Modified Oligonucleotides

Abstract

Disclosed herein are compositions comprising modified oligonucleotides. In some embodiments, the oligonucleotide comprises a hydrophobic moiety. In some embodiments, the oligonucleotide comprises a vinyl phosphonate. In some embodiments, the oligonucleotide comprises a modified oligonucleotide sugar, such as a 2' modified ribose molecule. In some embodiments, the oligonucleotide comprises a modified internucleoside linkage, such as a phosphorothioate linkage. In some embodiments, the oligonucleotide comprises an siRNA. In some embodiments, the siRNA comprises a duplex region with an overhang at either end.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 324,487, filed March 28, 2022, and U.S. Provisional Patent Application No. 63 / 429,756, filed December 2, 2022, both of which are incorporated by reference herein.

[0002] Sequence Listing This application is filed with a Sequence Listing in electronic format, which is provided as a file entitled 54462-739601_PCT.xml, created on March 20, 2023, and is 541 kilobytes in size. The information in the electronic Sequence Listing is incorporated by reference in its entirety. [Background technology]

[0003] There is a need in the art for improved oligonucleotides for reducing gene expression. Summary of the Invention

[0004] Described herein, in some embodiments, is a composition comprising an oligonucleotide that targets an mRNA and reduces the level of a target mRNA or protein when administered in an effective amount. Described herein, in some embodiments, is a composition comprising a sense strand, an antisense strand, and a small interfering RNA (siRNA) comprising a lipid moiety attached to an end of the sense strand or antisense strand, where the lipid moiety comprises a phenyl or cyclohexyl linker, and the linker is attached to the lipid and the end of the sense strand or antisense strand. The lipid moiety may be attached to the 5' or 3' end of the sense strand or antisense strand. In some embodiments, the lipid and the end of the sense strand or antisense strand are attached to the phenyl or cyclohexyl linker in a 1,4, 1,3, or 1,2 substitution pattern (e.g., para, meta, or ortho phenyl configuration). In some embodiments, the lipid and the end of the sense strand or antisense strand are attached to the phenyl or cyclohexyl linker in a 1,4 (para phenyl) configuration. In some embodiments, the lipid moiety has the following structure:

[0005] [ka] where the dashed line indicates a bond (e.g., a covalent bond) to the end of the sense or antisense strand, and R is an alkyl group containing 4-18 carbons. In some embodiments, R is not an octane. In some embodiments, R is an alkyl group containing 4-7 or 9-18 carbons. In some embodiments, the lipid moiety has the following structure:

[0006] [ka] where the dashed line indicates a bond (e.g., a covalent bond) to the end of the sense or antisense strand, n is 1-3, and R is an alkyl group containing 4-18 carbons. In some embodiments, the lipid moiety has the following structure:

[0007] [ka] where the dashed line indicates a bond (e.g., a covalent bond) to the end of the sense or antisense strand, n is 0 to 3, and R is an alkyl group containing 4 to 18 carbons. In some embodiments, the lipid moiety has the following structure:

[0008] [ka] where the dashed line indicates a bond (e.g., a covalent bond) to the end of the sense or antisense strand, and R is an alkyl group containing 4 to 18 carbons. In some embodiments, the lipid moiety has the following structure:

[0009] [ka] wherein the dashed line indicates a bond to the end of the sense or antisense strand, n is 0-3, and R is an alkyl group containing 4-18 carbons. In some embodiments, the lipid moiety comprises a lipid moiety as shown in Table 1. In some embodiments, the lipid moiety is bonded to the 5' end of the sense or antisense strand. In some embodiments, the lipid moiety is bonded to the 5' end of the sense strand. In some embodiments, the lipid moiety is bonded to the 5' end of the sense or antisense strand via a phosphate. In some embodiments, the lipid moiety is bonded to the end of the sense strand. In some embodiments, the antisense strand comprises a vinyl phosphonate. In some embodiments, the antisense strand comprises a 5' vinyl phosphonate. In some embodiments, the sense or antisense strand comprises one or two phosphorothioate linkages at the 5' or 3' end of the sense or antisense strand. In some embodiments, the antisense strand comprises one or two 5' phosphorothioate linkages. For example, there may be phosphorothioate between the first and second nucleotides from the 5' end of the antisense strand, or between the first, second, and third nucleotides from the 5' end of the antisense strand. In some embodiments, the antisense strand comprises one or two 3' phosphorothioate bonds. For example, there may be phosphorothioate between the first and second nucleotides from the 3' end of the antisense strand, or between the first, second, and third nucleotides from the 3' end of the antisense strand. In some embodiments, the sense strand comprises one or two 5' phosphorothioate bonds. For example, there may be phosphorothioate between the first and second nucleotides from the 5' end of the sense strand, or between the first, second, and third nucleotides from the 5' end of the sense strand.In some embodiments, the sense strand does not include one or two 5' phosphorothioate bonds. For example, in some embodiments, there is no phosphorothioate between the last three nucleotides at the 5' end of the sense strand. In some embodiments, the sense strand includes a 5' phosphate bond. In some embodiments, the sense strand includes one or two 3' phosphorothioate bonds. For example, there may be phosphorothioate between the first nucleotide and the second nucleotide from the 3' end of the sense strand, or there may be phosphorothioate between the first nucleotide, the second nucleotide, and the third nucleotide from the 3' end of the sense strand. In some embodiments, for the sense strand, all purine nucleosides include 2' fluoro and all pyrimidine nucleosides are modified with a mixture of 2' fluoro and 2'-O-methyl; all purine nucleosides include 2'-O-methyl and all pyrimidine nucleosides are modified with a mixture of 2' fluoro and 2'-O-methyl; all purine nucleosides include 2' fluoro and all pyrimidine nucleosides include 2'-O-methyl; all pyrimidine nucleosides include 2' fluoro. and all purine nucleosides are modified with a mixture of 2'fluoro and 2'-O-methyl, all pyrimidine nucleosides are modified with a mixture of 2'fluoro and 2'-O-methyl, or all pyrimidine nucleosides are modified with a mixture of 2'fluoro and 2'-O-methyl, with the proviso that in any of the above, the sense strand may include 2'deoxynucleosides. In some embodiments, the sense strand includes 2'deoxynucleosides. In some embodiments, the sense strand does not include 2'deoxynucleosides.In some embodiments, with respect to the antisense strand, any one of the following is true: all purine nucleosides contain 2' fluoro and all pyrimidine nucleosides are modified with a mixture of 2' fluoro and 2'-O-methyl; all purine nucleosides contain 2'-O-methyl and all pyrimidine nucleosides are modified with a mixture of 2' fluoro and 2'-O-methyl; all purine nucleosides contain 2' fluoro and all pyrimidine nucleosides are modified with a mixture of 2' fluoro and 2'-O-methyl; all pyrimidine nucleosides contain 2' fluoro and all purine nucleosides are modified with a mixture of 2' fluoro and 2'-O-methyl; all pyrimidine nucleosides contain 2' fluoro and all purine nucleosides are modified with a mixture of 2' fluoro and 2'-O-methyl; all pyrimidine nucleosides contain 2'-O-methyl and all purine nucleosides are modified with a mixture of 2' fluoro and 2'-O-methyl; or all pyrimidine nucleosides contain 2'-O-methyl and all purine nucleosides contain 2' fluoro. In some embodiments, with respect to the sense or antisense strand, any one of the following is true, provided that the sense or antisense strand may comprise deoxynucleosides: all purines comprise 2'fluoro modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines; all purines comprise 2'-O-methyl modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines; all purines comprise 2'fluoro modified purines and all pyrimidines comprise 2'-O-methyl modified pyrimidines; all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines; all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines; all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines; or all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise 2'-O-methyl modified purines.In some embodiments, with respect to the sense strand, any one of the following is true: all purines comprise 2'fluoro modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines; all purines comprise 2'-O-methyl modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines; all purines comprise 2'fluoro modified purines and all pyrimidines comprise 2'-O-methyl modified pyrimidines; all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines; all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines; all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines; or all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise 2'-O-methyl modified purines, with the proviso that in any of the foregoing, the sense strand may comprise deoxynucleosides. In some embodiments, the sense strand does not contain deoxynucleosides.In some embodiments, the sense strand is any one of the following: all purines comprise 2'fluoro modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines; all purines comprise 2'-O-methyl modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines; all purines comprise 2'fluoro modified purines and all pyrimidines comprise 2'-O-methyl modified pyrimidines; all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines; all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines; all pyrimidines comprise 2'-O-methyl modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines; or all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise 2'-O-methyl modified purines.In some embodiments, the antisense strand is any one of the following: all purines comprise 2'fluoro modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines, all purines comprise 2'-O-methyl modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines, all purines comprise 2'-O-methyl modified purines and all pyrimidines comprise 2'fluoro modified pyrimidines, all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines, all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines, all pyrimidines comprise 2'-O-methyl modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines, or all pyrimidines comprise 2'-O-methyl modified pyrimidines and all purines comprise 2'fluoro modified purines. The sense strand may comprise 2-deoxy modification at position 9.

[0010] Described herein, in some embodiments, is a composition comprising a small interfering RNA (siRNA) comprising a sense strand and an antisense strand, wherein the antisense strand comprises a 5' end comprising a vinyl phosphonate and two phosphorothioate linkages, and a 3' end comprising two phosphorothioate linkages, and the sense strand comprises a 5' end comprising a hydrophobic moiety, and a 3' end comprising two phosphorothioate linkages, and with respect to the sense strand, all purine nucleosides are provided, with the proviso that the sense strand may comprise a 2' deoxynucleoside. all purine nucleosides contain 2'-O-methyl and all pyrimidine nucleosides are modified with a mixture of 2'-fluoro and 2'-O-methyl; all purine nucleosides contain 2'-O-methyl and all pyrimidine nucleosides are modified with a mixture of 2'-fluoro and 2'-O-methyl; all purine nucleosides contain 2'-fluoro and all pyrimidine nucleosides contain 2'-O-methyl; all pyrimidine nucleosides contain 2'-fluoro and all purine nucleosides are modified with a mixture of 2'-fluoro and 2'-O-methyl all pyrimidine nucleosides contain 2'-O-methyl and all purine nucleosides are modified with a mixture of 2'-fluoro and 2'-O-methyl; or all pyrimidine nucleosides contain 2'-fluoro and all purine nucleosides contain 2'-O-methyl; and for the antisense strand, all purine nucleosides contain 2'-fluoro and all pyrimidine nucleosides are modified with a mixture of 2'-fluoro and 2'-O-methyl; all pyrimidine nucleosides contain 2'-O-methyl and all pyrimidine nucleosides are modified with a mixture of 2'fluoro and 2'-O-methyl; all purine nucleosides contain 2'fluoro; all pyrimidine nucleosides contain 2'fluoro and all purine nucleosides are modified with a mixture of 2'fluoro and 2'-O-methyl; all pyrimidine nucleosides contain 2'-O-methyl and all purine nucleosides are modified with a mixture of 2'fluoro and 2'-O-methyl;Or all pyrimidine nucleosides include 2'-O-methyl and all purine nucleosides include 2'fluoro. In some embodiments, the sense strand includes 2' deoxynucleosides. In some embodiments, the sense strand does not include 2' deoxynucleosides. Described herein, in some embodiments, is a composition comprising a small interfering RNA (siRNA) comprising a sense strand and an antisense strand, wherein the antisense strand comprises a 5' end comprising a vinyl phosphonate and two phosphorothioate linkages, and a 3' end comprising two phosphorothioate linkages, and the sense strand comprises a 5' end comprising a hydrophobic moiety, and a 3' end comprising two phosphorothioate linkages, and wherein, for the sense strand, all purines comprise 2'fluoro modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines, all purines comprise 2'-O-methyl modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines, all purines comprise 2'fluoro modified purines and all pyrimidines comprise 2'-O-methyl modified pyrimidines, all pyrimidines comprise 2'fluoro modified pyrimidines, and all pyrimidines comprise 2'fluoro modified pyrimidines. and all pyrimidines comprise 2'-fluoro and 2'-O-methyl modified purines, all pyrimidines comprise 2'-O-methyl modified pyrimidines and all purines comprise a mixture of 2'-fluoro and 2'-O-methyl modified purines, or all pyrimidines comprise 2'-fluoro modified pyrimidines and all purines comprise 2'-O-methyl modified purines, and with respect to the antisense strand, all purines comprise 2'-fluoro modified purines and all pyrimidines comprise a mixture of 2'-fluoro and 2'-O-methyl modified pyrimidines, all purines comprise 2'-O-methyl modified purines and all pyrimidines comprise a mixture of 2'-fluoro and 2'-O-methyl modified pyrimidines, all purines comprise 2'-O-methyl modified purines and all pyrimidines comprise 2'-fluoro modified pyrimidines, all pyrimidines comprise 2'-fluoro modified pyrimidines,Any one of the following applies: all purines include a mixture of 2'fluoro and 2'-O-methyl modified purines, all pyrimidines include 2'-O-methyl modified pyrimidines and all purines include a mixture of 2'fluoro and 2'-O-methyl modified purines, or all pyrimidines include 2'-O-methyl modified pyrimidines and all purines include 2'fluoro modified purines, with the proviso that in all cases the sense strand may include deoxynucleosides. In some embodiments, the sense strand does not include deoxynucleosides. Described herein, in some embodiments, is a composition comprising a small interfering RNA (siRNA) comprising a sense strand and an antisense strand, wherein the antisense strand comprises a 5' end comprising a vinyl phosphonate and two phosphorothioate linkages, and a 3' end comprising two phosphorothioate linkages, and the sense strand comprises a 5' end comprising a hydrophobic moiety, and a 3' end comprising two phosphorothioate linkages, and wherein, for the sense strand, all purines comprise 2'fluoro modified purines, all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines, all purines comprise 2'-O-methyl modified purines, all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines, all purines comprise 2'fluoro modified purines, all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines, all purines comprise 2'fluoro modified purines, and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines, all purines comprise 2'fluoro modified purines, and all pyrimidines comprise a mixture of 2'fluoro modified purines ... 2'fluoro modified purines. and wherein one of the following applies: all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines; all pyrimidines comprise 2'-O-methyl modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines; all pyrimidines comprise 2'-O-methyl modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines; or all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise 2'-O-methyl modified purines; and wherein, with respect to the antisense strand, all purines comprise 2'fluoro modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'O-methyl modified pyrimidines; all purines comprise 2'-O-methyl modified purines;Any one of the following applies: all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines; all purines comprise 2'-O-methyl modified purines and all pyrimidines comprise 2'fluoro modified pyrimidines; all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines; all pyrimidines comprise 2'-O-methyl modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines; or all pyrimidines comprise 2'-O-methyl modified pyrimidines and all purines comprise 2'fluoro modified purines. In some embodiments, the 5' end of the sense strand comprises two nucleotides linked by phosphate bond rather than by phosphorothioate bond. In some embodiments, the 5' end of the sense strand comprises two nucleosides linked by a phosphate bond rather than by a phosphorothioate bond, and the 3' end of the sense strand comprises two nucleosides linked by a phosphorothioate bond. In some embodiments, the 5' end of the sense strand further comprises one or two phosphorothioate bonds. In some embodiments, the hydrophobic moiety comprises a lipid attached to the 5' end of the sense strand by a phenyl or cyclohexyl linker. In some embodiments, the lipid and the 5' end of the sense strand are attached to a phenyl or cyclohexyl linker in a 1,4, 1,3, or 1,2 substitution pattern (e.g., para, meta, or ortho phenyl configuration). In some embodiments, the lipid and the 5' end of the sense strand are attached to a phenyl or cyclohexyl linker in a 1,4 (paraphenyl) configuration. In some embodiments, the hydrophobic moiety has the following structure:

[0011] [ka] where the dashed line indicates a bond to the end of the sense or antisense strand, n is 1-3, and R is an alkyl group containing 4-18 carbons. In some embodiments, the hydrophobic moiety has the following structure:

[0012] [ka] where the dashed line indicates a bond (e.g., a covalent bond) to the end of the sense or antisense strand, and R is an alkyl group containing 4-18 carbons. In some embodiments, R is not an octane. In some embodiments, R is an alkyl group containing 4-7 or 9-18 carbons. In some embodiments, the hydrophobic moiety has the following structure:

[0013] [ka] where the dashed line indicates a bond (e.g., a covalent bond) to the end of the sense or antisense strand, n is 1-3, and R is an alkyl group containing 4-18 carbons. In some embodiments, the hydrophobic moiety has the following structure:

[0014] [ka] where the dashed line indicates a bond (e.g., a covalent bond) to the end of the sense or antisense strand, n is 1-3, and R is an alkyl group containing 4-18 carbons. In some embodiments, the hydrophobic moiety has the following structure:

[0015] [ka] where the dashed line indicates a bond (e.g., a covalent bond) to the terminus of the sense or antisense strand, and R is an alkyl group containing 4 to 18 carbons. In some embodiments, the hydrophobic moiety comprises a lipid moiety as shown in Table 1. The sense strand may include a 2-deoxy modification at position 9.

[0016] In some embodiments, the sense strand and the antisense strand form a duplex. In some embodiments, the duplex comprises at least 10 base pairs, at least 11 base pairs, at least 12 base pairs, at least 13 base pairs, at least 14 base pairs, at least 15 base pairs, at least 16 base pairs, at least 17 base pairs, at least 18 base pairs, at least 19 base pairs, at least 20 base pairs, at least 21 base pairs, at least 22 base pairs, at least 23 base pairs, at least 24 base pairs, or at least 25 base pairs. In some embodiments, the duplex comprises 19 base pairs. In some embodiments, the duplex comprises a sense strand overhang. In some embodiments, the sense strand overhang comprises 1-4 nucleosides. In some embodiments, the sense strand overhang comprises 2 nucleosides. In some embodiments, the sense strand overhang comprises uracil. In some embodiments, the duplex comprises an antisense strand overhang. In some embodiments, the antisense strand overhang comprises 1-4 nucleosides. In some embodiments, the antisense strand overhang comprises two nucleosides. In some embodiments, the antisense strand overhang comprises uracil. In some embodiments, the sense strand and the antisense strand form a duplex comprising a sense strand 3' overhang of 1-3 nucleotides and an antisense strand 3' overhang of 1-3 nucleotides. In some embodiments, the sense strand and the antisense strand form a duplex comprising a sense strand 3' overhang of 2 uracil nucleotides and an antisense strand 3' overhang of 2 uracil nucleotides. Some embodiments include a pharma- ceutically acceptable carrier. Described herein, in some embodiments, is a method of treatment comprising administering to a subject in need thereof an effective amount of a composition. Described herein, in some embodiments, is a method of reducing the amount of RNA or protein in a cell comprising administering to the cell a composition. In some embodiments, administering to the cell a composition comprises administering to a subject comprising the cell a composition.

[0017] In some embodiments, the present disclosure describes a method of treatment, comprising administering to a subject in need thereof an effective amount of the composition or siRNA described herein.In some embodiments, the administration comprises subcutaneous administration, intravitreal administration, intrathecal administration, or intracerebroventricular administration.In some embodiments, the siRNA targets the mRNA that encodes the protein that mediates the subject's disease.In some embodiments, the mRNA is in the subject's tissue, including eye, liver, fat, brain, or spinal cord.

[0018] In some embodiments, described herein is a method of reducing the amount of target RNA or the amount of protein encoded by the RNA in a cell, comprising administering to the cell a composition or siRNA described herein, wherein the antisense strand comprises a sequence that is complementary to and binds to the RNA. In some embodiments, administering to the cell comprises administering to a subject comprising the cell the composition. In some embodiments, the administration comprises subcutaneous administration, intravitreal administration, intrathecal administration, or intracerebroventricular administration. In some embodiments, the administration reduces the measurement of the target RNA or the measurement of the protein encoded by the RNA by at least 10% compared to a baseline measurement or control. In some embodiments, the cell comprises an eye, liver, fat, brain, or spinal cord cell. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] In some embodiments, modified oligonucleotides are disclosed herein.Modified oligonucleotides may be siRNAs that contain modifications to ribose rings and phosphate bonds.Modifications may be specific patterns that maximize cell delivery, stability, and efficiency.siRNAs may further contain vinyl phosphonates and hydrophobic groups.These modifications can aid in delivery to cells or tissues in a subject.Modified oligonucleotides can be used in methods such as therapeutic methods or methods for reducing gene expression.

[0020] In some embodiments, the oligonucleotide comprises a duplex consisting of a 21 nucleotide single strand with base pairing between 19 base pairs. In some embodiments, the duplex comprises a single stranded 2 nucleotide overhang at the 3' end of each strand. One strand (the antisense strand) is complementary to the target mRNA. Each end of the antisense strand has one to two phosphorothioate linkages. The 5' end has an optional phosphate mimic such as a vinyl phosphonate. In some embodiments, the oligonucleotide is used to knock down a target mRNA or a target protein. In some embodiments, the sense strand has the same sequence as the target mRNA. In some embodiments, there are one to two phosphorothioates at the 3' end. In some embodiments, there is one phosphorothioate or no phosphorothioates at the 5' end. In some embodiments, there is a hydrophobic conjugate of 12 to 25 carbons attached to the 5' end via a phosphodiester bond.

[0021] I. Composition Disclosed herein in some embodiments is a composition comprising an oligonucleotide. In some embodiments, the composition comprises an oligonucleotide targeting a target oligonucleotide. The target oligonucleotide may comprise a target RNA. In some embodiments, the composition consists of an oligonucleotide targeting a target RNA. The target RNA may comprise a target mRNA. In some embodiments, the oligonucleotide reduces target mRNA expression in a subject. In some embodiments, the oligonucleotide reduces target protein expression in a subject. The oligonucleotide may comprise an RNA duplex. The oligonucleotide may comprise a small interfering RNA (siRNA). The oligonucleotide may comprise an antisense oligonucleotide (ASO). In some embodiments, the composition is used in a method of treating a disorder in a subject in need thereof. Some embodiments relate to a composition comprising an oligonucleotide for use in a method of treating a disorder. Some embodiments relate to the use of a composition comprising an oligonucleotide in a method of treating a disorder.

[0022] Targets can be identified by a variety of methods. In some cases, the target oligonucleotide comprises an mRNA that has an expression level associated with the occurrence of a disorder (e.g., an adipose-related or eye-related disorder). In some cases, the target oligonucleotide comprises an mRNA encoded by a gene that has a particular genotype associated with a disorder. Large-scale human genetic data can improve the success rate of drug discovery and development. Genome-wide association studies (GWAS) can detect associations between genetic variants and traits in population samples. GWAS can improve the biological understanding of disease and provide appropriate treatments. GWAS can utilize genotyping and / or sequencing data and often involve evaluating millions of genetic variants that are relatively evenly distributed across the genome. The most common GWAS design is a case-control study, which involves comparing variant frequencies in cases and controls. If a variant has a significantly different frequency in cases and controls, the variant is said to be associated with the disease. Association statistics that may be used in GWAS are p-values ​​as an indicator of statistical significance, odds ratios (ORs) as an indicator of effect size, or beta coefficients (β) as an indicator of effect size. Researchers often assume an additive genetic model and calculate allelic odds ratios, which indicate the increased (or decreased) risk of a given disease for each additional copy of an allele (compared to having no copies of that allele). An additional concept in the design and interpretation of GWAS is that of linkage disequilibrium, which is the non-random association of alleles. The presence of linkage disequilibrium can obscure which variants are "causative."

[0023] Functional annotation of variants and / or wet lab experiments are used to identify causative gene variants identified via GWAS, often leading to the identification of disease-causing genes. Notably, understanding the functional effect of a causative gene variant (e.g., loss of protein function, gain of protein function, increased gene expression, or decreased gene expression) can allow the variant to be used as a surrogate for therapeutic modulation of a target gene, gaining insight into the potential therapeutic efficacy and safety of therapeutics that modulate that target.

