, the percentage of residual murine mRNA was determined using the 2-ΔΔCt ("delta-delta Ct") method (Livak and Schmittgen (2001) METHODS 25:402-408).

The percentage of UGT8 mRNA expression measured in the frontal cortex, hippocampus, hypothalamus, cerebellum, medulla oblongata, and lumbar spinal cord for each treatment group are shown in Figures 28A to 28F, respectively. For most treatment groups, silencing of UGT8 mRNA expression was observed in all tissues evaluated. Incorporation of P-6 truncated lines reduced activity independently of the number of LNAs present. Lipid conjugation increases attenuated in deep (eg, frontal cortex and hypothalamus) tissues (compare compound 171 to compounds 162 to 165). Example 18: 5' P-6 truncation and locked nucleic acid on the sense strand for inhibition of CNS stellate cell target genes

Evaluation of RNAi oligonucleotide-lipid conjugates to determine the effect of blunt-ended passenger strand truncation versus locked nucleic acid on the exposed guide strand in stellate cells. Each oligonucleotide-lipid conjugate tested contained an antisense strand with a region complementary to the mRNA encoding GFAP, a protein expressed in stellate cells of the central nervous system (CNS).

Oligonucleotide-lipid conjugate systems evaluated included those having the structures of compounds 174 to 182 as shown in Figure 29, with the parent oligonucleotide-lipid conjugate compound 172 (having conjugation in four 173 (a blunt-end oligonucleotide with a C16 lipid conjugated to the 5' end nucleotide). Compounds 177 to 181 each contain a blunt end at the 3' end of the sense strand and a truncation at the 5' end of the sense strand, which results in a 6-nt truncation (i.e., an 8-nt truncation at the 3' end). sense strand overhang), and C16 lipid conjugated at the 5' end of the sense strand. Each of compounds 173 to 176 contains a blunt end at the 3' end of the sense strand and a C16 lipid conjugated at the 5' end of the sense strand. Compound 182 contains a blunt end at the 3' end of the sense strand and does not contain lipid conjugated at the 5' end of the sense strand. Compounds 173 to 176 and 182 contain phosphorothioate linkages between sense strands 1 and 2, 18 and 19 and 19 and 20; and antisense strands 1 and 2, 2 and 3, 3 and 4, 20 and 21 and 21 and 22. Compounds 177 to 181 contain phosphorothioate linkages between sense strands 1 and 2, 18 and 19, and 19 and 20; and antisense strands 1 and 2, 2 and 3, 3 and 4, 13 and 14, 14 and 15, 20 and 21 and 21 and 22. These compounds contain locked nucleic acids at different positions, as further described below: (i) Compounds 172, 173, 177 and 182 do not contain locked nucleic acids. (ii) Compounds 174 and 178 contain LNA at position 2 of the sense strand. (iii) Compound 175 contains LNA at positions 2 and 15. (iv) Compound 176 contains LNAs at positions 2, 15 and 16. (v) Compound 179 contains LNA at positions 2 and 9. (vi) Compound 180 contains LNAs at positions 2, 9 and 10. (vii) Compound 181 contains LNAs at positions 2, 9, 10, 12 and 13.

To evaluate these RNAi oligonucleotide-lipid conjugates, 6- to 10-week-old C57BL6 mice were given 500 µg of monomer of oligonucleotide-lipid conjugates formulated in artificial cerebrospinal fluid (aCSF). Intrathecal (it) injection. The RNAi oligonucleotide-lipid conjugate systems administered to each animal group are summarized in Table 18. Group A was control mice that received only artificial cerebrospinal fluid (aCSF).

Target attenuation was assessed 28 days after injection. RNA was extracted from CNS tissue and murine GFAP mRNA levels (normalized to the endogenous housekeeping gene Rp123) were measured by qPCR. In samples from treated mice, the percentage of residual murine mRNA was determined using the 2-ΔΔCt ("delta-delta Ct") method (Livak and Schmittgen (2001) METHODS 25:402-408).

The percentages of GFAP mRNA expression measured in the frontal cortex, hippocampus, cerebellum, medulla oblongata, lumbar root ganglia, hypothalamus, and lumbar spinal cord for each treatment group are shown in Figures 30A to 30F, respectively. For most treatment groups, silencing of GFAP mRNA expression was observed in all tissues evaluated. Regardless of the presence or absence of LNA, oligonucleotides containing the P-6 truncation showed equivalent attenuation to the full-length oligonucleotide.

Overall, the data of Examples 13, 16 and 17 demonstrate oligonucleotide chemistry for cell type specific targeting. For example, full-length blunt constructs with lipid conjugates can be used with enhanced targeting in stellate cells and oligodendritic cells, and some efficacy in neurons. If one is interested in targeting neurons and/or stellate cells rather than oligodendritic cells, a P-6 truncated oligonucleotide containing a sense strand containing at least one LNA can be used. Additionally, the use of full-length blunt-ended oligonucleotides allows targeting of oligodendritic cells and stellate cells, but potentially also neurons. To target stellate cells over oligodendritic cells, a P-6 sense strand truncated oligonucleotide can be used. Additionally, if interested in targeting oligodendritic cells and stellate cells but excluding neurons, oligonucleotides containing C16 lipids on the four-member ring can be used. Together, these examples describe specific oligonucleotide structures for targeting or excluding specific cell types of the CNS. Example 19: Positioning of truncated oligonucleotides on the sense strand for inhibition of CNS neuronal target genes Lipid walking

Evaluation of RNAi oligonucleotide-lipid conjugates to determine the effect of lipid position on truncated blunt-ended oligonucleotides. Each oligonucleotide-lipid conjugate tested contained an antisense strand with a region complementary to the mRNA encoding TUBB3.

Oligonucleotide-lipid conjugate systems evaluated included those having the structures of compounds 183 to 197 as shown in Figures 31A to 31D, compounds 137, 149, and 141 with the parent oligonucleotide-lipid conjugates. (blunt-ended oligonucleotides containing 2, 6, and 8 nucleotide antisense overhangs, respectively, as described above). Each of compounds 183 to 190 contains a blunt end at the 3' end of the sense strand and a 2-nt antisense strand overhang at the 3' end. Each of compounds 191 to 194 contains a blunt end at the 3' end of the sense strand and a 4-nt truncation at the 5' end of the sense strand (ie, a 6-nt antisense strand overhang at the 3' end). Compounds 195 and 196 contain a blunt end at the 3' end of the sense strand and a 6-nt truncation at the 5' end of the sense strand (ie, an 8-nt antisense strand overhang at the 3' end). Compound 197 contains a blunt end at the 3' end of the sense strand and an 8-nt truncation at the 5' end of the sense strand (i.e., a 10-nt antisense strand overhang at the 3' end).

Compounds 183 to 190 contain phosphorothioate linkages between sense strands 1 and 2, 18 and 19, and 19 and 20; and antisense strands 1 and 2, 2 and 3, 3 and 4, 20 and 21 and 21 and 22. Compounds 191 to 194 contain phosphorothioate linkages between sense strands 1 and 2, 14 and 15, and 15 and 16; and antisense strands 1 and 2, 2 and 3, 3 and 4, 20 and 21 and 21 and 22. Compounds 195 and 196 contain phosphorothioate linkages between sense strands 1 and 2, 12 and 13, and 13 and 14; and antisense strands 1 and 2, 2 and 3, 3 and 4, 13 and 14, 14 and 15, 20 and 21 and 21 and 22. Compound 197 contains phosphorothioate linkages between sense strands 1 and 2, 10 and 11, and 11 and 12; and antisense strands 1 and 2, 2 and 3, 3 and 4, 13, and 14, 14 and 15, 20 and 21 and 21 and 22.

These compounds comprise C16 lipids conjugated at different positions on the sense strand, as further described below: (i) Compounds 137, 149, 141 and 197 contain a C16 lipid at position 1. (ii) Compounds 183, 191 and 195 contain a C16 lipid at position 2. (iii) Compounds 184, 192 and 196 contain a C16 lipid at position 3. (iv) Compounds 185 and 193 contain a C16 lipid at position 4. (v) Compounds 186 and 194 contain a C16 lipid at position 5. (vi) Compound 187 contains a C16 lipid at position 6. (vii) Compound 188 contains a C16 lipid at position 7. (viii) Compound 189 contains a C16 lipid at position 8. (ix) Compound 190 contains a C16 lipid at position 9.

To evaluate these RNAi oligonucleotide-lipid conjugates, 6- to 10-week-old C57BL6 mice were given 500 µg of monomer of oligonucleotide-lipid conjugates formulated in artificial cerebrospinal fluid (aCSF). Intrathecal (it) injection. The RNAi oligonucleotide-lipid conjugate systems administered to each animal group are summarized in Table 19. Group A was control mice that received only artificial cerebrospinal fluid (aCSF).

Target attenuation was assessed 28 days after injection. RNA was extracted from CNS tissue and murine TUBB3 mRNA levels were determined as described in Example 2.

The percentage of TUBB3 mRNA expression measured in the frontal cortex, hippocampus, medulla oblongata, lumbar root ganglia, cerebellum, and lumbar spinal cord for each treatment group are shown in Figures 32A to 32F, respectively. Silencing of TUBB3 mRNA expression was observed in all tissues evaluated for several treatment groups. All structures containing the 5' lipid (position 1) enhanced neuronal degeneration in various tissues of the CNS. Example 20: Truncated sense strand oligonucleotides with 5' and 3' overhangs for inhibition of CNS stellate cell target genes

RNAi oligonucleotide-lipid conjugates were evaluated to determine the effect of 5' and/or 3' truncated lengths on the sense strand of the oligonucleotide. Each oligonucleotide-lipid conjugate tested contained an antisense strand with a region complementary to the mRNA encoding GFAP.

Oligonucleotide-lipid conjugate systems evaluated included those having the structures of compounds 180, 201 to 205, and 207 to 215 as shown in Figures 33A to 33C, compared with the parent oligonucleotide-lipid conjugate. Comparison of compounds 173, 176 and 200. Each of compounds 173, 176, and 200 contains a blunt end at the 3' end of the sense strand and a 2-nt overhang at the 3' end of the antisense strand. Compounds 201 to 205 contained a sense strand of 16 nucleotides in length. Compounds 180 and 207 to 210 contained a sense strand of 14 nucleotides in length. Compounds 211 to 215 contained a sense strand of 12 nucleotides in length.

Compounds 176, 180, 200 to 205 and 207 to 215 comprise locked nucleic acids on the sense strand at nucleotides complementary to nucleotides 5 and 6 of the antisense strand. Compounds 176, 180, 200 to 205 and 207 to 215 contain locked nucleic acids at position 2 of the sense strand.

Compounds 173, 176, and 200 contain phosphorothioate linkages between sense strands 1 and 2, 18 and 19, and 19 and 20; and antisense strands 1 and 2, 2 and 3, 3, and 4, 20 and 21 and 21 and 22. Compound 200 contains additional phosphorothioate linkages between positions 13 and 14 and 14 and 15 of the antisense strand. Compounds 201 to 205 contain phosphorothioate linkages between sense strands 1 and 2, 14 and 15, and 15 and 16; and antisense strands 1 and 2, 2 and 3, 3 and 4, 20 and 21 and 21 and 22. Compounds 180 and 207 to 210 contain phosphorothioate linkages between sense strands 1 and 2, 12 and 13, and 13 and 14; and antisense strands 1 and 2, 2 and 3, 3, and 4, 13 and 14, 14 and 15, 20 and 21 and 21 and 22. Compounds 211 to 215 contain phosphorothioate linkages between sense strands 1 and 2, 10 and 11, and 11 and 12; and antisense strands 1 and 2, 2 and 3, 3 and 4, 13 and 14, 14 and 15, 20 and 21 and 21 and 22.

Compounds 173, 176, 180, 200 to 205 and 207 to 215 comprise a C16 lipid conjugated at position 1 of the sense strand.

These compounds contain varying lengths of 5' and 3' overhangs of the antisense strand, as further described below: (i) Compounds 173, 176 and 200 contain a 2-nt antisense strand overhang at the 3' end. (ii) Compound 201 contains a 6-nt antisense strand overhang at the 3' end. (iii) Compound 202 contains a 5-nt antisense strand overhang at the 3' end and a 1-nt antisense strand overhang at the 5' end. (iv) Compound 203 contains a 4-nt antisense strand overhang at the 3' end and a 2-nt antisense strand overhang at the 5' end. (v) Compound 204 contains a 3-nt antisense strand overhang at the 3' end and a 3-nt antisense strand overhang at the 5' end. (vi) Compound 205 contains a 2-nt antisense strand overhang at the 3' end and a 4-nt antisense strand overhang at the 5' end. (vii) Compound 180 contains an 8-nt antisense strand overhang at the 3' end. (viii) Compound 207 contains a 7-nt antisense strand overhang at the 3' end and a 1-nt antisense strand overhang at the 5' end. (ix) Compound 208 contains a 6-nt antisense strand overhang at the 3' end and a 2-nt antisense strand overhang at the 5' end. (x) Compound 209 contains a 5-nt antisense strand overhang at the 3' end and a 3-nt antisense strand overhang at the 5' end. (xi) Compound 210 contains a 4-nt antisense strand overhang at the 3' end and a 4-nt antisense strand overhang at the 5' end. (xii) Compound 211 contains a 10-nt antisense strand overhang. (xiii) Compound 212 contains a 9-nt antisense strand overhang at the 3' end and a 1-nt antisense strand overhang at the 5' end. (xiv) Compound 213 contains an 8-nt antisense strand overhang at the 3' end and a 2-nt antisense strand overhang at the 5' end. (xv) Compound 214 contains a 7-nt antisense strand overhang at the 3' end and a 3-nt antisense strand overhang at the 5' end. (xvi) Compound 215 contains a 6-nt antisense strand overhang at the 3' end and a 4-nt antisense strand overhang at the 5' end.

To evaluate these RNAi oligonucleotide-lipid conjugates, 6- to 10-week-old C57BL6 mice were given 100 µg of monomer of oligonucleotide-lipid conjugates formulated in artificial cerebrospinal fluid (aCSF). Intrathecal (it) injection. The RNAi oligonucleotide-lipid conjugate systems administered to each animal group are summarized in Table 20. Control mice received only artificial cerebrospinal fluid (aCSF).

Target attenuation was assessed 28 days after injection. RNA was extracted from CNS tissue and murine GFAP mRNA levels were determined as described in Example 17.

The percentage of GFAP mRNA expression measured in the frontal cortex, hippocampus, medulla oblongata, and lumbar spinal cord for each treatment group is shown in Figures 34A to 34D, respectively. For most treatment groups, silencing of GFAP mRNA expression was observed in all tissues evaluated. Silencing of GFAP mRNA expression was observed in the lumbar spinal cord and medulla oblongata after treatment with all RNAi oligonucleotide-lipid conjugates tested. These results demonstrate the ability of RNAi oligonucleotide-lipid conjugates with truncated sense strands to inhibit stellate cell gene targets in various regions and tissues of the CNS. Example 21: Truncated sense strand oligonucleotides with 5' and 3' overhangs for inhibition of neuronal target genes in the CNS

RNAi oligonucleotide-lipid conjugates were evaluated to determine the effect of 5' and/or 3' truncated lengths on the sense strand of the oligonucleotide. Each oligonucleotide-lipid conjugate tested contained an antisense strand with a region complementary to the mRNA encoding TUBB3.

Oligonucleotide-lipid conjugate systems evaluated included those with the structures of compounds 144, 218 to 222, 224 to 232, and 279 to 285 as shown in Figures 35A to 35E, with the parent oligonucleotide-lipid Conjugate compounds 137, 140, 217 and 277 to 278 were compared. Each of compounds 137, 140, 217 and 277 to 278 contains a blunt end at the 3' end of the sense strand and a 2-nt antisense strand overhang at the 3' end. Compounds 218 to 222 and 277 to 278 contained a sense strand of 16 nucleotides in length. Compounds 140 and 224 to 227 contained a sense strand of 14 nucleotides in length. Compounds 228 to 232 contained a sense strand of 12 nucleotides in length. Compounds 279 to 285 contained a sense strand of 10 nucleotides in length.