[0024] Identification of such gene-disease associations provides insight into disease biology and is used to identify novel therapeutic targets for the pharmaceutical industry. To translate therapeutic insights gained from human genetics, disease biology in patients is exogenously "programmed" to recapitulate observations from human genetics. There are several options for therapeutic modalities that can be added when applying therapeutic targets identified by human genetics to new drugs. These include well-established therapeutic modalities such as small molecules and monoclonal antibodies, mature modalities such as oligonucleotides, as well as new modalities such as gene therapy and gene editing. The choice of therapeutic modality depends on factors such as the location of the target (e.g., intracellular, extracellular, or secreted), the relevant tissue (e.g., lung or eye), and the relevant indication. Such studies may be performed to identify specific disorder-associated targets for siRNA inhibition by the compositions or compounds described herein. In some cases, the target may be associated with a specific tissue.

[0025] Disclosed herein is a composition comprising an oligonucleotide that targets a target RNA. When inhibition or targeting of a target RNA is disclosed, it is contemplated that some embodiments may include inhibiting or targeting a target protein or a target RNA. For example, the target protein may be inhibited or targeted by using the oligonucleotides described herein to inhibit or target the RNA (e.g., mRNA) encoded by a target gene, resulting in less production of the target protein by translation of the target RNA, or the target protein may be targeted or inhibited by an oligonucleotide that binds to or interacts with the target RNA and reduces the production of the target protein from the target RNA. Thus, targeting may refer to the binding of the target RNA and the reduction of the level of the target RNA or protein. The oligonucleotide may include a small interfering RNA (siRNA) or an antisense oligonucleotide (ASO). Also provided herein is a method of treating a target disorder by providing an oligonucleotide that targets a target to a subject in need.

[0026] Some embodiments include compositions comprising oligonucleotides that target mRNA and that, when administered to a subject in an effective amount, reduce the level of a target mRNA or protein in a cell, body fluid, or tissue. In some embodiments, the compositions comprise oligonucleotides that target a target RNA and that, when administered to a subject in an effective amount, reduce the level of a target mRNA in a cell or tissue. In some embodiments, the target mRNA level is reduced by about 2.5% or more, about 5% or more, or about 7.5% or more compared to before administration. In some embodiments, the target mRNA level is reduced by about 10% or more compared to before administration. In some embodiments, the target mRNA level is reduced by about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 100% compared to before administration. In some embodiments, the target mRNA level is reduced by about 2.5% or less, about 5% or less, or about 7.5% or less compared to before administration. In some embodiments, the target mRNA level is reduced by about 10% or less compared to before administration. In some embodiments, the target mRNA level is reduced by about 20% or less, about 30% or less, about 40% or less, about 50% or less, about 60% or less, about 70% or less, about 80% or less, or about 90% or less compared to before administration. In some embodiments, the target mRNA level is reduced by 2.5%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or a range defined by any two of the aforementioned percentages.

[0027] In some embodiments, the composition comprises an oligonucleotide that targets a target mRNA and reduces target protein levels in cells, body fluids, or tissues when administered to a subject in an effective amount. In some embodiments, the target protein levels are reduced by about 2.5% or more, about 5% or more, or about 7.5% or more compared to before administration. In some embodiments, the target protein levels are reduced by about 10% or more compared to before administration. In some embodiments, the target protein levels are reduced by about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 100% compared to before administration. In some embodiments, the target protein levels are reduced by about 2.5% or less, about 5% or less, or about 7.5% or less compared to before administration. In some embodiments, the target protein levels are reduced by about 10% or less compared to before administration. In some embodiments, the target protein level is reduced by about 20% or less, about 30% or less, about 40% or less, about 50% or less, about 60% or less, about 70% or less, about 80% or less, or about 90% or less compared to before administration. In some embodiments, the target protein level is reduced by 2.5%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or a range defined by any two of the foregoing percentages.

[0028] In some embodiments, the composition comprises an oligonucleotide that targets mRNA and reduces a disease phenotype when administered to a subject in an effective amount. In some embodiments, the disease phenotype is reduced by about 2.5% or more, about 5% or more, or about 7.5% or more compared to before administration. In some embodiments, the disease phenotype is reduced by about 10% or more compared to before administration. In some embodiments, the disease phenotype is reduced by about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 100% compared to before administration. In some embodiments, the disease phenotype is reduced by about 2.5% or less, about 5% or less, or about 7.5% or less compared to before administration. In some embodiments, the disease phenotype is reduced by about 10% or less compared to before administration. In some embodiments, the disease phenotype is reduced by about 20% or less, about 30% or less, about 40% or less, about 50% or less, about 60% or less, about 70% or less, about 80% or less, or about 90% or less compared to before administration. In some embodiments, the disease phenotype is reduced by 2.5%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or a range defined by any two of the foregoing percentages.

[0029] In some embodiments, the composition comprises an oligonucleotide that targets an mRNA and enhances a protective phenotype against a disease in a subject when administered to the subject in an effective amount. In some embodiments, the protective phenotype is increased by about 2.5% or more, about 5% or more, or about 7.5% or more compared to before administration. In some embodiments, the protective phenotype is increased by about 10% or more compared to before administration. In some embodiments, the protective phenotype is increased by about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 100% or more compared to before administration. In some embodiments, the protective phenotype is increased by about 200% or more, about 300% or more, about 400% or more, about 500% or more, about 600% or more, about 700% or more, about 800% or more, about 900% or more, or about 1000% or more compared to before administration. In some embodiments, the protective phenotype is increased by about 2.5% or less, about 5% or less, or about 7.5% or less compared to before administration. In some embodiments, the protective phenotype is increased by about 10% or less compared to before administration. In some embodiments, the protective phenotype is increased by about 20% or less, about 30% or less, about 40% or less, about 50% or less, about 60% or less, about 70% or less, about 80% or less, about 90% or less, or about 100% or less compared to before administration. In some embodiments, the protective phenotype is increased by about 200% or less, about 300% or less, about 400% or less, about 500% or less, about 600% or less, about 700% or less, about 800% or less, about 900% or less, or about 1000% or less compared to before administration. In some embodiments, the protective phenotype is increased by 2.5%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, or a range defined by any two of the foregoing percentages.

[0030] A. siRNA In some embodiments, the composition comprises an oligonucleotide that targets a target RNA, such as an mRNA, and the oligonucleotide comprises a small interfering RNA (siRNA). In some embodiments, the composition comprises an oligonucleotide that targets a target mRNA, and the oligonucleotide comprises a small interfering RNA (siRNA) that comprises a sense strand and an antisense strand.

[0031] In some embodiments, the composition comprises an oligonucleotide that inhibits expression of a target mRNA, the oligonucleotide comprises an siRNA comprising a sense strand and an antisense strand, the sense strand being 12-30 nucleosides in length. In some embodiments, the composition comprises a sense strand that is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleosides in length, or a range defined by any two of the preceding numbers. The sense strand may be 14-30 nucleosides in length. In some embodiments, the composition comprises an antisense strand that is 12-30 nucleosides in length. In some embodiments, the composition comprises an antisense strand that is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleosides in length, or a range defined by any two of the foregoing numbers. The antisense strand may be 14-30 nucleosides in length.

[0032] In some embodiments, the composition comprises an oligonucleotide that inhibits expression of a target mRNA, the oligonucleotide comprises an siRNA comprising a sense strand and an antisense strand, each strand being independently about 12-30 nucleosides in length, and at least one of the sense strand and the antisense strand comprises a nucleoside sequence comprising about 12-30 contiguous nucleosides of a full-length human target mRNA sequence. In some embodiments, at least one of the sense strand and the antisense strand comprises a nucleoside sequence comprising at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more contiguous nucleosides of the target RNA.

[0033] In some embodiments, the composition comprises an oligonucleotide that inhibits the expression of target mRNA, the oligonucleotide comprises an siRNA that comprises a sense strand and an antisense strand, and the sense strand and the antisense strand form a double-stranded RNA duplex.In some embodiments, the first base pair of the double-stranded RNA duplex is an AU base pair.

[0034] In some embodiments, the sense strand further comprises a 3' overhang. In some embodiments, the 3' overhang comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleosides, or a range of nucleotides defined by any two of the preceding numbers. In some embodiments, the 3' overhang comprises 1, 2, or more nucleosides. In some embodiments, the 3' overhang comprises 2 nucleosides. In some embodiments, the sense strand further comprises a 5' overhang. In some embodiments, the 5' overhang comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleosides, or a range of nucleotides defined by any two of the preceding numbers. In some embodiments, the 5' overhang comprises 1, 2, or more nucleosides. In some embodiments, the 5' overhang comprises 2 nucleosides.

[0035] In some embodiments, the antisense strand further comprises a 3' overhang. In some embodiments, the 3' overhang comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleosides, or a range of nucleotides defined by any two of the preceding numbers. In some embodiments, the 3' overhang comprises 1, 2, or more nucleosides. In some embodiments, the 3' overhang comprises 2 nucleosides. In some embodiments, the antisense strand further comprises a 5' overhang. In some embodiments, the 5' overhang comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleosides, or a range of nucleotides defined by any two of the preceding numbers. In some embodiments, the 5' overhang comprises 1, 2, or more nucleosides. In some embodiments, the 5' overhang comprises 2 nucleosides.

[0036] In some embodiments, the composition comprises the oligonucleotide that inhibits the expression of target mRNA, the oligonucleotide comprises siRNA that comprises sense strand and antisense strand, and siRNA binds to 19-mer in human target mRNA.In some embodiments, siRNA binds to 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer or 25-mer in human target mRNA.

[0037] In some embodiments, the composition comprises the oligonucleotide that inhibits the expression of target mRNA, the oligonucleotide comprises siRNA that comprises sense strand and antisense strand, and the siRNA binds to 17-mer in non-human primate target mRNA.In some embodiments, the siRNA binds to 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer or 25-mer in non-human primate target mRNA.

[0038] In some embodiments, the composition comprises an oligonucleotide that inhibits expression of a target mRNA, the oligonucleotide comprises an siRNA comprising a sense strand and an antisense strand, the siRNA binds to a human target mRNA and 20 or less human off-targets, and has no more than two mismatches in the antisense strand. In some embodiments, the siRNA binds to a human target mRNA and 10 or less human off-targets, and has no more than two mismatches in the antisense strand. In some embodiments, the siRNA binds to a human target mRNA and 30 or less human off-targets, and has no more than two mismatches in the antisense strand. In some embodiments, the siRNA binds to a human target mRNA and 40 or less human off-targets, and has no more than two mismatches in the antisense strand. In some embodiments, the siRNA binds to a human target mRNA and 50 or less human off-targets, and has no more than two mismatches in the antisense strand. In some embodiments, the siRNA binds to a human target mRNA and 10 or less human off-targets, and has no more than three mismatches in the antisense strand. In some embodiments, the siRNA binds to a human target mRNA and 20 or less human off-targets, and the antisense strand has no more than 3 mismatches. In some embodiments, the siRNA binds to a human target mRNA and 30 or less human off-targets, and the antisense strand has no more than 3 mismatches. In some embodiments, the siRNA binds to a human target mRNA and 40 or less human off-targets, and the antisense strand has no more than 3 mismatches. In some embodiments, the siRNA binds to a human target mRNA and 50 or less human off-targets, and the antisense strand has no more than 3 mismatches.

[0039] In some embodiments, the composition comprises an oligonucleotide that inhibits expression of a target mRNA, the oligonucleotide comprises an siRNA comprising a sense strand and an antisense strand, the siRNA binds to a human target mRNA target site that does not carry a SNP, and the minor allele frequency (MAF) is 1% or more (positions 2-18). In some embodiments, the MAF is about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 16% or more, about 17% or more, about 18% or more, about 19% or more, or about 20% or more.

[0040] B.ASO In some embodiments, the composition comprises an oligonucleotide that inhibits expression of a target mRNA, the oligonucleotide comprising an antisense oligonucleotide (ASO). In some embodiments, the ASO is 12-30 nucleosides in length. In some embodiments, the ASO is 14-30 nucleosides in length. In some embodiments, the ASO is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleosides in length, or a range defined by any two of the foregoing numbers. In some embodiments, the ASO is 15-25 nucleosides in length. In some embodiments, the ASO is 20 nucleosides in length.

[0041] In some embodiments, the composition comprises an oligonucleotide that inhibits expression of a target mRNA, the oligonucleotide comprises an ASO of about 12-30 nucleosides in length, comprising a nucleoside sequence complementary to about 12-30 contiguous nucleosides of a full-length human target mRNA sequence, (i) the oligonucleotide comprises modifications including modified nucleosides and / or modified internucleoside linkages, and / or (ii) the composition comprises a pharma- ceutically acceptable carrier. In some embodiments, the ASO comprises a nucleoside sequence complementary to at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more contiguous nucleosides of the target mRNA.

[0042] C. Qualification In some embodiments, the composition comprises an oligonucleotide that inhibits expression of a target mRNA, wherein the oligonucleotide comprises a modification comprising a modified nucleoside and / or a modified internucleoside linkage, and / or (ii) the composition comprises a pharma- ceutically acceptable carrier. In some embodiments, the oligonucleotide comprises a modification comprising a modified nucleoside and / or a modified internucleoside linkage. In some embodiments, the oligonucleotide comprises a modified internucleoside linkage. In some embodiments, the modified internucleoside linkage comprises an alkyl phosphonate, phosphorothioate, methyl phosphonate, phosphorodithioate, alkyl phosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, or carboxymethyl ester, or a combination thereof. In some embodiments, the modified internucleoside linkage comprises one or more phosphorothioate linkages. The phosphorothioate may comprise a non-bridging oxygen atom in the phosphate backbone of the oligonucleotide that is replaced with sulfur. The modified internucleoside linkage may be comprised in an siRNA or an ASO. Advantages of modified internucleoside linkages may include reduced toxicity or improved pharmacokinetics.

[0043] In some embodiments, the composition comprises an oligonucleotide that inhibits expression of a target mRNA, the oligonucleotide comprises a modified internucleoside linkage, the oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 modified internucleoside linkages, or a range defined by any two of the preceding numbers. In some embodiments, the oligonucleotide comprises 18 or fewer modified internucleoside linkages. In some embodiments, the oligonucleotide comprises 20 or fewer modified internucleoside linkages. In some embodiments, the oligonucleotide has 2 or more modified internucleoside linkages, 3 or more modified internucleoside linkages, 4 or more modified internucleoside linkages, 5 or more modified internucleoside linkages, 6 or more modified internucleoside linkages, 7 or more modified internucleoside linkages, 8 or more modified internucleoside linkages, 9 or more modified internucleoside linkages, 10 or more modified internucleoside linkages, 11 or more modified internucleoside linkages, The modified internucleoside linkages include the above modified internucleoside linkages, 12 or more modified internucleoside linkages, 13 or more modified internucleoside linkages, 14 or more modified internucleoside linkages, 15 or more modified internucleoside linkages, 16 or more modified internucleoside linkages, 17 or more modified internucleoside linkages, 18 or more modified internucleoside linkages, 19 or more modified internucleoside linkages, or 20 or more modified internucleoside linkages.

[0044] In some embodiments, the composition comprises an oligonucleotide that inhibits expression of a target mRNA, the oligonucleotide comprises a modified nucleoside. In some embodiments, the modified nucleoside comprises a locked nucleic acid (LNA), a hexitol nucleic acid (HLA), a cyclohexene nucleic acid (CeNA), a 2'-methoxyethyl, a 2'-O-alkyl, a 2'-O-allyl, a 2'fluoro, or a 2'-deoxy, or a combination thereof. In some embodiments, the modified nucleoside comprises an LNA. In some embodiments, the modified nucleoside comprises a 2',4' constrained ethyl nucleic acid. In some embodiments, the modified nucleoside comprises an HLA. In some embodiments, the modified nucleoside comprises a CeNA. In some embodiments, the modified nucleoside comprises a 2'-methoxyethyl group. In some embodiments, the modified nucleoside comprises a 2'-O-alkyl group. In some embodiments, the modified nucleoside comprises a 2'-methoxyethyl. In some embodiments, the modified nucleoside comprises a methoxyethyl. For example, position 4 of the sense strand may include a methoxyethyl nucleoside, such as 2'-methoxyethylthymine. In some embodiments, the modified nucleoside includes a 2'-O-methyl. In some embodiments, the modified nucleoside includes a 2'-O-allyl group. In some embodiments, the modified nucleoside includes a 2'fluoro group. In some embodiments, the modified nucleoside includes a 2'-deoxy group. In some embodiments, the modified nucleoside includes a 2'-O-methyl nucleoside, a 2'-deoxyfluoro nucleoside, a 2'-ON-methylacetamide (2'-O-NMA) nucleoside, a 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleoside, a 2'-O-aminopropyl (2'-O-AP) nucleoside, or a 2'-ara-F, or a combination thereof. In some embodiments, the modified nucleoside includes a 2'-O-methyl nucleoside. In some embodiments, the modified nucleoside comprises a 2'-deoxyfluoronucleoside. In some embodiments, the modified nucleoside comprises a 2'-O-NMA nucleoside. In some embodiments, the modified nucleoside comprises a 2'-O-DMAEOE nucleoside.In some embodiments, the modified nucleoside comprises a 2'-O-aminopropyl (2'-O-AP) nucleoside. In some embodiments, the modified nucleoside comprises a 2'-ara-F. In some embodiments, the modified nucleoside comprises one or more 2'fluoro modified nucleosides. In some embodiments, the modified nucleoside comprises a 2'O-alkyl modified nucleoside. Advantages of the modified nucleoside may include reduced toxicity or improved pharmacokinetics.

[0045] In some embodiments, the oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 modified nucleosides, or a range defined by any two of the preceding numbers. In some embodiments, the oligonucleotide comprises 19 or fewer modified nucleosides. In some embodiments, the oligonucleotide comprises 21 or fewer modified nucleosides. In some embodiments, an oligonucleotide comprises 2 or more modified nucleosides, 3 or more modified nucleosides, 4 or more modified nucleosides, 5 or more modified nucleosides, 6 or more modified nucleosides, 7 or more modified nucleosides, 8 or more modified nucleosides, 9 or more modified nucleosides, 10 or more modified nucleosides, 11 or more modified nucleosides, 12 or more modified nucleosides, 13 or more modified nucleosides, 14 or more modified nucleosides, 15 or more modified nucleosides, 16 or more modified nucleosides, 17 or more modified nucleosides, 18 or more modified nucleosides, 19 or more modified nucleosides, 20 or more modified nucleosides, or 21 or more modified nucleosides.

[0046] In some embodiments, the composition comprises an oligonucleotide that inhibits expression of a target mRNA, the oligonucleotide comprises a moiety attached to the 3' or 5' end of the oligonucleotide. Exemplary moieties include hydrophobic moieties or sugar moieties, or combinations thereof. In some embodiments, the oligonucleotide is an siRNA having a sense strand, and the moiety is attached to the 5' end of the sense strand. In some embodiments, the oligonucleotide is an siRNA having a sense strand, and the moiety is attached to the 3' end of the sense strand. In some embodiments, the oligonucleotide is an siRNA having an antisense strand, and the moiety is attached to the 5' end of the antisense strand. In some embodiments, the oligonucleotide is an siRNA having an antisense strand, and the moiety is attached to the 3' end of the antisense strand. In some embodiments, the oligonucleotide is an ASO, and the moiety is attached to the 5' end of the ASO. In some embodiments, the oligonucleotide is an ASO, and the moiety is attached to the 3' end of the ASO.

[0047] In some embodiments, the composition includes an oligonucleotide that inhibits expression of a target mRNA, the oligonucleotide includes a hydrophobic moiety. An oligonucleotide that includes a hydrophobic moiety may include or be referred to as a hydrophobic conjugate. The hydrophobic moiety may be useful for enhancing cellular uptake. The hydrophobic moiety may be attached to the 3' or 5' end of the oligonucleotide. The hydrophobic moiety may include a lipid, such as a fatty acid. The hydrophobic moiety may include a hydrocarbon. The hydrocarbon may be linear. The hydrocarbon may be non-linear. The hydrophobic moiety may include a lipid moiety or a cholesterol moiety, or a combination thereof. In some embodiments, the hydrophobic moiety includes a cyclohexanyl. In some embodiments, the hydrophobic moiety includes a lipid. In some embodiments, the hydrophobic moiety is used in a specific format described herein. In some embodiments, the hydrophobic moiety is attached to the 5' end of the sense strand without a phosphorothioate group or phosphorothioate linkage at the 5' end of the sense strand. The hydrophobic moiety may include an esterified lipid.

[0048] The hydrophobic moiety may be or may include a lipid moiety. In some embodiments, the composition includes an oligonucleotide that inhibits expression of a target mRNA, the oligonucleotide includes a lipid attached to the 3' or 5' end of the oligonucleotide. In some embodiments, the lipid includes cholesterol, myristyl, palmityl, stearyl, lithocholyl, docosanoyl, docosahexaenoyl, myristyl, palmitylstearyl, or α-tocopherol, or a combination thereof. In some embodiments, the lipid includes stearol, t-butylphenol, n-butylphenol, octylphenyl, dodecylphenyl, phenyl n-dodecyl, octadecylbenzamide, hexadecylbenzamide, or octadecylcyclohexyl. In some embodiments, the lipid includes phenyl para C12. The lipid moiety may be esterified. In some embodiments, the oligonucleotide has the following structure:

[0049] [ka] In some embodiments, R is not an octane. In some embodiments, R is not an octane. In some embodiments, R is an alkyl group containing 4-7 or 9-18 carbons. In some embodiments, the oligonucleotide has the following structure:

[0050] [ka] In some embodiments, the oligonucleotide has the following structure:

[0051] [ka] In some embodiments, the oligonucleotide has the following structure:

[0052] [ka] The embodiments of the oligonucleotides include the entire structure or the lipid portion of any of the structures shown. In some embodiments, n is 1-3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, R is an alkyl group. In some embodiments, the alkyl group contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbons. In some embodiments, the alkyl group contains 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 carbons, or a range of carbons defined by any two of the preceding numbers. In some embodiments, the alkyl group contains 4-18 carbons. In some embodiments, R is not octane (C8). In some embodiments, R is not C8. 4-7 In some embodiments, R is C 9-18 In some embodiments, R is C 4-7 Alkyl and C 9-18In some embodiments, R is an alkyl group. In some embodiments, the alkyl group contains 11 carbons. In some embodiments, the alkyl group contains 12 carbons. In some embodiments, the alkyl group contains 13 carbons. In some embodiments, the alkyl group contains 14 carbons. In some embodiments, the alkyl group contains 15 carbons. In some embodiments, the alkyl group contains 16 carbons. In some embodiments, the alkyl group contains 17 carbons. In some embodiments, the alkyl group contains 18 carbons. In some embodiments, R is not octane (C8). In some embodiments, R comprises a branched carbon chain. In some embodiments, R comprises an unbranched carbon chain. In some embodiments, the lipid moiety comprises an alcohol or ether. In some embodiments, the degree of unsaturation of the lipid moiety is at least 1. In some embodiments, the lipid moiety is an omega fatty acid, such as an omega-3, omega-5, omega-6, omega-7, or omega-9 fatty acid.

[0053] In some embodiments, the lipid comprises a fatty acid. In some embodiments, the lipid comprises a lipid as shown in Table 1. The exemplary lipid moieties in Table 1 are shown attached to the 5' end of the oligonucleotide, and the 5' terminal phosphate of the oligonucleotide is shown with the lipid moiety. In some embodiments, the lipid moieties in Table 1 may be attached to a different attachment point than shown. For example, the attachment point of any of the lipid moieties in the table may be at the 3' oligonucleotide end. In some embodiments, the lipid is used to target the oligonucleotide to a non-hepatic cell or tissue.

[0054] [Table 1-1]

[0055] [Table 1-2]

[0056] In some embodiments, the lipid or lipid moiety contains 16-18 carbons. In some embodiments, the lipid contains 16 carbons. In some embodiments, the lipid contains 17 carbons. In some embodiments, the lipid contains 18 carbons. In some embodiments, the lipid moiety contains 16 carbons. In some embodiments, the lipid moiety contains 17 carbons. In some embodiments, the lipid moiety contains 18 carbons. In some embodiments, the lipid moiety contains 19 carbons. In some embodiments, the lipid moiety contains 20 carbons.

[0057] The hydrophobic moiety may include a linker that includes a carbocycle. The carbocycle may be six-membered. Some examples of carbocycles include phenyl or cyclohexyl. The linker may include phenyl. The linker may include cyclohexyl. The lipid may be attached to the carbocycle, which may in turn be attached to the phosphate (e.g., the 5' or 3' phosphate) of the oligonucleotide. In some embodiments, the lipid or hydrocarbon and the end of the sense strand are attached to the phenyl or cyclohexyl linker in a 1,4, 1,3, or 1,2 substitution pattern (e.g., para, meta, or ortho phenyl configuration). In some embodiments, the lipid or hydrocarbon and the end of the sense strand are attached to the phenyl or cyclohexyl linker in a 1,4 substitution pattern (e.g., para phenyl configuration). The lipid may be attached to the carbocycle in an ortho orientation relative to the oligonucleotide. The lipid may be attached to the carbocycle in a para orientation relative to the oligonucleotide. The lipid may be attached to the carbocycle in a meta orientation relative to the oligonucleotide. The lipid may be attached to the carbocycle in a 1,4 orientation relative to the oligonucleotide. The lipid may be attached to the carbocycle in a 1,3 orientation relative to the oligonucleotide.The lipid may be attached to the carbocycle in a 1,2 orientation relative to the oligonucleotide.

[0058] The lipid portion has the following structure:

[0059] [ka] In some embodiments, the lipid moiety may comprise or consist of the following structure:

[0060] [ka] In some embodiments, the lipid moiety comprises or consists of the structure:

[0061] [ka] In some embodiments, the lipid moiety comprises or consists of the structure:

[0062] [ka] or consists of the structure above. In some embodiments, the dashed line indicates a covalent bond. The covalent bond may be between the ends of the sense strand or the antisense strand. For example, the bond may be to the 5' end of the sense strand. In some embodiments, n is 0-3. In some embodiments, n is 1-3. In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, R comprises a branched carbon chain. In some embodiments, R comprises an unbranched carbon chain. In some embodiments, R is an alkyl group. In some embodiments, the alkyl group contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbons. In some embodiments, the alkyl group contains 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 carbons, or a range defined by any two of the preceding numbers. In some embodiments, the alkyl group contains 11 carbons. In some embodiments, the alkyl group contains 12 carbons. In some embodiments, the alkyl group contains 13 carbons. In some embodiments, the alkyl group contains 14 carbons. In some embodiments, the alkyl group contains 15 carbons. In some embodiments, the alkyl group contains 16 carbons. In some embodiments, the alkyl group contains 17 carbons. In some embodiments, the alkyl group contains 18 carbons. In some embodiments, R comprises or consists of an alkyl group containing 4 to 18 carbons. In some embodiments, the lipid moiety is not a phenyloctyl group. In some embodiments, R is not octane. In some embodiments, R is C 4-7 In some embodiments, R is C 9-18In some embodiments, R is C 4-7 Alkyl and C 9-18 In some embodiments, the lipid moiety is not a phenyloctyl group.

[0063] In some embodiments, the 5' hydrophobic portion has the following structure:

[0064] [ka] where the dashed line indicates a covalent bond to the 5' end of the sense strand, n is 1-3, and R is an alkyl group containing 4-18 carbons. In some embodiments, R is not octane. In some embodiments, the alkyl group contains 4-7 or 9-18 carbons. In some embodiments, the alkyl group contains 14 carbons. In some embodiments, the alkyl group contains 15 carbons. In some embodiments, the alkyl group contains 16 carbons. In some embodiments, the alkyl group contains 17 carbons. In some embodiments, the alkyl group contains 18 carbons. In some embodiments, the 5' hydrophobic portion comprises a hydrophobic portion of Table 1. In some embodiments, the 5' hydrophobic portion comprises a phenyl para C12. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In some embodiments, n is 0-3. In some embodiments, n is 1-3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, the hydrophobic moiety comprises an alcohol or an ether. In some embodiments, R is an unsaturated alkyl group. In some embodiments, the unsaturated alkyl group may be monounsaturated. In some embodiments, the unsaturated alkyl group may be unsaturated at the omega 3, omega 4, omega 5, omega 6, omega 7, omega 8, omega 9 positions, or combinations thereof. In some embodiments, the 5' hydrophobic moiety is not a phenyloctyl group.

[0065] The hydrophobic moiety may include a linker that includes a carbocycle. The carbocycle may be six-membered. Some examples of carbocycles include phenyl or cyclohexyl. The linker may include phenyl. The linker may include cyclohexyl. The lipid may be attached to the carbocycle, which may in turn be attached to the phosphate (e.g., the 5' or 3' phosphate) of the oligonucleotide. In some embodiments, the lipid or hydrocarbon and the end of the sense strand are attached to the phenyl or cyclohexyl linker in a 1,4, 1,3, or 1,2 substitution pattern (e.g., para, meta, or ortho phenyl configuration). In some embodiments, the lipid or hydrocarbon and the end of the sense strand are attached to the phenyl or cyclohexyl linker in a 1,4 substitution pattern (e.g., para phenyl configuration). The lipid may be attached to the carbocycle in an ortho orientation relative to the oligonucleotide. The lipid may be attached to the carbocycle in a para orientation relative to the oligonucleotide. The lipid may be attached to the carbocycle in a meta orientation relative to the oligonucleotide. The lipid may be attached to the carbocycle in a 1,4 orientation relative to the oligonucleotide. The lipid may be attached to the carbocycle in a 1,3 orientation relative to the oligonucleotide.The lipid may be attached to the carbocycle in a 1,2 orientation relative to the oligonucleotide.

[0066] The lipid moiety may be attached to the 5' end of the oligonucleotide. The 5' end may have one phosphate attached to the lipid moiety to the 5' carbon of the sugar of the oligonucleotide. The 5' end may have two phosphates attached to the lipid moiety to the 5' carbon of the sugar of the oligonucleotide. The 5' end may have three phosphates attached to the lipid moiety to the 5' carbon of the sugar of the oligonucleotide. The 5' end may have one phosphate attached to the 5' carbon of the sugar of the oligonucleotide, where one phosphate is attached to a lipid moiety. The 5' end may have two phosphates attached to the 5' carbon of the sugar of the oligonucleotide, where one of the two phosphates is attached to a lipid moiety. The 5' end may have three phosphates attached to the 5' carbon of the sugar of the oligonucleotide, where one of the three phosphates is attached to a lipid moiety. The sugar may comprise a ribose. The sugar may comprise a deoxyribose. The sugar may be modified, such as a 2' modified sugar (e.g., 2'O-methyl or 2'fluororibose). The 5' terminal phosphate may contain a modification, such as a sulfur instead of an oxygen. The 5' terminal two phosphates may contain a modification, such as a sulfur instead of an oxygen. The 5' terminal three phosphates may contain a modification, such as a sulfur instead of an oxygen. In some embodiments, the oligonucleotide is not d(pT-GGGGGG).

[0067] In some embodiments, the oligonucleotide comprises one lipid moiety. In some embodiments, the oligonucleotide comprises two lipid moieties. In some embodiments, the oligonucleotide comprises three lipid moieties. In some embodiments, the oligonucleotide comprises four lipid moieties.

[0068] Some embodiments relate to a method for making oligonucleotides that contain hydrophobic conjugates. The strategy for making hydrophobic conjugates may include the use of phosphoramidite reagents based on 6-membered alcohols such as phenol or cyclohexanol. The phosphoramidite may react with nucleotides to bind the nucleotides to hydrophobic moieties, thereby producing hydrophobic conjugates. Some examples of phosphoramidite reagents that can be used to produce hydrophobic conjugates are provided below.

[0069] [ka] In some embodiments, n is 1-3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, R is an alkyl group. In some embodiments, the alkyl group contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbons. In some embodiments, the alkyl group contains 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 carbons, or a range of carbons defined by any two of the preceding numbers. In some embodiments, R comprises or consists of an alkyl group containing 4-18 carbons. Any one of the phosphoramidite reagents may be reacted to the 5' end of the oligonucleotide to generate an oligonucleotide containing a hydrophobic moiety. In some embodiments, the phosphoramidite reagent is reacted to the 5' end of the sense strand of the siRNA. Then, the sense strand may be hybridized with the antisense strand to form a double strand.Hybridization may be performed by incubating the sense strand and the antisense strand in a solution at a predetermined temperature.The temperature may be gradually decreased.The temperature may include or may include the temperature that includes the annealing temperature of the sense strand and the antisense strand.The temperature may be below or may include the temperature that is below the annealing temperature of the sense strand and the antisense strand.The temperature may be below the melting temperature of the sense strand and the antisense strand.

[0070] In some embodiments, the oligonucleotide comprises a negatively charged group. The negatively charged group can aid in cell or tissue penetration. The negatively charged group may be attached to the 5' or 3' end (e.g., the 5' end) of the oligonucleotide. This is sometimes referred to as a terminal group. The terminal group may be or include phosphorothioate, phosphorodithioate, vinyl phosphonate, methyl phosphonate, cyclopropyl phosphonate, or deoxy-C-malonyl. The terminal group may include an extra 5' phosphate, e.g., an extra 5' phosphate. A combination of terminal groups may be used.

[0071] In some embodiments, the oligonucleotide comprises a phosphomimetic. In some embodiments, the phosphomimetic comprises a vinyl phosphonate. In some embodiments, the vinyl phosphonate comprises a trans-vinyl phosphonate. In some embodiments, the vinyl phosphonate comprises a cis-vinyl phosphonate. Examples of nucleotides that comprise vinyl phosphonates are shown below.

[0072] [ka]

[0073] In some embodiments, vinyl phosphonate increases the stability of oligonucleotide.In some embodiments, vinyl phosphonate increases the accumulation of oligonucleotide in tissue.In some embodiments, vinyl phosphonate protects oligonucleotide from exonucleases or phosphatases.In some embodiments, vinyl phosphonate improves the binding affinity of oligonucleotide with siRNA processing machinery.

[0074] In some embodiments, the oligonucleotide comprises one vinyl phosphonate. In some embodiments, the oligonucleotide comprises two vinyl phosphonates. In some embodiments, the oligonucleotide comprises three vinyl phosphonates. In some embodiments, the oligonucleotide comprises four vinyl phosphonates. In some embodiments, the antisense strand of the oligonucleotide comprises a vinyl phosphonate at the 5' end. In some embodiments, the antisense strand of the oligonucleotide comprises a vinyl phosphonate at the 3' end. In some embodiments, the sense strand of the oligonucleotide comprises a vinyl phosphonate at the 5' end. In some embodiments, the sense strand of the oligonucleotide comprises a vinyl phosphonate at the 3' end.

[0075] The oligonucleotide may include purines. Examples of purines include adenine (A) or guanine (G), or modified versions thereof. The oligonucleotide may include pyrimidines. Examples of pyrimidines include cytosine (C), thymine (T), or uracil (U), or modified versions thereof.

[0076] In some embodiments, the purines of the oligonucleotide comprise 2'fluoro modified purines. In some embodiments, the purines of the oligonucleotide comprise 2'-O-methyl modified purines. In some embodiments, the purines of the oligonucleotide comprise a mixture of 2'fluoro and 2'-O-methyl modified purines. In some embodiments, all of the purines of the oligonucleotide comprise 2'fluoro modified purines. In some embodiments, all of the purines of the oligonucleotide comprise 2'-O-methyl modified purines. In some embodiments, all of the purines of the oligonucleotide comprise a mixture of 2'fluoro and 2'-O-methyl modified purines. In some embodiments, 2'-O-methyl comprises 2'O-methyl.

[0077] In some embodiments, the pyrimidines of the oligonucleotide comprise 2'fluoro modified pyrimidines. In some embodiments, the pyrimidines of the oligonucleotide comprise 2'-O-methyl modified pyrimidines. In some embodiments, the pyrimidines of the oligonucleotide comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines. In some embodiments, all pyrimidines of the oligonucleotide comprise 2'fluoro modified pyrimidines. In some embodiments, all pyrimidines of the oligonucleotide comprise 2'-O-methyl modified pyrimidines. In some embodiments, all pyrimidines of the oligonucleotide comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines.

[0078] In some embodiments, the purines of the oligonucleotide comprise 2'fluoro modified purines and the pyrimidines of the oligonucleotide comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines. In some embodiments, the purines of the oligonucleotide comprise 2'-O-methyl modified purines and the pyrimidines of the oligonucleotide comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines. In some embodiments, the purines of the oligonucleotide comprise 2'fluoro modified purines and the pyrimidines of the oligonucleotide comprise 2'-O-methyl modified pyrimidines. In some embodiments, the purines of the oligonucleotide comprise 2'-O-methyl modified purines and the pyrimidines of the oligonucleotide comprise 2'fluoro modified pyrimidines. In some embodiments, the pyrimidines of the oligonucleotide comprise 2'fluoro modified pyrimidines and the purines of the oligonucleotide comprise a mixture of 2'fluoro and 2'-O-methyl modified purines. In some embodiments, the pyrimidines of the oligonucleotide comprise 2'-O-methyl modified pyrimidines and the purines of the oligonucleotide comprise a mixture of 2'fluoro and 2'-O-methyl modified purines. In some embodiments, the pyrimidines of the oligonucleotide comprise 2'-fluoro modified pyrimidines and the purines of the oligonucleotide comprise 2'-O-methyl modified purines. In some embodiments, the pyrimidines of the oligonucleotide comprise 2'-O-methyl modified pyrimidines and the purines of the oligonucleotide comprise 2'-fluoro modified purines.

[0079] In some embodiments, all purines of the oligonucleotide comprise 2'fluoro modified purines and all pyrimidines of the oligonucleotide comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines. In some embodiments, all purines of the oligonucleotide comprise 2'-O-methyl modified purines and all pyrimidines of the oligonucleotide comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines. In some embodiments, all purines of the oligonucleotide comprise 2'fluoro modified purines and all pyrimidines of the oligonucleotide comprise 2'-O-methyl modified pyrimidines. In some embodiments, all purines of the oligonucleotide comprise 2'-O-methyl modified purines and all pyrimidines of the oligonucleotide comprise 2'fluoro modified pyrimidines. In some embodiments, all pyrimidines of the oligonucleotide comprise 2'fluoro modified pyrimidines and all purines of the oligonucleotide comprise a mixture of 2'fluoro and 2'-O-methyl modified purines. In some embodiments, all pyrimidines of the oligonucleotide comprise 2'-O-methyl modified pyrimidines and all purines of the oligonucleotide comprise a mixture of 2'fluoro and 2'-O-methyl modified purines. In some embodiments, all of the pyrimidines of the oligonucleotide comprise 2'-fluoro modified pyrimidines and all of the purines of the oligonucleotide comprise 2'-O-methyl modified purines. In some embodiments, all of the pyrimidines of the oligonucleotide comprise 2'-O-methyl modified pyrimidines and all of the purines of the oligonucleotide comprise 2'-fluoro modified purines.

[0080] In some embodiments, position 9 of the sense strand comprises a 2'fluoro modified pyrimidine. In some embodiments, all purines of the sense strand comprise 2'-O-methyl modified purines. In some embodiments, 1, 2, 3, 4, or 5 pyrimidines between positions 5 and 11 comprise 2'fluoro modified pyrimidines, provided that there are not three 2'fluoro modified pyrimidines in a row. In some embodiments, odd-numbered positions of the antisense strand comprise 2'-O-methyl modified nucleotides. In some embodiments, even-numbered positions of the antisense strand comprise 2'fluoro modified nucleotides and unmodified deoxyribonucleotides. In some embodiments, position 9 of the sense strand comprises a 2' fluoro modified pyrimidine, all purines in the sense strand comprise 2'-O-methyl modified purines, 1, 2, 3, 4, or 5 pyrimidines between positions 5 and 11 comprise 2' fluoro modified pyrimidines, provided that there are not three 2' fluoro modified pyrimidines in a row, odd-numbered positions of the antisense strand comprise 2'-O-methyl modified nucleotides, and even-numbered positions of the antisense strand comprise 2' fluoro modified nucleotides and unmodified deoxyribonucleotides.

[0081] In some embodiments, position 9 of the sense strand comprises a 2'fluoro modified purine. In some embodiments, all pyrimidines of the sense strand comprise a 2'-O-methyl modified purine. In some embodiments, 1, 2, 3, 4, or 5 purines between positions 5 and 11 comprise a 2'fluoro modified purine, provided that there are not three 2'fluoro modified purines in a row. In some embodiments, odd-numbered positions of the antisense strand comprise 2'-O-methyl modified nucleotides. In some embodiments, even-numbered positions of the antisense strand comprise 2'fluoro modified nucleotides and unmodified deoxyribonucleotides. In some embodiments, position 9 of the sense strand comprises a 2'fluoro modified purine, all pyrimidines of the sense strand comprise 2'-O-methyl modified pyrimidines, 1, 2, 3, 4, or 5 purines between positions 5 and 11 comprise 2'fluoro modified purines, provided that there are no three 2'fluoro modified purines in a row, the odd positions of the antisense strand comprise 2'-O-methyl modified nucleotides, and the even positions of the antisense strand comprise 2'fluoro modified nucleotides and unmodified deoxyribonucleotides. In some embodiments, there are no three 2'fluoro modified purines in a row. In some embodiments, there are no three 2'fluoro modified pyrimidines in a row.

[0082] In some embodiments, position 9 of the sense strand comprises unmodified deoxyribonucleotides. In some embodiments, positions 5, 7, and 8 of the sense strand comprise 2'fluoro modified nucleotides. In some embodiments, all pyrimidines at positions 10-21 of the sense strand comprise 2'-O-methyl modified pyrimidines and all purines at positions 10-21 of the sense strand comprise 2'-O-methyl modified purines or 2'fluoro modified purines. In some embodiments, odd-numbered positions of the antisense strand comprise 2'-O-methyl modified nucleotides. In some embodiments, even-numbered positions of the antisense strand comprise 2'fluoro modified nucleotides and unmodified deoxyribonucleotides. In some embodiments, position 9 of the sense strand comprises unmodified deoxyribonucleotides, positions 5, 7, and 8 of the sense strand comprise 2' fluoro modified nucleotides, all pyrimidines at positions 10-21 of the sense strand comprise 2'-O-methyl modified pyrimidines and all purines at positions 10-21 comprise 2'-O-methyl modified purines or 2' fluoro modified purines, the odd positions of the antisense strand comprise 2'-O-methyl modified nucleotides, and the even positions of the antisense strand comprise 2' fluoro modified nucleotides and unmodified deoxyribonucleotides.

[0083] In some embodiments, position 9 of the sense strand comprises unmodified deoxyribonucleotides. In some embodiments, positions 5, 7, and 8 of the sense strand comprise 2'fluoro modified nucleotides. In some embodiments, all purines at positions 10-21 of the sense strand comprise 2'-O-methyl modified purines and all pyrimidines at positions 10-21 comprise 2'-O-methyl modified pyrimidines or 2'fluoro modified pyrimidines. In some embodiments, odd-numbered positions of the antisense strand comprise 2'-O-methyl modified nucleotides. In some embodiments, even-numbered positions of the antisense strand comprise 2'fluoro modified nucleotides and unmodified deoxyribonucleotides. In some embodiments, position 9 of the sense strand comprises unmodified deoxyribonucleotides, positions 5, 7, and 8 of the sense strand comprise 2' fluoro modified nucleotides, all purines at positions 10-21 of the sense strand comprise 2'-O-methyl modified purines and all pyrimidines at positions 10-21 comprise 2'-O-methyl modified pyrimidines or 2' fluoro modified pyrimidines, the odd positions of the antisense strand comprise 2'-O-methyl modified nucleotides, and the even positions of the antisense strand comprise 2' fluoro modified nucleotides and unmodified deoxyribonucleotides.

[0084] The modifications described herein may be useful for delivery, e.g., extrahepatic delivery or targeting of oligonucleotide compositions to cells or tissues. The modifications described herein may be useful for targeting oligonucleotide compositions to cells or tissues.