Compounds 140, 144, 217 to 222, 224 to 232, and 279 to 283 comprise locked nucleic acids on the sense strand at nucleotides complementary to nucleotides 5 and 6 of the antisense strand. Compounds 277 and 284 comprise locked nucleic acids on the sense strand at nucleotides complementary to nucleotides 6 and 7 of the antisense strand. Compounds 278 and 285 comprise locked nucleic acids on the sense strand at nucleotides complementary to nucleotides 7 and 8 of the antisense strand. Compounds 140, 144, 217 to 222, 224 to 232, and 277 to 285 contain locked nucleic acids at position 2 of the sense strand.

Compounds 137, 140, 217, 277, and 278 contain phosphorothioate linkages between: sense strands 1 and 2, 18 and 19, and 19 and 20; and antisense strands 1 and 2, 2, and 3, 3 and 4, 20 and 21 and 21 and 22. Compounds 217 and 277 to 278 contain additional phosphorothioate linkages between positions 13 and 14 and 14 and 15 of the antisense strand. Compounds 218 to 222 contain phosphorothioate linkages between sense strands 1 and 2, 14 and 15, and 15 and 16; and antisense strands 1 and 2, 2 and 3, 3 and 4, 20 and 21 and 21 and 22. Compounds 144 and 224 to 227 contain phosphorothioate linkages between sense strands 1 and 2, 12 and 13, and 13 and 14; and antisense strands 1 and 2, 2 and 3, 3, and 4, 13 and 14, 14 and 15, 20 and 21 and 21 and 22. Compounds 228 to 232 contain phosphorothioate linkages between sense strands 1 and 2, 10 and 11, and 11 and 12; and antisense strands 1 and 2, 2 and 3, 3 and 4, 13 and 14, 14 and 15, 20 and 21 and 21 and 22. Compounds 279 to 285 contain phosphorothioate linkages between sense strands 1 and 2, 8 and 9, and 9 and 10; and antisense strands 1 and 2, 2 and 3, 3 and 4, 13 and 14, 14 and 15, 20 and 21 and 21 and 22.

Compounds 137, 140, 144, 217 to 222, 224 to 232, and 278 to 285 comprised a C16 lipid conjugated at position 1 of the sense strand.

These compounds contain varying lengths of 5' and 3' overhangs of the antisense strand, as further described below: (i) Compounds 137, 140, 217, 277 and 278 contain a 2-nt antisense strand overhang at the 3' end. (ii) Compound 218 contains a 6-nt antisense strand overhang at the 3' end. (iii) Compound 219 contains a 5-nt antisense strand overhang at the 3' end and a 1-nt antisense strand overhang at the 5' end. (iv) Compound 220 contains a 4-nt antisense strand overhang at the 3' end and a 2-nt antisense strand overhang at the 5' end. (v) Compound 221 contains a 3-nt antisense strand overhang at the 3' end and a 3-nt antisense strand overhang at the 5' end. (vi) Compound 222 contains a 2-nt antisense strand overhang at the 3' end and a 4-nt antisense strand overhang at the 5' end. (vii) Compound 144 contains an 8-nt antisense strand overhang at the 3' end. (viii) Compound 224 contains a 7-nt antisense strand overhang at the 3' end and a 1-nt antisense strand overhang at the 5' end. (ix) Compound 225 contains a 6-nt antisense strand overhang at the 3' end and a 2-nt antisense strand overhang at the 5' end. (x) Compound 226 contains a 5-nt antisense strand overhang at the 3' end and a 3-nt antisense strand overhang at the 5' end. (xi) Compound 227 contains a 4-nt antisense strand overhang at the 3' end and a 4-nt antisense strand overhang at the 5' end. (xii) Compound 228 contains a 10-nt antisense strand overhang. (xiii) Compound 229 contains a 9-nt antisense strand overhang at the 3' end and a 1-nt antisense strand overhang at the 5' end. (xiv) Compound 230 contains an 8-nt antisense strand overhang at the 3' end and a 2-nt antisense strand overhang at the 5' end. (xv) Compound 231 contains a 7-nt antisense strand overhang at the 3' end and a 3-nt antisense strand overhang at the 5' end. (xvi) Compound 232 contains a 6-nt antisense strand overhang at the 3' end and a 4-nt antisense strand overhang at the 5' end. (xvii) Compound 279 contains a 12-nt antisense strand overhang at the 3' end. (xviii) Compound 280 contains an 11-nt antisense strand overhang at the 3' end and a 1-nt antisense strand overhang at the 5' end. (xix) Compound 281 contains a 10-nt antisense strand overhang at the 3' end and a 2-nt antisense strand overhang at the 5' end. (xx) Compound 282 contains a 9-nt antisense strand overhang at the 3' end and a 3-nt antisense strand overhang at the 5' end. (xxi) Compound 283 contains an 8-nt antisense strand overhang at the 3' end and a 4-nt antisense strand overhang at the 5' end. (xxii) Compound 284 contains a 7-nt antisense strand overhang at the 3' end and a 5-nt antisense strand overhang at the 5' end. (xxiii) Compound 285 contains a 6-nt antisense strand overhang at the 3' end and a 6-nt antisense strand overhang at the 5' end.

To evaluate these RNAi oligonucleotide-lipid conjugates, 6- to 10-week-old C57BL6 mice were given 500 µg of monomer of oligonucleotide-lipid conjugates formulated in artificial cerebrospinal fluid (aCSF). Intrathecal (it) injection. The RNAi oligonucleotide-lipid conjugate systems administered to each animal group are summarized in Table 21. Control mice received only artificial cerebrospinal fluid (aCSF).

Target mRNA attenuation was assessed 28 days after injection. RNA was extracted from CNS tissue and murine TUBB3 mRNA levels were determined as described in Example 2.

The percentage expression of TUBB3 mRNA in the frontal cortex, hippocampus, medulla oblongata, and lumbar spinal cord remnants after treatment with compounds 173, 176, and 217 to 232 are shown in Figures 36A to 36D, respectively. The percentage expression of TUBB3 mRNA in the frontal cortex, hippocampus, hypothalamus, cerebellum, brainstem and lumbar spinal cord remnant after treatment with compounds 140, 277, 278, 231 and 279 to 285, respectively, is shown in Figure 36E. For most treatment groups, silencing of TUBB3 mRNA expression was observed in all tissues evaluated. Silencing of TUBB3 mRNA expression was observed in the lumbar spinal cord after treatment with all RNAi oligonucleotide-lipid conjugates tested. Further inhibition was observed in the medulla oblongata, hippocampus, and frontal cortex after treatment with all tested RNAi oligonucleotide-lipid conjugates containing a sense strand of 12 nucleotides in length. Following treatment with Compounds 228 to 232, Compound 281 or Compound 283, silencing of TUBB3 mRNA expression was observed in all CNS regions evaluated. Together, these results demonstrate the ability of RNAi oligonucleotide-lipid conjugates with truncated sense strands to inhibit neuronal gene targets in various regions and tissues of the CNS. Example 22: Effect of Lipid Position on Truncated Sense Oligonucleotides with 5' and 3' Overhangs for Inhibition of CNS Star Cell Target Genes

RNAi oligonucleotide-lipid conjugates were evaluated to determine the effect of lipid position on oligonucleotides with 5' and/or 3' truncations on the sense strand. Each oligonucleotide-lipid conjugate tested contained an antisense strand with a region complementary to the mRNA encoding GFAP.

Oligonucleotide-lipid conjugate systems evaluated included those having the structures of compounds 234 to 245 as shown in Figures 37A to 37B, compounds 173, 176, and 233 with the parent oligonucleotide-lipid conjugates compared to. Compounds 173, 176, 233 to 236, and 240 to 242 each contain a blunt end at the 3' end of the sense strand. Compounds 173, 176 and 233 contain a 2-nt overhang on the 3' end of the antisense strand. Compounds 234 to 245 contain a 6-nt overhang on the 3' end of the antisense strand. Compounds 234 to 236 and 240 to 242 contained a sense strand of 16 nucleotides in length. Compounds 237 to 239 and 243 to 245 contained a sense strand of 14 nucleotides in length.

Compound 176 contains locked nucleic acids at nucleotide positions 2, 15 and 16 on the sense strand. Compound 233 contains locked nucleic acids at nucleotide positions 6, 15 and 16 on the sense strand. Compounds 240 to 245 comprise locked nucleic acids at nucleotide positions 2, 11 and 12 on the sense strand.

Compounds 173, 176, and 233 contain phosphorothioate linkages between: sense strands 1 and 2, 18 and 19, and 19 and 20; and antisense strands 1 and 2, 2 and 3, 3, and 4, 20 and 21 and 21 and 22. Compounds 234 to 236 and 240 to 242 contain phosphorothioate linkages between sense strands 1 and 2, 14 and 15, and 15 and 16; and antisense strands 1 and 2, 2 and 3, 3 and 4, 20 and 21 and 21 and 22. Compounds 237 to 239 and 243 to 245 contain phosphorothioate linkages between sense strands 1 and 2, 12 and 13, and 13 and 14; and antisense strands 1 and 2, 2 and 3, 3 and 4, 13 and 14, 14 and 15, 20 and 21 and 21 and 22. These compounds comprise C16 lipids conjugated at different positions on the sense strand, as further described below: (i) Compounds 173, 176, 233, 234, 237, 240 and 243 comprise a C16 lipid at position 1. (ii) Compounds 235, 238, 241 and 244 contain a C16 lipid at position 3. (iii) Compounds 236, 239, 242 and 245 contain a C16 lipid at position 5. To evaluate these RNAi oligonucleotide-lipid conjugates, 6- to 10-week-old C57BL6 mice were given 100 µg of monomer of oligonucleotide-lipid conjugates formulated in artificial cerebrospinal fluid (aCSF). Intrathecal (it) injection. The RNAi oligonucleotide-lipid conjugate systems administered to each animal group are summarized in Table 22. Control mice received only artificial cerebrospinal fluid (aCSF). Target attenuation was assessed 28 days after injection. RNA was extracted from CNS tissue and murine GFAP mRNA levels were determined as described in Example 17. The percentage of GFAP mRNA expression measured in the frontal cortex, hippocampus, medulla oblongata, and lumbar spinal cord for each treatment group are shown in Figures 38A to 38D, respectively. Silencing of GFAP mRNA expression was observed in the medulla oblongata and lumbar spinal cord after treatment with all RNAi oligonucleotide-lipid conjugates tested. Inclusion of locked nucleic acids in oligonucleotides containing 5' and 3' truncations improves attenuation of stellate cell targets. Example 23: Effect of Lipid Position on Truncated Sense Strands of Locked Nucleic Acids for Inhibition of CNS Star Cell Target Genes

RNAi oligonucleotide-lipid conjugates were evaluated to determine the effect of lipid position on oligonucleotides with locked nucleic acids and 5' and/or 3' truncations on the sense strand. Each oligonucleotide-lipid conjugate tested contained an antisense strand with a region complementary to the mRNA encoding GFAP.

Oligonucleotide-lipid conjugate systems evaluated included those having the structures of compounds 248 to 255 as shown in Figures 39A to 39B, compounds 173, 176, and 200 with the parent oligonucleotide-lipid conjugates Compared to 246 to 247. Compounds 173, 176 and 200 and each of 246 to 251 contain a blunt end at the 3' end of the sense strand. Compounds 252 to 255 contain a 2-nt overhang on the 5' end of the antisense strand. Compounds 248 to 255 contain an 8-nt overhang on the 3' end of the antisense strand. Compounds 173, 176, 200 and 246 to 247 contained a sense strand of 14 nucleotides in length. Compounds 248 to 250 contained a sense strand of 14 nucleotides in length. Compounds 253 to 255 contained a sense strand of 12 nucleotides in length.

Compounds 176, 200 comprise locked nucleic acids at nucleotide positions 2, 15 and 16 on the sense strand. Compound 247 contains locked nucleic acids at nucleotide positions 8, 15 and 16 on the sense strand. Compounds 250, 251, 254, 255 contain locked nucleic acids at nucleotide positions 2, 9 and 10 on the sense strand.

Compounds 173, 176, 200, 246, and 233 contain phosphorothioate linkages between sense strands 1 and 2, 18 and 19, and 19 and 20. Compounds 248, 250 to 252, and 254 to 255 contained phosphorothioate linkages between sense strands 1 and 2, 12 and 13, and 13 and 14. Compounds 249 and 253 contain phosphorothioate linkages between positions 12 and 13 and 13 and 14. Compounds 173, 176, 200, and 246 to 255 comprise phosphorothioates between positions 1 and 2, 2 and 3, 3 and 4, 13 and 14, 14 and 15, 20 and 21, and 21 and 22 of the antisense strand Ester linkage.

These compounds comprise C16 lipids conjugated at different positions on the sense strand, as further described below: (i) Compounds 173, 176, 200, 246 to 248, 250, 252 and 254 comprise a C16 lipid at position 1. (ii) Compounds 249, 251, 253 and 255 contain a C16 lipid at position 3.

To evaluate these RNAi oligonucleotide-lipid conjugates, 6- to 10-week-old C57BL6 mice were given 100 µg of monomer of oligonucleotide-lipid conjugates formulated in artificial cerebrospinal fluid (aCSF). Intrathecal (it) injection. The RNAi oligonucleotide-lipid conjugate systems administered to each animal group are summarized in Table 23. Control mice received only artificial cerebrospinal fluid (aCSF).

Target attenuation was assessed 28 days after injection. RNA was extracted from CNS tissue and murine GFAP mRNA levels were determined as described in Example 17.

The percentage of GFAP mRNA expression measured in the frontal cortex, hippocampus, medulla oblongata, and lumbar spinal cord for each treatment group is shown in Figures 40A to 40D, respectively. Silencing of GFAP mRNA expression was observed in the medulla oblongata and lumbar spinal cord after treatment with all RNAi oligonucleotide-lipid conjugates tested. These results demonstrate the ability of RNAi oligonucleotide-lipid conjugates with truncated sense strands to inhibit stellate cell gene targets in various regions and tissues of the CNS. Example 24: Effect of Lipid Position on Truncated Sense Oligonucleotides with 5' and 3' Overhangs for CNS Inhibition of Neuronal Target Genes

RNAi oligonucleotide-lipid conjugates were evaluated to determine the effect of lipid position on oligonucleotides with 5' and/or 3' truncations on the sense strand. Each oligonucleotide-lipid conjugate tested contained an antisense strand with a region complementary to the mRNA encoding TUBB3.

Oligonucleotide-lipid conjugate systems evaluated included those having the structures of compounds 257 to 268 as shown in Figures 41A to 41B, compounds 137, 140 and 256 with the parent oligonucleotide-lipid conjugates compared to. Each of compounds 137, 140, 152, 256 to 259, and 264 to 265 contains a blunt end at the 3' end of the sense strand. Compounds 137, 140 and 256 contain a 2-nt overhang on the 3' end of the antisense strand. Compounds 257 to 268 contain a 6-nt overhang on the 3' end of the antisense strand. Compounds 152, 257 to 259 and 264 to 265 contained a sense strand of 16 nucleotides in length. Compounds 260 to 262 and 266 to 268 contain a sense strand of 14 nucleotides in length.

Compound 140 contains locked nucleic acids at nucleotide positions 2, 15 and 16 on the sense strand. Compound 256 contains locked nucleic acids at nucleotide positions 6, 15 and 16 on the sense strand. Compounds 152 and 264 to 268 contain locked nucleic acids at nucleotide positions 2, 11 and 12 on the sense strand.