[0085] 1. siRNA In some embodiments, the composition comprises an oligonucleotide that inhibits expression of a target nucleic acid, the oligonucleotide comprises an siRNA comprising a sense strand and an antisense strand, the sense strand comprises a modification pattern 1S:5'-NfsnNfnNfnNfNfNfNfnNfnNfnNfnNfnNfnNfsnsn-3', where "Nf" is a 2' fluoro-modified nucleoside, "n" is a 2' O-methyl-modified nucleoside, and "s" is a phosphorothioate linkage. In some embodiments, the sense strand comprises a modification pattern 2S:5'-NfsnNfnNfnNfnNfnNfnNfnNfnNfnNfnNfnNfsnsn-3', where "Nf" is a 2' fluoro-modified nucleoside, "n" is a 2' O-methyl-modified nucleoside, and "s" is a phosphorothioate linkage. In some embodiments, the sense strand comprises the modification pattern 3S:5'-NfnNfnNfnNfnNfnNfnNfnNfnNfnNfsnsn-3', where "Nf" is a 2'fluoro modified nucleoside, "n" is a 2'O-methyl modified nucleoside, and "s" is a phosphorothioate linkage. In some embodiments, the sense strand comprises the modification pattern 4S:5'-NfsnsNfnNfnNfNfNfNfNfNfnNfnNfnNfnNfnNfsnsn-3', where "Nf" is a 2'fluoro modified nucleoside, "n" is a 2'O-methyl modified nucleoside, "s" is a phosphorothioate linkage, and N comprises one or more nucleosides. In some embodiments, the sense strand comprises the modification pattern 5S:5'-nnnnnnNfnNfnnnnnnnnnnsnsn-3', where "Nf" is a 2'fluoro modified nucleoside, "n" is a 2'O-methyl modified nucleoside, "s" is a phosphorothioate linkage, and N comprises one or more nucleosides. In some embodiments, the sense strand comprises the modification pattern 6S:5'-nnnnnnNfNfNfNfnnnnnnnnsnsn-3', where "Nf" is a 2'fluoro modified nucleoside, "n" is a 2'O-methyl modified nucleoside, "s" is a phosphorothioate linkage, and N comprises one or more nucleosides.In some embodiments, the sense strand comprises the modification pattern 7S:5'-nnnnNfnnnNfnNfnnnnnnnnsnsn-3', where "Nf" is a 2' fluoro modified nucleoside, "n" is a 2' O-methyl modified nucleoside, "s" is a phosphorothioate linkage, and N comprises one or more nucleosides. In some embodiments, the sense strand comprises the modification pattern 8S:5'-nnnnnnnNfNfnNfnnnnnnnnsnsn-3', where "Nf" is a 2' fluoro modified nucleoside, "n" is a 2' O-methyl modified nucleoside, "s" is a phosphorothioate linkage, and N comprises one or more nucleosides. In some embodiments, the sense strand comprises the modification pattern 9S:5'-nnnnnnNfNfNfNfNfnnnnnnnnsnsn-3', where "Nf" is a 2' fluoro modified nucleoside, "n" is a 2' O-methyl modified nucleoside, "s" is a phosphorothioate linkage, and N comprises one or more nucleosides. In some embodiments, the sense strand comprises the modification pattern 10S:5'-nnnnNfNfNfNfNfNfnnnnnnnnnsnsn-3', where "Nf" is a 2' fluoro modified nucleoside, "n" is a 2' O-methyl modified nucleoside, "s" is a phosphorothioate linkage, and N comprises one or more nucleosides. In some embodiments, the sense strand comprises the modification pattern 11S:5'-nnnnnNfNfNfNfNfnnnnnnnnsnsn-3', where "Nf" is a 2' fluoro modified nucleoside, "n" is a 2' O-methyl modified nucleoside, "s" is a phosphorothioate linkage, and N comprises one or more nucleosides. In some embodiments, the sense strand comprises the modification pattern 12S:5'-nnnnnNfNfNfNfnnnnnnnnnnsnsn-3', where "Nf" is a 2' fluoro modified nucleoside, "n" is a 2' O-methyl modified nucleoside, "s" is a phosphorothioate linkage, and N comprises one or more nucleosides.In some embodiments, the sense strand comprises the modification pattern 13S:5'-nnnnNfNfNfNfNfnnnnnnnnnnsnsn-3', where "Nf" is a 2' fluoro modified nucleoside, "n" is a 2' O-methyl modified nucleoside, "s" is a phosphorothioate linkage, and N comprises one or more nucleosides. In some embodiments, the sense strand comprises the modification pattern 14S:5'-nnnnnnnnNfNfnnnnnnnnnsnsn-3', where "Nf" is a 2' fluoro modified nucleoside, "n" is a 2' O-methyl modified nucleoside, "s" is a phosphorothioate linkage, and N comprises one or more nucleosides. In some embodiments, the sense strand comprises the modification pattern 15S:5'-nnnnnnNfNfNfNfnnnnnnnnnsnsn-3', where "Nf" is a 2' fluoro modified nucleoside, "n" is a 2' O-methyl modified nucleoside, "s" is a phosphorothioate linkage, and N comprises one or more nucleosides. In some embodiments, the sense strand comprises the modification pattern 16S:5'-nnnnNfNfnnNfnNfnnnnnnnnsnsn-3', where "Nf" is a 2' fluoro modified nucleoside, "n" is a 2' O-methyl modified nucleoside, "s" is a phosphorothioate linkage, and N comprises one or more nucleosides. In some embodiments, the sense strand comprises the modification pattern 17S:5'-nnnnNfnNfnNfnNfnnnnnnnnsnsn-3', where "Nf" is a 2' fluoro modified nucleoside, "n" is a 2' O-methyl modified nucleoside, "s" is a phosphorothioate linkage, and N comprises one or more nucleosides. In some embodiments, the sense strand comprises the modification pattern 18S:5'-nnnnnNfnnNfnNfnnnnnnnnsnsn-3', where "Nf" is a 2' fluoro modified nucleoside, "n" is a 2' O-methyl modified nucleoside, "s" is a phosphorothioate linkage, and N comprises one or more nucleosides.In some embodiments, the sense strand comprises the modification pattern 19S:5'-nnnnnNfNfnNfnnnnnnnnnnsnsn-3', where "Nf" is a 2' fluoro modified nucleoside, "n" is a 2' O-methyl modified nucleoside, "s" is a phosphorothioate linkage, and N comprises one or more nucleosides. In some embodiments, the sense strand comprises the modification pattern 20S:5'-nnnnnnNfnNfNfnnnnnnnnnsnsn-3', where "Nf" is a 2' fluoro modified nucleoside, "n" is a 2' O-methyl modified nucleoside, "s" is a phosphorothioate linkage, and N comprises one or more nucleosides. In some embodiments, the sense strand comprises the modification pattern 21S:5'-nNfnNfnNfnNfNfnnnnnNfnNfNfnsnsn-3', where "Nf" is a 2' fluoro modified nucleoside, "n" is a 2' O-methyl modified nucleoside, "s" is a phosphorothioate linkage, and N comprises one or more nucleosides. In some embodiments, the sense strand comprises the modification pattern 22S:5'-snnnnnNfNfNfNfNfnnnnnnnnsnsn-3', where "Nf" is a 2' fluoro modified nucleoside, "n" is a 2' O-methyl modified nucleoside, "s" is a phosphorothioate linkage, and N comprises one or more nucleosides. In some embodiments, the sense strand comprises the modification pattern 23S:5'-snnnnnNfNfNfNfnnnnnnnnnnsnsn-3', where "Nf" is a 2' fluoro modified nucleoside, "n" is a 2' O-methyl modified nucleoside, "s" is a phosphorothioate linkage, and N comprises one or more nucleosides. In some embodiments, the sense strand comprises the modification pattern 24S:5'-snnnnNfNfNfNfNfnnnnnnnnnnsnsn-3', where "Nf" is a 2' fluoro modified nucleoside, "n" is a 2' O-methyl modified nucleoside, "s" is a phosphorothioate linkage, and N comprises one or more nucleosides.In some embodiments, the sense strand comprises the modification pattern 25S:5'-snnnnnnnnNfNfnnnnnnnnnsnsn-3', where "Nf" is a 2'fluoro modified nucleoside, "n" is a 2'O-methyl modified nucleoside, "s" is a phosphorothioate linkage, and N comprises one or more nucleosides. In some embodiments, the sense strand comprises the modification pattern 26S:5'-snnnnNfNfnnNfnNfnnnnnnnnsnsn-3', where "Nf" is a 2'fluoro modified nucleoside, "n" is a 2'O-methyl modified nucleoside, "s" is a phosphorothioate linkage, and N comprises one or more nucleosides. In some embodiments, the sense strand comprises the modification pattern 27S:5'-snNfnNfnNfnNfNfnnnnnNfnNfNfnsnsn-3', where "Nf" is a 2' fluoro modified nucleoside, "n" is a 2' O-methyl modified nucleoside, "s" is a phosphorothioate linkage, and N comprises one or more nucleosides. In some embodiments, the sense strand comprises the modification pattern 28S:5'-NfnNfnNfnNfNfNfNfNfNfnNfnNfnNfnNfsnsn-3', where "Nf" is a 2' fluoro modified nucleoside, "n" is a 2' O-methyl modified nucleoside, "s" is a phosphorothioate linkage, and N comprises one or more nucleosides. In some embodiments, the sense strand comprises the modification pattern 29S:5'-nnNfnNfnNfnNfnNfnNfnnnNfnNfsnsn-3', where "Nf" is a 2' fluoro modified nucleoside, "n" is a 2' O-methyl modified nucleoside, "s" is a phosphorothioate linkage, and N comprises one or more nucleosides. In some embodiments, the sense strand comprises the modification pattern 30S:5'-nnnnnNfNfNfNfNfnnnnnnnsnsn-3', where "Nf" is a 2' fluoro modified nucleoside, "n" is a 2' O-methyl modified nucleoside, "s" is a phosphorothioate linkage, and N comprises one or more nucleosides.

[0086] In some embodiments, the composition comprises an oligonucleotide that inhibits expression of a target mRNA, the oligonucleotide comprises an siRNA comprising a sense strand and an antisense strand, the antisense strand comprises a modification pattern 1AS:5'-nsNfsnNfnNfnNfnNfnNfnNfnNfnNfnsnsn-3', where "Nf" is a 2'fluoro-modified nucleoside, "n" is a 2'O-methyl-modified nucleoside, and "s" is a phosphorothioate linkage. In some embodiments, the antisense strand comprises a modification pattern 2AS:5'-nsNfsnNfnNfnNfnNfnNfnnnNfnNfnsnsn-3', where "Nf" is a 2'fluoro-modified nucleoside, "n" is a 2'O-methyl-modified nucleoside, and "s" is a phosphorothioate linkage. In some embodiments, the antisense strand comprises the modification pattern 3AS:5'-nsnsnNfnNfnNfnNfnNfnNfnNfnNfnNfnsnsn-3', where "Nf" is a 2'fluoro modified nucleoside, "n" is a 2'O-methyl modified nucleoside, and "s" is a phosphorothioate linkage. In some embodiments, the antisense strand comprises the modification pattern 4AS:5'-nsNfsnnnNfnNfnnnnnNfnNfnnnsnsn-3', where "Nf" is a 2'fluoro modified nucleoside, "n" is a 2'O-methyl modified nucleoside, and "s" is a phosphorothioate linkage. In some embodiments, the antisense strand comprises the modification pattern 5AS:5'-nsNfsnnnNfnNfnNfnNfnNfnNfnNfnsnsn-3', where "Nf" is a 2' fluoro modified nucleoside, "n" is a 2' O-methyl modified nucleoside, and "s" is a phosphorothioate linkage.

[0087] In some embodiments, the composition comprises an oligonucleotide that inhibits expression of a target nucleic acid, the oligonucleotide comprises an siRNA comprising a sense strand and an antisense strand, the sense strand comprises pattern 1S and the antisense strand comprises pattern 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises pattern 2S and the antisense strand comprises pattern 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises pattern 3S and the antisense strand comprises 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises pattern 4S and the antisense strand comprises 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises pattern 5S and the antisense strand comprises 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises pattern 6S and the antisense strand comprises 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises pattern 7S and the antisense strand comprises 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises pattern 8S and the antisense strand comprises 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises pattern 9S and the antisense strand comprises 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises pattern 10S and the antisense strand comprises 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises pattern 11S and the antisense strand comprises pattern 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises pattern 12S and the antisense strand comprises pattern 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises pattern 13S and the antisense strand comprises 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises the pattern 14S and the antisense strand comprises 1AS, 2AS, 3AS, 4AS, or 5AS.In some embodiments, the sense strand comprises pattern 15S and the antisense strand comprises 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises pattern 16S and the antisense strand comprises 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises pattern 17S and the antisense strand comprises 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises pattern 18S and the antisense strand comprises 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises pattern 19S and the antisense strand comprises 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises pattern 20S and the antisense strand comprises 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises pattern 21S and the antisense strand comprises pattern 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises pattern 22S and the antisense strand comprises pattern 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises pattern 23S and the antisense strand comprises 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises pattern 24S and the antisense strand comprises 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises pattern 25S and the antisense strand comprises 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises pattern 26S and the antisense strand comprises 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises pattern 27S and the antisense strand comprises 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises pattern 28S and the antisense strand comprises 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises the pattern 29S and the antisense strand comprises 1AS, 2AS, 3AS, 4AS, or 5AS.In some embodiments, the sense strand comprises the pattern 30S and the antisense strand comprises 1AS, 2AS, 3AS, 4AS, or 5AS.

[0088] In some embodiments, the sense strand comprises pattern 1S, 2S, 3S, 4S, 5S, 6S, 7S, 8S, 9S, 10S, 11S, 12S, 13S, 14S, 15S, 16S, 17S, 18S, 19S, 20S, 21S, 22S, 23S, 24S, 25S, 26S, 27S, 28S, 29S, or 30S, and the antisense strand comprises pattern 1AS. In some embodiments, the sense strand comprises pattern 1S, 2S, 3S, 4S, 5S, 6S, 7S, 8S, 9S, 10S, 11S, 12S, 13S, 14S, 15S, 16S, 17S, 18S, 19S, 20S, 21S, 22S, 23S, 24S, 25S, 26S, 27S, 28S, 29S, or 30S, and the antisense strand comprises pattern 2AS. In some embodiments, the sense strand comprises pattern 1S, 2S, 3S, 4S, 5S, 6S, 7S, 8S, 9S, 10S, 11S, 12S, 13S, 14S, 15S, 16S, 17S, 18S, 19S, 20S, 21S, 22S, 23S, 24S, 25S, 26S, 27S, 28S, 29S, or 30S, and the antisense strand comprises pattern 3AS. In some embodiments, the sense strand comprises pattern 1S, 2S, 3S, 4S, 5S, 6S, 7S, 8S, 9S, 10S, 11S, 12S, 13S, 14S, 15S, 16S, 17S, 18S, 19S, 20S, 21S, 22S, 23S, 24S, 25S, 26S, 27S, 28S, 29S, or 30S, and the antisense strand comprises pattern 4AS. In some embodiments, the sense strand comprises pattern 1S, 2S, 3S, 4S, 5S, 6S, 7S, 8S, 9S, 10S, 11S, 12S, 13S, 14S, 15S, 16S, 17S, 18S, 19S, 20S, 21S, 22S, 23S, 24S, 25S, 26S, 27S, 28S, 29S, or 30S, and the antisense strand comprises pattern 5AS.

[0089] In some embodiments, the sense strand comprises a modification pattern 1AS, 2AS, 3AS, or 4AS. In some embodiments, the sense strand or the antisense strand comprises a modification pattern 1AS, 2AS, 3AS, 4AS, or 5S. In some embodiments, the antisense strand comprises a modification pattern 1S, 2S, 3S, 4S, 5S, 6S, 7S, 8S, 9S, 10S, 11S, 12S, 13S, 14S, 15S, 16S, 17S, 18S, 19S, 20S, 21S, 22S, 23S, 24S, 25S, 26S, 27S, 28S, or 29S. In some embodiments, the sense or antisense strand comprises modification pattern 1S, 2S, 3S, 4S, 5S, 6S, 7S, 8S, 9S, 10S, 11S, 12S, 13S, 14S, 15S, 16S, 17S, 18S, 19S, 20S, 21S, 22S, 23S, 24S, 25S, 26S, 27S, 28S, 29S, or 30AS.

[0090] In some embodiments, the purines in the sense strand comprise 2'fluoro modified purines. In some embodiments, the purines in the sense strand comprise 2'-O-methyl modified purines. In some embodiments, the purines in the sense strand comprise a mixture of 2'fluoro and 2'-O-methyl modified purines. In some embodiments, all of the purines in the sense strand comprise 2'fluoro modified purines. In some embodiments, all of the purines in the sense strand comprise 2'-O-methyl modified purines. In some embodiments, all of the purines in the sense strand comprise a mixture of 2'fluoro and 2'-O-methyl modified purines.

[0091] In some embodiments, the pyrimidines of the sense strand comprise 2'fluoro modified pyrimidines. In some embodiments, the pyrimidines of the sense strand comprise 2'-O-methyl modified pyrimidines. In some embodiments, the pyrimidines of the sense strand comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines. In some embodiments, all pyrimidines of the sense strand comprise 2'fluoro modified pyrimidines. In some embodiments, all pyrimidines of the sense strand comprise 2'-O-methyl modified pyrimidines. In some embodiments, all pyrimidines of the sense strand comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines.

[0092] In some embodiments, the purines of the sense strand include 2'fluoro modified purines and the pyrimidines of the sense strand include a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines. In some embodiments, the purines of the sense strand include 2'-O-methyl modified purines and the pyrimidines of the sense strand include a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines. In some embodiments, the purines of the sense strand include 2'fluoro modified purines and the pyrimidines of the sense strand include 2'-O-methyl modified pyrimidines. In some embodiments, the purines of the sense strand include 2'-O-methyl modified purines and the pyrimidines of the sense strand include 2'fluoro modified pyrimidines. In some embodiments, the pyrimidines of the sense strand include 2'fluoro modified pyrimidines and the purines of the sense strand include a mixture of 2'fluoro and 2'-O-methyl modified purines. In some embodiments, the pyrimidines of the sense strand include 2'-O-methyl modified pyrimidines and the purines of the sense strand include a mixture of 2'fluoro and 2'-O-methyl modified purines. In some embodiments, the pyrimidines of the sense strand comprise 2'-fluoro modified pyrimidines and the purines of the sense strand comprise 2'-O-methyl modified purines. In some embodiments, the pyrimidines of the sense strand comprise 2'-O-methyl modified pyrimidines and the purines of the sense strand comprise 2'-fluoro modified purines.

[0093] In some embodiments, all purines in the sense strand comprise 2'-fluoro modified purines and all pyrimidines in the sense strand comprise a mixture of 2'-fluoro and 2'-O-methyl modified pyrimidines. In some embodiments, all purines in the sense strand comprise 2'-O-methyl modified purines and all pyrimidines in the sense strand comprise a mixture of 2'-fluoro and 2'-O-methyl modified pyrimidines. In some embodiments, all purines in the sense strand comprise 2'-fluoro modified purines and all pyrimidines in the sense strand comprise 2'-O-methyl modified pyrimidines. In some embodiments, all purines in the sense strand comprise 2'-O-methyl modified purines and all pyrimidines in the sense strand comprise 2'-fluoro modified pyrimidines. In some embodiments, all pyrimidines in the sense strand comprise 2'-fluoro modified pyrimidines and all purines in the sense strand comprise a mixture of 2'-fluoro and 2'-O-methyl modified purines. In some embodiments, all pyrimidines in the sense strand comprise 2'-O-methyl modified pyrimidines and all purines in the sense strand comprise a mixture of 2'-fluoro and 2'-O-methyl modified purines. In some embodiments, all pyrimidines in the sense strand comprise 2'-fluoro modified pyrimidines and all purines in the sense strand comprise 2'-O-methyl modified purines. In some embodiments, all pyrimidines in the sense strand comprise 2'-O-methyl modified pyrimidines and all purines in the sense strand comprise 2'-fluoro modified purines.

[0094] In some embodiments, the purines in the antisense strand comprise 2'fluoro modified purines. In some embodiments, the purines in the antisense strand comprise 2'-O-methyl modified purines. In some embodiments, the purines in the antisense strand comprise a mixture of 2'fluoro and 2'-O-methyl modified purines. In some embodiments, all purines in the antisense strand comprise 2'fluoro modified purines. In some embodiments, all purines in the antisense strand comprise 2'-O-methyl modified purines. In some embodiments, all purines in the antisense strand comprise a mixture of 2'fluoro and 2'-O-methyl modified purines.

[0095] In some embodiments, the pyrimidines of the antisense strand comprise 2'fluoro modified pyrimidines. In some embodiments, the pyrimidines of the antisense strand comprise 2'-O-methyl modified pyrimidines. In some embodiments, the pyrimidines of the antisense strand comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines. In some embodiments, all pyrimidines of the antisense strand comprise 2'fluoro modified pyrimidines. In some embodiments, all pyrimidines of the antisense strand comprise 2'-O-methyl modified pyrimidines. In some embodiments, all pyrimidines of the antisense strand comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines.

[0096] In some embodiments, the purines of the antisense strand include 2'fluoro modified purines and the pyrimidines of the antisense strand include a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines. In some embodiments, the purines of the antisense strand include 2'-O-methyl modified purines and the pyrimidines of the antisense strand include a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines. In some embodiments, the purines of the antisense strand include 2'fluoro modified purines and the pyrimidines of the antisense strand include 2'-O-methyl modified pyrimidines. In some embodiments, the purines of the antisense strand include 2'-O-methyl modified purines and the pyrimidines of the antisense strand include 2'fluoro modified pyrimidines. In some embodiments, the pyrimidines of the antisense strand include 2'fluoro modified pyrimidines and the purines of the antisense strand include a mixture of 2'fluoro and 2'-O-methyl modified purines. In some embodiments, the pyrimidines of the antisense strand include 2'-O-methyl modified pyrimidines and the purines of the antisense strand include a mixture of 2'fluoro and 2'-O-methyl modified purines. In some embodiments, the pyrimidines of the antisense strand include 2'fluoro modified pyrimidines and the purines of the antisense strand include 2'-O-methyl modified purines. In some embodiments, the pyrimidines of the antisense strand include 2'-O-methyl modified pyrimidines and the purines of the antisense strand include 2'fluoro modified purines.

[0097] In some embodiments, all purines in the antisense strand include 2'fluoro modified purines and all pyrimidines in the antisense strand include a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines. In some embodiments, all purines in the antisense strand include 2'-O-methyl modified purines and all pyrimidines in the antisense strand include a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines. In some embodiments, all purines in the antisense strand include 2'fluoro modified purines and all pyrimidines in the antisense strand include 2'-O-methyl modified pyrimidines. In some embodiments, all purines in the antisense strand include 2'-O-methyl modified purines and all pyrimidines in the antisense strand include 2'fluoro modified pyrimidines. In some embodiments, all pyrimidines in the antisense strand include 2'fluoro modified pyrimidines and all purines in the antisense strand include a mixture of 2'fluoro and 2'-O-methyl modified purines. In some embodiments, all pyrimidines in the antisense strand include 2'-O-methyl modified pyrimidines and all purines in the antisense strand include a mixture of 2'fluoro and 2'-O-methyl modified purines. In some embodiments, all pyrimidines in the antisense strand comprise 2'-fluoro modified pyrimidines and all purines in the antisense strand comprise 2'-O-methyl modified purines. In some embodiments, all pyrimidines in the antisense strand comprise 2'-O-methyl modified pyrimidines and all purines in the antisense strand comprise 2'-fluoro modified purines.

[0098] In some embodiments, the siRNA comprises a sense strand, an antisense strand, and a lipid moiety attached to the end of the sense strand or the antisense strand, where the lipid moiety comprises a phenyl or cyclohexanyl linker, and the linker is attached to the lipid and the end of the sense strand or the antisense strand. In some embodiments, with respect to the sense strand, any one of the following is true: all purines comprise 2'fluoro modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines; all purines comprise 2'-O-methyl modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines; all purines comprise 2'fluoro modified purines and all pyrimidines comprise 2'-O-methyl modified pyrimidines; all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines; all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines; all pyrimidines comprise 2'-O-methyl modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines; or all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise 2'-O-methyl modified purines. In some embodiments, with respect to the antisense strand, any one of the following is true: all purines comprise 2'fluoro modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines; all purines comprise 2'-O-methyl modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines; all purines comprise 2'-O-methyl modified purines and all pyrimidines comprise 2'fluoro modified pyrimidines; all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines; all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines; all pyrimidines comprise 2'-O-methyl modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines; or all pyrimidines comprise 2'-O-methyl modified pyrimidines and all purines comprise 2'fluoro modified purines.In some embodiments, the siRNA comprises a sense strand and an antisense strand, wherein the antisense strand comprises a 5' end comprising a vinyl phosphonate and two phosphorothioate linkages and a 3' end comprising two phosphorothioate linkages, and the sense strand comprises a 5' end comprising a hydrophobic moiety and a 3' end comprising two phosphorothioate linkages, and wherein, for the sense strand, all purines comprise 2' fluoro modified purines and all pyrimidines comprise a mixture of 2' fluoro and 2'-O-methyl modified pyrimidines, and all proline modified purines comprise a mixture of 2' fluoro and 2'-O-methyl modified pyrimidines. all purines contain 2'-O-methyl modified purines and all pyrimidines contain a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines, all purines contain 2'fluoro modified purines and all pyrimidines contain 2'-O-methyl modified pyrimidines, all pyrimidines contain 2'fluoro modified pyrimidines and all purines contain a mixture of 2'fluoro and 2'-O-methyl modified purines, all pyrimidines contain 2'-O-methyl modified pyrimidines and all purines contain a mixture of 2'fluoro and 2'-O-methyl modified purines or all pyrimidines include 2'fluoro modified pyrimidines and all purines include 2'-O-methyl modified purines, and with respect to the antisense strand, all purines include 2'fluoro modified purines and all pyrimidines include a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines, all purines include 2'-O-methyl modified purines and all pyrimidines include a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines, all purines include 2'-O-methyl modified purines and all pyrimidines include a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines, all purines include 2'-O-methyl modified all pyrimidines comprise 2'-fluoro modified pyrimidines and all purines comprise a mixture of 2'-fluoro and 2'-O-methyl modified purines; all pyrimidines comprise 2'-O-methyl modified pyrimidines and all purines comprise a mixture of 2'-fluoro and 2'-O-methyl modified purines; or all pyrimidines comprise 2'-O-methyl modified pyrimidines and all purines comprise a mixture of 2'-fluoro and 2'-O-methyl modified purines.

[0099] In some embodiments, with respect to the sense strand, any one of the following is true: all purines comprise 2'fluoro modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines; all purines comprise 2'-O-methyl modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines; all purines comprise 2'fluoro modified purines and all pyrimidines comprise 2'-O-methyl modified pyrimidines; all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines; all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines; all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines; or all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise 2'-O-methyl modified purines, with the proviso that in any of the foregoing, the sense strand may comprise deoxynucleosides. In some embodiments, all purines include 2'-fluoro modified purines and all pyrimidines include a mixture of 2'-fluoro and 2'-O-methyl modified pyrimidines, with the proviso that deoxynucleosides may be included in the sense strand. In some embodiments, in the sense strand, all purines include 2'-O-methyl modified purines and all pyrimidines include a mixture of 2'-fluoro and 2'-O-methyl modified pyrimidines, with the proviso that deoxynucleosides may be included in the sense strand. In some embodiments, in the sense strand, all purines include 2'-fluoro modified purines and all pyrimidines include 2'-O-methyl modified pyrimidines, with the proviso that deoxynucleosides may be included in the sense strand. In some embodiments, all pyrimidines include 2'-fluoro modified pyrimidines and all purines include a mixture of 2'-fluoro and 2'-O-methyl modified purines, with the proviso that deoxynucleosides may be included in the sense strand.In some embodiments, in the sense strand, all pyrimidines include 2'-O-methyl modified pyrimidines and all purines include a mixture of 2'fluoro and 2'-O-methyl modified purines, with the proviso that deoxynucleosides may be included in the sense strand. In some embodiments, in the sense strand, all pyrimidines include 2'fluoro modified pyrimidines and all purines include 2'-O-methyl modified purines, with the proviso that deoxynucleosides may be included in the sense strand. In some embodiments, the sense strand includes deoxynucleosides. The deoxynucleosides may be at the nucleoside 9 position of the sense strand. In some embodiments, the sense strand does not include deoxynucleosides. Otherwise, the deoxynucleosides of the sense strand may be unmodified.

[0100] In some embodiments, with respect to the antisense strand, any one of the following is true: all purines comprise 2'fluoro modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines; all purines comprise 2'-O-methyl modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines; all purines comprise 2'-O-methyl modified purines and all pyrimidines comprise 2'fluoro modified pyrimidines; all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines; all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines; all pyrimidines comprise 2'-O-methyl modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines; or all pyrimidines comprise 2'-O-methyl modified pyrimidines and all purines comprise 2'fluoro modified purines, with the proviso that in any of the foregoing, the sense strand may comprise deoxynucleosides. In some embodiments, all purines include 2'-fluoro modified purines and all pyrimidines include a mixture of 2'-fluoro and 2'-O-methyl modified pyrimidines, with the proviso that deoxynucleosides may be included in the antisense strand. In some embodiments, in the antisense strand, all purines include 2'-O-methyl modified purines and all pyrimidines include a mixture of 2'-fluoro and 2'-O-methyl modified pyrimidines, with the proviso that deoxynucleosides may be included in the antisense strand. In some embodiments, in the antisense strand, all purines include 2'-O-methyl modified purines and all pyrimidines include 2'-fluoro modified pyrimidines, with the proviso that deoxynucleosides may be included in the antisense strand. In some embodiments, in the antisense strand, all pyrimidines include 2'-fluoro modified pyrimidines and all purines include a mixture of 2'-fluoro and 2'-O-methyl modified purines, with the proviso that deoxynucleosides may be included in the antisense strand.In some embodiments, in the antisense strand, all pyrimidines include 2'-O-methyl modified pyrimidines and all purines include a mixture of 2'fluoro and 2'-O-methyl modified purines, with the proviso that deoxynucleosides may be included in the antisense strand. In some embodiments, in the antisense strand, all pyrimidines include 2'-O-methyl modified pyrimidines and all purines include 2'fluoro modified purines, with the proviso that deoxynucleosides may be included in the antisense strand. In some embodiments, the antisense strand includes deoxynucleosides. The deoxynucleosides may be at nucleoside 9 position of the antisense strand. In some embodiments, the antisense strand does not include deoxynucleosides. Otherwise, the deoxynucleosides of the antisense strand may be unmodified.

[0101] In some embodiments, for the sense strand, all purines comprise 2'fluoro modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines, all purines comprise 2'-O-methyl modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines, all purines comprise 2'fluoro modified purines and all pyrimidines comprise 2'-O-methyl modified pyrimidines, all pyrimidines comprise 2'fluoro modified pyrimidines and ... pyrimidines In some embodiments, the sense strand may include deoxynucleosides or 2'-O-methoxyethyl nucleosides, in which case all pyrimidines include 2'-fluoro modified purines and all purines include a mixture of 2'-fluoro and 2'-O-methyl modified purines, all pyrimidines include 2'-O-methyl modified pyrimidines and all purines include a mixture of 2'-fluoro and 2'-O-methyl modified purines, or all pyrimidines include 2'-fluoro modified pyrimidines and all purines include 2'-O-methyl modified purines, with the proviso that in any of the foregoing, the sense strand may include deoxynucleosides or 2'-O-methoxyethyl nucleosides. In some embodiments, all purines include 2'-fluoro modified purines and all pyrimidines include a mixture of 2'-fluoro and 2'-O-methyl modified pyrimidines, with the proviso that deoxynucleosides or 2'-O-methoxyethyl nucleosides may be included in the sense strand. In some embodiments, in the sense strand, all purines comprise 2'-O-methyl modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines, with the proviso that deoxynucleosides or 2'-O-methoxyethyl nucleosides may be included in the sense strand. In some embodiments, in the sense strand, all purines comprise 2'fluoro modified purines and all pyrimidines comprise 2'-O-methyl modified pyrimidines, with the proviso that deoxynucleosides or 2'-O-methoxyethyl nucleosides may be included in the sense strand.In some embodiments, all pyrimidines include 2'-fluoro modified pyrimidines and all purines include a mixture of 2'-fluoro and 2'-O-methyl modified purines, with the proviso that deoxynucleosides or 2'-O-methoxyethyl nucleosides may be included in the sense strand. In some embodiments, in the sense strand, all pyrimidines include 2'-O-methyl modified pyrimidines and all purines include a mixture of 2'-fluoro and 2'-O-methyl modified purines, with the proviso that deoxynucleosides or 2'-O-methoxyethyl nucleosides may be included in the sense strand. In some embodiments, in the sense strand, all pyrimidines include 2'-fluoro modified pyrimidines and all purines include a mixture of 2'-O-methyl modified purines, with the proviso that deoxynucleosides or 2'-O-methoxyethyl nucleosides may be included in the sense strand. In some embodiments, the sense strand includes deoxynucleosides. The deoxynucleosides may be at the nucleoside 9 position of the sense strand. In some embodiments, the sense strand does not include a deoxynucleoside. The deoxynucleosides of the sense strand may otherwise be unmodified. In some embodiments, the sense strand includes a 2'-O-methoxyethyl nucleoside. The 2'-O-methoxyethyl nucleoside may be at the nucleoside 4 position of the sense strand. The 2'-O-methoxyethyl nucleoside may include a 2'-O-methoxyethyl thymine nucleoside. In some embodiments, the sense strand does not include a 2'-O-methoxyethyl nucleoside. The 2'-O-methoxyethyl nucleoside of the sense strand may otherwise be unmodified.

[0102] In some embodiments, for the antisense strand, all purines comprise 2'fluoro modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines, all purines comprise 2'-O-methyl modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines, all purines comprise 2'-O-methyl modified purines and all pyrimidines comprise 2'fluoro modified pyrimidines, all pyrimidines comprise 2'fluoro modified pyrimidines and all Any one of the following applies: the purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines, all pyrimidines comprise 2'-O-methyl modified pyrimidines, all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines, or all pyrimidines comprise 2'-O-methyl modified pyrimidines and all purines comprise 2'fluoro modified purines, with the proviso that in any of the above, the sense strand may comprise deoxynucleosides or 2'-O-methoxyethyl nucleosides. In some embodiments, all purines comprise 2'fluoro modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines, with the proviso that deoxynucleosides or 2'-O-methoxyethyl nucleosides may be included in the antisense strand. In some embodiments, in the antisense strand, all purines comprise 2'-O-methyl modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines, with the proviso that deoxynucleosides or 2'-O-methoxyethyl nucleosides may be included in the antisense strand. In some embodiments, in the antisense strand, all purines comprise 2'-O-methyl modified purines and all pyrimidines comprise 2'fluoro modified pyrimidines, with the proviso that deoxynucleosides or 2'-O-methoxyethyl nucleosides may be included in the antisense strand.In some embodiments, in the antisense strand, all pyrimidines include 2'-fluoro modified pyrimidines and all purines include a mixture of 2'-fluoro and 2'-O-methyl modified purines, with the proviso that deoxynucleosides or 2'-O-methoxyethyl nucleosides may be included in the antisense strand. In some embodiments, in the antisense strand, all pyrimidines include 2'-O-methyl modified pyrimidines and all purines include a mixture of 2'-fluoro and 2'-O-methyl modified purines, with the proviso that deoxynucleosides or 2'-O-methoxyethyl nucleosides may be included in the antisense strand. In some embodiments, in the antisense strand, all pyrimidines include 2'-O-methyl modified pyrimidines and all purines include a mixture of 2'-fluoro and 2'-O-methyl modified purines, with the proviso that deoxynucleosides or 2'-O-methoxyethyl nucleosides may be included in the antisense strand. In some embodiments, the antisense strand includes deoxynucleosides. The deoxynucleoside may be at nucleoside 9 position of the antisense strand. In some embodiments, the antisense strand does not include a deoxynucleoside. Otherwise, the deoxynucleosides of the antisense strand may be unmodified. In some embodiments, the antisense strand includes a 2'-O-methoxyethyl nucleoside. The 2'-O-methoxyethyl nucleoside may be at nucleoside 4 position of the sense strand. The 2'-O-methoxyethyl nucleoside may include a 2'-O-methoxyethyl thymine nucleoside. In some embodiments, the antisense strand does not include a 2'-O-methoxyethyl nucleoside. Otherwise, the 2'-O-methoxyethyl nucleoside of the antisense strand may be unmodified.

[0103] In some embodiments, with respect to the sense strand, any one of the following is true: all purines comprise 2'fluoro modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines; all purines comprise 2'-O-methyl modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines; all purines comprise 2'fluoro modified purines and all pyrimidines comprise 2'-O-methyl modified pyrimidines; all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines; all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines; all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines; or all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise 2'-O-methyl modified purines, with the proviso that in any of the foregoing, the sense strand may comprise 2'-O-methoxyethyl nucleosides. In some embodiments, all purines include 2'-fluoro modified purines and all pyrimidines include a mixture of 2'-fluoro and 2'-O-methyl modified pyrimidines, with the proviso that 2'-O-methoxyethyl nucleosides may be included in the sense strand. In some embodiments, in the sense strand, all purines include 2'-O-methyl modified purines and all pyrimidines include a mixture of 2'-fluoro and 2'-O-methyl modified pyrimidines, with the proviso that 2'-O-methoxyethyl nucleosides may be included in the sense strand. In some embodiments, in the sense strand, all purines include 2'-fluoro modified purines and all pyrimidines include 2'-O-methyl modified pyrimidines, with the proviso that 2'-O-methoxyethyl nucleosides may be included in the sense strand. In some embodiments, all pyrimidines include 2'-fluoro modified pyrimidines and all purines include a mixture of 2'-fluoro and 2'-O-methyl modified purines, with the proviso that 2'-O-methoxyethyl nucleosides may be included in the sense strand.In some embodiments, in the sense strand, all pyrimidines comprise 2'-O-methyl modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines, with the proviso that 2'-O-methoxyethyl nucleosides may be included in the sense strand. In some embodiments, in the sense strand, all pyrimidines comprise 2'fluoro modified pyrimidines and all purines comprise 2'-O-methyl modified purines, with the proviso that 2'-O-methoxyethyl nucleosides may be included in the sense strand. In some embodiments, the sense strand comprises 2'-O-methoxyethyl nucleosides. The 2'-O-methoxyethyl nucleosides may be at the nucleoside 4 position of the sense strand. The 2'-O-methoxyethyl nucleosides may comprise 2'-O-methoxyethyl thymine nucleosides. In some embodiments, the sense strand does not comprise 2'-O-methoxyethyl nucleosides. Otherwise, the 2'-O-methoxyethyl nucleosides in the sense strand can be unmodified.

[0104] In some embodiments, for the antisense strand, all purines comprise 2'fluoro modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines, all purines comprise 2'-O-methyl modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines, all purines comprise 2'-O-methyl modified purines and all pyrimidines comprise 2'fluoro modified pyrimidines, all pyrimidines comprise 2'fluoro modified pyrimidines and all Any one of the following applies: the purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines, all pyrimidines comprise 2'-O-methyl modified pyrimidines and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines, or all pyrimidines comprise 2'-O-methyl modified pyrimidines and all purines comprise 2'fluoro modified purines, with the proviso that in any of the above, the sense strand may comprise deoxynucleosides or 2'-O-methoxyethyl nucleosides. In some embodiments, all purines comprise 2'fluoro modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines, with the proviso that 2'-O-methoxyethyl nucleosides may be included in the antisense strand. In some embodiments, in the antisense strand, all purines comprise 2'-O-methyl modified purines and all pyrimidines comprise a mixture of 2'fluoro and 2'-O-methyl modified pyrimidines, with the proviso that 2'-O-methoxyethyl nucleosides may be included in the antisense strand. In some embodiments, in the antisense strand, all purines comprise 2'-O-methyl modified purines and all pyrimidines comprise 2'fluoro modified pyrimidines, with the proviso that 2'-O-methoxyethyl nucleosides may be included in the antisense strand.In some embodiments, in the antisense strand, all pyrimidines include 2'-fluoro modified pyrimidines and all purines include a mixture of 2'-fluoro and 2'-O-methyl modified purines, with the proviso that 2'-O-methoxyethyl nucleosides may be included in the antisense strand. In some embodiments, in the antisense strand, all pyrimidines include 2'-O-methyl modified pyrimidines and all purines include a mixture of 2'-fluoro and 2'-O-methyl modified purines, with the proviso that 2'-O-methoxyethyl nucleosides may be included in the antisense strand. In some embodiments, in the antisense strand, all pyrimidines include 2'-O-methyl modified pyrimidines and all purines include a mixture of 2'-fluoro and 2'-O-methyl modified purines, with the proviso that 2'-O-methoxyethyl nucleosides may be included in the antisense strand. In some embodiments, the antisense strand includes 2'-O-methoxyethyl nucleosides. The 2'-O-methoxyethyl nucleosides may be at the nucleoside 4 position of the sense strand. The 2'-O-methoxyethyl nucleosides may include 2'-O-methoxyethyl thymine nucleosides. In some embodiments, the antisense strand does not include 2'-O-methoxyethyl nucleosides. Otherwise, the 2'-O-methoxyethyl nucleosides of the antisense strand may be unmodified.

[0105] In some embodiments, with respect to the sense strand, all purine nucleosides include 2' fluoro and all pyrimidine nucleosides are modified with a mixture of 2' fluoro and 2'-O-methyl, all purine nucleosides include 2'-O-methyl and all pyrimidine nucleosides are modified with a mixture of 2' fluoro and 2'-O-methyl, all purine nucleosides include 2' fluoro ... with the proviso that the sense strand may include 2' deoxynucleosides. In some embodiments, in the sense strand, all purine nucleosides include 2'-O-methyl, all pyrimidine nucleosides include 2'-fluoro and all purine nucleosides are modified with a mixture of 2'-fluoro and 2'-O-methyl, all pyrimidine nucleosides include 2'-O-methyl and all purine nucleosides are modified with a mixture of 2'-fluoro and 2'-O-methyl, or all pyrimidine nucleosides include 2'-fluoro and all purine nucleosides include 2'-O-methyl. In some embodiments, in the sense strand, all purine nucleosides include 2'-fluoro and all pyrimidine nucleosides are modified with a mixture of 2'-fluoro and 2'-O-methyl, with the proviso that the sense strand may include 2'-deoxynucleosides. In some embodiments, in the sense strand, all purine nucleosides include 2'-O-methyl and all pyrimidine nucleosides are modified with a mixture of 2'fluoro and 2'-O-methyl, with the proviso that the sense strand may also include 2'deoxynucleosides. In some embodiments, in the sense strand, all purine nucleosides include 2'fluoro and all pyrimidine nucleosides include 2'-O-methyl, with the proviso that the sense strand may also include 2'deoxynucleosides. In some embodiments, in the sense strand, all pyrimidine nucleosides include 2'fluoro and all purine nucleosides are modified with a mixture of 2'fluoro and 2'-O-methyl, with the proviso that the sense strand may also include 2'deoxynucleosides.In some embodiments, in the sense strand, all pyrimidine nucleosides include 2'-O-methyl and all purine nucleosides are modified with a mixture of 2'fluoro and 2'-O-methyl, with the proviso that the sense strand may include 2'deoxynucleosides. In some embodiments, in the sense strand, all pyrimidine nucleosides include 2'fluoro and all purine nucleosides include 2'-O-methyl, with the proviso that the sense strand may include 2'deoxynucleosides. In some embodiments, the sense strand includes 2'deoxynucleosides. In some embodiments, the sense strand does not include 2'deoxynucleosides. Some embodiments include the proviso that the sense strand may include 2'-O-methoxyethyl nucleosides (e.g., at the 4th position, counting from 5' to 3'). Some embodiments include 2'-O-methoxyethyl nucleosides in the sense strand. Some embodiments do not include 2'-O-methoxyethyl nucleosides in the sense strand.

[0106] In some embodiments, with respect to the sense strand, any one of the following is true: all purine nucleosides include 2' fluoro and all pyrimidine nucleosides are modified with a mixture of 2' fluoro and 2'-O-methyl; all purine nucleosides include 2'-O-methyl and all pyrimidine nucleosides are modified with a mixture of 2' fluoro and 2'-O-methyl; all purine nucleosides include 2' fluoro and all pyrimidine nucleosides include 2'-O-methyl; all pyrimidine nucleosides include 2' fluoro and all purine nucleosides are modified with a mixture of 2' fluoro and 2'-O-methyl; all pyrimidine nucleosides include 2' fluoro and all purine nucleosides are modified with a mixture of 2' fluoro and 2'-O-methyl; all pyrimidine nucleosides include 2'-O-methyl and all purine nucleosides are modified with a mixture of 2' fluoro and 2'-O-methyl; or all pyrimidine nucleosides include 2' fluoro and all purine nucleosides include 2'-O-methyl. In some embodiments, in the sense strand, all purine nucleosides include 2'fluoro and all pyrimidine nucleosides are modified with a mixture of 2'fluoro and 2'-O-methyl. In some embodiments, in the sense strand, all purine nucleosides include 2'-O-methyl and all pyrimidine nucleosides are modified with a mixture of 2'fluoro and 2'-O-methyl. In some embodiments, in the sense strand, all purine nucleosides include 2'fluoro and all pyrimidine nucleosides include 2'-O-methyl. In some embodiments, in the sense strand, all pyrimidine nucleosides include 2'fluoro and all purine nucleosides are modified with a mixture of 2'fluoro and 2'-O-methyl. In some embodiments, in the sense strand, all pyrimidine nucleosides include 2'-O-methyl and all purine nucleosides are modified with a mixture of 2'fluoro and 2'-O-methyl. In some embodiments, in the sense strand, all pyrimidine nucleosides include 2'fluoro and all purine nucleosides include 2'-O-methyl. Some embodiments include the proviso that the sense strand may include a 2'-O-methoxyethyl nucleoside (e.g., at the 4th position, counting from 5' to 3').Some embodiments include 2'-O-methoxyethyl nucleosides in the sense strand.Some embodiments do not include 2'-O-methoxyethyl nucleosides in the sense strand.

[0107] In some embodiments, with respect to the antisense strand, all purine nucleosides include 2' fluoro and all pyrimidine nucleosides are modified with a mixture of 2' fluoro and 2'-O-methyl, all purine nucleosides include 2'-O-methyl ... with the proviso that the antisense strand may include 2' deoxynucleosides. Any one of the following applies: all pyrimidine nucleosides include 2'fluoro, all pyrimidine nucleosides include 2'fluoro and all purine nucleosides are modified with a mixture of 2'fluoro and 2'-O-methyl, all pyrimidine nucleosides include 2'-O-methyl and all purine nucleosides are modified with a mixture of 2'fluoro and 2'-O-methyl, or all pyrimidine nucleosides include 2'-O-methyl and all purine nucleosides include 2'fluoro.In some embodiments, in the antisense strand, all purine nucleosides include 2'fluoro and all pyrimidine nucleosides are modified with a mixture of 2'fluoro and 2'-O-methyl, with the proviso that the antisense strand may include 2'deoxynucleosides. In some embodiments, in the antisense strand, all purine nucleosides include 2'-O-methyl and all pyrimidine nucleosides are modified with a mixture of 2'fluoro and 2'-O-methyl, with the proviso that the antisense strand may also include 2'deoxynucleosides. In some embodiments, in the antisense strand, all purine nucleosides include 2'fluoro and all pyrimidine nucleosides include 2'-O-methyl, with the proviso that the antisense strand may also include 2'deoxynucleosides. In some embodiments, in the antisense strand, all pyrimidine nucleosides include 2'fluoro and all purine nucleosides are modified with a mixture of 2'fluoro and 2'-O-methyl, with the proviso that the antisense strand may also include 2'deoxynucleosides.In some embodiments, in the antisense strand, all pyrimidine nucleosides include 2'-O-methyl and all purine nucleosides are modified with a mixture of 2'fluoro and 2'-O-methyl, with the proviso that the antisense strand may include 2'deoxynucleosides. In some embodiments, in the antisense strand, all pyrimidine nucleosides include 2'fluoro and all purine nucleosides include 2'-O-methyl, with the proviso that the antisense strand may include 2'deoxynucleosides. In some embodiments, the antisense strand includes 2'deoxynucleosides. In some embodiments, the antisense strand does not include 2'deoxynucleosides. Some embodiments include the proviso that the antisense strand may include 2'-O-methoxyethyl nucleosides (e.g., at the 4th position, counting from 5' to 3'). Some embodiments include 2'-O-methoxyethyl nucleosides in the antisense strand. Some embodiments do not include 2'-O-methoxyethyl nucleosides in the antisense strand.

[0108] In some embodiments, with respect to the antisense strand, any one of the following is true: all purine nucleosides contain 2' fluoro and all pyrimidine nucleosides are modified with a mixture of 2' fluoro and 2'-O-methyl; all purine nucleosides contain 2'-O-methyl and all pyrimidine nucleosides are modified with a mixture of 2' fluoro and 2'-O-methyl; all purine nucleosides contain 2' fluoro and all pyrimidine nucleosides are modified with a mixture of 2' fluoro and 2'-O-methyl; all pyrimidine nucleosides contain 2' fluoro and all purine nucleosides are modified with a mixture of 2' fluoro and 2'-O-methyl; all pyrimidine nucleosides contain 2' fluoro and all purine nucleosides are modified with a mixture of 2' fluoro and 2'-O-methyl; all pyrimidine nucleosides contain 2'-O-methyl and all purine nucleosides are modified with a mixture of 2' fluoro and 2'-O-methyl; or all pyrimidine nucleosides contain 2'-O-methyl and all purine nucleosides contain 2' fluoro. In some embodiments, in the antisense strand, all purine nucleosides include 2'fluoro and all pyrimidine nucleosides are modified with a mixture of 2'fluoro and 2'-O-methyl. In some embodiments, in the antisense strand, all purine nucleosides include 2'-O-methyl and all pyrimidine nucleosides are modified with a mixture of 2'fluoro and 2'-O-methyl. In some embodiments, in the antisense strand, all purine nucleosides include 2'fluoro and all pyrimidine nucleosides include 2'-O-methyl. In some embodiments, in the antisense strand, all pyrimidine nucleosides include 2'fluoro and all purine nucleosides are modified with a mixture of 2'fluoro and 2'-O-methyl. In some embodiments, in the antisense strand, all pyrimidine nucleosides include 2'fluoro and all purine nucleosides are modified with a mixture of 2'fluoro and 2'-O-methyl. In some embodiments, in the antisense strand, all pyrimidine nucleosides include 2'-O-methyl and all purine nucleosides are modified with a mixture of 2'fluoro and 2'-O-methyl. In some embodiments, in the antisense strand, all pyrimidine nucleosides contain 2' fluoro and all purine nucleosides contain 2'-O-methyl.Some embodiments include the proviso that the antisense strand may include a 2'-O-methoxyethyl nucleoside (e.g., at the 4th position, counting from 5' to 3'). Some embodiments include a 2'-O-methoxyethyl nucleoside in the antisense strand. Some embodiments do not include a 2'-O-methoxyethyl nucleoside in the antisense strand.