Compounds 137, 140, and 256 contain phosphorothioate linkages between sense strands 1 and 2, 18 and 19, and 19 and 20; and antisense strands 1 and 2, 2 and 3, 3, and 4, 20 and 21 and 21 and 22. Compounds 152, 257 to 259, and 264 to 265 contain phosphorothioate linkages between: sense strands 1 and 2, 14 and 15, and 15 and 16; and antisense strands 1 and 2, 2, and 3, 3 and 4, 20 and 21 and 21 and 22. Compounds 260 to 262 and 266 to 268 contain phosphorothioate linkages between sense strands 1 and 2, 12 and 13, and 13 and 14; and antisense strands 1 and 2, 2 and 3, 3 and 4, 13 and 14, 14 and 15, 20 and 21 and 21 and 22.

These compounds comprise C16 lipids conjugated at different positions on the sense strand, as further described below: (i) Compounds 137, 140, 256, 257, 260, 152 and 266 contain a C16 lipid at position 1. (ii) Compounds 258, 261, 264 and 267 contain a C16 lipid at position 3. (iii) Compounds 259, 262, 265 and 268 contain a C16 lipid at position 5.

To evaluate these RNAi oligonucleotide-lipid conjugates, 6- to 10-week-old C57BL6 mice were given 500 µg of monomer of oligonucleotide-lipid conjugates formulated in artificial cerebrospinal fluid (aCSF). Intrathecal (it) injection. The RNAi oligonucleotide-lipid conjugate systems administered to each animal group are summarized in Table 24. Control mice received only artificial cerebrospinal fluid (aCSF).

Target attenuation was assessed 28 days after injection. RNA was extracted from CNS tissue and murine TUBB3 mRNA levels were determined as described in Example 2.

The percentage of TUBB3 mRNA expression measured in the frontal cortex, hippocampus, medulla oblongata, and lumbar spinal cord for each treatment group are shown in Figures 42A to 42D, respectively. Silencing of TUBB3 mRNA expression was observed in the lumbar spinal cord and medulla oblongata after treatment with all RNAi oligonucleotide-lipid conjugates tested. After treatment with all tested RNAi oligonucleotide-lipid conjugates containing sense strands with 5' and 3' truncations and LNA (e.g., compounds 266 to 268), further studies in the hippocampus and frontal cortex Inhibition was observed. Example 25: Effect of Lipid Position on Truncated Sense Strands of Locked Nucleic Acids for Inhibition of Neuronal Target Genes in the CNS

RNAi oligonucleotide-lipid conjugates were evaluated to determine the effect of lipid position on oligonucleotides with or without locked nucleic acids and 5' and/or 3' truncations on the sense strand. Each oligonucleotide-lipid conjugate tested contained an antisense strand with a region complementary to the mRNA encoding TUBB3.

Oligonucleotide-lipid conjugate systems evaluated included those having the structures of compounds 144 and 272 to 276 as shown in Figures 43A to 43B, and compounds 137, 140 of the parent oligonucleotide-lipid conjugates. , 217 and 269 compared to 270. Compounds 137, 140, 217 and each of 269 to 272 contain a blunt end at the 3' end of the sense strand. Compounds 137, 140, 217, 269 and 270 contain a 2-nt overhang on the 5' end of the antisense strand. Compounds 144 and 272 to 276 contained an 8-nt overhang on the 3' end of the antisense strand. Compounds 144 and 272 contained a sense strand of 14 nucleotides in length. Compounds 273 to 276 contained a sense strand of 12 nucleotides in length.

Compounds 140 and 217 contain locked nucleic acids at nucleotide positions 2, 15 and 16 on the sense strand. Compound 270 contains locked nucleic acids at nucleotide positions 8, 15 and 16 on the sense strand. Compounds 144, 272, 275, 276 contain locked nucleic acids at nucleotide positions 2, 9 and 10 on the sense strand.

Compounds 137, 140, 217, 269, and 270 contain phosphorothioate linkages between sense strands 1 and 2, 18 and 19, and 19 and 20. Compounds 144 and 272 contain phosphorothioate linkages between sense strands 1 and 2, 12 and 13, and 13 and 14. Compounds 273 to 276 contain phosphorothioate linkages between sense strands 1 and 2, 11 and 12, and 12 and 13. Compounds 137 and 140 contain phosphorothioate linkages between positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22 of the antisense strand. Compounds 217 and 269 to 276 contain phosphorothioate linkages between positions 1 and 2, 2 and 3, 3 and 4, 13 and 14, 14 and 15, 20 and 21, and 21 and 22 of the antisense strand.

These compounds comprise C16 lipids conjugated at different positions on the sense strand, as further described below: (i) Compounds 137, 140, 217, 269 to 270, 144, 273 and 275 comprise a C16 lipid at position 1. (ii) Compounds 272, 274 and 276 contain a C16 lipid at position 3.

To evaluate these RNAi oligonucleotide-lipid conjugates, 6- to 10-week-old C57BL6 mice were given 500 µg of monomer of oligonucleotide-lipid conjugates formulated in artificial cerebrospinal fluid (aCSF). Intrathecal (it) injection. The RNAi oligonucleotide-lipid conjugate systems administered to each animal group are summarized in Table 25. Control mice received only artificial cerebrospinal fluid (aCSF).

Target attenuation was assessed 28 days after injection. RNA was extracted from CNS tissue and murine TUBB3 mRNA levels were determined as described in Example 2.

The percentage of TUBB3 mRNA expression measured in the frontal cortex, hippocampus, medulla oblongata, and lumbar spinal cord remnants for each treatment group are shown in Figures 44A to 44D, respectively. For most treatment groups, silencing of TUBB3 mRNA expression was observed in all tissues evaluated. Silencing of TUBB3 mRNA expression was observed in the lumbar spinal cord and medulla oblongata with all RNAi oligonucleotide-lipid conjugates tested. Adding locked nucleic acid increases the inhibition of TUBB3 and decreases it. Furthermore, conjugation of lipids at the 5' end of the sense strand provides enhanced attenuation across tissues. Together, this data demonstrates the ability of RNAi oligonucleotide-lipid conjugates with truncated sense strands and/or LNA to inhibit neuronal targets in various regions and tissues of the CNS. Example 26: Effect of 3' sense strand truncation on inhibiting target genes in tissues outside the central nervous tissue

RNAi oligonucleotide-lipid conjugates were evaluated to determine the effect of 3' truncation on the sense strand. Each oligonucleotide-lipid conjugate tested contained an antisense strand with a region complementary to the mRNA encoding ALDH2.

Oligonucleotide-lipid conjugate systems evaluated included those having the structures of compounds 287 to 292 as shown in Figure 45, compared to the parent oligonucleotide-lipid conjugate compound 286. Each of compounds 286 to 292 contains a 2-nt overhang on the 5' end of the antisense strand. Compound 287 contains a 1-nt overhang on the 3' end of the antisense strand. Compound 288 contains a 2-nt overhang on the 3' end of the antisense strand. Compound 289 contains a 3-nt overhang on the 3' end of the antisense strand. Compounds 290 and 291 contain a 4-nt overhang on the 3' end of the antisense strand.

Compounds 286 and 292 contain phosphorothioate linkages between sense strands 1 and 2, 18 and 19, and 19 and 20. Compound 287 contains phosphorothioate linkages between sense strands 1 and 2, 17 and 18, and 18 and 19. Compound 288 contains phosphorothioate linkages between sense strands 1 and 2, 16 and 17, and 17 and 18. Compound 289 contains phosphorothioate linkages between positions 1 and 2, 15 and 16, and 16 and 17 of the sense strand. Compounds 290 and 291 contain phosphorothioate linkages between positions 1 and 2, 14 and 15, and 15 and 16 of the sense strand. Compounds 286 to 290 contain phosphorothioate linkages between positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22 of the antisense strand. Compounds 291 and 292 contain phosphorothioate linkages between positions 1 and 2, 2 and 3, 3 and 4, 4 and 5, 20 and 21, and 21 and 22 of the antisense strand.

Compounds 286 to 292 comprise a C22 lipid conjugated at position 1 of the sense strand.

To evaluate these RNAi oligonucleotide-lipid conjugates, 6- to 10-week-old C57BL6 mice were dosed with a single dose of 15 mg/kg of oligonucleotide-lipid conjugates in phosphate-buffered saline (PBS). Subcutaneous (sc) injection of yoke. The RNAi oligonucleotide-lipid conjugate systems administered to each animal group are summarized in Table 26. Control mice received PBS only.

Target attenuation was assessed 13 days after injection. RNA was extracted from liver, quadriceps, heart, and gonadal white adipose tissue (gWAT), and murine ALDH2 mRNA levels were determined as described in Example 11.

The percentage of ALDH2 mRNA expression measured in liver, quadriceps, heart, and gWAT residual for each treatment group is shown in Figures 46A to 46D, respectively. Silencing of ALDH2 mRNA expression was observed in all tissues. Specifically, truncation of the 3' end of the sense strand can inhibit gene expression across various tissues. Together, this data demonstrates the ability of truncated molecules to reduce gene expression in tissues outside the CNS. Example 27: Truncated sense strand oligonucleotides with 5' and 3' overhangs for CNS inhibition of oligodendritic cell target genes

RNAi oligonucleotides with 5' and/or 3' sense strand truncations were evaluated for their ability to reduce the expression of oligodendritic cell targets in the CNS. Each oligonucleotide-lipid conjugate tested contained an antisense strand with a region complementary to the mRNA encoding UGT8.

Oligonucleotide-lipid conjugate systems evaluated included those with structures as shown in Figure 47 for compounds 167, 293, and 294, compared to the parent oligonucleotide-lipid conjugate compound 162. Each of compounds 162, 167, 293, and 294 contained a C16 lipid at position 1 of the sense strand. Compounds 162 and 167 each contain a blunt end at the 3' end of the sense strand. Compounds 293 and 294 contain a 3-nt overhang on the 5' end of the antisense strand. Compounds 167 and 294 contain an 8-nt overhang on the 3' end of the antisense strand.

Compound 162 contains phosphorothioate linkages between sense strands 1 and 2, 18 and 19, and 19 and 20. Compound 167 contains phosphorothioate linkages between sense strands 1 and 2, 12 and 13, and 13 and 14. Compound 293 contains phosphorothioate linkages between sense strands 1 and 2, 15 and 16, and 16 and 17. Compound 294 contains phosphorothioate linkages between sense strands 1 and 2, 9 and 10, and 10 and 11. Compounds 162 and 293 contain phosphorothioate linkages between positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22 of the antisense strand. Compounds 167 and 294 contain phosphorothioate linkages between positions 1 and 2, 2 and 3, 3 and 4, 13 and 14, 14 and 15, 20 and 21, and 21 and 22 of the antisense strand.

To evaluate these RNAi oligonucleotide-lipid conjugates, 6- to 10-week-old C57BL6 mice were given 300 µg of monomer of oligonucleotide-lipid conjugates formulated in artificial cerebrospinal fluid (aCSF). Intrathecal (it) injection. The RNAi oligonucleotide-lipid conjugate systems administered to each animal group are summarized in Table 27. Control mice received only artificial cerebrospinal fluid (aCSF).

Target attenuation was assessed 28 days after injection. RNA was extracted from CNS tissue and murine UGT8 mRNA levels were determined as described in Example 2. The percentage of UGT8 mRNA expression measured in the frontal cortex, hippocampus, brainstem, and lumbar spinal cord remnant for each treatment group is shown in Figures 48A to 48D, respectively. Silencing of UGT8 mRNA expression was observed in all tissues. Specifically, truncations of the sense strand greater than 2 nucleotides behave similarly to blunt-ended oligonucleotides with 2-nt truncations (compound 162) throughout the CNS. Together, these data demonstrate the ability of truncated molecules to reduce oligodendritic cell target genes in the CNS.

[Figs. 1A to 1C] Schematic diagrams of RNAi oligonucleotide-lipid conjugates targeting TUBB3 mRNA having the structures of compounds 1 to 14 are provided.

[Fig. 2A to 2F] Provided are the frontal cortex (Fig. 2A), hippocampus ( Graph of measured percentage (%) of residual murine TUBB3 mRNA in the cerebellum (Figure 2C), brainstem (Figure 2D), lumbar root ganglia (DRG) (Figure 2E), and lumbar spinal cord (Figure 2F) .

[Fig. 3] A schematic diagram of an RNAi oligonucleotide-lipid conjugate targeting TUBB3 mRNA having the structure of compounds 15 to 18 is provided.

[Fig. 4A to 4F] Provided are mice in which compounds 7 to 9 were administered via intrathecal injection to control mice (group A) or mice (groups B to D, respectively) or compounds 15 to 18 (groups E to 18, respectively). Group H) frontal cortex (Fig. 4A), hippocampus (Fig. 4B), medulla oblongata (Fig. 4C), cerebellum (Fig. 4D), lumbar root ganglion (DRG) (Fig. 4E) and lumbar spinal cord (Fig. 4F) Graph of measured percentage (%) of residual murine TUBB3 mRNA.

[Fig. 5] A schematic diagram of an RNAi oligonucleotide-lipid conjugate targeting TUBB3 mRNA having the structure of compounds 18 to 28 is provided.

[Fig. 6A to 6B] Provided are the cerebellum (Fig. 6A) and lumbar dorsal root ganglia (DRG) (Fig. 6B) Graph of measured percentage (%) of residual murine TUBB3 mRNA.

[Fig. 7] A schematic diagram of an RNAi oligonucleotide-lipid conjugate targeting TUBB3 mRNA having the structures of compounds 18 and 29 to 38 is provided.

[Fig. 8A to 8B] Provided are the cerebellum (Fig. 8A) and lumbar root ganglia (DRG) of control mice (Group A) or mice administered compound 18 or 29 to 38 (Groups B to L, respectively). (Figure 8B) Graph of measured percentage (%) of residual murine TUBB3 mRNA.

[Fig. 9] A schematic diagram of an RNAi oligonucleotide-lipid conjugate targeting reticulin 4 (RTN4) mRNA having the structure of compounds 39 to 49 is provided.

[Figures 10A to 10B] Provided are retinal (Figure 10A) and optic nerve (Figure 8B) tissues in control rats (Group A) or rats administered Compounds 39 to 49 via intravitreal injection (Groups B to L, respectively) Graph of measured percentage (%) of rat RTN4 mRNA remaining.

[Fig. 10C] A schematic diagram of an Aldh2 mRNA-targeting RNAi oligonucleotide-GalNAc conjugate having the structures of compounds 57 and 98 to 108 is provided.

[Fig. 10D] Provides the measured percentage of residual murine Aldh2 mRNA in liver tissue of control mice administered PBS (Group A) or mice administered Compound 57 or 98 to 108 (Groups B to N, respectively) %) chart.

[Fig. 10E] Schematic diagrams of RNAi oligonucleotide-lipid conjugates targeting PECAM-1 mRNA having the structures of compounds 120 and 121 and targeting CD68 mRNA having the structure of compound 122 are provided.

[Figures 10F to 10G] Provided are residual murine PECAM mRNA ( Graph of measured percentage (%) of CD68 mRNA (Fig. 10F) and CD68 mRNA (Fig. 10G).

[Figures 10H to 10I] Provided are liver tissue residuals measured by qPCR and immunohistochemistry (IHC) in control mice administered PBS or mice administered Compound 122 at the indicated doses via subcutaneous injection, respectively. Graph of measured percentage (%) of CD68-like mRNA (Fig. 10H) and CD68 protein (Fig. 10I).

[Figure 10J] Provides representative images of liver tissue obtained from PBS control mice as described in Figures 10H to 10I, imaged after CD68 immunostaining. Arrows indicate representative cells that appear morphologically as hepatocytes or macrophages. Dark staining indicates CD68 protein expression.

[Figure 10K] Provides representative images of liver tissue obtained from mice as described in Figures 10H to 10I, imaged after CD68 immunostaining. Dark staining indicates CD68 protein expression.

[Figs. 11A to 11B] Schematic diagrams of CD68 mRNA-targeting RNAi oligonucleotide-lipid conjugates having the structures of compounds 58 to 97 are provided.