[0109] In some embodiments, the antisense strand comprises one or two 3' phosphorothioate bonds. For example, there may be a phosphorothioate bond between the first and second nucleotides from the 3' end of the antisense strand, or there may be a phosphorothioate bond between the first, second, and third nucleotides from the 3' end of the antisense strand. In some embodiments, the sense strand comprises one or two 5' phosphorothioate bonds. For example, there may be a phosphorothioate bond between the first and second nucleotides from the 5' end of the sense strand, or there may be a phosphorothioate bond between the first, second, and third nucleotides from the 5' end of the sense strand. In some embodiments, the sense strand does not comprise one or two 5' phosphorothioate bonds. For example, in some embodiments, there is no phosphorothioate bond between the last three nucleotides at the 5' end of the sense strand. In some embodiments, the sense strand comprises a 5' phosphate bond. In some embodiments, the sense strand comprises one or two 3' phosphorothioate bonds. For example, there may be a phosphorothioate bond between the first and second nucleotides from the 3' end of the sense strand, or there may be a phosphorothioate bond between the first, second, and third nucleotides from the 3' end of the sense strand.

[0110] In some embodiments, the antisense strand comprises a 5' end comprising two phosphorothioate bonds. The 5' end may comprise three nucleosides separated by two phosphorothioate bonds. In some embodiments, the antisense strand comprises a 3' end comprising two phosphorothioate bonds. The 3' end may comprise three nucleosides separated by two phosphorothioate bonds.

[0111] In some embodiments, the composition comprises an oligonucleotide that inhibits expression of a target nucleic acid, the oligonucleotide comprises an siRNA comprising a sense strand and an antisense strand, and the oligonucleotide comprises a hydrophobic moiety. In some embodiments, the hydrophobic moiety may be attached to the 5' end of the sense strand. In some embodiments, the hydrophobic moiety may be attached to the 3' end of the sense strand. In some embodiments, the hydrophobic moiety may be attached to the 5' end of the antisense strand. In some embodiments, the hydrophobic moiety may be attached to the 3' end of the antisense strand.

[0112] In some embodiments, the composition comprises an oligonucleotide that inhibits expression of a target nucleic acid, the oligonucleotide comprises an siRNA that comprises a sense strand and an antisense strand, and the oligonucleotide comprises one or more vinyl phosphonates. In some embodiments, the one or more vinyl phosphonates may be bound to the 5' end of the sense strand. In some embodiments, the one or more vinyl phosphonates may be bound to the 3' end of the sense strand. In some embodiments, the one or more vinyl phosphonates may be bound to the 5' end of the antisense strand. In some embodiments, the one or more vinyl phosphonates may be bound to the 3' end of the antisense strand.

[0113] In some embodiments, the sense strand comprises or consists of RNA or modified RNA nucleotides. In some embodiments, the sense strand comprises deoxynucleosides. The deoxynucleosides may comprise DNA nucleosides. In some embodiments, the deoxynucleosides comprise or consist of 2' deoxynucleosides. The deoxynucleosides may be at positions (5' to 3', with the 5' position being 1) in the sense strand. The positions in the sense strand may be or include positions 2, 4, 6, 8, 9, 10, 12, 14, 16, or 18, or combinations of the aforementioned positions. The positions in the sense strand may be or include positions 2, 4, 6, 8, 10, 12, 14, 16, or 18, or combinations of the aforementioned positions. The positions in the sense strand may be or include positions 2, 6, 9, 10, 14, or 18, or combinations of the aforementioned positions. The positions in the sense strand may be or include positions 2, 6, 10, 14, or 18, or combinations of the aforementioned positions. The positions in the sense strand may be or include positions 4, 8, 9, 12, or 16, or combinations of the aforementioned positions. The positions in the sense strand may be or include positions 4, 8, 12, or 16, or combinations of the aforementioned positions. The positions in the sense strand may include position 9. The positions in the sense strand may be position 9. The sense strand may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 deoxynucleosides. In some embodiments, the sense strand comprises one deoxynucleoside. The sense strand may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 deoxynucleosides, or a range of deoxynucleosides defined by any two of the preceding numbers. The sense strand may comprise deoxynucleosides at all even-numbered positions. The sense strand may comprise deoxynucleosides at some even-numbered positions.The sense strand may include deoxynucleosides at every other even-numbered position. The sense strand may include one deoxynucleoside. The sense strand may include at least one deoxynucleoside. The sense strand may include at least two deoxynucleosides. The sense strand may include at least three deoxynucleosides. The sense strand may include at least four deoxynucleosides. The sense strand may include at least five deoxynucleosides. The sense strand may include at least six deoxynucleosides. The sense strand may include at least seven deoxynucleosides. The sense strand may include at least eight deoxynucleosides. The sense strand may include at least nine deoxynucleosides. The sense strand may include at least ten deoxynucleosides. The sense strand may include no more than two deoxynucleosides. The sense strand may include 3 deoxynucleosides or less. The sense strand may include 4 deoxynucleosides or less. The sense strand may include 5 deoxynucleosides or less. The sense strand may include 6 deoxynucleosides or less. The sense strand may include 7 deoxynucleosides or less. The sense strand may include 8 deoxynucleosides or less. The sense strand may include 9 deoxynucleosides or less. The sense strand may include 10 deoxynucleosides or less.

[0114] In some embodiments, the antisense strand comprises or consists of RNA or modified RNA nucleotides. In some embodiments, the antisense strand comprises deoxynucleosides. The deoxynucleosides may comprise DNA nucleosides. In some embodiments, the deoxynucleosides comprise or consist of 2' deoxynucleosides. The antisense strand may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 deoxynucleosides, or a range of deoxynucleosides defined by any two of the aforementioned numbers.

[0115] In some embodiments where the sense strand contains a deoxynucleoside (e.g., at the 9th nucleotide counting from the 5' end), the nucleosides at positions 1-8 contain a mixture of 2'fluoro and 2'-O-methyl modified nucleosides. In some embodiments where the sense strand contains a deoxynucleoside, the purines at positions 1-8 contain a mixture of 2'fluoro and 2'-O-methyl modified nucleosides. In some embodiments where the sense strand contains a deoxynucleoside, the pyrimidines at positions 1-8 contain a mixture of 2'fluoro and 2'-O-methyl modified nucleosides. In some embodiments where the sense strand contains a deoxynucleoside, all of the nucleosides at positions 1-8 contain 2'-O-methyl modified nucleosides. In some embodiments where the sense strand contains a deoxynucleoside, all of the purines at positions 1-8 contain 2'-O-methyl modified nucleosides. In some embodiments where the sense strand includes deoxynucleosides, the pyrimidines at positions 1-8 all include 2'-O-methyl modified nucleosides. In some embodiments where the sense strand includes deoxynucleosides, the purines at positions 1-8 all include 2'-O-methyl modified nucleosides and the pyrimidines at positions 1-8 all include 2'-O-methyl modified nucleosides. In some embodiments where the sense strand includes deoxynucleosides, the pyrimidines at positions 1-8 all include a mixture of 2'-fluoro and 2'-O-methyl modified nucleosides and the purines at positions 1-8 all include 2'-O-methyl modified nucleosides.

[0116] The antisense strand may begin with an alternating pattern, where the even-numbered nucleosides contain 2'-fluoro modified nucleosides and the odd-numbered nucleosides contain 2'-O-methyl modified nucleosides.The sense strand may then be optimized by replacing some of the 2'-fluoro modifications with 2'-O-methyl modifications.The antisense strand may contain a mixture of 2'-fluoro and 2'-O-methyl modifications.

[0117] 2. Qualified ASO In some embodiments, the composition comprises an oligonucleotide that inhibits expression of a target nucleic acid, wherein the oligonucleotide comprises an antisense oligonucleotide (ASO). In some embodiments, the ASO comprises modification pattern 1S, 2S, 3S, 4S, 1AS, or 2AS, or any other combination of modifications described herein. In some embodiments, the ASO comprises modification pattern 1S, 2S, 3S, 4S, 5S, 6S, 7S, 8S, 9S, 10S, 11S, 12S, 13S, 14S, 15S, 16S, 17S, 18S, 19S, 20S, 21S, 22S, 23S, 24S, 25S, 26S, 27S, 28S, 29S, 30S, 1AS, 2AS, 3AS, 4AS, or 5AS.

[0118] D. Preparation In some embodiments, the composition comprises a pharmaceutical composition. In some embodiments, the composition is sterile. In some embodiments, the composition further comprises a pharma- ceutically acceptable carrier.

[0119] In some embodiments, the pharma- ceutically acceptable carrier comprises water. In some embodiments, the pharma- ceutically acceptable carrier comprises a buffer. In some embodiments, the pharma- ceutically acceptable diluent is saline. In some embodiments, the pharma- ceutically acceptable carrier comprises water, a buffer, or saline. In some embodiments, the composition comprises a liposome. In some embodiments, the pharma- ceutically acceptable carrier comprises a liposome, a lipid, a nanoparticle, a protein, a protein-antibody complex, a peptide, cellulose, a nanogel, or a combination thereof.

[0120] In some embodiments, the composition is formulated to cross the blood-brain barrier. In some embodiments, the composition is formulated to be delivered to the central nervous system (CNS). In some embodiments, the composition comprises a lipophilic compound. The lipophilic compound may be useful for crossing the blood-brain barrier or for delivery to the CNS.

[0121] In some embodiments, the composition is formulated for administration. Administration may be systemic. In some embodiments, administration is intravenous. In some embodiments, administration is by injection. In some embodiments, injection is subcutaneous. In some embodiments, injection is subcutaneous. In some embodiments, injection is intraperitoneal. In some embodiments, injection is intramuscular. In some embodiments, injection is intrathecal. Administration may be to the eye (e.g., intravitreal). Administration may be to neural tissue. Administration may be to the brain. Administration may be intracerebroventricular. Administration may be intrathecal. Administration may be to the spinal cord or spinal canal. In some embodiments, the formulation allows for delivery of a compound, such as an oligonucleotide, to an ocular cell. In some embodiments, the formulation allows for delivery of a compound, such as an oligonucleotide, to a neuronal cell.

[0122] E.Kit In some embodiments, a kit is described herein. The kit may include an oligonucleotide, such as an siRNA, as described herein. The oligonucleotide may be conjugated to a lipid moiety. The kit may include a lipid moiety as described herein. The oligonucleotide may include a nucleoside modification or a modified internucleoside bond. The oligonucleotide may include any of the modifications described herein, such as modifications of the base sequence. The kit may include a delivery reagent, such as a needle. The kit may include instructions for use, such as a method for use in the methods described herein.

[0123] II. Methods and Uses Disclosed herein, in some embodiments, is a method of administering the compositions described herein to a subject. Some embodiments relate to the use of the compositions described herein, such as administering the compositions to a subject.

[0124] Some embodiments relate to a method of treating a disorder in a subject in need thereof. Some embodiments relate to the use of a composition described herein in a method of treatment. Some embodiments include administering a composition described herein to a subject suffering from the disorder. In some embodiments, the administration treats the disorder in the subject. In some embodiments, the composition treats the disorder in the subject.

[0125] In some embodiments, the treatment includes preventing, inhibiting, or reversing a disorder in a subject. Some embodiments relate to the use of a composition described herein in a method of preventing, inhibiting, or reversing a disorder. Some embodiments relate to a method of preventing, inhibiting, or reversing a disorder in a subject in need thereof. Some embodiments include administering a composition described herein to a subject suffering from a disorder. In some embodiments, the administration prevents, inhibits, or reverses a disorder in the subject. In some embodiments, the composition prevents, inhibits, or reverses a disorder in the subject.

[0126] Some embodiments relate to a method of preventing a disorder in a subject in need thereof. Some embodiments relate to the use of a composition described herein in a method of preventing a disorder. Some embodiments include administering a composition described herein to a subject suffering from the disorder. In some embodiments, the administration prevents the disorder in the subject. In some embodiments, the composition prevents the disorder in the subject.

[0127] Some embodiments relate to a method of inhibiting a disorder in a subject in need thereof. Some embodiments relate to the use of a composition described herein in a method of inhibiting a disorder. Some embodiments include administering a composition described herein to a subject suffering from the disorder. In some embodiments, the administration inhibits the disorder in the subject. In some embodiments, the composition inhibits the disorder in the subject.

[0128] Some embodiments relate to a method of reversing a disorder in a subject in need thereof. Some embodiments relate to the use of a composition described herein in a method of reversing a disorder. Some embodiments include administering a composition described herein to a subject suffering from a disorder. In some embodiments, the administration reverses the disorder in the subject. In some embodiments, the composition reverses the disorder in the subject.

[0129] In some embodiments, administration is systemic. In some embodiments, administration is intravenous. In some embodiments, administration is by injection. In some embodiments, injection is subcutaneous. In some embodiments, injection is subcutaneous. In some embodiments, injection is intraperitoneal. In some embodiments, injection is intramuscular. Administration may be to the eye (e.g., intravitreal). Administration may be to neural tissue. Administration may be to the brain. Administration may be intracerebroventricular. In some embodiments, injection is intrathecal. Administration may be intrathecal. Administration may be to the spinal cord or spinal canal.

[0130] In some embodiments, disclosed herein is a method for targeting siRNA to cells or tissues.The method may comprise conjugating siRNA (e.g., the sense strand of siRNA) to lipid moiety as described herein.The method may comprise delivering the siRNA conjugated to lipid moiety to tissue or cells.The method may comprise contacting the siRNA conjugated to lipid moiety to tissue or cells.

[0131] In some embodiments, the present specification describes a method for reducing the amount of target RNA or protein in cells or tissues, comprising administering or delivering the composition described herein to cells or tissues.The composition may comprise siRNA having an antisense strand with a sequence complementary to RNA.The antisense strand may bind to RNA.

[0132] In some embodiments, the present disclosure provides a method for making siRNA compositions.The method may include conjugating siRNA to lipid moiety.The siRNA and lipid moiety may include any siRNA or lipid moiety described herein.The method may include synthesizing siRNA.The method may include synthesizing lipid moiety.

[0133] A. Disability Some embodiments of the methods described herein include treating a disorder in a subject in need thereof. In some embodiments, the disorder is an eye disorder. In some embodiments, the disorder is a fat-related disorder. In some embodiments, the disorder is a kidney disorder. In some embodiments, the disorder is a brain disorder. In some embodiments, the disorder is a vascular disorder. In some embodiments, the disorder is a muscle disorder. In some embodiments, the disorder is a pulmonary disorder.

[0134] B. Target Some embodiments of the methods described herein include treatment of a subject. Non-limiting examples of subjects include vertebrates, animals, mammals, dogs, cats, cows, rodents, mice, rats, primates, monkeys, and humans. In some embodiments, the subject is a vertebrate. In some embodiments, the subject is an animal. In some embodiments, the subject is a mammal. In some embodiments, the subject is a dog. In some embodiments, the subject is a cat. In some embodiments, the subject is a cow. In some embodiments, the subject is a mouse. In some embodiments, the subject is a rat. In some embodiments, the subject is a primate. In some embodiments, the subject is a monkey. In some embodiments, the subject is an animal, mammal, dog, cat, cow, rodent, mouse, rat, primate, or monkey. In some embodiments, the subject is a human.

[0135] In some embodiments, the subject is male. In some embodiments, the subject is female. In some embodiments, the subject is an adult (e.g., at least 18 years of age).

[0136] C. Baseline Measurements Some embodiments of the methods described herein include obtaining a baseline measurement from the subject. For example, in some embodiments, the baseline measurement is obtained from the subject before treating the subject.

[0137] In some embodiments, the baseline measurements are obtained directly from the subject. In some embodiments, the baseline measurements are obtained by observation, e.g., by observation of the subject or tissues of the subject. In some embodiments, the baseline measurements are obtained non-invasively using an imaging device.

[0138] In some embodiments, the baseline measurements are obtained on a sample from the subject. In some embodiments, the baseline measurements are obtained on one or more histological tissue sections. In some embodiments, the baseline measurements are obtained by performing an assay, such as an immunoassay, a colorimetric assay, or a fluorescent assay, on a sample obtained from the subject. In some embodiments, the baseline measurements are obtained by an immunoassay, a colorimetric assay, a fluorescent assay, or a chromatographic (e.g., HPLC) assay. In some embodiments, the baseline measurements are obtained by PCR.

[0139] In some embodiments, the baseline measurement is a baseline target protein measurement. In some embodiments, the baseline target protein measurement comprises a baseline target protein level. In some embodiments, the baseline target protein level is expressed as mass or percentage of target protein per sample weight. In some embodiments, the baseline target protein level is expressed as mass or percentage of target protein per sample volume. In some embodiments, the baseline target protein level is expressed as mass or percentage of target protein per total protein in the sample. In some embodiments, the baseline target protein measurement is a baseline blood / tissue target protein measurement. In some embodiments, the baseline target protein measurement is obtained by an assay such as an immunoassay, a colorimetric assay, or a fluorescent assay.

[0140] In some embodiments, the baseline measurement is a baseline target mRNA measurement. In some embodiments, the baseline target mRNA measurement is a baseline target mRNA level. In some embodiments, the baseline target mRNA level is presented as the amount or percentage of target mRNA per sample weight. In some embodiments, the baseline target mRNA level is presented as the amount or percentage of target mRNA per sample volume. In some embodiments, the baseline target mRNA level is presented as the amount or percentage of target mRNA per total mRNA in the sample. In some embodiments, the baseline target mRNA level is presented as the amount or percentage of target mRNA per total nucleic acid in the sample. In some embodiments, the baseline target mRNA level is presented relative to another mRNA level, such as the mRNA level of a housekeeping gene in the sample. In some embodiments, the baseline target mRNA measurement is a baseline tissue target mRNA measurement. In some embodiments, the baseline target mRNA measurement is obtained by an assay, such as a polymerase chain reaction (PCR) assay. In some embodiments, the PCR comprises quantitative PCR (qPCR). In some embodiments, the PCR comprises reverse transcription of the target mRNA.

[0141] Some embodiments of the methods described herein include obtaining a sample from a subject. In some embodiments, a baseline measurement is obtained in a sample obtained from the subject. In some embodiments, a sample is obtained from a subject prior to administration or treatment of the subject with a composition described herein. In some embodiments, a baseline measurement is obtained in a sample obtained from a subject prior to administration of a composition to the subject. In some embodiments, a sample is obtained from a subject in a fasted state. In some embodiments, a sample is obtained from a subject after an overnight fasting period. In some embodiments, a sample is obtained from a subject in a fed state.

[0142] In some embodiments, the sample comprises a bodily fluid. In some embodiments, the sample is a bodily fluid sample. In some embodiments, the sample is a sample of blood, plasma, or serum. In some embodiments, the sample comprises blood. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a whole blood sample. In some embodiments, the blood is fractionated or centrifuged. In some embodiments, the sample comprises plasma. In some embodiments, the sample may be a plasma sample. In some embodiments, the blood sample may be a plasma sample. In some embodiments, the sample comprises serum. In some embodiments, the sample is a serum sample. The blood sample may be a serum sample. In some embodiments, the fluid comprises cerebrospinal fluid (CSF). For example, the bodily fluid may be obtained by spinal tap. In some embodiments, the bodily fluid comprises spinal fluid. In some embodiments, the bodily fluid comprises cerebrospinal fluid. In some embodiments, the bodily fluid comprises cerebrospinal fluid.

[0143] In some embodiments, the sample comprises a tissue. In some embodiments, the sample is a tissue sample. In some embodiments, the tissue comprises eye, kidney, muscle, adipose, brain, lung, or vascular tissue. For example, the baseline target mRNA measurements or baseline target protein measurements may be obtained in a kidney, adipose, brain, lung, or vascular sample obtained from the patient. In some embodiments, the tissue comprises liver tissue. The kidney tissue may comprise renal medullary cells or renal proximal tubule cells. In some embodiments, the tissue comprises adipose tissue. In some embodiments, the adipose tissue comprises white adipose tissue. The adipose tissue may comprise adipocytes. In some embodiments, the tissue comprises liver tissue. The liver may comprise hepatocytes. In some embodiments, the tissue is a non-liver tissue. In some embodiments, the tissue comprises ocular tissue. In some embodiments, the ocular tissue comprises retinal tissue. In some embodiments, the tissue comprises brain tissue. The brain tissue may comprise neurons or glial cells. The brain tissue may comprise the cerebellum. The brain tissue may comprise the frontal cortex. The brain tissue may include the temporal cortex. The brain tissue may include tissue from the right hemisphere of the brain (e.g., the right frontal cortex or the right temporal cortex). The brain tissue may include tissue from the left hemisphere of the brain (e.g., the left frontal cortex or the left temporal cortex). The brain tissue may include hippocampal tissue. In some embodiments, the tissue includes neural tissue. In some embodiments, the tissue includes nerves. In some embodiments, the tissue includes spinal cord tissue. In some embodiments, the tissue includes spinal cord or spinal canal tissue. In some embodiments, the tissue includes lung tissue. In some embodiments, the tissue includes vascular tissue. The vascular tissue may include vascular endothelial cells.

[0144] In some embodiments, the sample comprises a cell. In some embodiments, the sample comprises a cell. In some embodiments, the cell comprises an ocular cell, a renal cell, a muscle cell, an adipocyte, a brain cell, or a vasculature cell. In some embodiments, the cell is a renal cell. In some embodiments, the renal cell is a renal medullary cell. In some embodiments, the renal cell is a renal proximal tubule cell. In some embodiments, the cell is an adipocyte. In some embodiments, the cell is a liver cell. In some embodiments, the liver cell is a hepatocyte. In some embodiments, the cell is a non-liver cell or is not a hepatocyte. In some embodiments, the cell is an ocular cell. In some embodiments, the ocular cell is a retinal cell. In some embodiments, the ocular cell is a rod cell. In some embodiments, the ocular cell is a cone cell. In some embodiments, the cell is a brain cell. In some embodiments, the brain cell is from brain tissue, such as cerebellar, cortical, or hippocampal tissue. In some embodiments, the brain cell is a neuron. In some embodiments, the brain cell is a glial cell. In some embodiments, the cell is a spinal cell. In some embodiments, the cell is a spinal cord cell. In some embodiments, the cell is a lung cell. In some embodiments, the cell is a vasculature cell. In some embodiments, the vasculature cell is an endothelial cell.

[0145] D. Effects In some embodiments, the composition or administration of the composition affects a measurement, such as a target protein measurement or a target mRNA measurement, as compared to a baseline measurement.

[0146] Some embodiments of the method described herein include obtaining a measurement from a subject. For example, the measurement may be obtained from the subject after the subject is treated. In some embodiments, the measurement is obtained in a second sample (such as a body fluid or tissue sample described herein) obtained from the subject after the composition is administered to the subject. In some embodiments, the measurement is indicative of the disorder being treated.