[Fig. 12] Provides measurements of the percentage (%) of murine CD68 mRNA remaining in liver tissue of control mice administered PBS or mice administered compounds 58 to 97 via subcutaneous injection (groups B to AO, respectively) chart.

[Fig. 13] A schematic diagram of an Aldh2 mRNA-targeting RNAi oligonucleotide-lipid conjugate having the structure of compounds 109 to 117 is provided.

[Fig. 14A to 14D] Provided are liver (Fig. 14A), fat (Fig. 14B), skeletal muscle (Figure 14C) and adrenal gland (Figure 14D) tissue residual measured percentage (%) of murine Aldh2 mRNA.

[Fig. 15] 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) are provided schematic diagram.

[Figures 16A to 16D] provide graphs showing the measured percentage of residual murine STAT3 mRNA in liver tissue of mice administered PBS (Group A) or Compound 123 or 127 (Groups B1 and C1, respectively) ( %) (Figure 16A); Measured percentage (%) of residual murine SLC25A1 mRNA in liver tissue of mice administered PBS (Group A) or Compound 124 or 128 (Groups B2 and C2, respectively) (Figure and Measured percentage (%) of murine ALDH2 mRNA remaining in liver tissue of mice administered PBS (Group A) or Compound 126 or 130 (Groups B4 and C4, respectively) (Figure 16D). RNAi oligonucleotide-lipid conjugates were administered to mice via subcutaneous injection.

[Figure 17] Provides a schematic diagram of RNAi oligonucleotide-lipid conjugates targeting TUBB3, in which the oligonucleotide contains a P-4, P-6 or P-8 truncated sense strand (i.e., antisense strand 6, 8 and 10 nucleotide overhangs) (compounds 131 to 133) or a p-4, p-6 or p-8 truncated sense strand containing phosphorothioate linkages (compounds 134 to 136).

[Figures 18A to 18F] Provided are mice in which compounds 131 to 136 were administered to control mice (aCSF) or via intrathecal injection (parental C16, P-4, P-6, P-8, P-, respectively). 4 PS, P-6 PS and P-8 PS) in the frontal cortex (Fig. 18A), hippocampus (Fig. 18B), cerebellum (Fig. 18C), lumbar root ganglion (DRG) (Fig. 18D), medulla oblongata (Fig. 18D) Figure 18E) and lumbar spinal cord (SC) (Figure 18F). Graph of measured percentage (%) of residual murine TUBB3 mRNA.

[Fig. 19] Provides a schematic diagram of blunt-ended RNAi oligonucleotide-lipid conjugates targeting TUBB3, in which the oligonucleotide contains the sense strand (compounds 137 to 140) with different amounts of locked nucleic acid (LNA) or P-6 truncated sense strand (i.e., 8 nucleotide overhang of the antisense strand) containing varying amounts of LNA (compounds 141 to 145).

[Figures 20A to 20F] Provided are the frontal cortex (Figure 20A), hippocampus in control mice (aCSF) or mice administered Compounds 1 and 137 to 145 via intrathecal injection (groups B to K, respectively). Measured percentage (%) of residual murine TUBB3 mRNA in (Fig. 20B), cerebellum (Fig. 20C), medulla oblongata (Fig. 20D), lumbar root ganglion (DRG) (Fig. 20E), and lumbar spinal cord (SC) (Fig. 20F) ) chart.

[Figure 21] Provides a schematic diagram of blunt-ended RNAi oligonucleotide-lipid conjugates targeting TUBB3, in which the oligonucleotides include sense strands containing different amounts of locked nucleic acid (LNA) (compounds 138, 139, 140 , 147 and 148).

[Figures 22A to 22F] Provided are the frontal values of Compounds 1, 137, 146, 138, 139, 140, 147 and 148 in control mice (aCSF) or mice administered via intrathecal injection (B to I, respectively). The remaining parts of the cortex (Fig. 22A), hippocampus (Fig. 22B), medulla oblongata (Fig. 22C), lumbar root ganglion (DRG) (Fig. 22D), cerebellum (Fig. 22E) and lumbar spinal cord (SC) (Fig. 22F) Graph of measured percentage (%) of murine TUBB3 mRNA.

[Figure 23] Provides a schematic diagram of blunt-ended RNAi oligonucleotide-lipid conjugates targeting TUBB3, in which the oligonucleotides comprise P-4 truncated sense strands containing varying amounts of locked nucleic acid (LNA) ( Compounds 149 to 154).

[Figures 24A to 24F] Provided are the frontal cortex (Figure 24A), hippocampus in control mice (aCSF) or mice administered compounds 1, 137, and 149 to 154 via intrathecal injection (B to I, respectively) Measured percentages of residual murine TUBB3 mRNA in the gyrus (Fig. 24B), medulla oblongata (Fig. 24C), lumbar root ganglion (DRG) (Fig. 24D), cerebellum (Fig. 24E), and lumbar spinal cord (SC) (Fig. 24F) %) chart.

[Figure 25] provides a schematic diagram of blunt-ended RNAi oligonucleotide-lipid conjugates targeting TUBB3, in which the oligonucleotides comprise P-8 truncated sense strands containing varying amounts of locked nucleic acid (LNA) ( Compounds 155 to 160).

[Figures 26A to 26F] Provided are the frontal cortex (Figure 26A), hippocampus in control mice (aCSF) or mice administered compounds 1, 137, and 155 to 160 via intrathecal injection (B to I, respectively) Measured percentage of residual murine TUBB3 mRNA in the gyrus (Fig. 26B), medulla oblongata (Fig. 26C), lumbar root ganglion (DRG) (Fig. 26D), cerebellum (Fig. 26E), and lumbar spinal cord (SC) (Fig. 26F) %) chart.

[Fig. 27] Provides a schematic diagram of blunt-ended RNAi oligonucleotide-lipid conjugates targeting UGT8, in which the oligonucleotides comprise the sense strand (compounds 162 to 165) containing different amounts of locked nucleic acid (LNA) or P-6 truncated sense strands containing varying amounts of LNA (compounds 166 to 171).

[Figures 28A to 28F] Provided are the frontal cortex (Figure 28A), hippocampus (Figure 28B) in control mice (aCSF) or mice administered compounds 161 to 171 via intrathecal injection (B to L, respectively) ), hypothalamus (Fig. 28C), cerebellum (Fig. 28D), medulla oblongata (Fig. 28E), and lumbar spinal cord (Fig. 26F). Graph of measured percentage (%) of residual murine UGT8 mRNA.

[Fig. 29] Provides a schematic diagram of blunt-ended RNAi oligonucleotide-lipid conjugates targeting GFAP, in which the oligonucleotides comprise the sense strand (compounds 173 to 176) containing different amounts of locked nucleic acid (LNA) or P-6 truncated sense strands containing varying amounts of LNA (compounds 177 to 182).

[Figures 30A to 30F] Provided are the frontal cortex (Figure 30A), hippocampus (Figure 30B) in control mice (aCSF) or mice administered Compounds 172 to 182 via intrathecal injection (B to L, respectively) ), cerebellum (Fig. 30C), medulla oblongata (Fig. 30D), hypothalamus (Fig. 30E), and lumbar spinal cord (Fig. 30F). Graph of measured percentage (%) of residual murine GFAP mRNA.

[Figures 31A to 31D] A schematic representation of blunt-ended RNAi oligonucleotide-lipid conjugates targeting TUBB3 is provided, where the oligonucleotides contain different sense strand truncations, including: no truncations (Figure 31A; Compounds 137 and 183 to 190); P-4 truncated sense strand (Figure 31B; Compounds 149 and 191 to 194); P-6 truncated sense strand (Figure 31C; Compounds 141, 195 and 196); and P- 8 truncated sense strand (Figure 31D; compound 197). Each compound contains a C16 lipid conjugated to a different position on the sense strand as indicated in the schematic.

[Figures 32A to 32F] Provided are the frontal cortex of control mice (aCSF) or mice administered compounds 137, 183 to 190, 149, 191 to 194, 141, and 195 to 197 via intrathecal injection (Fig. 32A), hippocampus (Fig. 32B), medulla oblongata (Fig. 32C), lumbar dorsal root ganglia (Fig. 32D), cerebellum (Fig. 32E) and lumbar spinal cord (Fig. 32F). Measured percentage (%) of residual murine TUBB3 mRNA chart.

[Figures 33A to 33C] Schematic diagrams of RNAi oligonucleotide-lipid conjugates targeting GFAP mRNA having the structures of compounds 173, 176, 180, 200 to 205, and 207 to 215 are provided.

[Figures 34A to 34D] Provided are the frontal lobes of control mice (aCSF) or mice administered compounds 173, 176, 180, 200 to 205, and 207 to 215 (as identified in Table 20) via intrathecal injection Graph of measured percentage (%) of murine GFAP mRNA remaining in the cortex (Fig. 34A), hippocampus (Fig. 34B), medulla oblongata (Fig. 34C), and lumbar spinal cord (Fig. 34D).

[Figures 35A to 35E] Schematic diagrams of RNAi oligonucleotide-lipid conjugates targeting TUBB3 mRNA having the structures of compounds 137, 140, 144, 217 to 222, 224 to 232, and 277 to 285 are provided.

[Figures 36A to 36D] Provided are the forehead values of Compounds 137, and 140, 144, 217 to 222, 224 to 232 (as identified in Table 21) in control mice (aCSF) or mice administered via intrathecal injection. Graph of measured percentage (%) of murine TUBB3 mRNA remaining in the lobe cortex (Fig. 36A), hippocampus (Fig. 36B), medulla oblongata (Fig. 36C), and lumbar spinal cord (Fig. 36D).

[Fig. 36E] Figure 36E] shows the frontal cortex, hippocampus, inferior cortex of control mice (aCSF) or mice administered compounds 140, 277 to 278, 231, and 279 to 285 via intrathecal injection (B to L, respectively). Graph of mouse TUBB3 mRNA measurements (%) in the optic thalamus, cerebellum, brainstem and lumbar spinal cord remnants.

[Figures 37A to 37B] Schematic diagrams of RNAi oligonucleotide-lipid conjugates targeting GFAP mRNA having the structures of compounds 173, 176, and 233 to 245 are provided.

[Figures 38A to 38D] Provided are the frontal cortex (Figure 38A), hippocampus in control mice (aCSF) or mice administered compounds 173, 176, and 233 to 245 via intrathecal injection (B to P, respectively). Graph of measured percentage (%) of murine GFAP mRNA in the gyrus (Fig. 38B), medulla oblongata (Fig. 38C), and lumbar spinal cord (Fig. 38D).

[Figures 39A to 39B] Schematic diagrams of GFAP mRNA-targeting RNAi oligonucleotide-lipid conjugates having the structures of compounds 173, 176, 200, and 246 to 255 are provided.

[Figures 40A to 40D] Provided are the frontal cortex of control mice (aCSF) or mice administered compounds 173, 176, 200, and 246 to 255 via intrathecal injection (B to N, respectively) (Figure 40A) , graph of measured percentage (%) of residual murine GFAP mRNA in the hippocampus (Fig. 40B), medulla oblongata (Fig. 40C), and lumbar spinal cord (Fig. 40D).

[Figures 41A to 41B] Schematic diagrams of RNAi oligonucleotide-lipid conjugates targeting TUBB3 mRNA having the structures of compounds 137, 140, and 257 to 268 are provided.

[Figures 42A to 42D] Provided are the frontal cortex (Figure 42A), hippocampus in control mice (aCSF) or mice administered compounds 137, 140, and 257 to 268 via intrathecal injection (B to P, respectively). Graph of measured percentage (%) of murine TUBB3 mRNA in the gyrus (Fig. 42B), medulla oblongata (Fig. 42C), and lumbar spinal cord (Fig. 42D).

[Figures 43A to 43B] Schematic representations of RNAi oligonucleotide-lipid conjugates with structural targeting of TUBB3 mRNA by compounds 137, 140, 217, and 269 to 276 are provided.

[Figures 44A to 44D] Provided are the frontal cortex of control mice (aCSF) or mice administered compounds 137, 140, 217, and 269 to 276 via intrathecal injection (B to F and I to N, respectively) Graph of measured percentage (%) of murine TUBB3 mRNA remaining in (Fig. 44A), hippocampus (Fig. 44B), medulla oblongata (Fig. 44C), and lumbar spinal cord (Fig. 44D).

[Fig. 45] A schematic diagram of an RNAi oligonucleotide-lipid conjugate targeting GFAP mRNA having the structure of compounds 286 to 292 is provided.

[Figures 46A to 46D] Provided are liver (Figure 46A), quadriceps femoris (quad) muscle (Figure 46A) in control mice (PBS) or mice (A to G, respectively) administered compounds 286 to 292 via subcutaneous injection 46B), myocardium (Figure 46C) and gonadal white adipose tissue (gWAT) (Figure 46D). Graph of measured percentage (%) of residual murine ALDH2 mRNA.

[Fig. 47] A schematic diagram of RNAi oligonucleotide-lipid conjugates targeting UGT8 mRNA having the structures of compounds 162, 167, 293, and 294 is provided.

[Figures 48A to 48D] Provided are mice administered compounds 162, 167, 293 and 294 (obtuse end, 5' p-6, 3' p-3, respectively) in control mice (aCSF) or via intrathecal injection. Measurement of residual murine UGT8 mRNA in the frontal cortex (Figure 48A), hippocampus (Figure 48B), brainstem (Figure 48C) and lumbar spinal cord (Figure 48D) and 5' p-6 & 3' p-3) Percentage (%) chart.

TW202333750A_111142550_SEQL.xml

Claims (381)