[0147] In some embodiments, the measurements are obtained directly from the subject. In some embodiments, the measurements are obtained non-invasively using an imaging device. In some embodiments, the measurements are obtained in a second sample from the subject. In some embodiments, the measurements are obtained in one or more histological tissue sections. In some embodiments, the measurements are obtained by performing an assay on the second sample obtained from the subject. In some embodiments, the measurements are obtained by an assay such as the assays described herein. In some embodiments, the assay is an immunoassay, a colorimetric assay, a fluorescent assay, a chromatographic (e.g., HPLC) assay, or a PCR assay. In some embodiments, the measurements are obtained by an assay such as an immunoassay, a colorimetric assay, a fluorescent assay, or a chromatographic (e.g., HPLC) assay. In some embodiments, the measurements are obtained by PCR. In some embodiments, the measurements are obtained by histological examination. In some embodiments, the measurements are obtained by observation. In some embodiments, further measurements are provided in a third sample, a fourth sample, a fifth sample, etc.

[0148] In some embodiments, the measurements are obtained within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, or 24 hours after administration of the composition. In some embodiments, the measurements are obtained within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after administration of the composition. In some embodiments, the measurements are obtained within 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, 3 years, 4 years, or 5 years after administration of the composition. In some embodiments, the measurements are obtained 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, or 24 hours after administration of the composition. In some embodiments, the measurements are obtained 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after administration of the composition. In some embodiments, measurements are obtained 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, 3 years, 4 years, or 5 years after administration of the composition.

[0149] In some embodiments, the composition reduces the measurement compared to a baseline measurement. In some embodiments, the reduction is measured in a second sample obtained from the subject after administration of the composition to the subject. In some embodiments, the reduction is measured directly in the subject after administration of the composition to the subject. In some embodiments, the measurement is reduced by about 2.5% or more, about 5% or more, or about 7.5% or more compared to the baseline measurement. In some embodiments, the measurement is reduced by about 10% or more compared to the baseline measurement. In some embodiments, the measurement is reduced by about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more compared to the baseline measurement. In some embodiments, the measurement is reduced by about 2.5% or less, about 5% or less, or about 7.5% or less compared to the baseline measurement. In some embodiments, the measurement is reduced by about 10% or less compared to the baseline measurement. In some embodiments, the measurement is decreased by about 20% or less, about 30% or less, about 40% or less, about 50% or less, about 60% or less, about 70% or less, about 80% or less, about 90% or less, or about 100% or less compared to the baseline measurement, hi some embodiments, the measurement is decreased by 2.5%, 5%, 7.5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or a range defined by any two of the foregoing percentages.

[0150] In some embodiments, the composition increases the measurement compared to a baseline measurement. In some embodiments, the increase is measured in a second sample obtained from the subject after administering the composition to the subject. In some embodiments, the increase is measured directly in the subject after administering the composition to the subject. In some embodiments, the measurement increases by about 2.5% or more, about 5% or more, or about 7.5% or more compared to the baseline measurement. In some embodiments, the measurement increases by about 10% or more compared to the baseline measurement. In some embodiments, the measurement increases by about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more compared to the baseline measurement. In some embodiments, the measurement increases by about 100% or more, about 250% or more, about 500% or more, about 750% or more, or about 1000% or more compared to the baseline measurement. In some embodiments, the measurement increases by about 2.5% or less, about 5% or less, or about 7.5% or less compared to the baseline measurement. In some embodiments, the measurement increases by about 10% or less compared to the baseline measurement. In some embodiments, the measurement increases by about 20% or less, about 30% or less, about 40% or less, about 50% or less, about 60% or less, about 70% or less, about 80% or less, about 90% or less, or about 100% or less compared to the baseline measurement. In some embodiments, the measurement increases by about 100% or less, about 250% or less, about 500% or less, about 750% or less, or about 1000% or less compared to the baseline measurement. In some embodiments, the measurement increases by 2.5%, 5%, 7.5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 250%, 500%, 750%, or 1000%, or a range defined by any two of the foregoing percentages.

[0151] In some embodiments, the measurements are target protein measurements. In some embodiments, the target protein measurements include target protein levels. In some embodiments, the target protein levels are presented as mass or percentage of target protein per sample weight. In some embodiments, the target protein levels are presented as mass or percentage of target protein per sample volume. In some embodiments, the target protein levels are presented as mass or percentage of target protein per total protein in the sample. In some embodiments, the target protein measurements are blood / tissue target protein measurements. In some embodiments, the target protein measurements are obtained by assays such as immunoassays, colorimetric assays, or fluorescent assays.

[0152] In some embodiments, the composition reduces the target protein measurement compared to a baseline target protein measurement. In some embodiments, the composition reduces blood target protein levels compared to a baseline target protein measurement. In some embodiments, the composition reduces tissue target protein levels compared to a baseline target protein measurement. In some embodiments, the reduction in target protein levels is measured in a second sample obtained from the subject after administering the composition to the subject. In some embodiments, the target protein measurement is reduced by about 2.5% or more, about 5% or more, or about 7.5% or more compared to the baseline target protein measurement. In some embodiments, the target protein measurement is reduced by about 10% or more compared to the baseline target protein measurement. In some embodiments, the target protein measurement is reduced by about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 100% compared to the baseline target protein measurement. In some embodiments, the target protein measurement is reduced by about 2.5% or less, about 5% or less, or about 7.5% or less compared to the baseline target protein measurement. In some embodiments, the target protein measurement is decreased by about 10% or less compared to the baseline target protein measurement. In some embodiments, the target protein measurement is decreased by about 20% or less, about 30% or less, about 40% or less, about 50% or less, about 60% or less, about 70% or less, about 80% or less, about 90% or less, or about 100% or less compared to the baseline target protein measurement. In some embodiments, the target protein measurement is decreased by 2.5%, 5%, 7.5%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or a range defined by any two of the foregoing percentages.

[0153] In some embodiments, the measurements are target mRNA measurements. In some embodiments, the target mRNA measurements include target mRNA levels. In some embodiments, the target mRNA levels are presented as the amount or percentage of target mRNA per sample weight. In some embodiments, the target mRNA levels are presented as the amount or percentage of target mRNA per sample volume. In some embodiments, the target mRNA levels are presented as the amount or percentage of target mRNA per total mRNA in the sample. In some embodiments, the target mRNA levels are presented as the amount or percentage of target mRNA per total nucleic acid in the sample. In some embodiments, the target mRNA levels are presented relative to another mRNA level, such as the mRNA level of a housekeeping gene in the sample. In some embodiments, the target mRNA measurements are blood / tissue target mRNA measurements. In some embodiments, the target mRNA measurements are obtained by an assay, such as a PCR assay. In some embodiments, the PCR comprises qPCR. In some embodiments, the PCR comprises reverse transcription of the target mRNA.

[0154] In some embodiments, the composition reduces the target mRNA measurement compared to a baseline target mRNA measurement. In some embodiments, the target mRNA measurement is obtained in a second sample obtained from the subject after administering the composition to the subject. In some embodiments, the composition reduces the target mRNA level relative to a baseline target mRNA level. In some embodiments, the reduction in the target mRNA level is measured in a second sample obtained from the subject after administering the composition to the subject. In some embodiments, the second sample is a liver sample. In some embodiments, the second sample is an adipose sample. In some embodiments, the target mRNA measurement is reduced by about 2.5% or more, about 5% or more, or about 7.5% or more compared to the baseline target mRNA measurement. In some embodiments, the target mRNA measurement is reduced by about 10% or more compared to the baseline target mRNA measurement. In some embodiments, the target mRNA measurement is reduced by about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 100% compared to the baseline target mRNA measurement. In some embodiments, the target mRNA measurement is decreased by about 2.5% or less, about 5% or less, or about 7.5% or less compared to the baseline target mRNA measurement. In some embodiments, the target mRNA measurement is decreased by about 10% or less compared to the baseline target mRNA measurement. In some embodiments, the target mRNA measurement is decreased by about 20% or less, about 30% or less, about 40% or less, about 50% or less, about 60% or less, about 70% or less, about 80% or less, about 90% or less, or about 100% or less compared to the baseline target mRNA measurement. In some embodiments, the target mRNA measurement is decreased by 2.5%, 5%, 7.5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or a range defined by any two of the foregoing percentages.

[0155] III. Definition Unless otherwise defined, all terminology, notations, and other technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be construed as indicating that they are substantially different from those commonly understood in the art.

[0156] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Thus, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and individual numerical values ​​within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0157] As used in this specification and claims, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise. For example, the term "a sample" includes a plurality of samples, including mixtures thereof.

[0158] The terms "determining," "measuring," "evaluating," "assessing," "assaying," and "analyzing" are often used interchangeably herein to refer to forms of measurement. The terms include determining whether an element is present (e.g., detecting). Such terms can include quantitative, qualitative, or quantitative and qualitative determinations. Assessing can be relative or absolute. "Detecting the presence of" can include determining the amount of something present in addition to determining whether something is present or absent depending on the context.

[0159] The terms "subject" and "patient" may be used interchangeably herein. A "subject" may be a biological entity that contains expressed genetic material. The biological entity may be, for example, a plant, an animal, or a microorganism, including bacteria, viruses, fungi, and protozoa. The subject may be a mammal. The mammal may be a human. The subject may be diagnosed or suspected to be at high risk for a disease. In some cases, the subject is not necessarily diagnosed or suspected to be at high risk for a disease.

[0160] As used herein, a number followed by the term "about" refers to a number that is plus or minus 10% of that number. A range followed by the term "about" refers to a range of minus 10% of its minimum value and plus 10% of its maximum value.

[0161] As used herein, the term "treatment" or "treating" is used in reference to a pharmaceutical or other interventional regimen to obtain a beneficial or desired outcome in a recipient. A beneficial or desired outcome includes, but is not limited to, a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit may also refer to the eradication or amelioration of the condition or underlying disease being treated. Similarly, a therapeutic benefit may be achieved by the eradication or amelioration of one or more of the physiological symptoms associated with an underlying disease, such that an improvement is observed in a subject, even though the subject may still be affected by the underlying disease. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or illness, delaying or eliminating the onset of symptoms of a disease or illness, slowing, stopping, or reversing the progression of a disease or illness, or any combination thereof. With regard to a prophylactic benefit, a subject at risk of developing a particular disease or reporting one or more physiological symptoms of a disease may be treated, even if a diagnosis of the disease has not been made.

[0162] "Treatment" or "treating" may include an approach to obtain a beneficial or desired result with respect to a disease, disorder, or medical condition, including, but not limited to, a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit may include, for example, eradication or amelioration of the underlying disease being treated. Similarly, a therapeutic benefit may include, for example, eradication or amelioration of one or more physiological symptoms associated with an underlying disease, such that an improvement is observed in a subject, even though the subject may still be affected by the underlying disease. In certain embodiments, with respect to a prophylactic benefit, the composition is administered to a subject at risk of developing a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even if a diagnosis of the disease has not been made. Treatment by administration of the compounds described herein does not require the involvement of a medical professional.

[0163] Some embodiments refer to nucleic acid sequence information. It is contemplated that in some embodiments, thymine (T) may be replaced with uracil (U) or vice versa. For example, some sequences in the sequence listing may list T, but in some embodiments, these may be replaced with U. In some oligonucleotides having nucleic acid sequences that include uracil, uracil may be replaced with thymine. Similarly, in some oligonucleotides having nucleic acid sequences that include thymine, thymine may be replaced with uracil. In some embodiments, oligonucleotides such as siRNAs may include or consist of RNA. In some embodiments, oligonucleotides may include DNA. For example, oligonucleotides may include 2' deoxyribonucleotides. ASOs may include or consist of DNA. To the extent that the sequence listing conflicts with the disclosure herein, the present specification controls.

[0164] Some embodiments include sequences with nucleotide modifications or modified internucleoside linkages. Generally, unless otherwise specified, Nf (e.g., Af, Cf, Gf, Tf, or Uf) refers to 2'fluoro-modified nucleosides, dN (e.g., dA, dC, dG, dT, or dU) refers to 2'deoxynucleosides, n (e.g., a, c, g, t, or u) refers to 2'O-methyl-modified nucleosides, and "s" refers to phosphorothioate linkages.

[0165] A pyrimidine may include cytosine (C), thymine (T), or uracil (U). A pyrimidine may include C or U. A pyrimidine may include C or T. A reference to a pyrimidine may include a nucleoside or nucleotide comprising a pyrimidine. A purine may include guanine (G) or adenine (A). A reference to a purine may include a nucleoside or nucleotide comprising a purine.

[0166] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0167] "C x-y " or "C x- C y The term "alkyl," when used with a chemical moiety such as alkyl, alkenyl, or alkynyl, means that the group contains from x to y carbons in the chain. For example, "C 1-6 The term "alkyl" refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain and branched-chain alkyl groups containing 1 to 6 carbons.

[0168] "C x-y alkenyl" and "C x-y The term "alkynyl" refers to substituted or unsubstituted unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one double or triple bond respectively.

[0169] The term "carbocycle" as used herein refers to a saturated, unsaturated, or aromatic ring in which each atom of the ring is carbon. Carbocycles include 3-10 membered monocyclic rings, 5-12 membered bicyclic rings, 5-12 membered spiro bicyclic rings, and 5-12 membered bridged rings. Each ring of a bicyclic carbocycle may be selected from a saturated ring, an unsaturated ring, and an aromatic ring. In an exemplary embodiment, an aromatic ring, e.g., phenyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Bicyclic carbocycles include any combination of saturated, unsaturated, and aromatic bicyclic rings, as valences permit. Bicyclic carbocycles further include spiro bicyclic rings, e.g., spiropentane. Bicyclic carbocycles include any combination of ring sizes such as a 3-3 spiro ring system, a 4-4 spiro ring system, a 4-5 fused ring system, a 5-5 fused ring system, a 5-6 fused ring system, a 6-6 fused ring system, a 5-7 fused ring system, a 6-7 fused ring system, a 5-8 fused ring system, and a 6-8 fused ring system. Exemplary carbocycles include cyclopentyl, cyclohexyl, cyclohexenyl, adamantyl, phenyl, indanyl, naphthyl, and bicyclo[1.1.1]pentanyl.

[0170] The term "aryl" refers to an aromatic monocyclic or aromatic polycyclic hydrocarbon ring system. An aromatic monocyclic or aromatic polycyclic hydrocarbon ring system contains only hydrogen and carbon from 5 to 18 carbon atoms, and at least one of the rings in the ring system is aromatic, i.e., contains a cyclic delocalized (4n+2) π-electron system according to the Hückel theory. Ring systems from which aryl groups are derived include, but are not limited to, groups such as benzene, fluorene, indane, indene, tetralin, and naphthalene.

[0171] The term "cycloalkyl" refers to a saturated ring in which each atom of the ring is carbon. Cycloalkyls may include monocyclic and polycyclic rings, such as 3-10 membered monocyclic rings, 5-12 membered bicyclic rings, 5-12 membered spiro bicyclic rings, and 5-12 membered bridged rings. In certain embodiments, cycloalkyls contain 3-10 carbon atoms. In other embodiments, cycloalkyls contain 5-7 carbon atoms. Cycloalkyls can be attached to the remainder of the molecule by a single bond. Examples of monocyclic cycloalkyls include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic cycloalkyl radicals include, for example, adamantyl, spiropentane, norbornyl (i.e., bicyclo[2.2.1]heptanyl), decalinyl, 7,7-dimethylbicyclo[2.2.1]heptanyl, bicyclo[1.1.1]pentanyl, and the like.

[0172] The term "cycloalkenyl" refers to a saturated ring in which each atom of the ring is carbon and there is at least one double bond between two ring carbons. Cycloalkenyls may include monocyclic and polycyclic rings, such as 3-10 membered monocyclic rings, 6-12 membered bicyclic rings, and 5-12 membered bridged rings. In other embodiments, cycloalkenyls contain 5-7 carbon atoms. Cycloalkenyls can be attached to the remainder of the molecule by a single bond. Examples of monocyclic cycloalkenyls include, for example, cyclopentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl.

[0173] The term "halo," or alternatively, "halogen" or "halide," means fluoro, chloro, bromo, or iodo. In some embodiments, halo is fluoro, chloro, or bromo.

[0174] The term "haloalkyl" refers to an alkyl radical, as defined above, that is substituted by one or more halo radicals, e.g., trifluoromethyl, dichloromethyl, bromomethyl, 2,2,2-trifluoroethyl, 1-chloromethyl-2-fluoroethyl, etc. In some embodiments, the alkyl portion of the haloalkyl radical is optionally further substituted as described herein.

[0175] The term "heterocycle" refers to a saturated, unsaturated, or aromatic ring containing one or more heteroatoms. Exemplary heteroatoms include N, O, Si, P, B, and S atoms. Heterocycles include 3-10 membered monocyclic rings, 6-12 membered bicyclic rings, 5-12 membered spiro bicyclic rings, and 5-12 membered bridged rings. Bicyclic heterocycles include any combination of saturated, unsaturated, and aromatic bicyclic rings, as valences permit. In exemplary embodiments, an aromatic ring, e.g., pyridyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, morpholine, piperidine, or cyclohexene. Bicyclic heterocycles include any combination of ring sizes such as 4-5 fused ring systems, 5-5 fused ring systems, 5-6 fused ring systems, 6-6 fused ring systems, 5-7 fused ring systems, 6-7 fused ring systems, 5-8 fused ring systems, and 6-8 fused ring systems. Bicyclic heterocycles further include spiro bicyclic rings, for example, 5-12 membered spiro bicycles, for example, 2-oxa-6-azaspiro[3.3]heptane.

[0176] The term "heteroaryl" refers to a radical derived from a 5-18 membered aromatic ring radical containing 2-17 carbon atoms and 1-6 heteroatoms selected from nitrogen, oxygen, and sulfur. As used herein, a heteroaryl radical is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, in which at least one of the rings in the ring system is aromatic, i.e., contains a cyclic delocalized (4n+2) π-electron system according to the Hückel theory. Heteroaryl includes fused or bridged ring systems. The heteroatoms in a heteroaryl radical are optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. Heteroaryl is attached to the remainder of the molecule through any atom of the ring. Examples of heteroaryl include azepinyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzodioxolyl, benzofuranyl, benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzothieno[3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, cyclopenta[d]pyrimidinyl, 6,7-dihydro-5H- Cyclopenta[4,5]thieno[2,3-d]pyrimidinyl, 5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, furo[3,2-c]pyridinyl, 5,6,7,8,9,10-hexa Hydrocycloocta[d]pyrimidinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridazinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridinyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, 5,8-Methano-5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl, 1,6-naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 5,6,6a,7,8,9,10,10a-octahydrobenzo[h]quinazolinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyridinyl, pyrido[3,2-d]pyrimidinyl, pyrido do) [3,4-d]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 6,7,8,9-tetrahydro-5H-cyclohepta[4,5 ]thieno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pyridinyl, and thiophenyl (i.e., thienyl).

[0177] The term "heterocycloalkyl" refers to a saturated ring having carbon atoms and at least one heteroatom. Exemplary heteroatoms include N, O, Si, P, B, and S atoms. Heterocycloalkyls may include monocyclic and polycyclic rings, such as 3-10 membered monocyclic rings, 6-12 membered bicyclic rings, 5-12 membered spiro bicyclic rings, and 5-12 membered bridged rings. The heteroatoms in the heterocycloalkyl radical are optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. The heterocycloalkyl is attached to the remainder of the molecule through any atom of the heterocycloalkyl, such as any carbon or nitrogen atom of the heterocycloalkyl, where valence allows. Examples of heterocycloalkyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, 2-oxa-6-azaspiro[3.3]heptane, and 1,1-dioxo-thiomorpholinyl.

[0178] The term "heterocycloalkenyl" refers to an unsaturated ring having carbon atoms and at least one heteroatom, and at least one double bond exists between two ring carbons. Heterocycloalkenyl does not include heteroaryl rings. Exemplary heteroatoms include N, O, Si, P, B, and S atoms. Heterocycloalkenyl may include monocyclic and polycyclic rings, such as 3-10 membered monocyclic rings, 6-12 membered bicyclic rings, and 5-12 membered bridged rings. In other embodiments, heterocycloalkenyl contains 5-7 ring atoms. Heterocycloalkenyl can be attached to the remainder of the molecule by a single bond. Examples of monocyclic cycloalkenyls include, for example, pyrroline (dihydropyrrole), pyrazoline (dihydropyrazole), imidazoline (dihydroimidazole), triazoline (dihydrotriazole), dihydrofuran, dihydrothiophene, oxazoline (dihydrooxazole), isoxazoline (dihydroisoxazole), thiazoline (dihydrothiazole), isothiazolin (dihydroisothiazole), oxadiazoline (dihydrooxadiazole), thiadiazoline (dihydrothiadiazole), dihydropyridine, tetrahydropyridine, dihydropyridazine, tetrahydropyridazine, dihydropyrimidine, tetrahydropyrimidine, dihydropyrazine, tetrahydropyrazine, pyran, dihydropyran, thiopyran, dihydrothiopyran, dioxin, dihydrodioxin, oxazine, dihydrooxazine, thiazine, and dihydrothiazine.

[0179] The term "substituted" refers to a moiety having a substituent replacing a hydrogen on one or more carbons or substitutable heteroatoms, e.g., NH or NH2 of a compound. It will be understood that "substituted" or "substituted with" includes the implicit proviso that such substitution results in a stable compound, subject to the permissible valences of the replaced atoms and substituents, and does not spontaneously undergo transformation, e.g., by rearrangement, cyclization, elimination, and the like. In certain embodiments, substituted refers to a moiety having a substituent replacing two hydrogen atoms on the same carbon atom, such as replacing two hydrogen atoms on a single carbon with an oxo, imino, or thioxo group. As used herein, the term "substituted" is intended to include all permissible substituents of an organic compound. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. The permissible substituents may be one or more and the same or different for appropriate organic compounds.

[0180] In some embodiments, a substituent may be any of the substituents described herein, for example, halogen, hydroxy, oxo (=O), thioxo (=S), cyano (-CN), nitro (-NO2), imino (=NH), oximo (=N-OH), hydrazino (=N-NH2), -R b -OR a , -R b -OC(O)-R a , -R b -OC(O)-OR a , -R b -OC(O)-N(R a )2, -R b -N(R a )2, -R b -C(O)R a , -R b -C(O)OR a , -R b -C(O)N(R a )2, -R b -OR c -C(O)N(R a )2, -R b-N(R a )C(O)OR a , -R b -N(R a )C(O)R a , -R b -N(R a )S(O) t R a (t is 1 or 2), -R b -S(O) t R a (t is 1 or 2), -R b -S(O) t OR a (t is 1 or 2), and -R b -S(O) t N(R a )2 (where t is 1 or 2), as well as alkyl, alkenyl, alkynyl, aryl, aralkyl, aralkenyl, aralkynyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, and heteroarylalkyl, any of which may be alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, oxo (=O), thioxo (=S), cyano (-CN), nitro (-NO2), imino (=NH), oximo (=N-OH), hydrazine (=N-NH2), -R b -OR a , -R b -OC(O)-R a , -R b -OC(O)-OR a , -R b -OC(O)-N(R a )2, -R b -N(R a )2, -R b -C(O)R a , -R b -C(O)OR a , -R b -C(O)N(R a )2, -R b -OR c -C(O)N(R a )2, -R b -N(R a )C(O)ORa , -R b -N(R a )C(O)R a , -R b -N(R a )S(O) t R a (t is 1 or 2), -R b -S(O) t R a (t is 1 or 2), -R b -S(O) t OR a (t is 1 or 2), and -R b -S(O) t N(R a )2 (t is 1 or 2), where R a are each independently selected from hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, or heteroarylalkyl; R a are alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, oxo (=O), thioxo (=S), cyano (-CN), nitro (-NO2), imino (=NH), oximo (=N-OH), hydrazine (=N-NH2), -R, where valence allows. b -OR a , -R b -OC(O)-R a , -R b -OC(O)-OR a , -R b -OC(O)-N(R a )2, -R b -N(R a )2, -R b -C(O)R a , -R b -C(O)OR a , -R b -C(O)N(R a )2, -R b -OR c -C(O)N(R a )2, -R b -N(R a)C(O)OR a , -R b -N(R a )C(O)R a , -R b -N(R a )S(O) t R a (t is 1 or 2), -R b -S(O) t R a (t is 1 or 2), -R b -S(O) t OR a (t is 1 or 2), and -R b -S(O) t N(R a )2 (where t is 1 or 2), and R b are each independently selected from a direct bond, or a straight or branched alkylene, alkenylene, or alkynylene chain; R c is a straight or branched alkylene chain, alkenylene chain, or alkynylene chain.