A double-stranded oligonucleotide comprising an antisense strand of about 20 to 22 nucleotides in length and a sense strand of about 8 to 20 nucleotides in length, wherein the antisense strand and the sense strand form about 8 to 20 nucleotides in length. duplex region of base pairs, wherein the antisense strand includes a 5' to 3' direction, and wherein the antisense strand includes a 5' overhang of at least one nucleotide and a 3' overhang of at least one nucleotide , wherein the antisense strand comprises a region complementary to the mRNA target sequence, and wherein the sense strand comprises at least one lipid moiety conjugated to a nucleotide on the sense strand. The oligonucleotide of claim 1, wherein the 5' overhang is about 1 to 10 nucleotides. The oligonucleotide of claim 1, wherein the 5' overhang is about 2 to 10 nucleotides. The oligonucleotide of claim 1, wherein the 5' overhang is about 1 to 6 nucleotides. The oligonucleotide of any one of claims 1-4, wherein the 3' overhang is about 2 to 12 nucleotides. The oligonucleotide of any one of claims 1-4, wherein the 3' overhang is about 2 to 8 nucleotides. The oligonucleotide of any one of claims 1-5, wherein the 5' overhang is 2 nucleotides and the 3' overhang is about 3 to 7 nucleotides. The oligonucleotide of any one of claims 1-5, wherein the 3' overhang is 2 nucleotides and the 5' overhang is about 2 to 8 nucleotides. The oligonucleotide of any one of claims 1-5, wherein the 3' overhang is 6 to 8 nucleotides and the 5' overhang is 2 to 4 nucleotides. 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. 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. 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 long. 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 long. 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 long. The oligonucleotide of any one of claims 1-14, wherein the lipid portion is selected from: ,and . The oligonucleotide of any one of claims 1-14, wherein the lipid moiety is a hydrocarbon chain. The oligonucleotide of claim 16, wherein the hydrocarbon chain is a C8-C30 hydrocarbon chain. The oligonucleotide of claim 17, wherein the hydrocarbon chain is a C16 hydrocarbon chain. Such as the oligonucleotide of claim 18, wherein the C16 hydrocarbon chain is represented by the following formula: . The oligonucleotide of claim 17, wherein the hydrocarbon chain is a C22 hydrocarbon chain. Such as the oligonucleotide of claim 20, wherein the C22 hydrocarbon chain is represented by the following formula: . The oligonucleotide of any one of claims 1-21, wherein the lipid part is conjugated to the 5' end nucleotide of the sense strand. The oligonucleotide of any one of claims 1-22, wherein the sense strand is 22 nucleotides, and the positions are numbered from 1 to 22 from 5' to 3'. The oligonucleotide of claim 23, wherein the nucleotide conjugated to the lipid moiety forms a base pair with the nucleotide at position 16, 14 or 12 of the antisense strand, wherein the position is from 5' to 3' number. The oligonucleotide of claim 23, wherein the nucleotide conjugated to the lipid moiety forms a base pair with the nucleotide at position 14 of the antisense strand, wherein the positions are numbered from 5' to 3'. The oligonucleotide of any one of claims 1-25, wherein the oligonucleotide comprises at least one modified nucleotide. The oligonucleotide of claim 26, wherein the modified nucleotide comprises a 2' modification. The oligonucleotide of claim 27, wherein each of the nucleotides of the sense strand and the antisense strand includes a 2' modification. The oligonucleotide of claim 27 or 28, wherein the 2' modification is selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl and 2'-deoxy-2'-fluoro-β-d-arabinose nucleic acid modification. The oligonucleotide of any one of claims 27-29, wherein the sense strand includes nucleotide positions numbered from 5' to 3', wherein each of positions 8 to 11 includes a 2'-fluorine modification. The oligonucleotide of any one of claims 27-29, wherein each of the nucleotides forming a base pair with the nucleotides at positions 10 to 13 of the sense strand includes 2' -Fluorine modification, where positions are numbered from 5' to 3'. The oligonucleotide of any one of claims 27-31, wherein the antisense strand includes 22 nucleotides from 5' to 3' from positions 1 to 22, and wherein positions 2, 3, 4, and 5 Each of , 7, 10 and 14 contains a 2'-fluoro modification. The oligonucleotide of any one of claims 30-32, wherein the residual nucleotide contains a 2'-O-methyl modification, provided that the nucleoside of the sense strand is conjugated to the at least one lipid moiety The acid does not contain 2'-O-methyl modification. The oligonucleotide of any one of the preceding claims, wherein the oligonucleotide comprises at least one modified inter-nucleotide linkage. The oligonucleotide of claim 34, wherein the at least one modified inter-nucleotide linkage is a phosphorothioate linkage. The oligonucleotide of claim 35, wherein the antisense strand is (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 contains a phosphorothioate linkage between positions 3 and 4, with positions numbered 1 to 4 from 5' to 3'. The oligonucleotide of claim 35 or 36, wherein the length of the antisense strand is 22 nucleotides, and wherein the antisense strand contains phosphorothioate between positions 20 and 21 and between positions 21 and 22 Ester linkage, where positions are numbered 1 to 22 from 5' to 3'. The oligonucleotide of claim 37, wherein the antisense strand comprises a phosphorothioate linkage between positions 13 and 14 and between positions 14 and 15. The oligonucleotide of any one of claims 35-38, wherein the sense strand is between positions 1 and 2, between the penultimate nucleotide and the third nucleotide from the 3' end, and A phosphorothioate linkage is included between the penultimate nucleotide and the final nucleotide. The oligonucleotide of any one of claims 1-39, wherein the sense strand contains at least one nucleotide that increases _Tm . The oligonucleotide of claim 40, wherein the sense strand contains up to four nucleotides that increase _Tm . The oligonucleotide of claim 40 or 41, wherein the _Tm -increasing nucleotide is a bicyclic nucleotide. The oligonucleotide of claim 40 or 41, wherein the _Tm -increasing nucleotide is a locked nucleic acid. 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 strand and the sense strand form a duplex region of 18 base pairs , wherein the antisense strand includes a direction from 5' to 3', wherein the antisense strand includes a 5' overhang and a 3' overhang, the 5' overhang includes at least two nucleotides and the 3' overhang includes at least two nucleotides. nucleotides, wherein the antisense strand comprises a region complementary to the mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to the 5' end nucleotide of the sense strand, and wherein the antisense strand and Each of the sense strands includes at least one 2' modified nucleotide and at least one modified inter-nucleotide linkage. The oligonucleotide of claim 44, wherein the at least one modified inter-nucleotide linkage is a phosphorothioate linkage. The oligonucleotide of claim 45, wherein the sense strand comprises a phosphorothioate linkage between nucleotides at positions 1 and 2, 16 and 17, and 17 and 18, numbered from 5' to 3'. The oligonucleotide of claim 45 or 46, wherein the antisense strand has nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21 and 21 and 22 numbered from 5' to 3' Contains phosphorothioate linkages. The oligonucleotide of any one of claims 44-47, wherein the 5' overhang is 2 nucleotides and the 3' overhang is 2 nucleotides. 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. The oligonucleotide of claim 49, wherein the CNS target sequence is a stellate cell mRNA target sequence. 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 Sinus endothelial cell mRNA target sequences. 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 strand and the sense strand form a duplex region of 17 base pairs , wherein the antisense strand includes a direction from 5' to 3', wherein the antisense strand includes a 5' overhang and a 3' overhang, the 5' overhang includes at least three nucleotides and the 3' overhang includes at least two nucleotides, wherein the antisense strand comprises a region complementary to the mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to the 5' end nucleotide of the sense strand, and wherein the antisense strand and Each of the sense strands includes at least one 2' modified nucleotide and at least one modified inter-nucleotide linkage. The oligonucleotide of claim 52, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage. The oligonucleotide of claim 53, wherein the sense strand comprises a phosphorothioate linkage between nucleotides at positions 1 and 2, 15 and 16, and 16 and 17, numbered from 5' to 3'. The oligonucleotide of claim 53 or 54, wherein the antisense strand is at the nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22, numbered from 5' to 3' Contains phosphorothioate linkages. The oligonucleotide of any one of claims 52-55, wherein the 5' overhang is 3 nucleotides and the 3' overhang is 2 nucleotides. 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 strand and the sense strand form a duplex region of 16 base pairs , wherein the antisense strand includes a direction from 5' to 3', wherein the antisense strand includes a 5' overhang and a 3' overhang, the 5' overhang includes at least four nucleotides and the 3' overhang includes at least two Nucleotides, wherein the antisense strand comprises a region complementary to the mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to the 5' end nucleotide of the sense strand, wherein the sense strand comprises at most five nucleotides that increase the _Tm , and wherein each of the antisense strand and the sense strand includes at least one 2' modified nucleotide and at least one modified inter-nucleotide linkage. The oligonucleotide of any one of claims 44-57, wherein the sense strand contains a 2'-fluorine modification at positions 8 to 11 numbered from 5' to 3'. The oligonucleotide of claim 58, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage. The oligonucleotide of claim 59, wherein the sense strand comprises a phosphorothioate linkage between nucleotides at positions 1 and 2, 14 and 15, and 15 and 16, numbered from 5' to 3'. The oligonucleotide of claim 59 or 60, wherein the antisense strand is at the nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22, numbered from 5' to 3' Contains phosphorothioate linkages. The oligonucleotide of any one of claims 57-61, wherein the 5' overhang is 4 nucleotides and the 3' overhang is 2 nucleotides. The oligonucleotide of any one of claims 57-62, comprising: (i) 1 to 5, 1 to 4, 1 to 3 or 1 to 2 nucleotides that increase T _m ; (ii) 1 , 2, 3, 4 or 5 nucleotides that increase _Tm ; or (iii) up to three nucleotides that increase _Tm . The oligonucleotide of claim 63, which contains two nucleotides that increase _Tm . The oligonucleotide of claim 63, which contains three nucleotides that increase _Tm . The oligonucleotide of any one of claims 57-65, wherein the sense strand contains _Tm -increasing nucleotides at positions 2, 15 and 16 numbered from 5' to 3'. The oligonucleotide of any one of claims 57-66, wherein the _Tm -increasing nucleotide is a bicyclic nucleotide, optionally a locked nucleic acid (LNA). 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. 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 strand and the sense strand form a duplex region of 13 base pairs , wherein the antisense strand includes a direction from 5' to 3', wherein the antisense strand includes a 5' overhang and a 3' overhang, the 5' overhang includes at least two nucleotides and the 3' overhang includes at least seven nucleotides. nucleotides, wherein the antisense strand comprises a region complementary to the 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 additional _Tm nucleotides, and wherein each of the antisense and sense strands includes at least one 2' modified nucleotide and at least one modified inter-nucleotide linkage. The oligonucleotide of claim 69, wherein the sense strand contains a 2'-fluorine modification at positions 3 to 6, numbered from 5' to 3'. The oligonucleotide of claim 69 or 70, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage. The oligonucleotide of claim 71, wherein the sense strand comprises a phosphorothioate linkage between nucleotides at positions 1 and 2, 11 and 12, and 12 and 13, numbered from 5' to 3'. The oligonucleotide of claim 71 or 72, wherein the antisense strand is at the nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21 and 21 and 22, numbered from 5' to 3' Contains phosphorothioate linkages. The oligonucleotide of any one of claims 69-73, wherein the lipid moiety is conjugated to the nucleotide at position 2 of the sense strand numbered from 5' to 3'. The oligonucleotide of any one of claims 69-74, wherein the 5' overhang is 2 nucleotides and the 3' overhang is 7 nucleotides. The oligonucleotide of any one of claims 69-75, comprising: (i) 1 to 3 or 1 to 2 nucleotides that increase T _m ; or (ii) 1, 2 or 3 nucleotides that increase T _m is the nucleotide. The oligonucleotide of any one of claims 69-76, wherein the sense strand comprises an increased _Tm at (i) positions 1 and 10 or (ii) positions 1, 10 and 11 numbered from 5' to 3' of nucleotides. The oligonucleotide of any one of claims 69-77, wherein the _Tm -increasing nucleotide is a bicyclic nucleotide, optionally a locked nucleic acid (LNA). 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 strand and the sense strand form a duplex region of 12 base pairs , wherein the antisense strand includes a direction from 5' to 3', wherein the antisense strand includes a 5' overhang and a 3' overhang, the 5' overhang includes at least two nucleotides and the 3' overhang includes at least seven nucleotides. nucleotides, wherein the antisense strand comprises a region complementary to the mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to the 5' end nucleotide of the sense strand, and wherein the antisense strand and Each of the sense strands includes at least one 2' modified nucleotide and at least one modified inter-nucleotide linkage. The oligonucleotide of claim 79, wherein the sense strand contains a 2'-fluoro modification at positions 3 to 6 or 4 to 7 numbered from 5' to 3'. The oligonucleotide of any one of claims 79-80, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage. The oligonucleotide of claim 81, wherein the sense strand comprises a phosphorothioate linkage between nucleotides at positions 1 and 2, 10 and 11, and 11 and 12, numbered from 5' to 3'. The oligonucleotide of claim 81 or 82, wherein the antisense strand is at positions 1 and 2, 2 and 3, 3 and 4, 12 and 14, 14 and 15, 20 and 21 numbered from 5' to 3' and contains a phosphorothioate linkage between nucleotides 21 and 22. The oligonucleotide of any one of claims 79-83, wherein the 5' overhang is 2 nucleotides and the 3' overhang is 8 nucleotides. The oligonucleotide of any one of claims 79-83, wherein the 5' overhang is 3 nucleotides and the 3' overhang is 7 nucleotides. The oligonucleotide of any one of claims 79-85, comprising: (i) 1 to 3 or 1 to 2 nucleotides that increase T _m ; or (ii) 1, 2 or 3 nucleotides that increase T _m is the nucleotide. The oligonucleotide of any one of claims 79-86, wherein the sense strand contains an increase in (i) positions 2, 10 and 11 or (ii) positions 2, 11 and 12 numbered from 5' to 3' _Tm is the nucleotide. The oligonucleotide of any one of claims 79-87, wherein the _Tm -increasing nucleotide is a bicyclic nucleotide, optionally a locked nucleic acid (LNA). 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 strand and the sense strand form a duplex region of 10 base pairs , wherein the antisense strand contains a direction from 5' to 3', wherein the antisense strand contains a 5' overhang and a 3' overhang, the 5' overhang contains one nucleotide and the 3' overhang contains eleven nucleosides acid, wherein the antisense strand comprises a region complementary to the mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to the 5' end nucleotide of the sense strand, and wherein the antisense strand and the sense strand Each of them includes at least one 2' modified nucleotide and at least one modified inter-nucleotide linkage. The oligonucleotide of claim 89, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage. The oligonucleotide of claim 90, wherein the sense strand comprises a phosphorothioate linkage between nucleotides at positions 1 and 2, 8 and 9, and 9 and 10, numbered from 5' to 3'. The oligonucleotide of claim 90 or 91, wherein the antisense strand is at positions 1 and 2, 2 and 3, 3 and 4, 12 and 14, 14 and 15, 20 and 21 numbered from 5' to 3' and contains a phosphorothioate linkage between nucleotides 21 and 22. The oligonucleotide of any one of claims 89-92, comprising: (i) 1 to 3 nucleotides that increase _Tm ; or (ii) 1, 2 or 3 nucleosides that increase _Tm acid. The oligonucleotide of any one of claims 89-93, wherein the sense strand comprises nucleotides at positions 2, 6 and 7 that increase _Tm . The oligonucleotide of any one of claims 89-94, wherein the _Tm -increasing nucleotide is a bicyclic nucleotide, optionally a locked nucleic acid (LNA). The oligonucleotide of any one of claims 44-95, wherein the antisense strand contains 2'-fluorine modifications at positions 2 to 5, 7, 10 and 14 numbered from 5' to 3'. For example, the oligonucleotide of any one of claims 58-68, 70-78, 80-88 and 96, wherein the residual nucleotide contains a 2'-O-methyl modification, with the restriction that the sense strand is The nucleotide to which at least one lipid moiety is conjugated does not contain a 2'-O-methyl modification. The oligonucleotide of any one of claims 44-68 and 79-98, wherein the lipid moiety is a C16 hydrocarbon represented by the formula: . The oligonucleotide of any one of claims 44-51, 69-78 and 96-97, wherein the lipid moiety is a C22 hydrocarbon represented by the following formula: . 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. The oligonucleotide of any one of claims 1-100, wherein the antisense strand comprises a phosphorylated nucleotide at the 5' end, wherein the phosphorylated nucleotide is selected from uridine and adenosine. The oligonucleotide of claim 101, wherein the phosphorylated nucleotide is uridine. 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. The oligonucleotide of claim 103, wherein the phosphate analog is oxymethylphosphonate, vinylphosphonate or malonylphosphonate. The oligonucleotide of any one of claims 102-104, wherein the phosphorylated nucleotide is 4'-O-monomethylphosphonate-2'-O-methyluridine. The oligonucleotide of any one of claims 1-105, wherein the complementary region is completely complementary to the mRNA target sequence. The oligonucleotide of any one of claims 1-105, wherein the complementary region is partially complementary to the mRNA target sequence. The oligonucleotide of claim 107, wherein the complementary region contains no more than four mismatches to the mRNA target sequence. The oligonucleotide of any one of claims 69-108, wherein the mRNA target sequence is a liver mRNA target sequence, optionally a liver hepatocyte mRNA target sequence, a liver macrophage mRNA target sequence or a liver Sinus endothelial cell mRNA target sequences. The oligonucleotide of any one of claims 1-109, wherein the oligonucleotide is an endonuclease (Dicer) substrate. The oligonucleotide of any one of claims 1-110, wherein the oligonucleotide reduces the expression of the mRNA target sequence in cells or cell populations in vitro and/or in vivo. A pharmaceutical composition comprising the oligonucleotide of any one of claims 1-111 and a pharmaceutically acceptable carrier, delivery agent or excipient. 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. 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. 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. 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. 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. A pharmaceutical composition comprising the oligonucleotide of any one of claims 89-111 and a pharmaceutically acceptable carrier, delivery agent or excipient. A method for treating an individual suffering from a disease, disorder or condition associated with the expression of a target mRNA, the method comprising administering to the individual a therapeutically effective amount of an oligonucleotide according to any one of claims 1-111 Acid or a pharmaceutical composition according to any one of claims 112-118. A method of reducing the expression of target mRNA in an individual, comprising administering to the individual an oligonucleotide according to any one of claims 1-111 or a pharmaceutical composition according to any one of claims 112-118. The method of claim 119 or 120, wherein the target mRNA is expressed in the central nervous system, optionally wherein the central nervous system includes the frontal cortex, hippocampus, medulla oblongata, cerebellum, lumbar root ganglia and/or lumbar spinal cord. The method of claim 121, wherein the target mRNA is expressed in neurons of the central nervous system. The method of claim 119 or 120, wherein the target mRNA is expressed in the liver, optionally in hepatocytes, liver macrophages or liver sinusoidal endothelial cells. The method of claim 123, wherein the target mRNA is expressed in liver macrophages. The method of claim 119 or 120, wherein the target mRNA is expressed in eye tissue. The method of claim 119 or 120, wherein the target mRNA is expressed in central nervous system tissue, liver tissue, eye tissue, adipose tissue, muscle tissue, adrenal tissue, heart tissue, lung tissue or any combination thereof. A method for treating an individual suffering from a disease, disorder or condition associated with the expression of central nervous system, hepatic, or ocular mRNA, the method comprising administering to the individual a therapeutically effective amount of claim 44- The oligonucleotide of any one of 51, 68 and 96-111 or the pharmaceutical composition of claim 113, optionally wherein the central nervous system includes the frontal cortex, hippocampus, medulla oblongata, cerebellum, and lumbar root nerves spinal cord and/or lumbar spinal cord. The method of claim 127, wherein the central nervous system mRNA is neuronal mRNA. A method for treating an individual suffering from a disease, disorder or condition associated with expression of mRNA in the central nervous system, the method comprising administering to the individual a therapeutically effective amount of any of claims 52-68 and 96-111 The oligonucleotide of one item or the pharmaceutical composition of claim 114 or 115, optionally wherein the central nervous system includes the frontal cortex, hippocampus, medulla oblongata, cerebellum, lumbar root ganglia and/or lumbar spinal cord. The method of claim 129, wherein the central nervous system mRNA is neuronal mRNA. A method for treating an individual suffering from a disease, disorder or condition associated with expression of macrophage mRNA, the method comprising administering to the individual a therapeutically effective amount of any one of claims 69-78 and 96-111 The oligonucleotide of claim 116 or the pharmaceutical composition of claim 116. The method of claim 131, wherein the macrophage mRNA is expressed in the liver. A method of delivering oligonucleotides to cells or cell populations in the central nervous system, liver tissue or eye tissue, the method comprising administering a pharmaceutical composition according to any one of claims 112-118. A method for reducing the expression of a target mRNA expressed in the central nervous system of an individual, the target mRNA expressed in the liver of an individual, or the expression of a target mRNA expressed in eye tissue of an individual, comprising administering to the individual an agent as claimed The oligonucleotide of any one of items 44-51, 68 and 96-111 or the pharmaceutical composition of claim 113, optionally wherein the central nervous system includes the frontal cortex, hippocampus, medulla oblongata, cerebellum, lumbar dorsal root ganglion and/or lumbar spinal cord. The method of claim 134, wherein the target mRNA is expressed in neurons of the central nervous system. A method for reducing the expression of a target mRNA expressed in the central nervous system of an individual, comprising administering to the individual a therapeutically effective amount of an oligonucleotide according to any one of claims 52-68 and 96-111 Or the pharmaceutical composition of claim 114 or 115, optionally wherein the central nervous system includes the frontal cortex, hippocampus, medulla oblongata, cerebellum, lumbar root ganglia and/or lumbar spinal cord. The method of claim 136, wherein the target mRNA is expressed in neurons of the central nervous system. A method for reducing the expression of target mRNA expressed in macrophages of an individual, comprising administering to the individual a therapeutically effective amount of an oligonucleotide according to any one of claims 69-78 and 96-111 Or a pharmaceutical composition as claimed in claim 116. The method of claim 138, wherein the macrophages are in the liver of the individual. A method for reducing the expression of a target mRNA expressed in the central nervous system of an individual, comprising administering to the individual a therapeutically effective amount of an oligonucleotide according to any one of claims 79-88 and 96-111 Or the pharmaceutical composition of claim 117, optionally wherein the central nervous system includes the frontal cortex, hippocampus, medulla oblongata, cerebellum, lumbar root ganglia and/or lumbar spinal cord. A method for reducing the expression of target mRNA expressed in the central nervous system of an individual, comprising administering to the individual a therapeutically effective amount of an oligonucleotide as claimed in any one of claims 89-111 or as claimed The pharmaceutical composition of 118, optionally wherein the central nervous system includes the frontal cortex, hippocampus, medulla oblongata, cerebellum, lumbar root ganglia and/or lumbar spinal cord. A kit, which contains the oligonucleotide of any one of claims 1-111, a pharmaceutically acceptable carrier if necessary, and a drug instruction sheet, which drug instruction sheet contains a drug for patients with a target mRNA. Instructions for administration to individuals with excessive manifestations of a disease, disorder, or condition related to the disease. A kit, which contains the oligonucleotide of any one of claims 1-111, a pharmaceutically acceptable carrier if necessary, and a drug instruction sheet, which drug instruction sheet contains a drug for patients with a target mRNA. Instructions for administration to individuals showing reduction in associated disease, disorder, or condition. An oligonucleotide according to any one of claims 1 to 111 or a pharmaceutical composition according to any one of claims 112 to 118 for use in the treatment of diseases, disorders or conditions associated with reduced expression of target mRNA The purpose of the medicine. An oligonucleotide according to any one of claims 1 to 111 or a pharmaceutical composition according to any one of claims 112 to 118 for use in the treatment of diseases, disorders or conditions associated with overexpression of target mRNA The purpose of the medicine. For example, the oligonucleotide according to any one of claims 1 to 111 or the pharmaceutical composition according to any one of claims 112 to 118, which is used or can be applied to the treatment of diseases related to the expression of target mRNA, Disease or condition. Such as 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, neurons of the central nervous system, macrophages Cells, optionally macrophages of the liver, ocular tissue, or any combination thereof, optionally wherein the central nervous system includes the frontal cortex, hippocampus, medulla oblongata, cerebellum, lumbar root ganglia, and/or lumbar spinal cord . A method of activating target-specific RNA interference (RNAi) in an organism, comprising administering to the organism an oligonucleotide as claimed in claim 1, 44, 52, 57 or 69, the oligonucleotide being An amount sufficient to cause degradation of the target mRNA is administered, thereby activating target-specific RNAi in the organism. A method of activating target-specific RNA interference (RNAi) in an organism, comprising administering to the organism an oligonucleotide as claimed in claim 1, 44, 52, 57, 69, 79 or 89, the oligonucleotide The nucleotide is administered in an amount sufficient to cause degradation of the target mRNA, thereby activating target-specific RNAi in the organism. The method of claim 148 or 149, wherein the target mRNA is specifically related to or expected to be related to the amino acid sequence of a protein of a human disease or disorder. The method of claim 150, wherein the disease or condition is selected from the group consisting of viral infection, bacterial infection, parasitic infection, cancer, allergy, autoimmune disease, immune deficiency and immunosuppression. A double-stranded oligonucleotide comprising an antisense strand of about 15 to 30 nucleotides in length and a sense strand of about 15 to 50 nucleotides in length, wherein the antisense strand and the sense strand form about 15 to 30 nucleotides in length A duplex region of base pairs, wherein the antisense strand includes a region complementary to the mRNA target sequence, and wherein the sense strand includes (i) at least one lipid moiety conjugated to a nucleotide of the sense strand and ( ii) Backbone loop, wherein the backbone loop includes a nucleotide sequence represented by the formula: 5'-S1-L-S2-3', wherein S1 is complementary to S2, and wherein L is between S1 and S2 A loop is formed, and the sense strand and the antisense strand each include a direction from 5' to 3', and the backbone loop is tied to the 5' end of the sense strand. The oligonucleotide of claim 152, comprising a blunt end. The oligonucleotide of claim 153, wherein the blunt end includes the 3' end of the sense strand and the 5' end of the antisense strand. The oligonucleotide of claim 152, comprising an overhang of at least two nucleotides. The oligonucleotide of claim 155, wherein the overhang includes the 5' end of the antisense strand. The oligonucleotide of any one of claims 152-156, wherein the sense strand is about 28 to 38 nucleotides. The oligonucleotide of any one of claims 152-157, wherein the antisense strand is 22 nucleotides. The oligonucleotide of any one of claims 152-158, wherein the lipid moiety is conjugated to a nucleotide comprising the loop. The oligonucleotide of any one of claims 152-159, wherein: (i) The sense strand is 28 nucleotides, and the lipid part is conjugated to the nucleotide at position 4, and the positions are numbered from 5' to 3'; (ii) The sense strand is 30 nucleotides, and the lipid part is conjugated to the nucleotide at position 4, and the positions are numbered from 5' to 3'; (iii) The sense strand is 34 nucleotides, and the lipid moiety is conjugated to the nucleotide at position 6 or position 15, with the positions numbered from 5' to 3'; or (iv) The sense strand is 38 nucleotides long, and the lipid part is conjugated to the nucleotide at position 8, and the positions are numbered from 5' to 3'. The oligonucleotide of any one of claims 152-160, wherein the lipid portion is selected from: and . The oligonucleotide of any one of claims 152-160, wherein the lipid moiety is a hydrocarbon chain. The oligonucleotide of claim 162, wherein the hydrocarbon chain is a C8-C30 hydrocarbon chain. The oligonucleotide of claim 163, wherein the hydrocarbon chain is a C16 hydrocarbon chain. Such as the oligonucleotide of claim 164, wherein the C16 hydrocarbon chain is represented by the following formula: . 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. The oligonucleotide of any one of claims 152-165, wherein the mRNA target sequence is an eye mRNA target sequence. The oligonucleotide of any one of claims 152-167, wherein the loop sequence is 5'-GAAA-3'. The oligonucleotide of any one of claims 152-167, wherein the loop sequence is 5'-UNCG-3', wherein N is any nucleotide. The oligonucleotide of claim 169, wherein the loop sequence is 5'-UACG-3'. The oligonucleotide of any one of claims 152-170, wherein the oligonucleotide comprises at least one modified nucleotide. The oligonucleotide of claim 171, wherein the modified nucleotide comprises a 2' modification. The oligonucleotide of claim 171, wherein each of the nucleotides of the sense strand and the antisense strand comprises a 2' modification. The oligonucleotide of claim 172 or 173, wherein the 2' modification is selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl and 2'-deoxy-2'-fluoro-β-d-arabinose nucleic acid modification. The oligonucleotide of any one of claims 172-174, wherein the antisense strand includes 22 nucleotides from 5' to 3' from positions 1 to 22, and wherein positions 2, 3, 4, and 5 Each of , 7, 10 and 14 contains a 2'-fluoro modification. The oligonucleotide of any one of claims 172-175, wherein: (i) The sense strand is 38 nucleotides from positions 1 to 38 from 5' to 3', and each of positions 26 to 29 includes a 2'-fluorine modification; (ii) The sense strand is 34 nucleotides from positions 1 to 34 from 5' to 3', and each of positions 22 to 25 includes a 2'-fluorine modification; (iii) The sense strand is 30 nucleotides from 5' to 3' from positions 1 to 30, and each of positions 18 to 21 contains a 2'-fluoro modification; or (iv) The sense strand is 28 nucleotides from positions 1 to 28 from 5' to 3', and each of positions 18 to 21 includes a 2'-fluorine modification. The oligonucleotide of any one of claims 152-176, wherein the oligonucleotide comprises at least one modified inter-nucleotide linkage. The oligonucleotide of claim 177, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage. The oligonucleotide of claim 178, wherein the antisense strand is (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 A phosphorothioate linkage is included between positions 3 and 4, with positions numbered 1 to 4 from 5' to 3'. The oligonucleotide of claim 177 or 178, wherein the antisense strand is 22 nucleotides in length, and wherein the antisense strand contains a phosphorothioate between positions 20 and 21 and between positions 21 and 22 Ester linkage, where positions are numbered 1 to 22 from 5' to 3'. The oligonucleotide of any one of claims 177-180, wherein the sense strand is between the penultimate nucleotide and the third nucleotide from the 3' end and the penultimate nucleotide Contains a phosphorothioate linkage to the final nucleotide. The oligonucleotide of any one of claims 177-180, wherein: (i) the sense strand is 38 nucleotides from 5' to 3' from positions 1 to 38, and wherein the sense strand contains a phosphorothioate linkage between positions 36 and 37 and 37 and 38; (ii) the sense strand is 34 nucleotides from 5' to 3' from positions 1 to 34, and wherein the sense strand contains a phosphorothioate linkage between positions 32 and 33 and 33 and 34; (iii) the sense strand is 30 nucleotides from 5' to 3' from positions 1 to 30, and wherein the sense strand contains a phosphorothioate linkage between positions 28 and 29 and 29 and 30; or (iv) The sense strand is 28 nucleotides from positions 1 to 28 from 5' to 3', and wherein the sense strand contains a phosphorothioate linkage between positions 26 and 27 and 27 and 28. The oligonucleotide of any one of claims 152-182, wherein the sense strand contains at least one nucleotide that increases _Tm . The oligonucleotide of claim 183, wherein the sense strand contains up to six _Tm -increasing nucleotides. The oligonucleotide of claim 183 or 184, wherein the _Tm -increasing nucleotide is a bicyclic nucleotide or a locked nucleic acid. The oligonucleotide of any one of claims 152-185, wherein S1 and S2 each comprise 1 to 6 nucleotides. The oligonucleotide of claim 186, wherein S1 and S2 each contain 4 nucleotides. The oligonucleotide of claim 186, wherein S1 and S2 each contain 2 nucleotides. The oligonucleotide of any one of claims 182-188, wherein S1 and S2 each comprise at least one nucleotide that increases _Tm . Such as the oligonucleotide of claim 189, wherein each of S1 and S2 is 4 nucleotides, and 1 to 3 nucleotides of each of S1 and S2 are nucleotides that increase _Tm . The oligonucleotide of any one of claims 175-190, wherein the residual nucleotide contains a 2'-O-methyl modification, provided that the nucleoside of the sense strand is conjugated to the at least one lipid moiety The acid does not contain 2'-O-methyl modification. A double-stranded oligonucleotide comprising an antisense strand of about 20 to 22 nucleotides in length and a sense strand of about 32 to 34 nucleotides in length, wherein the antisense strand and the sense strand form about 20 to 22 nucleotides in length. base pair duplex region and the oligonucleotide is blunt-ended, wherein the antisense strand contains a region complementary to the mRNA target sequence, wherein the sense strand contains (i) a backbone loop, wherein the backbone loop The circle includes a nucleotide sequence represented by the formula: 5'-S1-L-S2-3', wherein S1 is complementary to S2, and wherein L forms a loop between S1 and S2; and (ii) with the loop at least one lipid moiety of a nucleotide conjugate of a loop, wherein the sense strand and the antisense strand each include a 5' to 3' direction, wherein the backbone loop is tethered to the 