[0181] A double bond to an oxygen atom, such as an oxo group, is represented herein as both "=O" and "(O)". A double bond to a nitrogen atom is represented herein as both "=NR" and "(NR)". A double bond to a sulfur atom is represented herein as both "=S" and "(S)".

[0182] The phrases "parenteral administration" and "administered parenterally" as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and substernal injection and infusion.

[0183] The phrase "pharmacologically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit-risk ratio.

[0184] The phrases "pharmacologically acceptable excipient" or "pharmacologically acceptable carrier" as used herein refer to a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Some examples of substances that can function as pharma- ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and the like. (10) glycols, such as propylene glycol, (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol, (12) esters, such as ethyl oleate and ethyl laurate, (13) agar, (14) buffers, such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) phosphate buffer, and (21) other non-toxic compatible substances utilized in pharmaceutical formulations.

[0185] The term "salt" or "pharmaceutically acceptable salt" refers to salts derived from various organic and inorganic counterions well known in the art. Pharmaceutically acceptable acid addition salts can be formed with inorganic and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Pharmaceutically acceptable acid addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Organic bases from which salts may be derived include, for example, primary, secondary, and tertiary amines, naturally occurring substituted amines, cyclic amines, substituted amines including basic ion exchange resins, and the like, specifically, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In some embodiments, the pharma-ceutically acceptable base addition salts are selected from ammonium, potassium, sodium, calcium, and magnesium salts.

[0186] VI. Working Examples Example 1: Intravitreal injection of siRNA duplexes For intravitreal injections into mice (4-7 week old ICR mice, Envigo), a 33 gauge needle on a glass microsyringe (10 μL volume, Hamilton Company) was used. The eye was proptotically positioned and the needle was inserted through the scleral equator and into the vitreous chamber at an approximately 45 degree angle, taking care not to touch the posterior lens or retina. The vitreous was injected with 5 μg of siRNA duplex in 1 μL of phosphate buffered saline vehicle. The siRNA duplexes injected are shown in Table 2.

[0187] [Table 2]

[0188] Mice were euthanized 14 days after injection. Both eyes of each animal were enucleated and dissected along the equator, and the anterior and posterior hemispheres were placed in RNAlater. Total RNA was extracted from homogenized tissues and reverse transcribed into cDNA using the First-Strand III cDNA Synthesis kit. Normalized cDNA quantification was performed by real-time TaqMan PCR using fluorescently labeled TaqMan probe / primer sets for selected genes (ANGPTL7, MYOC, COL1A1, COL5A1, VCAN, FN1, and PPIA). Reactions were performed in 20 μL aliquots using TaqMan Universal PCR Master Mix No AmpErase UNG on an ABI Prism 7500 Fast Real-Time PCR System Sequence Detection System and analyzed by the 7500 System software. Relative quantification (RQ) values ​​between treated and untreated samples were calculated by the formula 2-ΔΔCT, where CT is the cycle of the threshold (automated measurement), ΔCT is the CT of the assayed gene minus the CT of the endogenous control (PPIA), and ΔΔCT is the ΔCT of the normalized assayed gene in treated samples minus the ΔCT of the same gene (calibrator) in untreated samples.

[0189] The results are shown in Table 3. ETD1208 was more effective than ETD1207, which was more effective than ETD1206. This indicates that having fewer phosphorothioates (or no phosphorothioates) at the 5' end of the sense strand to which the hydrophobic group is attached was more effective than having two phosphorothioates at the end.

[0190] ETD1588 was more active than ETD1589, meaning that some sequences showed greater activation with a 5' vinyl phosphonate on the antisense strand than without a 5' vinyl phosphonate on the antisense strand.

[0191] The ETD1716-ETD1787 series allows for different hydrophobic groups with 12 to 25 carbons. All were conjugated with phosphorothioate at the 5' end of the sense strand.

[0192] [Table 3]

[0193] Example 2: Intravitreal injection of mRNA and quantification of siRNA Intravitreal injection was performed with 20μg siRNA in 1μL as described above.Eyes were enucleated 14 days after injection, and mRNA was quantified as described in the previous example.The amount of siRNA was quantified using stem-loop assay according to published procedures (Cheng A, Li M, Liang Y, Wang Y, Wong L, Chen C, Vlassov AV, Magdaleno S 2009.Stem-loop RT-PCR quantification of siRNAs in vitro and in vivo.Oligonucleotides 19:203-208).

[0194] The results are shown in Table 4. There was a correlation between the size of the hydrophobic group, the percent knockdown, and the levels of siRNA observed in tissues. In general, hydrophobic groups with more carbons resulted in an increased percent knockdown and an increased level of siRNA observed in tissues.

[0195] [Table 4]

[0196] Example 3: siRNA-mediated knockdown of PLIN1 in mouse adipose tissue Four- to seven-week-old ICR mice (Envigo Labs) in group 1 (n=4) were administered 100 μL of phosphate-buffered saline (PBS) or 500 μg of siRNA targeting mouse PLIN1 in 100 μL of PBS by subcutaneous injection. Mice were then euthanized on day 14 and abdominal white fat samples were collected from each and placed in RNAlater (ThermoFisher catalog no. AM7020). Total liver RNA was prepared by homogenizing liver tissue in homogenization buffer (Maxwell RSC simplyRNA Tissue Kit) using a Percellys 24 tissue homogenizer (Bertin Instruments) set at 5000 rpm for two 10-s cycles. Total RNA from the lysates was purified on a Maxwell RSC 48 platform (Promega Corporation) following the manufacturer's recommendations. Hepatic PLIN1 mRNA levels were assessed by RT-qPCR using TaqMan assays for mouse PLIN1 and the mouse housekeeping gene PPIA (ThermoFisher, assay number Mm02342430_g1). Data were normalized to levels in PBS-treated animals.

[0197] The results are shown in Table 5. The addition of 5' vinyl phosphonate increased efficacy and enabled delivery to adipose tissue.

[0198] [Table 5]

[0199] Example 4: Injection for central nervous system delivery siRNAs with the modifications and hydrophobic conjugates described herein are injected intracerebroventricularly or intrathecally according to published procedures (Alterman, JF, Godinho, BMDC, Hassler, MR et al. A divalent siRNA chemical scaffold for potent and sustained modulation of gene expression throughout the central nervous system. Nat Biotechnol 37, 884-894 (2019). https: / / doi.org / 10.1038 / s41587-019-0205-0, Njoo, C., Heinl, C., Kuner, R. In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice. J. Vis. Exp. (85), e51229, doi:10.3791 / 51229 (2014)). Mice were euthanized 14 days after injection and brain hemispheres were removed, frozen, and then homogenized and tested for target mRNA and protein expression.

[0200] Example 5: siRNA Description in Bullet Points An example of an siRNA is illustrated as follows: 21-mer: a.19 base pairs b. 2nt overhang Antisense strand: a. Vinylphosphonate (VP) at the 5' end b. Two phosphorothioate bonds (PS) at each end Sense strand: a. Hydrophobic group (C16-C18) at the 5' end b. Optionally, 0 to 2 PS at the 5' end c. Two PS at the 3' end ●Modification pattern of 2' fluoro group and 2' methyl group.

[0201] Example 6: Modification motif 1 Examples of siRNAs include combinations of the following modifications: • Position 9 (5' to 3') of the sense strand is 2'F. If position 9 is a pyrimidine, then all purines in the sense strand are 2'OMe and 1 to 5 pyrimidines between positions 5 and 11 are 2'F, provided that there are not three 2'F modifications in a row. If position 9 is a purine, then all pyrimidines in the sense strand are 2'OMe and 1 to 5 purines between positions 5 and 11 are 2'F, provided that there are not three 2'F modifications in a row. • The odd-numbered positions of the antisense strand are 2'OMe, and the even-numbered positions are a mixture of 2'F and 2'deoxy.

[0202] Example 7: Modification motif 2 Examples of siRNAs include combinations of the following modifications: • Position 9 (5' to 3') of the sense strand is 2' deoxy. • Positions 5, 7, and 8 of the sense strand are 2'F. ● All pyrimidines at positions 10-21 are 2'OMe and the purines are a mixture of 2'OMe and 2'F. Alternatively, all purines at positions 10-21 are 2'OMe and all pyrimidines at positions 10-21 are a mixture of 2'OMe and 2'F. • The odd-numbered positions of the antisense strand are 2'OMe, and the even-numbered positions are a mixture of 2'F and 2'deoxy.

[0203] Example 8: Synthesis of ETL phosphoramidites

[0204] [ka]

[0205] Example 8A: Synthesis of ETL20 phosphoramidite

[0206] [ka]

[0207] Synthesis of N-(4-hydroxyphenethyl)palmitamide (5):

[0208] 12.82 grams of 1 (palmitic acid) was weighed out and dissolved in 450 mL of CHCl. ​​To the solution of 1 was added 16.3 mL of diisopropylethylamine (DIEA). Then 12.88 mL of 2 (perfluorophenyl 2,2,2-trifluoroacetic acid, "PFP") was added dropwise and the reaction was stirred for 10 minutes after addition was complete. To the solution of PFP activated acid was added 8.26 grams of 4 (4-(2-aminoethyl)phenol) via addition funnel, which was rinsed with 50 mL of CHCl. ​​The reaction was placed under argon and stirred overnight. After stirring overnight, a precipitate formed due to 5. The precipitate was collected via filtration and washed with 75 mL of MTBE that had been pre-chilled to -20°C. The white to off-white solid was dried under high vacuum overnight. The product was used in the next step without further purification.

[0209] [ka]

[0210] Synthesis of ETL20 phosphoramidite (6): To N-(4-hydroxyphenethyl)palmitamide 5 (5.2 grams) was added 100 mL of anhydrous ethyl acetate, followed by 250 mg of 3 angstrom molecular sieves. The mixture was stirred for 1 hour. The mixture was heated at 50° C. to obtain a clear solution. 7.3 mL of DIEA was added and the mixture was placed in an ice bath, causing the solution to turn cloudy. To this cloudy solution was slowly added 3-((chloro(diisopropylamino)phosphaneyl)oxy)propanenitrile (3.5 mL). After the addition was complete, the reaction mixture was removed from the ice bath and stirred overnight at room temperature under Ar. The reaction mixture was then diluted with ethyl acetate (200 mL) and washed with saturated NaHCO3 solution (2×50 mL) followed by brine (50 mL). The solution was dried over Na2SO4, filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography, eluting with 5%-30% ethyl acetate in hexane containing 2% triethylamine.

[0211] Hydroxybenzylamine and lauric acid (C 12 ) or myristic acid (C 14 ) to generate ETL20 phosphoramidites with ETL18 (C 14 ) and ETL19(C 12 ) phosphoramidite was synthesized.

[0212] Example 9: Intravitreal injection of siRNA duplexes For intravitreal injections into mice (4-7 week old ICR mice, Envigo), a 33 gauge needle on a glass microsyringe (10 μL volume, Hamilton Company) was used. The eye was proptotically positioned and the needle was inserted through the scleral equator and into the vitreous chamber at an approximately 45 degree angle, taking care not to touch the posterior lens or retina. The vitreous was injected with 2.5 or 1 μg of siRNA duplex in 1 μL of phosphate buffered saline vehicle. The siRNA duplexes injected are listed in Table 1.

[0213] [Table 6]

[0214] Mice were euthanized 14 days after injection. Both eyes of each animal were enucleated and dissected along the equator, and the anterior and posterior hemispheres were placed in RNAlater. Total RNA was extracted from homogenized tissues and reverse transcribed into cDNA using the First-Strand III cDNA Synthesis kit. Normalized cDNA quantification was performed by real-time TaqMan PCR using fluorescently labeled TaqMan probe / primer sets for selected genes (ANGPTL7, MYOC, COL1A1, COL5A1, VCAN, FN1, and PPIA). Reactions were performed in 20 μL aliquots using TaqMan Universal PCR Master Mix No AmpErase UNG on an ABI Prism 7500 Fast Real-Time PCR System Sequence Detection System and analyzed by the 7500 System software. Relative quantification (RQ) values ​​between treated and untreated samples were calculated by the formula 2-ΔΔCT, where CT is the cycle of the threshold (automated measurement), ΔCT is the CT of the assayed gene minus the CT of the endogenous control (PPIA), and ΔΔCT is the ΔCT of the normalized assayed gene in treated samples minus the ΔCT of the same gene (calibrator) in untreated samples.

[0215] In particular, the ETD1928-1930 series demonstrates the utility of different hydrophobic groups with 12 to 16 carbons attached to a tyramine. The hydrophobic group was conjugated to the 5' end of the sense strand without phosphorothioate.

[0216] [Table 7]

[0217] Example 10: Intraventricular injection Mice were induced into anesthesia in an induction chamber with 1.2% isoflurane vaporized in 1.0 L / min oxygen, then transferred to a stereotaxic frame and immobilized while still anesthetized with 0.8% isoflurane through a mask. After the skull was exposed and needle placement, a single intraventricular injection (5 μl, artificial cerebrospinal fluid as vehicle) was administered in 500 nl min at coordinates of −0.2 mm anteroposterior, 0.8 mm mediolateral, and −2.5 mm dorsoventral from bregma using a Standard U-Frame Stereotaxic Instrument for Mouse (Harvard Apparatus 75-1808) and Stereotaxic Anesthesia Adapters with Anesthesia Masks (Harvard Apparatus 75-1856). -1 So I went.

[0218] [Table 8]

[0219] Mice were euthanized 14 days after injection. The brain was removed from each animal and dissected into right and left hemispheres.

[0220] Total RNA was extracted from homogenized tissues and reverse transcribed into cDNA using the First-Strand III cDNA Synthesis Kit. Normalized cDNA quantification was performed by real-time TaqMan PCR using fluorescently labeled TaqMan probe / primer sets for selected genes (MTRES1, MYOC, COL1A1, COL5A1, VCAN, FN1, and PPIA). Reactions were performed in 20 μL aliquots using TaqMan Universal PCR Master Mix No AmpErase UNG on an ABI Prism 7500 Fast Real-Time PCR System Sequence Detection System and analyzed by the 7500 System software. Relative quantification (RQ) values ​​between treated and untreated samples were calculated by the formula 2-ΔΔCT, where CT is the cycle of the threshold (automated measurement), ΔCT is the CT of the assayed gene minus the CT of the endogenous control (PPIA), and ΔΔCT is the ΔCT of the normalized assayed gene in treated samples minus the ΔCT of the same gene (calibrator) in untreated samples.

[0221] [Table 9]

[0222] Example 11: Intrathecal injection in rats Male Sprague Dawley rats (n=2) were used for intrathecal (IT) injection of siRNAs. Duplex ETD02273 was administered at 30 mg ml -1 and administered as a 30-μl IT injection via lumbar puncture into the area dorsal to the spine between the L3-L5 vertebral bodies of rats. Duplex ETD02210 was administered at 9 mg ml -1 and administered as a 100-μl IT injection via lumbar puncture into the area dorsal to the spine between the L3-L5 vertebral bodies of rats.

[0223] Rats were anesthetized with isoflurane and then placed on a warm heating pad. The IT injection site was shaved and disinfected. An incision was made to expose the spinal column. siRNA was administered using a 30-gauge insulin syringe. Proper needle placement was confirmed by CSF reflux at the needle hub or tail flick. After completion of siRNA administration, gentle, constant pressure on the plunger was held for 30 seconds. The incision was sutured and secured with tissue adhesive. Rats were placed in sternal recumbency on a heating pad until recovery.

[0224] Rats were euthanized 14 days after injection. Liver, right / left (R / L) frontal cortex, R / L temporal cortex, hippocampus, cerebellum, and spinal cord samples were collected from each rat and placed in RNAlater (ThermoFisher Cat. No. AM7020) until processing.

[0225] Total liver RNA was prepared by homogenizing liver tissue in homogenization buffer (Maxwell RSC simplyRNA Tissue Kit) using a Percellys 24 tissue homogenizer (Bertin Instruments) set at 5000 rpm for two 10-second cycles. Total RNA from the lysate was purified on a Maxwell RSC 48 platform (Promega Corporation) according to the manufacturer's recommendations. cDNA preparation was performed using Quanta qScript cDNA SuperMix (VWR, catalog no. 95048-500) according to the manufacturer's instructions.

[0226] Relative levels of hepatic MTRES1 mRNA were assessed by RT-qPCR in triplicate on a QuantStudio™ 6 Pro Real-Time PCR System using TaqMan assays for rat MTRES1 (ThermoFisher, Assay No. Rn01441122_m1) and the rat housekeeping gene PPIA (ThermoFisher, Assay No. Rn03302269_gH), as well as PerfeCTa® qPCR FastMix®, Low ROX™ (VWR, Cat. No. 101419-222).

[0227] Data was normalized to the average MTRES1 mRNA levels in untreated animals. The results are shown in Tables 10 and 11 below.

[0228] [Table 10]

[0229] [Table 11]

[0230] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present invention. It is understood that various alternatives to the embodiments of the present invention described herein may be utilized in the practice of the present invention. The following claims define the scope of the present invention, and it is intended that methods and compositions within the scope of the claims, and equivalents thereof, be covered thereby.

Claims

1. A composition, said composition is Sense strand, antisense strand, and the following structure: 【Chemistry 1】 ligands containing, small interfering RNAs (siRNAs) containing Includes, A composition in which, in the formula, the dashed line indicates a bond to the 5' or 3' end of the sense chain or the antisense chain, n is 0 to 3, and R is an alkyl group containing 4 to 20 carbon atoms.

2. The composition has the following structure: 【Chemistry 2】 The composition according to claim 1, comprising, wherein the binding site of any of the ligands may be at the 3' oligonucleotide terminus or the 5' oligonucleotide terminus.

3. The ligand is (a) bonded to the 5' end of the sense chain or the antisense chain, (b) By binding to the 5' end of the sense chain or the antisense chain via a phosphate, or (c) The composition according to claim 1, wherein the sense chain is bonded to the end of the sense chain.

4. (a) The antisense chain is (i) 5' vinylphosphonate, (ii) One or two 5' phosphorothioate bonds, (iii) One or two 3'-phosphorothioate bonds Includes, (b) The sense chain is (i) One or two 5' phosphorothioate bonds, (ii) 5' phosphate bond, or (iii) One or two 3'-phosphorothioate bonds The composition according to claim 1, comprising:

5. The composition according to claim 1, wherein the sense chain does not contain one or two 5'-phosphorothioate bonds.

6. (a) With respect to the sense chain, All purine nucleosides contain a 2'-fluoro compound, and all pyrimidine nucleosides are modified with a mixture of a 2'-fluoro compound and a 2'-O-methyl compound. All purine nucleosides contain 2'-O-methyl, and all pyrimidine nucleosides are modified with a mixture of 2'-fluoro and 2'-O-methyl. All purine nucleosides contain a 2'-fluoro compound, and all pyrimidine nucleosides contain a 2'-O-methyl compound. All pyrimidine nucleosides contain a 2'-fluoro compound, and all purine nucleosides are modified with a mixture of a 2'-fluoro compound and a 2'-O-methyl compound. All pyrimidine nucleosides contain 2'-O-methyl, and all purine nucleosides are modified with a mixture of 2'-fluoro and 2'-O-methyl, or One of the following is true: all pyrimidine nucleosides contain a 2'-fluoro compound, and all purine nucleosides contain a 2'-O-methyl compound. However, in all of the above, the sense chain may contain a 2'-deoxynucleoside. (b) With respect to the antisense chain, All purine nucleosides contain a 2'-fluoro compound, and all pyrimidine nucleosides are modified with a mixture of a 2'-fluoro compound and a 2'-O-methyl compound. All purine nucleosides contain 2'-O-methyl, and all pyrimidine nucleosides are modified with a mixture of 2'-fluoro and 2'-O-methyl. All purine nucleosides contain 2'-O-methyl, and all pyrimidine nucleosides contain 2'-fluoro. All pyrimidine nucleosides contain a 2'-fluoro compound, and all purine nucleosides are modified with a mixture of a 2'-fluoro compound and a 2'-O-methyl compound. All pyrimidine nucleosides contain 2'-O-methyl, and all purine nucleosides are modified with a mixture of 2'-fluoro and 2'-O-methyl, or The composition according to claim 1, wherein all pyrimidine nucleosides contain 2'-O-methyl and all purine nucleosides contain 2'-fluoro.

7. A composition, wherein the composition is Sense strand and antisense strand, as well as the following structure: 【Transformation 3】 ligands containing, small interfering RNAs (siRNAs) containing Includes, A composition in which, in the formula, the dashed line indicates a bond to the 5' or 3' end of the sense chain or the antisense chain, and R is an alkyl group containing 4 to 20 carbon atoms, provided that R is not octane.

8. The composition according to claim 7, wherein the ligand is bound to the 5' end of the sense chain.

9. With respect to the sense chain or the antisense chain, provided that the sense chain or the antisense chain may contain a 2'-deoxynucleoside, All purine nucleosides contain a 2'-fluoro compound, and all pyrimidine nucleosides are modified with a mixture of a 2'-fluoro compound or a 2'-O-methyl compound. All purine nucleosides contain 2'-O-methyl, and all pyrimidine nucleosides are modified with a mixture of 2'-fluoro or 2'-O-methyl. All purine nucleosides contain a 2'-fluoro compound, and all pyrimidine nucleosides contain a 2'-O-methyl compound. All pyrimidine nucleosides contain a 2'-fluoro compound, and all purine nucleosides are modified with a mixture of a 2'-fluoro compound and a 2'-O-methyl compound. All pyrimidine nucleosides contain 2'-O-methyl, and all purine nucleosides are modified with a mixture of 2'-fluoro and 2'-O-methyl. All pyrimidine nucleosides contain 2'-O-methyl, and all purine nucleosides contain 2'-fluoro, or The composition according to claim 7, wherein all pyrimidine nucleosides contain a 2'-fluoro compound, and all purine nucleosides contain a 2'-O-methyl compound.

10. A pharmaceutical composition comprising the composition according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier, excipient, or diluent. 【Request Item 11】 【Chemistry 4】 A compound containing [this compound].

12. A composition for use in reducing a target mRNA or target protein of a subject that requires a reduction of the target mRNA or target protein, wherein the composition is (a) The following structure: 【Transformation 5】 Compounds represented by (wherein the formula, the dashed line indicates a bond to the 5' or 3' end of the sense or antisense chain, n is 0 to 3, and R is an alkyl group containing 4 to 20 carbon atoms), or (b) The following structure: 【Transformation 6】 A composition comprising a compound represented by (wherein the formula, the dashed line indicates a bond to the 5' or 3' end of a sense or antisense chain, and R is an alkyl group containing 4 to 20 carbon atoms, provided that R is not octane).

13. The composition according to claim 12, wherein the composition is administered by subcutaneous, intravitreous, intrathecal, or intraventricular administration.

14. The following structure: 【Transformation 7】 A composition comprising, in the formula, R contains an alkyl group containing 4 to 20 carbon atoms. A composition in which n is 1 to 3.

15. (a) The following structure: 【Transformation 8】 Compounds represented by (wherein the formula, the dashed line indicates a bond to the 5' or 3' end of the sense or antisense chain, n is 0 to 3, and R is an alkyl group containing 4 to 20 carbon atoms), or (b) The following structure: 【Chemistry 9】 Compounds represented by (wherein the formula, the dashed line indicates a bond to the 5' or 3' end of the sense or antisense chain, and R is an alkyl group containing 4 to 20 carbon atoms, provided that R is not octane) A method for synthesizing, A method comprising the step of reacting the composition according to claim 14 with an oligonucleotide.

Images