5' end of the sense strand, and wherein the Each of the antisense and sense strands includes at least one 2' modified nucleotide and at least one modified inter-nucleotide linkage. The oligonucleotide of claim 192, wherein the sense strand is 34 nucleotides and includes a 2'-fluoro modification at positions 22 to 25, numbered from 5' to 3'. The oligonucleotide of any one of claims 192-193, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage. The oligonucleotide of claim 194, wherein the sense strand is 34 nucleotides and is at positions 1 and 2, 2 and 3, 32 and 33, and 33 and 34 nucleotides numbered from 5' to 3' Contains phosphorothioate linkages. Such as claim 194 or 195 oligonucleotide, wherein the antisense strand is 22 nucleotides and is numbered from 5' to 3' at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21 and There is a phosphorothioate linkage between nucleotides 21 and 22. The oligonucleotide of any one of claims 192-196, wherein the lipid moiety is conjugated to the nucleotide at position 6 of the sense strand numbered from 5' to 3'. The oligonucleotide of any one of claims 192-197, wherein S1 and S2 each comprise 1 to 6 nucleotides. Such as the oligonucleotide of claim 198, wherein S1 and S2 each contain 4 nucleotides. The oligonucleotide of any one of claims 192-199, wherein L is 4 nucleotides. The oligonucleotide of claim 200, wherein L includes the sequence 5'-GAAA-3'. A double-stranded oligonucleotide comprising an antisense strand of about 20 to 22 nucleotides in length and a sense strand of about 26 to 28 nucleotides in length, wherein the antisense strand and the sense strand form about 20 to 22 nucleotides in length an asymmetric duplex region of base pairs, the asymmetric duplex region comprising a 3' overhang of at least 2 nucleotides of the antisense strand, wherein the antisense strand comprises a sequence complementary to the mRNA target sequence A region, wherein the sense strand includes: (i) a backbone loop, wherein the backbone loop includes a nucleotide sequence represented by the formula: 5'-S1-L-S2-3', wherein S1 is complementary to S2, and wherein L forms a loop between S1 and S2 and includes the sequence UNCG, and wherein S1 and S2 each include at least one nucleotide that increases T _m ; and (ii) at least one nucleotide conjugated to the loop A lipid moiety, wherein the sense strand contains a direction from 5' to 3', wherein the backbone loop is tied to the 5' end of the sense strand, and wherein each of the antisense strand and the sense strand contains at least one 2' Modified nucleotides and at least one modified inter-nucleotide linkage. The oligonucleotide of claim 202, wherein the sense strand is 28 nucleotides and includes a 2'-fluoro modification at positions 18 to 21, numbered from 5' to 3'. The oligonucleotide of any one of claims 202-203, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage. The oligonucleotide of claim 204, wherein the sense strand is 28 nucleotides and includes a phosphorothioate bond between nucleotides at positions 26 and 27 and 27 and 28, numbered from 5' to 3' Union. Such as claim 204 or 205 oligonucleotide, wherein the antisense strand is 22 nucleotides and is numbered from 5' to 3' at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21 and There is a phosphorothioate linkage between nucleotides 21 and 22. The oligonucleotide of any one of claims 202-206, wherein the 3' end overhang is 2 nucleotides. The oligonucleotide of any one of claims 202-207, wherein the lipid moiety is conjugated to the nucleotide at position 4 of the sense strand numbered from 5' to 3'. The oligonucleotide of any one of claims 202-208, wherein S1 and S2 each comprise 1 to 6 nucleotides. Such as the oligonucleotide of claim 209, wherein S1 and S2 each contain 2 nucleotides. The oligonucleotide of claim 210, wherein S1 and S2 each comprise a nucleotide that increases _Tm . The oligonucleotide of any one of claims 202-211, wherein the _Tm -increasing nucleotide is a bicyclic nucleotide. The oligonucleotide of any one of claims 202-211, wherein the _Tm -increasing nucleotide is a locked nucleic acid (LNA). The oligonucleotide of any one of claims 202-213, wherein L is 4 nucleotides. The oligonucleotide of claim 214, wherein L comprises the sequence 5'-UNCG-3', wherein N is any nucleotide. The oligonucleotide of claim 215, wherein L includes the sequence 5'-UACG-3'. A double-stranded oligonucleotide comprising an antisense strand of about 20 to 22 nucleotides in length and a sense strand of about 32 to 34 nucleotides in length, wherein the antisense strand and the sense strand form about 20 to 22 nucleotides in length. base pair duplex region, and the oligonucleotide is blunt-ended, wherein the antisense strand includes a region complementary to the mRNA target sequence, wherein the sense strand includes: (i) a backbone loop, wherein the The backbone loop includes a nucleotide sequence represented by the formula: 5'-S1-L-S2-3', wherein S1 is complementary to S2, and wherein L forms a loop between S1 and S2, and wherein S1 and S2 each includes at least one nucleotide that increases _Tm ; and (ii) at least one lipid moiety conjugated to a nucleotide of the loop, wherein the sense strand includes a direction from 5' to 3', wherein the backbone A loop is attached to the 5' end of the sense strand, and wherein each of the antisense and sense strands includes at least one 2' modified nucleotide and at least one modified inter-nucleotide linkage. The oligonucleotide of claim 217, wherein the sense strand is 34 nucleotides and includes a 2'-fluoro modification at positions 22 to 25, numbered from 5' to 3'. The oligonucleotide of any one of claims 217-218, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage. The oligonucleotide of claim 219, wherein the sense strand is 34 nucleotides and includes a phosphorothioate bond between nucleotides at positions 32 and 33 and 33 and 34, numbered from 5' to 3' Union. Such as claim 219 or 220 oligonucleotide, wherein the antisense strand is 22 nucleotides and is numbered from 5' to 3' at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21 and There is a phosphorothioate linkage between nucleotides 21 and 22. The oligonucleotide of any one of claims 217 to 221, wherein the oligonucleotide includes a blunt end, the blunt end includes the 5' end of the antisense strand and the 3' end of the sense strand. The oligonucleotide of any one of claims 217-222, wherein the lipid moiety is conjugated to the nucleotide at position 6 of the sense strand numbered from 5' to 3'. The oligonucleotide of any one of claims 217-223, wherein S1 and S2 each comprise 1 to 6 nucleotides. The oligonucleotide of claim 224, wherein S1 and S2 each contain 4 nucleotides. The oligonucleotide of claim 225, wherein S1 and S2 each comprise a nucleotide that increases _Tm . The oligonucleotide of any one of claims 217-226, wherein the _Tm -increasing nucleotide is a bicyclic nucleotide. The oligonucleotide of any one of claims 217-226, wherein the _Tm -increasing nucleotide is a locked nucleic acid (LNA). The oligonucleotide of any one of claims 217-228, wherein L is 4 nucleotides. The oligonucleotide of claim 229, wherein L includes the sequence 5'-GAAA-3'. The oligonucleotide of any one of claims 192-230, wherein the antisense strand is 22 nucleotides and includes 2' at positions 2 to 5, 7, 10 and 14 numbered from 5' to 3' -Fluorine modification. The oligonucleotide of any one of claims 193-201, 203-216 and 218-231, wherein the residual nucleotide contains a 2'-O-methyl modification, with the restriction that the sense strand is the same as the at least one The nucleotide to which the lipid moiety is conjugated does not contain 2'-O-methyl modification. The oligonucleotide of any one of claims 192-232, wherein the lipid moiety is a C16 hydrocarbon represented by the following formula: . The oligonucleotide of any one of claims 152-233, wherein the antisense strand comprises a phosphorylated nucleotide at the 5' end, wherein the phosphorylated nucleotide is selected from uridine and adenosine. The oligonucleotide of claim 234, wherein the phosphorylated nucleotide is uridine. 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. The oligonucleotide of claim 236, wherein the phosphate analog is oxymethylphosphonate, vinylphosphonate or malonylphosphonate. The oligonucleotide of any one of claims 235-237, wherein the phosphorylated nucleotide is 4'-O-monomethylphosphonate-2'-O-methyluridine. 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. The oligonucleotide of any one of claims 152-239, wherein the complementary region is completely complementary to the mRNA target sequence. The oligonucleotide of any one of claims 152-239, wherein the complementary region is partially complementary to the mRNA target sequence. The oligonucleotide of claim 241, wherein the complementary region contains no more than four mismatches to the mRNA target sequence. 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 eye mRNA target sequence. target sequence; or (ii) eye tissue mRNA target sequence. Such as the oligonucleotide of any one of claims 152-242, wherein the target mRNA is expressed in central nervous system tissue, liver tissue, eye tissue, adipose tissue, muscle tissue, adrenal gland tissue, heart tissue, lung tissue or any combination thereof. The oligonucleotide of any one of claims 152-244, wherein the oligonucleotide is an endonuclease (Dicer) substrate. The oligonucleotide of any one of claims 152-245, wherein the oligonucleotide reduces the expression of the mRNA target sequence in a cell or cell population in vitro and/or in vivo. A pharmaceutical composition comprising the oligonucleotide of any one of claims 152-246 and a pharmaceutically acceptable carrier, delivery agent or excipient. A method of treating an individual suffering from a disease, disorder or condition associated with expression of a target mRNA, the method comprising administering to the individual a therapeutically effective amount of an oligonucleotide of any one of claims 152-246 or Such as the pharmaceutical composition of claim 247. The method of claim 248, wherein the target mRNA is expressed in the central nervous system, optionally neurons of the central nervous system. The method of claim 248, wherein the target mRNA is expressed in eye tissue, optionally the optic nerve and/or retina. A method of delivering an oligonucleotide to cells or cell populations in the central nervous system or eye tissue, the method comprising administering a pharmaceutical composition as claimed in claim 247. A method of reducing the expression of target mRNA in an individual, comprising administering to the individual an oligonucleotide according to any one of claims 152-246 or a pharmaceutical composition according to claim 247. The method of claim 252, wherein the target mRNA is expressed in the central nervous system, optionally neurons of the central nervous system. The method of claim 253, wherein the target mRNA is expressed in eye tissue, optionally the optic nerve and/or retina. A kit, which contains the oligonucleotide of any one of claims 152-246, a pharmaceutically acceptable carrier if necessary, and a drug instruction sheet, which drug instruction sheet contains a drug for patients with a target mRNA. Instructions for administration to individuals exhibiting related diseases, disorders, or conditions. Use of an oligonucleotide according to any one of claims 152 to 246 or a pharmaceutical composition according to claim 247 in the manufacture of a medicament for the treatment of a disease, disorder or condition associated with the expression of target mRNA. The oligonucleotide of any one of claims 152-246 or the pharmaceutical composition of claim 122 is for use or is applicable for the treatment of diseases, disorders or conditions related to the expression of target mRNA. 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 neurons of the central nervous system. The kit of claim 255, the use of claim 256 or the oligonucleotide of claim 257, wherein the target mRNA is expressed in eye tissue, optionally the retina and/or optic nerve. The method of any one of claims 249, 251, and 253, wherein the central nervous system includes the frontal cortex, hippocampus, cerebellum, brainstem, lumbar root ganglia, lumbar spinal cord, or a combination thereof. For example, the kit, use or oligonucleotide of claim 258, wherein the central nervous system includes frontal cortex, hippocampus, cerebellum, brainstem, lumbar root ganglia, lumbar spinal cord or a combination thereof. A method of activating target-specific RNA interference (RNAi) in an organism, comprising administering to the organism a dsRNA oligonucleotide as claimed in claim 152, 192, 202 or 217, the oligonucleotide being sufficient to An amount is administered that causes degradation of the target mRNA, thereby activating target-specific RNAi in the organism. The method of claim 262, wherein the target mRNA is specifically related to or expected to be related to the amino acid sequence of a protein of a human disease or disorder. The method of claim 263, wherein the disease or condition is selected from the group consisting of viral infection, bacterial infection, parasitic infection, cancer, allergy, autoimmune disease, immune deficiency and immunosuppression. A double-stranded oligonucleotide comprising an antisense strand of about 13 to 30 nucleotides in length and a sense strand of about 10 to 50 nucleotides in length, wherein the antisense strand and the sense strand are separate strands, The strands form an asymmetric duplex region having an overhang of about 2 to 10 nucleotides at the 3' end of the antisense strand, wherein the duplex region is about 10 to 30 nucleotides, wherein the The antisense strand includes a region complementary to the mRNA target sequence, and wherein the sense strand includes at least one lipid moiety conjugated to a nucleotide on the sense strand. A double-stranded oligonucleotide comprising an antisense strand of about 13 to 30 nucleotides in length and a sense strand of about 10 to 50 nucleotides in length, wherein the antisense strand and the sense strand are separate strands, The strands form an asymmetric duplex region having an overhang of about 2 to 12 nucleotides at the 3' end of the antisense strand, wherein the duplex region is about 10 to 30 nucleotides, wherein the The antisense strand includes a region complementary to the mRNA target sequence, and wherein the sense strand includes at least one lipid moiety conjugated to a nucleotide on the sense strand. The oligonucleotide of claim 265 or 266, which includes a blunt end. The oligonucleotide of claim 267, wherein the blunt end includes the 3' end of the sense strand and the 5' end of the antisense strand. The oligonucleotide of claim 265 or 266, which includes a backbone loop, wherein the backbone loop includes a nucleotide sequence represented by the formula: 5'-S1-L-S2-3', wherein S1 is with S2 is complementary, and wherein L forms a loop between S1 and S2, and wherein the sense strand includes a direction from 5' to 3', and wherein the backbone loop is tied to the 3' end of the sense strand. 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. 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. 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. Such as 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. The oligonucleotide of any one of claims 265-273, wherein the antisense strand is 22 nucleotides numbered from positions 1 to 22 from 5' to 3'. The oligonucleotide of any one of claims 270-274, wherein the lipid moiety is conjugated to a nucleotide that forms a base pair with the nucleotide at position 14 of the sense strand and the antisense strand. The oligonucleotide of any one of claims 270-274, wherein the lipid moiety is conjugated to a nucleotide that forms a base pair with the nucleotide at position 12 of the sense strand and the antisense strand. Such as the oligonucleotide according to any one of claims 270-274, wherein the lipid part is at position 20, position 19, position 18, position 17, position 16, position 15, of the sense strand and the antisense strand. The nucleotide at position 14, position 13, or position 12 forms a base pair nucleotide conjugate. The oligonucleotide of claim 269 or 274, wherein the lipid moiety is conjugated to the nucleotide of the loop. The oligonucleotide of any one of claims 265-278, wherein the lipid portion is conjugated to the 3' end nucleotide of the sense strand. The oligonucleotide of any one of claims 265-278, wherein the lipid portion is conjugated to the 5' end nucleotide of the sense strand. The oligonucleotide of any one of claims 265-280, wherein the lipid portion is selected from: and . The oligonucleotide of any one of claims 265-280, wherein the lipid moiety is a hydrocarbon chain. The oligonucleotide of claim 282, wherein the hydrocarbon chain is a C8-C30 hydrocarbon chain. The oligonucleotide of claim 283, wherein the hydrocarbon chain is a C16 hydrocarbon chain. Such as the oligonucleotide of claim 284, wherein the C16 hydrocarbon chain is represented by the following formula: . The oligonucleotide of claim 283, wherein the hydrocarbon chain is a C22 hydrocarbon chain. Such as the oligonucleotide of claim 286, wherein the C22 hydrocarbon chain is represented by the following formula: . 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. The oligonucleotide of any one of claims 265-288, wherein the complementary region is completely complementary to the mRNA target sequence. The oligonucleotide of any one of claims 265-288, wherein the complementary region is partially complementary to the mRNA target sequence. The oligonucleotide of claim 290, wherein the complementary region contains no more than four mismatches to the mRNA target sequence. 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 Sinus endothelial cell mRNA target sequences. The oligonucleotide of any one of claims 265-291, wherein the mRNA target sequence is an eye mRNA target sequence. 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. 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. The oligonucleotide of claim 295, wherein the at least one tissue of the central nervous system is selected from the group consisting of frontal cortex, medulla oblongata, hippocampus, hypothalamus, cerebellum, lumbar spinal cord, lumbar dorsal root ganglia, and any of them combination. The oligonucleotide of any one of claims 265-296, wherein the oligonucleotide comprises at least one modified nucleotide. The oligonucleotide of claim 297, wherein the modified nucleotide comprises a 2' modification. The oligonucleotide of claim 298, wherein each of the nucleotides of the sense strand and the antisense strand comprises a 2' modification. The oligonucleotide of claim 297 or 298, wherein the 2' modification is selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl and 2'-deoxy-2'-fluoro-β-d-arabinose nucleic acid modification. The oligonucleotide of any one of claims 298-300, wherein the antisense strand is 22 nucleotides numbered from positions 1 to 22 from 5' to 3', and wherein the sense strand is between Each of the nucleotides forming a base pair at positions 10 to 13 of the antisense strand contains a 2'-fluoro modification. The oligonucleotide of any one of claims 298-300, wherein the antisense strand is 22 nucleotides numbered from positions 1 to 22 from 5' to 3', and wherein the sense strand is between Each of the nucleotides forming a base pair at positions 10, 11, 12, 13, or any combination thereof, of the antisense strand contains a 2'-fluoro modification. The oligonucleotide of any one of claims 298-301, wherein the antisense strand includes 22 nucleotides from 5' to 3' from positions 1 to 22, and wherein positions 2, 3, 4, and 5 Each of , 7, 10 and 14 contains a 2'-fluoro modification. The oligonucleotide of any one of claims 301-303, wherein the residual nucleotide contains a 2'-O-methyl modification, provided that the nucleoside of the sense strand is conjugated to the at least one lipid moiety The acid does not contain 2'-O-methyl modification. The oligonucleotide of any one of claims 265-304, wherein the oligonucleotide comprises at least one modified internucleotide linkage. The oligonucleotide of claim 305, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage. The oligonucleotide of claim 306, wherein the antisense strand is (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 A phosphorothioate linkage is included between positions 3 and 4, with positions numbered 1 to 4 from 5' to 3'. The oligonucleotide of claim 306 or 307, wherein the antisense strand is 22 nucleotides in length, and wherein the antisense strand contains a phosphorothioate between positions 20 and 21 and between positions 21 and 22 Ester linkage, where positions are numbered 1 to 22 from 5' to 3'. The oligonucleotide of claim 308, wherein the antisense strand comprises a phosphorothioate linkage between positions 13 and 14 and between positions 14 and 15. 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 . The oligonucleotide of claim 308, wherein the antisense strand is 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 , contains phosphorothioate linkages between positions 18 and 19 and between positions 19 and 20. The oligonucleotide of claim 308, wherein the antisense strand is 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, include phosphorothioate linkages. The oligonucleotide of any one of claims 306-312, wherein the sense strand comprises a phosphorothioate linkage between positions 1 and 2. The oligonucleotide of any one of claims 306-312, wherein the sense strand is between positions 1 and 2, between the penultimate nucleotide and the third nucleotide from the 3' end, and A phosphorothioate linkage is included between the penultimate nucleotide and the final nucleotide. The oligonucleotide of any one of claims 265-314, wherein the antisense strand comprises a phosphorylated nucleotide at the 5' end, wherein the phosphorylated nucleotide is selected from uridine and adenosine. The oligonucleotide of claim 315, wherein the phosphorylated nucleotide is uridine. 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. The oligonucleotide of claim 317, wherein the phosphate analog is oxymethylphosphonate, vinylphosphonate or malonylphosphonate. The oligonucleotide of any one of claims 316-318, wherein the phosphorylated nucleotide is 4'-O-monomethylphosphonate-2'-O-methyluridine. The oligonucleotide of any one of claims 265-319, wherein the sense strand contains at least one nucleotide that increases _Tm . The oligonucleotide of claim 320, wherein the sense strand contains up to nine _Tm -increasing nucleotides. The oligonucleotide of claim 321, wherein the sense strand contains 1 to 3 nucleotides that increase _Tm . The oligonucleotide of any one of claims 269 and 279-322, wherein S1 and S2 each comprise at least one nucleotide that increases _Tm . Such as the oligonucleotide of claim 323, wherein S1 and S2 are each 3 nucleotides, and each nucleotide is a nucleotide that increases _Tm . The oligonucleotide of claim 323 or 324, wherein the sense strand contains at most three _Tm -increasing nucleotide positions at nucleotide positions, with the proviso that these nucleotide positions are not in the backbone loop. The oligonucleotide of any one of claims 267-268 and 270-322, wherein the sense strand contains up to five _Tm- increasing nucleotides. The oligonucleotide of any one of claims 320-326, wherein the _Tm -increasing nucleotide is a bicyclic nucleotide. The oligonucleotide of any one of claims 320-326, wherein the _Tm -increasing nucleotide is a locked nucleic acid. Such as the oligonucleotide of claim 328, wherein the length of the sense strand is 20 nucleotides, wherein the nucleotides are numbered from 5' to 3', and wherein the sense strand is located at the following position The nucleotides contain locked nucleic acids: (i) Position 2; (ii) Position 2 and Position 15; (iii) Position 2, Position 15 and Position 16; (iv) Position 2, Position 15, Position 16 and Position 18; or (v) Position 2, Position 15, Position 16, Position 18 and Position 19. Such as the oligonucleotide of claim 328, wherein the length of the sense strand is 16 nucleotides, wherein the nucleotides are numbered from 5' to 3', and wherein the sense strand is located at the following position The nucleotides contain locked nucleic acids: (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. Such as the oligonucleotide of claim 328, wherein the length of the sense strand is 14 nucleotides, wherein the nucleotides are numbered from 5' to 3', and wherein the sense strand is located at the following position The nucleotides contain locked nucleic acids: (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. Such as the oligonucleotide of claim 328, wherein the length of the sense strand is 12 nucleotides, wherein the nucleotides are numbered from 5' to 3', and wherein the sense strand is located at the following position The nucleotides contain locked nucleic acids: (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. The oligonucleotide of any one of claims 265-332, wherein the oligonucleotide is an endonuclease (Dicer) substrate. The oligonucleotide of any one of claims 265-333, wherein the oligonucleotide reduces the expression of the mRNA target sequence in a cell or cell population in vitro and/or in vivo. A pharmaceutical composition comprising the oligonucleotide of any one of claims 265-334 and a pharmaceutically acceptable carrier, delivery agent or excipient. A method of delivering oligonucleotides to cells or cell populations in the central nervous system, liver tissue, skeletal muscle tissue, adipose tissue, adrenal tissue and/or eye tissue, the method comprising administering a pharmaceutical composition according to claim 69. A method for treating an individual suffering from a disease, disorder or condition associated with expression of a target mRNA, the method comprising administering to the individual a therapeutically effective amount of an oligonucleotide of any one of claims 265-334 acid or a pharmaceutical composition as claimed in claim 335. A method of reducing the expression of target mRNA in an individual, comprising administering to the individual an oligonucleotide according to any one of claims 265-334 or a pharmaceutical composition according to claim 335. The method of claim 337 or 338, wherein the target mRNA is expressed in the liver, optionally in macrophages of the liver. The method of claim 337 or 338, wherein the target mRNA is expressed in at least one cell type of the central nervous system. The method of claim 340, wherein the at least one cell type of the central nervous system is a stellate cell, a neuron, or an oligodendritic cell. The method of claim 337 or 338, wherein the target mRNA is expressed in stellate cells, neurons, oligodendritic cells, or any combination thereof. The method of claim 340-342, wherein the target mRNA is expressed in the frontal cortex, medulla oblongata, hippocampus, hypothalamus, cerebellum, lumbar spinal cord, lumbar dorsal root ganglia, or any combination thereof . The method of claim 337 or 338, wherein the target mRNA is expressed in eye tissue, optionally retina or optic nerve, liver tissue, skeletal muscle tissue, adipose tissue and/or adrenal tissue. A kit, which contains the oligonucleotide of any one of claims 265-334, a pharmaceutically acceptable carrier if necessary, and a drug instruction sheet, the drug instruction sheet containing Instructions for administration to individuals exhibiting related diseases, disorders, or conditions. Use of an oligonucleotide according to any one of claims 265-334 or a pharmaceutical composition according to claim 335 in the manufacture of a medicament for the treatment of a disease, disorder or condition associated with the expression of target mRNA. The oligonucleotide of any one of claims 265-334 or the pharmaceutical composition of claim 335 is for use or is applicable for the treatment of diseases, disorders or conditions associated with the expression of target mRNA. The method of claim 347, wherein the target mRNA is expressed in at least one cell type of the central nervous system. The method of claim 348, wherein the at least one cell type of the central nervous system is a stellate cell, a neuron, or an oligodendritic cell, optionally wherein the target mRNA is expressed in a stellate cell, a neuron, an oligodendritic cell, or a stellate cell. dendritic cells or any combination thereof. The method of claim 347-349, wherein the target mRNA is expressed in the frontal cortex, medulla oblongata, hippocampus, hypothalamus, cerebellum, lumbar spinal cord, lumbar dorsal root ganglia, or any combination thereof . The kit of claim 345, the use of claim 346, or the oligonucleotide of claim 347, wherein the target mRNA is expressed in liver, optionally wherein the target mRNA is expressed in liver macrophages. , liver hepatocytes or liver sinusoidal endothelial cells. The kit of claim 345, the use of claim 346, or the oligonucleotide of claim 347, wherein the target mRNA is expressed in eye tissue, optionally the retina or optic nerve. 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. 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. The kit, use or oligonucleotide of claim 354, wherein the at least one tissue of the central nervous system is selected from the group consisting of frontal cortex, medulla oblongata, hippocampus, hypothalamus, cerebellum, lumbar spinal cord, and lumbar dorsal root ganglia. and any combination thereof. The method of claim 337, wherein the disease or condition is selected from the group consisting of viral infection, bacterial infection, parasitic infection, cancer, allergy, autoimmune disease, immune deficiency, and immunosuppression. A double-stranded oligonucleotide containing: (i) a sense strand of 14 to 20 nucleotides in length, wherein the sense strand includes at least one lipid moiety conjugated to the 5' end, and wherein the sense strand includes at least one locked nucleic acid; and (ii) an antisense strand of 22 nucleotides in length, wherein the antisense strand contains a region complementary to the stellate cell target mRNA, and wherein the sense strand and the antisense strand are separate strands forming an asymmetric duplex region having an overhang of about 2 to 8 nucleotides at the 3' end of the antisense strand, wherein the duplex The region is approximately 14 to 20 nucleotides in length. A double-stranded oligonucleotide containing: (i) a sense strand of 14 to 20 nucleotides in length, wherein the sense strand includes at least one lipid moiety conjugated to the 5' end, and wherein the sense strand includes at least one locked nucleic acid; and (ii) an antisense strand of 22 nucleotides in length, wherein the antisense strand contains a region complementary to the neuronal target mRNA, and wherein the sense strand and the antisense strand are separate strands forming an asymmetric duplex region having an overhang of about 2 to 8 nucleotides at the 3' end of the antisense strand, wherein the duplex The region is approximately 14 to 20 nucleotides in length. A double-stranded oligonucleotide containing: (i) a sense strand of 14 to 20 nucleotides in length, wherein the sense strand includes at least one lipid moiety conjugated to the 5' end, and wherein the sense strand includes at least one locked nucleic acid; and (ii) an antisense strand of 22 nucleotides in length, wherein the antisense strand contains a region complementary to the oligodendritic cell target mRNA, and wherein the sense strand and the antisense strand are separate strands forming an asymmetric duplex region having an overhang of about 2 to 8 nucleotides at the 3' end of the antisense strand, wherein the duplex The region is approximately 14 to 20 nucleotides in length. The oligonucleotide of any one of claims 357-359, wherein the length of the sense strand is 14 nucleotides. The oligonucleotide of claim 360, wherein the sense strand includes a locked nucleic acid at one or more of position 2, position 9, position 10, position 12, or position 13, numbered from 5' to 3'. The oligonucleotide of claim 360 or 361, wherein the antisense strand is 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 Phosphorothioate linkages are included between, between positions 20 and 21, and between positions 21 and 22. The oligonucleotide of any one of claims 357-359, wherein the length of the sense strand is 20 nucleotides. 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 of positions numbered from 5' to 3'. The oligonucleotide of claim 363 or 364, wherein the antisense strand is 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 Contains phosphorothioate linkages. A method of reducing the expression of target mRNA in stellate cells, comprising administering a double-stranded oligonucleotide, wherein the oligonucleotide comprises: (i) a sense strand, wherein the sense strand is 14 to 20 nucleotides in length, wherein the sense strand includes at least one lipid moiety conjugated to the 5' end, and wherein the sense strand includes 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 complementary to the target mRNA of the stellate cell, and wherein the sense strand and the antisense strand are separate strands forming an asymmetric duplex region having an overhang of about 2 to 8 nucleotides at the 3' end of the antisense strand, wherein the duplex The region is approximately 14 to 20 nucleotides, thereby reducing the expression of the target mRNA in the stellate cells. The method of claim 366, wherein the oligonucleotide includes a blunt end comprising the 3' end of the sense strand and the 5' end of the antisense strand. The method of any one of claims 366-367, wherein the sense strand contains no more than 3 locked nucleic acids. The method of any one of claims 366-368, wherein the length of the sense strand is 20 nucleotides, and wherein the reduction of the target mRNA in stellate cells is increased compared to the reduction in neurons, depending on Desirably wherein the target mRNA reduction is increased by at least 5%. The method of any one of claims 366-368, wherein the length of the sense strand is 14 nucleotides, and wherein the reduction of the target mRNA in stellate cells is increased compared to the reduction in oligodendritic cells , optionally wherein the target mRNA reduction is increased by at least 5%. The method of any one of claims 366-368, wherein the length of the sense strand is 20 nucleotides, and wherein the target mRNA is reduced to the same or similar levels in stellate cells and oligodendritic cells. The method of any one of claims 366-368, wherein the length of the sense strand is 14 nucleotides, wherein the target mRNA is reduced to the same or similar levels in stellate cells and neurons, and wherein the target The reduction of target mRNA in stellate cells and neurons is increased compared to the reduction in oligodendritic cells, optionally wherein the reduction of target mRNA is increased by at least 5%. A method of reducing the expression of target mRNA in oligodendritic cells, comprising administering a double-stranded oligonucleotide, wherein the oligonucleotide comprises: (i) a sense strand, wherein the sense strand is 14 to 40 nucleotides in length, wherein the sense strand includes at least one lipid moiety conjugated to the 5' end, and wherein the sense strand includes 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 complementary to the target mRNA of the oligodendritic cell, and wherein the sense strand and the antisense strand are separate strands forming an asymmetric duplex region having an overhang of about 2 to 8 nucleotides at the 3' end of the antisense strand, wherein the duplex The region is approximately 14 to 20 nucleotides, thereby reducing the expression of the target mRNA in the oligodendritic cells. The method of claim 373, wherein the oligonucleotide includes a blunt end that includes the 3' end of the sense strand and the 5' end of the antisense strand. The method of claim 374, wherein the sense strand is 20 nucleotides in length. The method of claim 373, wherein the sense strand is 36 nucleotides in length, and wherein the oligonucleotide includes a backbone loop. The method of claim 376, wherein the target mRNA is reduced to the same or similar level in stellate cells and oligodendritic cells, and wherein the reduction of the target mRNA in stellate cells and oligodendritic cells is the same as in neurons. The decrease is compared to the increase, optionally wherein the decrease in the target mRNA is increased by at least 5%. A method of reducing the expression of target mRNA in neurons, the method comprising administering a double-stranded oligonucleotide, wherein the oligonucleotide comprises: (i) a sense strand, wherein the sense strand is 14 to 20 nucleotides in length, wherein the sense strand includes at least one lipid moiety conjugated to the 5' end, and wherein the sense strand includes 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 complementary to the target mRNA of the neuron, and wherein the sense strand and the antisense strand are separate strands forming an asymmetric duplex region having an overhang of about 2 to 8 nucleotides at the 3' end of the antisense strand, wherein the duplex The region is approximately 14 to 20 nucleotides, thereby reducing expression of the target mRNA in the neuron. The method of claim 378, wherein the oligonucleotide includes a blunt end comprising the 3' end of the sense strand and the 5' end of the antisense strand. The method of claim 378 or 379, wherein the sense strand contains no more than 5 locked nucleic acids. The method of any one of claims 378-380, wherein the sense strand is 14 nucleotides in length.